Methods and systems for menstrual cycle analysis

Abstract

The paper provides samples, a system for collecting samples, and a method for preserving samples from menstrual fluid.

Description

[0001] Cross-references

[0002] This application claims the benefits of U.S. Patent Application No. 62 / 929,579, filed November 1, 2019; U.S. Patent Application No. 62 / 930,465, filed November 4, 2019; and U.S. Patent Application No. 63 / 061,709, filed August 5, 2020, all of which are incorporated herein by reference in their entirety. Background Technology

[0003] Chronic pelvic pain (CPP), dysmenorrhea, and infertility are symptoms that prompt women to seek medical care for conditions associated with menstrual cycle abnormalities, such as undiagnosed endometriosis. For example, the prevalence of endometriosis is as high as 70% in women with CPP and 30-50% in women presenting to IVF clinics for infertility. The presentation of these symptoms, along with the type of endometriosis, often determines the treatment options. Treatment options include surgery and pain management via hormone therapy and / or GnRH analogs. However, reimbursement for GnRH analogs varies considerably and can be limited by surgical confirmation of the disease, making a surgical diagnosis of endometriosis a necessary step to receive appropriate care. This not only significantly increases costs for the healthcare system but also for individuals with the disease and contributes to an overall lag in time to diagnosis. Even with surgical intervention, 50% of patients experience recurrence; highlighting that endometriosis exhibits a cyclical reactivation state regardless of surgical or treatment interventions.

[0004] On average, it takes ten years from the onset of symptoms to diagnosis of endometriosis, which allows adhesions and scar tissue to form in the reproductive system, hindering function and often causing severe pain. Furthermore, the combination of painful menstruation and a lack of clear diagnosis can lead to psychological distress and depression in affected women. Summary of the Invention

[0005] In some embodiments, methods for preparing a menstrualome fingerprint are disclosed herein. In some embodiments, the method includes: (a) obtaining a first sample and a second sample from a subject, wherein the first sample and the second sample comprise cervical vaginal fluid or menstrual fluid collected onto a first absorbent sample collector and a second absorbent sample collector; (b) eluting the first sample and the second sample from the first sample collector and the second sample collector, respectively, into an aqueous buffer; (c) isolating biological material from each of the first sample and the second sample; and (d) constructing a sample menstrualome fingerprint, wherein the sample menstrualome fingerprint includes levels and / or differences in the presence of multiple menstrualome biomarkers from the biological material of the first sample and the second sample. In some embodiments, the biological material includes one or more biological materials selected from: RNA, DNA, methylated nucleic acids, miRNA, proteins, protein-nucleic acid complexes, microorganisms, and mammalian cell types. In some embodiments, constructing the sample menstrualome fingerprint in (d) includes measuring the extracted biological material from the first sample and the second sample to identify multiple biomarkers. In some embodiments, the plurality of menstrual group biomarkers includes biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid between two or more health states. In some embodiments, the plurality of menstrual group biomarkers includes biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid compared to peripheral blood, cervical vaginal tissue, or longitudinal menstrual samples. In some embodiments, the method further includes (e) comparing the sample menstrual group fingerprint with a reference menstrual group fingerprint. In some embodiments, the reference menstrual group fingerprint includes threshold levels or presence of the plurality of menstrual group biomarkers associated with health states. In some embodiments, the first sample and the second sample comprise biological material collected from the subject at different time points. In some embodiments, the time points are separated by time intervals between about 15 minutes and about 30 days, about 60 days, or about 90 days. In some embodiments, the time points include different numbers of days within the subject's menstrual cycle. In some embodiments, the time points are within a single menstrual cycle. In some embodiments, the time points include days within different menstrual cycles. In some embodiments, the time point occurs during one or more days of the subject's menstrual cycle. In some embodiments, one time point occurs during the subject's menstrual cycle while another time point does not occur during the subject's menstrual cycle. In some embodiments, the sample collector is an intravaginal sample collector. In some embodiments, the sample collector preserves the biological material in its intact state.In some embodiments, the sample collector is capable of absorbing at least 3 ml of fluid. In some embodiments, the sample collector is placed in a buffer solution after the sample is collected. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers include the methylation status of multiple loci. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the expression levels of multiple genes. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the presence and / or levels of multiple miRNAs. In some embodiments, the biological material is cells, and the multiple menstrual biomarkers measure the presence and / or quantity of one or more cell types. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure the presence and / or level of one or more microorganisms. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure microbial diversity. In some embodiments, the two or more health states include those prior to and after medical treatment. In some embodiments, the health state includes a preoperative health state. In some embodiments, the reference state includes a postoperative health state. In some embodiments, the health status includes menstrual disorders. In some embodiments, the health status includes endometriosis. In some embodiments, the health status includes a healthy patient. In some embodiments, the healthy reference menstrual group fingerprint includes principal component analysis, t-distributed random nearest neighbor embedding, heatmap, diversity index, or a combination thereof.

[0006] On the other hand, this document discloses a method for preparing a menstrual fingerprint. In some embodiments, the method includes: (a) obtaining a first sample and a second sample from a subject, wherein the first sample and the second sample comprise cervical vaginal fluid or menstrual fluid collected on a first absorbent sample collector and a second absorbent sample collector; (b) eluting the first sample and the second sample from the first sample collector and the second sample collector, respectively, into an aqueous buffer; (c) isolating biological material from each of the first sample and the second sample; and (d) constructing a sample menstrual fingerprint, wherein the sample menstrual fingerprint includes differences in the levels and / or presence of multiple menstrual biomarkers in the biological material from the first sample and / or the second sample compared to a reference menstrual fingerprint. In some embodiments, the biological material includes one or more biological materials selected from: RNA, DNA, methylated nucleic acids, miRNA, proteins, protein-nucleic acid complexes, microorganisms, and mammalian cell types. In some embodiments, constructing the sample menstrual fingerprint in (d) includes measuring extracted biological material from the first and second samples to identify multiple biomarkers. In some embodiments, the multiple menstrual biomarkers include biomarkers showing different presence or levels in cervical vaginal fluid or menstrual fluid between two or more health states. In some embodiments, the multiple menstrual biomarkers include biomarkers showing different presence or levels in cervical vaginal fluid or menstrual fluid compared to peripheral blood, cervical vaginal tissue, or longitudinal menstrual samples. In some embodiments, the reference menstrual fingerprint includes threshold levels or presence of the multiple menstrual biomarkers associated with a health state. In some embodiments, the first and second samples comprise biological material collected from the subject at different time points. In some embodiments, the time points are separated by time intervals between approximately 15 minutes and approximately 30 days, approximately 60 days, or approximately 90 days. In some embodiments, the time points include different numbers of days within the subject's menstrual cycle. In some embodiments, the time points are within a single menstrual cycle. In some embodiments, the time points include days within different menstrual cycles. In some embodiments, the time points occur during one or more days of the subject's menstrual cycle. In some embodiments, one time point occurs during the subject's menstrual cycle while another time point does not occur during the subject's menstrual cycle. In some embodiments, the sample collector is an intravaginal sample collector. In some embodiments, the sample collector preserves the biological material in its intact state. In some embodiments, the sample collector is capable of absorbing at least 3 ml of fluid. In some embodiments, the sample collector is placed in a buffer solution after collecting the sample.In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers include the methylation status of multiple loci. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the expression levels of multiple genes. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the presence and / or levels of multiple miRNAs. In some embodiments, the biological material is cells, and the multiple menstrual biomarkers measure the presence and / or quantity of one or more cell types. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure the presence and / or level of one or more microorganisms. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure microbial diversity. In some embodiments, the two or more health states include those prior to and after medical treatment. In some embodiments, the health state includes a preoperative health state. In some embodiments, the reference state includes a postoperative health state. In some embodiments, the health state includes menstrual disorders. In some embodiments, the health state includes endometriosis. In some embodiments, the health state includes a healthy patient. In some implementations, the healthy reference menstrual group fingerprint includes principal component analysis, t-distributed random nearest neighbor embedding, heatmap, diversity index, or a combination thereof.

[0007] On the other hand, this document discloses a method for preparing a menstrual fingerprint. In some embodiments, the method includes: (a) obtaining a first sample from a subject, wherein the first sample comprises cervical vaginal fluid or menstrual fluid collected on an absorbent sample collector; (b) eluting the first sample from the sample collector into an aqueous buffer; (c) isolating biological material from the first sample; (d) constructing a sample menstrual fingerprint, wherein the sample menstrual fingerprint includes the levels and / or presence of multiple menstrual biomarkers in the biological material from the first sample; and (e) comparing the sample menstrual fingerprint with a reference fingerprint. In some embodiments, the reference fingerprint includes the levels and / or presence of multiple menstrual biomarkers in a subject reference group. In some embodiments, the reference fingerprint includes the levels and / or presence of multiple menstrual biomarkers in the subject at a previous time point. In some embodiments, the reference menstrual fingerprint includes threshold levels or presence of the multiple menstrual biomarkers associated with health status. In some embodiments, the reference menstrual fingerprint includes threshold levels or presence of the plurality of menstrual biomarkers associated with a health status. In some embodiments, the plurality of menstrual biomarkers includes biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid between two or more health statuses. In some embodiments, the two or more health statuses include before and after medical treatment. In some embodiments, the health status includes preoperative health status. In some embodiments, the reference status includes postoperative health status. In some embodiments, the health status includes menstrual disorders. In some embodiments, the health status includes endometriosis. In some embodiments, the health status includes a healthy patient. In some embodiments, the biological material includes one or more biological materials selected from: RNA, DNA, methylated nucleic acids, miRNA, proteins, protein-nucleic acid complexes, microorganisms, and mammalian cell types. In some embodiments, constructing the sample menstrual fingerprint in (d) includes determining extracted biological material from the first and second samples to identify multiple biomarkers. In some embodiments, the plurality of menstrual group biomarkers include biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid compared to peripheral blood, cervical vaginal tissue, or longitudinal menstrual samples. In some embodiments, the first sample and reference sample comprise biological material collected from the subject at different time points. In some embodiments, the sample collector is an intravaginal sample collector. In some embodiments, the sample collector preserves the biological material in an intact state. In some embodiments, the sample collector is capable of absorbing at least 3 ml of fluid.In some embodiments, the sample collector is placed in a buffer solution after the sample is collected. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers include the methylation status of multiple loci. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the expression levels of multiple genes. In some embodiments, the biological material is RNA, and the multiple menstrual biomarkers include the presence and / or levels of multiple miRNAs. In some embodiments, the biological material is cells, and the multiple menstrual biomarkers measure the presence and / or quantity of one or more cell types. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure the presence and / or level of one or more microorganisms. In some embodiments, the biological material is DNA, and the multiple menstrual biomarkers measure microbial diversity. In some embodiments, the healthy reference menstrual fingerprint includes principal component analysis, t-distributed random nearest neighbor embedding, heatmap, diversity index, or a combination thereof.

[0008] In a further aspect, this document discloses a method for preparing a menstrual fingerprint. In some embodiments, the method includes: (a) obtaining a first sample and a second sample from a subject with or suspected of having endometriosis, wherein the first sample and the second sample comprise cervical vaginal fluid or menstrual fluid collected on an absorbent sample collector; (b) eluting the first sample and the second sample from the first sample collector and the second sample collector, respectively, into an aqueous buffer; (c) isolating biological material from each of the first sample and the second sample; and (d) constructing a sample menstrual fingerprint, wherein the sample menstrual fingerprint includes levels and / or differences in the presence of multiple menstrual biomarkers from the biological material of the first sample and the second sample. In some embodiments, the biological material includes one or more biological materials selected from: RNA, DNA, methylated nucleic acids, miRNA, proteins, protein-nucleic acid complexes, microorganisms, and mammalian cell types. In some embodiments, constructing the sample menstrual fingerprint in (d) includes measuring the extracted biological material from the first sample and the second sample to identify multiple biomarkers. In some embodiments, the biological material is miRNA and the plurality of biomarkers include those selected from let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2-3p, miR-17-3p, and m iR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,miR-200c-3p,m iR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-222-3p,miR- 224-5p,miR-23b-3p,miR-27b-3p,miR-28-3p,miR-30a-3p,miR-30a-5p,miR-34 a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409 and miR-98-5p miRNAs.According to the method of claim 93, the miRNA is selected from miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p. In some embodiments, the plurality of biomarkers includes methylation profiles of one or more CpG sites selected from the CpG sites in Table 4. In some embodiments, the microorganism is a bacterial genera selected from the following genera: *Atopobium*, *Propionibacterium*, *Dialister*, *Porphyromonas*, *Streptococcus*, *Dermabacter*, *Moraxella*, *Anaerococcus*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Facklamia*, and *Klebsiella*. In some embodiments, the mammalian cell type is selected from endothelial cells, epithelial cells, leukocytes, mesenchymal cells, and combinations thereof. In some embodiments, the method further includes (e) comparing the sample menstrual fingerprint with a reference menstrual fingerprint. In some embodiments, the reference menstrual fingerprint includes threshold levels or presence of the plurality of menstrual biomarkers associated with a health status. In some embodiments, the health status includes preoperative health status. In some embodiments, the reference status includes postoperative health status. In some embodiments, the first sample and the second sample comprise biological material collected from the subject at different time points. In some embodiments, the time points are separated by a time interval between approximately 15 minutes and approximately 30 days. In some embodiments, the time points include different numbers of days within the subject's menstrual cycle. In some embodiments, the time points are within a single menstrual cycle. In some embodiments, the time points include days within different menstrual cycles. In some embodiments, the time points occur during one or more days of the subject's menstrual cycle. In some embodiments, one time point occurs during the subject's menstrual cycle while another time point does not occur during the subject's menstrual cycle. In some embodiments, the sample collector is an intravaginal sample collector. In some embodiments, the sample collector preserves the biological material in its intact state. In some embodiments, the sample collector is capable of absorbing at least 3 ml of fluid.In some implementations, the sample collector is placed in a buffer solution after the sample is collected.

[0009] Incorporation

[0010] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference.

[0011] Brief description of the attached figures

[0012] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of the invention will be gained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing this principle, in which:

[0013] Figure 1A-Figure 1B The figure illustrates the time-series data of RNA-Seq. Figure 1A Principal component analyses comparing menstrual blood, whole blood, and cervical vaginal fluid are presented. Figure 1B Principal component analysis comparing menstrual blood and whole blood is shown. Figure 1C The tSNE dimension analysis comparing menstrual blood, whole blood, and cervical vaginal fluid is shown. Figure 1D The tSNE dimension analysis comparing menstrual blood and whole blood is shown.

[0014] Figure 2A The diagram illustrates the timeline of the menstrual cycle. Figure 2B The diagram illustrates the changes in gene expression of cell-specific markers over time. MUC21 and ALOX12 represent cervical and vaginal specific expression. SPRR2F represents ovarian and fallopian tube specific expression. PAEP represents endometrial specific expression. The vertical dashed line represents day 2 of the woman's cycle.

[0015] Figure 3 This study showed that endometriosis and EMT share 11 Kegg pathways.

[0016] Figures 4A-4E The diagram illustrates bacterial diversity in cervical and vaginal fluids and menstrual fluids from patients who were considered "truly healthy," "suspected unhealthy," had endometriosis, and PCOS. A total of 79 patients were analyzed (5 individuals with PCOS, 19 with endometriosis, 5 truly healthy individuals, and 50 suspected unhealthy individuals). Box plots represent β-diversity, while individual points represent α-diversity in a single sample. Figure 4A The illustration shows the bacterial diversity present in cervical vaginal fluid. Figure 4B The illustration shows the diversity of bacteria present in menstrual fluid. Figure 4CThe bacterial genera described were found to be more abundant in menstrual fluid than in cervical vaginal fluid. Figure 4D The correlation between the number of excess bacterial strains and the health status in the patient cohort was depicted. Figure 4E A comparison of bacterial genera abundance in menstrual blood was depicted in a healthy cohort.

[0017] Figure 5 A cross-sectional view of an embodiment of the system described herein is shown.

[0018] Figures 6A-6D The illustration shows a perspective view of an implementation of the system. Figure 6A The diagram illustrates a complete perspective view of the system implementation. Figure 6B The diagram shows... Figure 6A A perspective view of the upper and central portions of the system implementation. Figure 6C The diagram shows... Figure 6A The bottom perspective view of the implementation of the system. Figure 6D The diagram shows... Figure 6A Additional perspective views of the implementation of the system.

[0019] Figures 7A-7C The diagram illustrates the implementation of the system. Figure 7A The central and lower portions of the system implementation before coupling the upper portion are shown. Figure 7B This shows what happens after starting the upper part. Figure 7A The implementation method of the system. Figure 7C This shows what happens after the lower part is started. Figure 7A The implementation method of the system.

[0020] Figures 8A-8C A cross-sectional view is shown during an implementation of the system. Figure 8A A cross-sectional view of an implementation of the system after the sample collector has been inserted is shown. Figure 8B This shows what happens after starting the upper part. Figure 8A A cross-sectional view of an implementation of the system. Figure Figure 8C This shows what happens after the lower part is started. Figure 8A A cross-sectional view of the implementation method of the system.

[0021] Figure 9 This is a heatmap diagram illustrating the clustering of cervical vaginal fluid samples and menstrual fluid samples over a period of time.

[0022] Figures 10A-10E Depicting by Figure 9 The five cluster-regulated Kegg pathways are shown in the figure.

[0023] Figure 11A It is a principal component analysis of the differential methylation sites in menstrual blood and whole blood. Figure 11BThis is a tSNE dimension analysis of the differential methylation sites in menstrual blood and whole blood.

[0024] Figure 12A The location of differentially methylated CpGs is shown when whole blood is compared with menstrual blood. Figure 12B The differentially methylated regions between whole blood and menstrual blood were shown.

[0025] Figure 13A It is principal component analysis of miRNA sequencing in menstrual blood and whole blood. Figure 13B This is a tSNE dimension analysis of miRNA sequencing in menstrual blood and whole blood.

[0026] Figure 13C A volcano plot depicting gene expression changes between all controls and all endometriosis patients was created. Figure 13D Volcano plots depicting gene expression changes between healthy patients and patients with endometriosis before surgery (left panel) and between healthy patients and patients with endometriosis after surgery (right panel).

[0027] Figure 14 The KEGG pathway associated with differentially regulated miRNAs was depicted.

[0028] Figure 15A The characteristics of bacterial genera that are uniquely present in patients with preoperative endometriosis were described. Figure 15B The characteristics of bacterial genera that are unique to postoperative endometriosis patients were described.

[0029] Figure 16A tSNE clustering depicts methylation patterns in menstrual blood samples from different patients. Figure 16B Methylation clusters are depicted as distinct queues. Figure 16C The abundance of Lactobacillus spp. in menstrual blood samples from each patient was depicted. Detailed Implementation

[0030] This article provides non-invasive methods for detecting menstrual disorders (such as early detection of endometriosis) and for analyzing menstrual and non-menstrual vaginal fluids. In some implementations, non-invasive methods for detecting endometriosis reduce the need for surgical diagnosis, provide clinicians with the ability to inform them about patient management, and / or allow monitoring of the effectiveness of interventions. This article also provides samples collected from menstrual fluids, systems for collecting samples, and methods for detecting endometriosis from samples collected from menstrual fluids.

[0031] The terminology used herein is for descriptive purposes only and is not intended to be limiting. In some embodiments, the following terms are discussed to illustrate the meaning of terms used in this specification, in addition to their understanding by those skilled in the art. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. It is also noted that the drafting of the claims excludes any optional elements. Therefore, this statement is intended as a priori basis for the use of exclusive terms such as “unique,” ​​“only,” etc., when referencing elements of a claim or using the “negative” limitation.

[0032] Certain ranges are presented herein with numerical values ​​beginning with the term “about.” The term “about” is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximate to the number that follows the term. In some embodiments, in determining whether a number is close to or approximates a specifically listed number, the close or approximate unlisted number is a substantially equivalent number to the specifically listed number provided in the context of its presentation. Where numerical ranges are provided, it should be understood that each intermediate value between the upper and lower limits of the range up to one-tenth of the lower limit unit (unless otherwise expressly specified in the context), as well as any other specified value or intermediate value within the range, is included in the methods and compositions described herein. In some embodiments, the upper and lower limits of these smaller ranges are independently included within the smaller range and are also included in the methods and compositions described herein, but are subject to any express exclusions within the range. Where the range includes one or both of the included limits, the range excluding one or both of those included limits is also included in the methods and compositions described herein.

[0033] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably. No term should be construed as requiring supervision by a medical professional (e.g., a physician, nurse, physician assistant, healthcare worker, or hospice worker). As used herein, a subject is any animal, including mammals (e.g., humans or non-human animals). In one embodiment of the methods and compositions provided herein, the mammal is a human. In some embodiments, the subject is female.

[0034] As used herein, the term "nucleic acid" can generally refer to a polymer of nucleotides of any length, ribonucleotides and / or deoxyribonucleotides. Therefore, these terms include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), mitochondrial DNA (mtDNA), mitochondrial RNA (mtRNA), guide RNA (gRNA), messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free DNA (cfDNA), cell-free RNA (cfRNA), DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0035] As used herein, the term "menstrual community" generally refers to the following as a whole: molecules found in menstrual fluid, molecules separated from cells found in menstrual fluid, and cells found in menstrual fluid, as well as information determined from these molecules and cells. In some cases, molecules are nucleic acids such as DNA or RNA, proteins, metabolites, or combinations thereof. In some cases, cells are endometrial cells, non-endometrial cells such as immune cells and stem cells, bacterial cells, or combinations thereof. In some embodiments, molecules or cells are derived from an individual or an individual's vaginal microbiome. Information determined from molecules includes, but is not limited to, for example, the sequence and / or methylation pattern of a DNA sequence, the expression level, abundance, or presence of the molecule of interest. Information determined from cells includes, but is not limited to, for example, the presence or abundance of the cell of interest, including its cell surface markers.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions described herein pertain. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the methods and compositions described herein, representative illustrative methods and materials are described hereafter.

[0037] menstrual fluid sample

[0038] In some embodiments of the methods and systems provided herein, a sample collector that collects fluids from the vaginal cavity is used to collect fluid biofluid samples, such as menstrual fluid samples or samples of another fluid, from a subject. In some embodiments, the sample collector is placed inside or outside the vagina for sample collection. In some embodiments, the sample collector collects samples by pooling, holding, capturing, guiding, or absorbing the sample. In some embodiments, the sample collector is absorbent, semi-absorbent, or non-absorbent. In some embodiments, the sample collector is soluble in a buffer solution. In some embodiments, the sample collector is degraded, for example, by exposing it to an acidic environment, an alkaline environment, or an enzyme. In some embodiments, the sample collector includes a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. In some embodiments, more than one type of sample collector is used.

[0039] In some embodiments of the methods and systems provided herein, the sample collector is held in place for a predetermined period of time to collect biological samples. In some embodiments, this takes at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours. In some embodiments, the sample collection device remains in place for a maximum of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours. In some embodiments, the sample collector remains in place for approximately 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0040] In some embodiments of the methods and systems provided herein, samples are collected during the subject's menstrual window (menstrual period). In some embodiments, the sample collector is disposable. In some embodiments, the disposable sample collector is discarded or decomposed after use. In some embodiments, the disposable sample collector is soluble, biodegradable, recyclable, or compostable. In some embodiments, one disposable sample collector is used to collect one sample from one subject. In some embodiments, the sample collector is reusable. In some embodiments, the reusable sample collector is washable, sterilizable, or autoclaved. In some embodiments, the reusable sample collector is resistant to degradation, tearing, pore formation, or dissolution. In some embodiments, the reusable sample collector includes antimicrobial, antibacterial, antiviral, or antifungal properties. In some embodiments, the reusable sample collector is used once or multiple times to collect one or more samples. In some embodiments, the reusable sample collector is used about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more times to collect one or more biological samples. In some implementations, a reusable sample collector is used to repeatedly collect biological samples from a single subject. In some implementations, a reusable sample collector is used to collect samples from multiple subjects.

[0041] In some implementations of the methods and systems provided herein, one or more samples are collected during one or more menstrual periods (menstrual windows) of the subject. In some implementations, one sample is collected within one menstrual cycle; two samples are collected within one menstrual cycle; three samples are collected within one menstrual cycle; four samples are collected within one menstrual cycle; more than four samples are collected within one menstrual cycle; two samples are collected within two menstrual cycles; three samples are collected within two menstrual cycles; four samples are collected within two menstrual cycles; five samples are collected within two menstrual cycles; six samples are collected within two menstrual cycles; seven samples are collected within two menstrual cycles; eight samples are collected within two menstrual cycles; more than eight samples are collected within two menstrual cycles; three samples are collected within three menstrual cycles; four samples are collected within three menstrual cycles; five samples are collected within three menstrual cycles; six samples are collected within three menstrual cycles; seven samples are collected within three menstrual cycles; eight samples are collected within three menstrual cycles; and so on. Nine samples were collected within one menstrual cycle; ten samples were collected within three menstrual cycles; eleven samples were collected within three menstrual cycles; twelve samples were collected within three menstrual cycles; more than twelve samples were collected within three menstrual cycles; four samples were collected within four menstrual cycles; five samples were collected within four menstrual cycles; six samples were collected within four menstrual cycles; seven samples were collected within four menstrual cycles; eight samples were collected within four menstrual cycles; nine samples were collected within four menstrual cycles; ten samples were collected within four menstrual cycles; eleven samples were collected within four menstrual cycles; twelve samples were collected within four menstrual cycles; thirteen samples were collected within four menstrual cycles; fourteen samples were collected within four menstrual cycles; fifteen samples were collected within four menstrual cycles; sixteen samples were collected within four menstrual cycles; or more than sixteen samples were collected within four menstrual cycles. In some implementations, multiple samples are collected over more than four menstrual cycles.

[0042] In some embodiments, samples are collected outside the menstrual window, for example, between the subject's menstrual periods. In some embodiments, a sample collector is used to collect non-menstrual fluids. In some embodiments, the collected non-menstrual fluids include vaginal secretions, cervical mucus, cervicovaginal fluid, spotting blood (i.e., from between menstrual periods), amniotic fluid, mucus plugs, or other vaginal discharges. In some embodiments, protocols for collecting and analyzing menstrual fluids are used to collect and analyze non-menstrual fluids.

[0043] In some embodiments, samples are collected after the menstrual window has closed, for example, after the end of menstruation. In some embodiments, samples are collected on the same day the menstrual window closes. In some embodiments, samples are collected approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, approximately 28 days, approximately 29 days, or approximately 30 days after the menstrual window has closed. In some implementations, samples are collected at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days after the menstrual window closes. In some implementations, samples are collected no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days after the menstrual window closes. In some implementations, samples are collected between 1 and 30 days after the menstrual window closes, between 1 and 25 days, between 1 and 20 days, between 1 and 5 days, between 1 and 10 days, between 1 and 5 days, between 5 and 30 days, between 5 and 25 days, between 5 and 20 days, between 5 and 15 days, between 5 and 10 days, between 10 and 30 days, between 10 and 25 days, between 10 and 20 days, between 10 and 15 days, between 15 and 30 days, between 15 and 25 days, between 15 and 20 days, between 20 and 30 days, between 20 and 25 days, or between 25 and 30 days.

[0044] In some embodiments, non-menstrual fluid collected between two menstrual windows is collected at different points during the reproductive cycle. Non-menstrual fluid is collected during the pre-ovulatory, ovulatory, or post-ovulatory phases. In some embodiments, non-menstrual fluid is collected during the proliferative, luteal, or secretory phases. In some embodiments, a phase of the reproductive cycle is an abnormal phase. In some embodiments, both menstrual and non-menstrual fluids are collected from the same subject.

[0045] In some implementations, samples are collected between two menstrual windows. In other implementations, samples are collected approximately midway between two menstrual windows, before the midpoint between two menstrual windows, or after the midpoint between two menstrual windows.

[0046] In some implementations, multiple samples are collected between two menstrual windows. In some implementations, 2, 3, 4, 5, 6, 7, or 8 samples are collected between two menstrual windows. In some such cases, multiple samples are collected at different times between two menstrual windows.

[0047] In some implementations, a sample is collected between two menstrual windows, and a second sample is collected between the second and third menstrual windows. In other cases, a third sample is collected between the third and second menstrual windows. In general, the nth sample is collected between n and second menstrual windows, where n is a positive integer equal to or greater than 1.

[0048] In some implementations, biological samples are collected from subjects both during and between menstrual windows. In some implementations, one biological sample is collected from a subject during a menstrual window, and a second biological sample is collected from the same subject between two menstrual windows. In some implementations, one biological sample is collected from a subject during a menstrual window, and a second biological sample is collected from the same subject after the end of that menstrual window and before the next menstrual window. In some implementations, one biological sample is collected from a subject before the start of a menstrual window, and a second biological sample is collected from the same subject during that menstrual window.

[0049] In some embodiments, the volume of fluid, such as menstrual fluid or other fluids collected from the vaginal cavity, is determined using a sample collector. In some embodiments, the volume of menstrual fluid in the sample collector is determined, for example, by reading a scale on the sample collector. The scale is at least 0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, 0.05 mL, 0.06 mL, 0.07 mL, 0.08 mL, 0.09 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL. In some embodiments, the volume of menstrual fluid in the sample collector is determined by measuring the mass of the fluid within the sample collector.

[0050] In some embodiments, the collected fluid, such as menstrual fluid, is extracted from the sample collector. In some embodiments, extraction is performed by pouring, transferring, or aspirating the fluid, which is suitable, for example, when the sample collector includes a menstrual cup or other non-absorbent reservoir. In some embodiments, extraction is performed by dissolving or otherwise breaking down the sample collector and removing it from the sample, which is suitable, for example, when the sample collector includes a sponge, tampons, pads, or other absorbent material. In some embodiments, extraction is performed by squeezing or compressing the sample collector, or by eluting the collector from the sample collector in a buffer solution such as an aqueous buffer. In some embodiments, the sample is extracted from the sample collector using the systems, methods, and apparatus described herein.

[0051] In some embodiments, the samples described herein include one or more biomarkers, including but not limited to nucleic acids, proteins, and cells. In some embodiments, one or more biomarkers include cells. In some embodiments, the cells are derived from menstrual fluid samples and preservation solutions (e.g., Biomatrica). In some embodiments, the sample is an endometrial cell sample comprising one or more endometrial cells. In some embodiments, the sample is an enriched cell sample. In some embodiments, the sample is collected using the system or apparatus described herein. In some embodiments, the biomarker shows a different presence or level in cervical vaginal fluid or menstrual fluid compared to peripheral blood or cervical vaginal tissue.

[0052] In some embodiments, one or more cells are derived from a biological sample. In some embodiments, the biological sample is taken from a woman. In some embodiments, the biological sample is taken from an individual suffering from a reproductive disorder (such as chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof). In some embodiments, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual is suspected of having endometriosis. In some embodiments, the individual has not yet undergone a surgical diagnosis of endometriosis. In some embodiments, the biological sample is obtained on the second day of the individual's menstrual cycle. In some embodiments, the biological sample is obtained on the same day of the individual's menstrual cycle when the individual is experiencing heavy menstrual flow. In some embodiments, the biological sample is obtained from the individual before administering treatment (such as surgery or administration of a treatment composition). In some embodiments, the treatment or intervention is a treatment for endometriosis. In some embodiments, the biological sample is obtained from the individual after administering treatment. In some embodiments, a first biological sample is obtained before administering treatment and a second biological sample is obtained after administering treatment. In some embodiments, the method includes determining differences between a first biological sample and a second biological sample in the expression of one or more microRNAs, methylation profiles of one or more CpG sites selected from CpG sites in Table 4, a measure of bacterial diversity, or a combination thereof.

[0053] In some embodiments, the biological sample includes menstrual fluid. In some embodiments, the biological sample includes cervical-vaginal fluid, cervical fluid, or vaginal fluid. In some embodiments, the biological sample comprises one or more endometrial cells. In some embodiments, the endometrial cells include endometrial stromal cells, endometrial epithelial cells, or combinations thereof. In some embodiments, the endometrial cells include endometrial stem cells. In some embodiments, endometrial stem cells include mesenchymal stem cells from menstrual blood. In some embodiments, the biological sample includes non-endometrial cells of an individual. In some embodiments, the non-endometrial cells of an individual include macrophages, glandular cells, squamous cells, cervical columnar cells, leukocytes, lymphocytes, non-endometrial stromal cells, non-endometrial endothelial cells, fibroblasts, erythrocytes, mesenchymal stem cells, ovaries, or combinations thereof. In some embodiments, the biological sample includes one or more sperm.

[0054] In some embodiments, the biological sample comprises one or more bacterial cells. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the phyla Bacteroidetes, Proteobacteria, Actinobacteria, Cyanobacteria, Fusobacteria, Spirochates, Tenericulates, Acidobacteria, TM7, or Sygerstetes. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the following genera: *Lactobacillus*, *Gardnerella*, *Fusobacterium*, *Staphylococcus*, *Streptococcus*, *Mageeibacillus*, *Mobiluncus*, *Mycoplasma*, *Bacteroides*, *Prevotella*, *Porphyeromonas*, *Alistella*, *Megacoccus*, *Propionibacterium*, *Porphyromonas*, *Dermatobacter*, *Moraxella*, *Anaerobes*, *Peptostreptococcus*, *Campylobacter*, *Corynebacterium*, *Franklinella*, *Klebsiella*, and *Peptone*. The bacteria include *Peptoniphilis*, *Sneathia*, *Ureaplasma*, *Finegoldia*, *Actinomyces*, *Clostridium*, *Veillonella*, *Peptinophilus*, *Adlercreurzia*, *Faecalibacterium*, *Haemophilus*, *Sphingomonas*, *Aerococcus*, *Weeksella*, *Biffidobacterium*, *Blautia*, or combinations thereof. In some embodiments, one or more bacteria include those from... Figure 4C , Figure 4D , Figure 4EBacteria from the genera described in the genus *Lactobacillus*, or combinations thereof. In some embodiments, one or more bacteria from the genus *Lactobacillus* are *L. acidophilus*, *L. amylovorus ultunensis*, *L. colonohominis*, *L. crispatus*, *L. fermentum*, *L. gasseri*, *L. iners*, *L. jensenii*, *L. kitasatonis*, *L. mucosa*, *L. paracaseirhamnosus*, *L. plantarum*, *L. pontis*, *L. reuteri frumenti*, species 3 of the genus *Lactobacillus*, species 9 of the genus *Lactobacillus*, or combinations thereof.

[0055] In some embodiments, one or more bacteria from the genus *Gardnerella* are *Gardnerella vaginalis*. In some embodiments, one or more bacteria from the genus *Streptococcus* are *Streptococcus agalactiae* or *Streptococcus gallolyticus*. In some embodiments, one or more bacteria from the genus *Sneathia* are *Sneathia sanguinegens*. In some embodiments, one or more bacteria from the genus *Mobiluncus* are *Mobiluncus curtisii*, *Mobiluncus mulieris*, or combinations thereof. In some embodiments, one or more bacteria from the genus *Mageeibacillus* are *Mageeibacillus indolicus*. In some embodiments, one or more bacteria from the genus *Megashaera* are *Megashaera elsdenii micronuciformis*, species 1 of the genus *Megashaera*, species 2 of the genus *Megashaera*, or combinations thereof. In some embodiments, one or more bacteria from the genus *Dialister* are *Dialister micraerophilus*. In some embodiments, one or more bacteria from the genus *Propionibacterium* are *Propionibacterium acnes*. In some embodiments, one or more bacteria from the genus *Porphyromonas* are *Porphyromonas somerae*. In some embodiments, one or more bacteria from the genus *Dermatobacter* are *Dermabacter vaginalis*. In some embodiments, one or more bacteria from the genus *Moraxella* are *Moraxella catarrhalis*.

[0056] In some embodiments, one or more bacteria from the genus *Anaerococcus* are *Anaerococcus tetradius* or *Anaerococcus prevotii*. In some embodiments, one or more bacteria from the genus *Peptostreptococcus* are *Peptostreptococcus magnus* or *Peptostreptococcus anaerobius*. In some embodiments, one or more bacteria from the genus *Campylobacter* are *Campylobacter ureolyticus* or *Campylobacter fetus*. In some embodiments, one or more bacteria from the genus *Corynebacterium* are *Corynebacterium amycolatum* or *Corynebacterium fournierii*. In some embodiments, one or more bacteria from the genus *Facklamia* are *Facklamia hominis* or *Facklamia massiliensis*. In some embodiments, one or more bacteria from the genus *Klebsiella* are *Klebsiella pneumoniae*. In some embodiments, one or more bacteria from the genus *Peptoniphilus* are *Peptoniphilus harei*. In some embodiments, one or more bacteria from the genus *Porphyromonas* are *Porphyeromonas asaccharolytica*. In some embodiments, one or more bacteria from the genus *Prevotella* are *Prevotella buccalis*, *Prevotella amnii*, *Prevotella bivia*, *Prevotella disiens*, *Prevotella melaninogenica*, or *Prevotella timonensis*. In some embodiments, one or more bacteria from the genus *A. deltae*, *A. minutum*, *A. parvulum*, *A. vaginae*, or combinations thereof. In some embodiments, the biological sample comprises one or more fungal cells. In some embodiments, the fungal cells are yeast.In some embodiments, the yeast is a yeast of the genus *Candida*. In some embodiments, the yeast of the genus *Candida* is *Candida albicans*, *Candida glabrata*, *Candida parapsilosis*, *Candida fomata*, or a combination thereof.

[0057] In some embodiments, the sample comprises at least one protein or fragment thereof derived from endometrial cells, non-endometrial cells from an individual, sperm, bacterial cells, fungal cells, or combinations thereof. In some embodiments, the sample comprises at least one nucleic acid derived from endometrial cells, non-endometrial cells from an individual, sperm, bacterial cells, fungal cells, or combinations thereof. In some embodiments, the at least one nucleic acid is a cell-free nucleic acid. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, rRNA, miRNA, or siRNA. In some embodiments, the nucleic acid is a nucleic acid encoding at least one protein or fragment thereof described herein.

[0058] In some embodiments, the sample includes a portion of a sample collector. In some embodiments, a portion of the sample collector dissolves or decomposes into the sample. In some embodiments, the sample collector is a tampon, pad, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad. In some embodiments, the tampon is a low-absorbency tampon. In some embodiments, the tampon includes an applicator.

[0059] In some embodiments, the sample volume is between 2 ml and 15 ml. In some embodiments, the sample volume is between approximately 7 ml and 10 ml. In some embodiments, the sample volume is less than 20 ml, less than 15 ml, less than 10 ml, or less than 8 ml. In some embodiments, the sample volume is between 1 ml and 4 ml. In some embodiments, the volume of menstrual fluid in the sample is between 2 ml and 3 ml. In some embodiments, the volume of menstrual fluid in the sample is less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, or less than 1 ml. In some embodiments, the volume of menstrual fluid in the sample is between 2 ml and 15 ml. In some embodiments, the sample volume is between approximately 7 ml and 10 ml. In some embodiments, the sample volume is less than 20 ml, less than 15 ml, less than 10 ml, or less than 8 ml. In some embodiments, the volume of menstrual fluid in the sample is between 1 ml and 4 ml. In some embodiments, the volume of menstrual fluid in the sample is between 2 ml and 3 ml. In some embodiments, the volume of menstrual fluid in the sample is less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, or less than 1 ml. In some embodiments, the sample contains less than 10 ml of menstrual fluid. 5 Cells, less than 10 6 Cells, less than 10 7 Cells, less than 10 8 10 cells or less 9 Cells. In some embodiments, the sample comprises fewer than 10 5 fewer than 10 endometrial cells 6 fewer than 10 endometrial cells 7 fewer than 10 endometrial cells 8 10 or fewer endometrial cells 9 10 endometrial cells. In some embodiments, the sample contains more than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 One cell or more than 10 9 10 cells. In some embodiments, the sample contains more than 10 5 Endometrial cells, greater than 10 6 Endometrial cells, greater than 10 7 Endometrial cells, greater than 10 8 10 or more endometrial cells 9 10 endometrial cells. In some embodiments, the sample contains fewer than 10 5 10 endothelial cells, less than 10 6 10 endothelial cells, less than 10 7 10 endothelial cells, less than 108个 10 or fewer endothelial cells 9 10 endothelial cells. In some embodiments, the sample contains more than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 One cell or more than 10 9 Cells. In some embodiments, the sample contains more than 10 5 10 endothelial cells, more than 10 6 10 endothelial cells, more than 10 7 10 endothelial cells, more than 10 8 One or more endothelial cells 9 10 endothelial cells. In some embodiments, the sample contains fewer than 10 5 fewer than 10 epithelial cells 6 fewer than 10 epithelial cells 7 fewer than 10 epithelial cells 8 10 or fewer epithelial cells 9 10 epithelial cells. In some embodiments, the sample contains more than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 One cell or more than 10 9 Cells. In some embodiments, the sample contains more than 10 5 Epithelial cells, more than 10 6 Epithelial cells, more than 10 7 Epithelial cells, more than 10 8 Each epithelial cell or more than 10 9 10 epithelial cells. In some embodiments, the sample contains fewer than 10 5 fewer than 10 white blood cells 6 fewer than 10 white blood cells 7 fewer than 10 white blood cells 8 10 or fewer white blood cells 9 10 white blood cells. In some embodiments, the sample contains more than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 One cell or more than 10 9 10 cells. In some embodiments, the sample contains more than 10 5 White blood cells, greater than 10 6 White blood cells, greater than 10 7 White blood cells, greater than 10 8Each white blood cell or more than 10 9 10 white blood cells. In some embodiments, the sample contains fewer than 10 5 fewer than 10 mesenchymal cells 6 fewer than 10 mesenchymal cells 7 fewer than 10 mesenchymal cells 8 Each mesenchymal cell or fewer than 10 9 Individual mesenchymal cells. In some embodiments, the sample contains more than 10 mesenchymal cells. 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 One cell or more than 10 9 10 cells. In some embodiments, the sample contains more than 10 5 Mesenchymal cells, more than 10 6 Mesenchymal cells, more than 10 7 Mesenchymal cells, more than 10 8 One or more mesenchymal cells 9 Mesenchymal cells.

[0060] In some cases, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells in the sample are intact. In some embodiments, the target cells are endometrial cells. In some embodiments, the target cells are endothelial cells, epithelial cells, leukocytes, mesenchymal cells, or combinations thereof. In some cases, at least 95% of the target cells in the sample are intact. An intact cell is a cell whose cell membrane has not ruptured. An intact cell is a cell in its native state. An intact cell is a living cell, wherein the living cells are cultured in a cell culture.

[0061] In some cases, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the target cells in the sample are viable. In some embodiments, the term "viable" refers to intact, living, and / or proliferating cells. The viability of multiple cells is assessed by measuring membrane permeability, enzyme activity, metabolic activity, DNA synthesis, membrane potential, expression of proliferation markers, or combinations thereof.

[0062] In some embodiments, the preservation solution includes Biomatrica Or Biomatrica In some embodiments, the preservation solution preserves RNA at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days. In some embodiments, the preservation solution prevents at least 50%, 60%, 70%, or 80% of RNA degradation. In some embodiments, the pH range of the preservation solution is pH 3 to pH 8, or more preferably pH 3 to pH 6.5. In some embodiments, the preservation solution preserves DNA at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days. In some embodiments, the preservation solution prevents at least 50%, 60%, 70%, or 80% of DNA degradation. In some embodiments, the pH range of the preservation solution is pH 5 to pH 10, or more preferably pH 6 to pH 9.

[0063] In some embodiments of the methods and systems provided herein, the preservation solution preserves nucleic acids at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days. In some embodiments, the preservation solution prevents at least 50%, 60%, 70%, or 80% degradation of the nucleic acids. In some embodiments, the pH range of the preservation solution is pH 3 to pH 8, or more preferably pH 3 to pH 6.5. In some embodiments, the preservation solution preserves RNA at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days. In some embodiments, the preservation solution prevents at least 50%, 60%, 70%, or 80% degradation of the RNA. In some embodiments, the pH range of the preservation solution is pH 3 to pH 8, or more preferably pH 3 to pH 6.5. In some embodiments, the preservation solution preserves DNA at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days. In some embodiments, the preservation solution prevents at least 50%, 60%, 70%, or 80% degradation of the DNA. In some embodiments, the pH range of the preservation solution is pH 5 to pH 10, or more preferably pH 6 to pH 9. In some embodiments, the preservation solution includes Biomatrica. Or Biomatrica

[0064] In some embodiments, the preservation solution includes a spike-in. As used herein, an "incorporation" is a molecule, such as a nucleic acid, cell, or group of molecules or cells, added to a sample, wherein the incorporation is used for quantitative or qualitative assessment or normalization of the sample. In some embodiments, the incorporation is a nucleic acid incorporation. In some embodiments, the nucleic acid incorporation includes DNA incorporations, RNA incorporations, bacterial incorporations, or combinations thereof. In some embodiments, the DNA incorporation includes synthetic DNA or multiple synthetic DNA molecules. In some embodiments, the RNA incorporation includes synthetic RNA or multiple synthetic RNA molecules. In some embodiments, the RNA incorporation includes a group of RNA transcripts developed by the External RNA Controls Consortium (ERCC).

[0065] In some embodiments, the preservation solution includes a mucolytic agent. In some embodiments, the mucolytic agent dissociates (e.g., "disaggregates") at least a portion of cell aggregates in the cervical vaginal sample. In some embodiments, the mucolytic agent includes acetylcysteine, ambroxol, bromhexine, carboxycysteine, domiocol, α-chain enzyme, ipramone, erdosteine, letostan, mannitol, mesna, netecoxib, sobrreol, stipronin, thiopronin, N-acetyl-L-cysteine, L-acetylcysteine / Liberase TM , or a combination thereof.

[0066] In some embodiments, the preservation solution includes an expectorant. In some embodiments, the expectorant includes marshmallow root, antimony pentasulfide, creosote oil, guaiacol sulfonate, guaiacol glyceryl ether (+ oxomazine), ipecac, levamisole, potassium iodide, polygala tenuifolia, tylosap, ammonium chloride, or combinations thereof.

[0067] In some embodiments, the preservation solution includes a surfactant. In some embodiments, the surfactant includes polyethylene glycol octylphenol ether; polyethylene glycol alkylphenol ether; polyethylene glycol alkyl sorbitol ester; alkyl sorbitol ester; polyethylene glycol; polypropylene glycol; carboxylates; sulfonates; petroleum sulfonates; alkylbenzene sulfonates; naphthalene sulfonates; olefin sulfonates; alkyl sulfates; sulfates; sulfated esters; sulfated alkanolamides; alkylphenols; ethoxylated fatty alcohols; polyoxyethylene surfactants; carboxylates; polyethylene glycol esters; sorbitol esters; ethylene glycol esters; carboxamides; monoalkanolamine condensates; polyoxyethylene fatty acid amides; quaternary ammonium salts; polyoxyethylene alkyl and alicyclic amines; N,N,N',N' tetrasubstituted ethylenediamine; 2-alkyl-1-hydroxyethyl-2-imidazoline; or combinations thereof.

[0068] In some embodiments, the preservation solution includes a nuclease. In some embodiments, the nuclease includes... DNA enzyme I, DNA enzyme II, exonuclease III, micrococcal nuclease, nuclease P1, nuclease S1, phosphodiesterase I, phosphodiesterase II, RNase A, RNase H, RNase T1, or combinations thereof.

[0069] In some embodiments, the preservation solution includes a protease. In some embodiments, the protease includes lipase II, trypsin, streptomycin, collagenase 1, collagenase 2, collagenase 3, collagenase 4, hyaluronidase, pepsin, papain, chemical trypsin, chymotrypsin, clostridium protease, complement C1r, complement C1s, complement factor D, complement factor I, cucumber extract, dipeptidyl peptidase, elastase, endopeptidase, enterokinase, activated factor X, caspase, cathepsin, matrix metalloproteinase, or combinations thereof.

[0070] In some embodiments, the osmolar concentration of the preservation solution includes about 310 to about 410 mOsm kg. -1 In some embodiments, the osmolar concentration of the preservation solution comprises approximately 95 to approximately 210 mOsm kg. -1 .

[0071] In some embodiments, the preservation solution does not include a fixative. In some embodiments, the fixative includes alcohols, aldehydes, oxidizing agents, metal fixatives, or combinations thereof. In some embodiments, the alcohol includes methanol, ethanol, propanol, isopropanol, butanol, or combinations thereof. In some embodiments, the aldehyde includes formaldehyde, glutaraldehyde, or combinations thereof. In some embodiments, the oxidizing agent includes osmium tetroxide, potassium permanganate, potassium dichromate, or combinations thereof. In some embodiments, the metal fixative includes mercuric chloride, picric acid, or combinations thereof. In some embodiments, the preservation solution does not include alcohols, aldehydes, oxidizing agents, metal fixatives, or combinations thereof.

[0072] In some embodiments, the preservation solution includes a binding agent. In some embodiments, the binding agent selectively binds to target cells or non-target cells of an individual. In some embodiments, target cells include endothelial cells, epithelial cells, leukocytes, mesenchymal cells, or combinations thereof. In some embodiments, non-target cells include endothelial cells, epithelial cells, leukocytes, mesenchymal cells, sperm, bacterial cells, fungal cells, or combinations thereof. In some embodiments, non-target cells include those different from target cells. In some embodiments, the binding agent selectively binds to at least one protein or fragment thereof. In some embodiments, at least one protein or fragment thereof includes a biomarker of endometriosis. In some embodiments, the binding agent selectively binds to nucleic acids. In some embodiments, nucleic acids include a biomarker of endometriosis. In some embodiments, the binding agent is immobilized to the surface of, for example, beads or components of the system described herein. In some embodiments, the binding agent is coupled to the surface of beads or the system. In some embodiments, the binding agent is reversibly or irreversibly coupled to the surface of beads or the system. In some embodiments, the binding agent includes a cleavable portion, such as a cleavable connector. In some embodiments, the cleavable connector is photolyzed, chemically cleaved, thermally cleaved, or enzymatically cleaved.

[0073] In some embodiments, the preservation solution is diluted by 0.1 ml to 0.9 ml, 0.3 ml to 0.7 ml, or 0.4 ml to 0.6 ml to form a diluted preservation solution. In some embodiments, the preservation solution includes Biomatrica. Or Biomatrica In some embodiments, the preservation solution is diluted to a second solution of 4.5 ml to 12.5 ml, 6.5 ml to 10.5 ml, or 7.5 ml to 9.5 ml. In some embodiments, the second solution is distilled water. In some embodiments, the diluted preservation solution is used in the methods and / or systems provided herein. In some embodiments, the diluted preservation solution is added to the sample collector at a rate of 2 ml to 6 ml or 3 ml to 5 ml of diluted preservation solution per gram of fluid absorbed into the sample collector. In some embodiments, the sample collector absorbs up to 6 g of fluid, therefore, about 18 ml to about 30 ml of diluted preservation solution is added to the low-absorbency tampons. In some embodiments, the diluted preservation solution is added to the sample collector in the system described herein after the destructible component has broken. Therefore, as the absorbency of the sample collector increases, the amount of diluted preservation solution to be added increases.

[0074] In other embodiments, the preservation solution is not diluted. In some embodiments, the undiluted preservation solution is used in the methods and / or systems provided herein. In some embodiments, the undiluted preservation solution is added to the sample collector at a rate of about 3 to about 5 ml of undiluted preservation solution per gram of fluid absorbed into the sample collector. In some embodiments, the absorbent tampon absorbs up to 6 g of fluid, therefore, about 18 ml to about 30 ml of undiluted preservation solution is added to the low-absorbency tampon. In some embodiments, the undiluted preservation solution is added to the sample collector in the system described herein after the destructible component has broken. Therefore, as the absorbency of the sample collector increases, the amount of undiluted preservation solution to be added increases.

[0075] In some embodiments, the binder includes an antibody. In some embodiments, the term "antibody," as used herein, refers to an immunoglobulin molecule and the immunoactive portion of an immunoglobulin molecule, i.e., a molecule that includes an antigen-binding site that specifically binds to an antigen. In some embodiments, the term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as various forms including full-length antibodies and portions thereof; including, for example, immunoglobulin molecules, polyclonal antibodies, monoclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, polymeric antibodies, CDR transplantation antibodies, F(ab)2, Fv, scFv, IgGΔCH2, F(ab')2, scFv2CH3, F(ab), VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, disulfide-linked Fv, single-domain antibodies (dAb), bispecific antibodies, multispecific antibodies, bispecific antibodies, anti-idiotype antibodies, bispecific antibodies, any isotype (including but not limited to IgA, IgD, IgE, IgG, or IgM), modified antibodies, and synthetic antibodies (including but not limited to non-consumable IgG antibodies, T antibodies, or other Fc or Fab variants of antibodies). In some embodiments, antibodies include polymeric antibodies. In some embodiments, the antibody is configured to selectively bind to target cells rather than non-target cells.

[0076] In some embodiments, this document describes a method for preserving cells from a menstrual fluid sample. In some embodiments, the cells include endometrial cells or non-endometrial cells. In some embodiments, the method includes placing the menstrual fluid in a preservation solution to form a mixture of the menstrual fluid sample and the preservation solution. In various embodiments, placing the menstrual fluid sample in the preservation solution to form the mixture includes placing a sample collector in a first central cavity of the system, wherein the sample collector is compressed or squeezed, for example, to remove at least a portion of the sample from the sample collector. In some embodiments, the sample collector includes a tampon, pad, menstrual disc, cervical cup, cervical disc, sponge, interlabial pad, or other suitable sample collector. In some cases, the placement of the sample collector into the first central cavity is performed by the individual from whom the menstrual fluid sample was collected. In some cases, the placement of the sample collector into the first central cavity is performed by a medical professional, such as an obstetrician or nurse. When the sample collector is compressed, the endometrial cells in the menstrual fluid sample are disrupted or sheared, causing the contents of the endometrial cells (e.g., nucleic acids) to be released into the mixture. In some cases, the compression of the sample collector is performed by the individual from whom the menstrual fluid sample was collected. In other cases, the compression of the sample collector is performed in the laboratory or at other locations where the sample collector is processed to determine the collected sample.

[0077] In some embodiments, the method described herein includes contacting cells in a menstrual fluid sample with an antibody that binds to a cell surface antigen of a target cell in the cells of the menstrual fluid sample. In some implementations, when the target cells include endothelial cells, the cell surface antigens include CD31 / PECAM-1, CD34, CD36 / SR-B3, CD39, CD44, CD47, CD54 / ICAM-1, CD61, CD62E, CD62P, CD80, CD86, CD93, CD102, CD105, CD106, CD112, CD117, ESAM, endothelial mucin, CXCL16, CD121a, CD141, CD142, CD143, CD144, CD146, CD147, CD151, CD160, CD201, CD213a, CD248, CD309, ADAM 8, ADAM 9, ADAM 10, ADAM 11, ADAM 12, ADAM 13, ADAM 14, ADAM 15, ADAM 16, ADAM 17. ADAM 33, ADAMTS-13, ADAMTS-18, VWF, TEM8, NOTCH, or KLF4. In some embodiments, when the target cells are epithelial cells, the cell surface antigen is epithelial cell adhesion molecule (EpCAM), E-cadherin, or CD326. In some embodiments, when the target cells are leukocytes, the cell surface antigen is CD45. In some embodiments, when the target cells are mesenchymal cells, the cell surface antigen is N-cadherin, OB-cadherin, α-5β-1 integrin, α-Vβ-6 integrin, or multiligand proteoglycan-1.

[0078] In some embodiments, the method described herein includes contacting cells in a menstrual fluid sample with an antibody that binds to target cells in the menstrual fluid sample. In some embodiments, the antibody comprises a monoclonal antibody. In some embodiments, the antibody is attached to a solid support. In some embodiments, the solid support is a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the antibody is conjugated to a detectable marker. In some embodiments, the detectable marker includes an optically detectable marker. In some embodiments, the optically detectable marker includes a fluorophore. In some embodiments, the fluorophore includes a dye, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or perCP. In some embodiments, the fluorophore includes a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), or mCHERRY. In some embodiments, the fluorophore emits light with a wavelength of 355 nm to 650 nm.

[0079] In some embodiments, the method herein further includes enriching at least one target cell in the sample to produce an enriched cell sample. In some embodiments, the cell sample includes a menstrual fluid cell sample. In some embodiments, the enriched sample includes an enriched menstrual fluid cell sample. In some embodiments, at least one target cell includes endometrial cells. In some embodiments, at least one target cell includes endothelial cells, epithelial cells, leukocytes, mesenchymal cells, or combinations thereof. In some embodiments, at least one non-target cell includes endothelial cells, epithelial cells, leukocytes, mesenchymal cells, or combinations thereof. In some embodiments, the endothelial cells include endometrial endothelial cells.

[0080] In some embodiments, enriching at least one target cell includes increasing the amount of at least one target cell in the enriched cell sample relative to the amount of at least one target cell in the cell sample before enrichment. Enriching at least one target cell includes increasing the ratio of at least one target cell to at least one non-target cell in the enriched cell sample relative to the ratio of at least one target cell to at least one non-target cell before enrichment. In some embodiments, enriching at least one target cell includes separating at least one target cell bound to at least one antibody. In some embodiments, the separated antibody-bound at least one target cell comprises an enriched cell sample. Enriching at least one target cell includes removing at least one non-target cell from the cell sample, wherein the at least one non-target cell is bound to at least one antibody. In some embodiments, the cell sample after removing at least one non-target cell thus produces an enriched cell sample. Separating antibody-bound target cells or antibody-bound non-target cells includes using flow cytometry. Separating antibody-bound target cells or antibody-bound non-target cells includes using fluorescence-activated cell sorting (FACS), magnetically activated cell sorting (MACS), or a combination thereof.

[0081] In some embodiments, methods for preserving nucleic acids, proteins, and / or metabolites from menstrual fluid samples are described herein. In some embodiments, the method includes placing the menstrual fluid in a preservation solution to form a mixture of the menstrual fluid sample and the preservation solution, wherein the preservation solution preserves the integrity of the nucleic acids (DNA or RNA) or one or more metabolites or proteins. In various embodiments, placing the menstrual fluid sample in the preservation solution to form a mixture includes placing a sample collector in a first central cavity of the system, wherein the sample collector is compressed or squeezed, for example, to remove at least a portion of the sample from the sample collector. In some embodiments, the sample collector includes a tampon, pad, menstrual tray, cervical cup, cervical disc, sponge, interlabial pad, or other suitable sample collector. In some cases, placement of the sample collector into the first central cavity is performed by the individual from whom the menstrual fluid sample was collected. In some cases, placement of the sample collector into the first central cavity is performed by a medical professional, such as an obstetrician or nurse. In some cases, compression of the sample collector is performed by the individual from whom the menstrual fluid sample was collected. In some cases, the compression of the sample collector is performed in the laboratory or at other locations where the sample collector is processed to determine the collected sample.

[0082] In some embodiments, once collected in the system described herein, the collected samples are stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the samples are stored at room temperature for up to 2 weeks. In some embodiments, the method includes transporting the mixture. In some embodiments, incubation occurs before, during, or after the transport of the mixture, or any combination thereof. In some embodiments, the mixture is transported, for example, to a testing facility or the office of a healthcare provider.

[0083] In some embodiments, the preservation solution comprises 1-methyl-3-carboxyethyl-imidazolium bromide, 1-hexyl-3-methylimidazolium bromide, 1-octyl-3-methylimidazolium bromide, 1-decyl-3-methylimidazolium bromide, or 1-(2-hydroxyethyl)-3-methylimidazolium bromide. In some embodiments, the concentration of 1-methyl-3-carboxyethyl-imidazolium bromide, 1-hexyl-3-methylimidazolium bromide, 1-octyl-3-methylimidazolium bromide, 1-decyl-3-methylimidazolium bromide, or 1-(2-hydroxyethyl)-3-methylimidazolium bromide in the preservation solution is about 0.1% to 10% (w / v). In some embodiments, the preservation solution further comprises a precipitant, a lower alcohol, a dissociating agent, a chelating agent, a reducing agent, a pH buffer, water, a surfactant, or a combination thereof. In some embodiments, the preservation solution includes at least one of the following: a precipitant, a lower alcohol, and a dissociation agent. In some embodiments, the preservation solution includes at least one of the following: a chelating agent, a reducing agent, and a pH buffer.

[0084] In some embodiments, the preservative solution includes a surfactant. In some embodiments, the surfactant is a detergent. In some embodiments, the precipitant is 5-(4-dimethyl)aminobenzylmethylrhodanine, sulfosalicylic acid, lithium chloride, or lithium hydroxide. In some embodiments, the lower alcohol includes methanol, ethanol, n-propanol, isopropanol, n-butanol, or isobutanol (2-methylprop-1-ol). In some embodiments, the dissociating agent includes guanidine hydrochloride, guanidine thiocyanate, potassium thiocyanate, sodium thiocyanate, or urea. In some embodiments, the chelating agent includes diethylenetriaminepentaacetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); ethylene glycol tetraacetic acid (EGTA); trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid; or nitrilotriacetic acid (NTA). In some embodiments, the reducing agent includes 2-mercaptoethanol, thiosulfate, TCEP (tris-(2-carboxyethyl)phosphine), dithiothreitol, or dithioerythritol. In some embodiments, the pH buffer includes citric acid; tartaric acid; malic acid; sulfosalicylic acid; sulfoisophthalic acid; oxalic acid; borate; CAPS (3-(cyclohexylamino)-1-propanesulfonic acid); CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid); EPPS (4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid); HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid); MES (2-(N-morpholino)ethanesulfonic acid); MOPS (3-(N-morpholino)propanesulfonic acid); MOPSO (3-morpholino-2-hydroxypropanesulfonic acid); PIP ES (1-4-piperazine diethanesulfonic acid); TAPS (N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid); TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid); TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid); bicine (N,N-bis(2-hydroxyethyl)glycine); tricine (N-[tris(hydroxymethyl)methyl]glycine); tris (tris(hydroxymethyl)aminomethane), or bis-tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol). In some embodiments, the detergent includes X-100 P40 Stain remover Ethoxylated amine detergents and Detergent. In some embodiments, the detergent includes bis-(2-hydroxyethyl)isodecoxypropylamine, poly(5)oxyethylene isodexoxypropylamine, bis-(2-hydroxyethyl)isotridecoxypropylamine, poly(5)oxyethylene isodexoxypropylamine, bis-(2-hydroxyethyl) linear alkoxypropylamine, bis(2-hydroxyethyl) daidzeinamine, poly(15)oxyethylene daidzeinamine, bis(2-hydroxyethyl)octadecylamine, poly(5)oxyethylene octadecylamine, poly(8)oxyethylene octadecylamine, poly(10)oxyethylene octadecylamine, poly(15)oxyethylene octadecylamine, bis(2-hydroxyethyl)octadecyloxypropylamine, bis-(2-hydroxyethyl) tallow amine, poly(5)oxyethylene tallow amine, poly(15)oxyethylene tallow amine, poly(3)oxyethylene 1,3-diaminopropane, bis(2-hydroxyethyl)tartrazine, poly ... 1-hydroxyethyl)cocoamine, bis-(2-hydroxyethyl)isodecoxypropylamine, poly(5)oxyethylene isodexoxypropylamine, bis-(2-hydroxyethyl)isodecoxypropylamine, poly(5)oxyethylene isotriadecoxypropylamine, bis-(2-hydroxyethyl)linear alkyloxypropylamine, bis(2-hydroxyethyl)soyamine, poly(15)oxyethylenesoyamine, bis(2-hydroxyethyl)octadecylamine, poly(5)oxyethylenesoctadecylamine, poly(8)oxyethylenesoctadecylamine, poly(10)oxyethylenesoctadecylamine, poly(15)oxyethylenesoctadecylamine, bis(2-hydroxyethyl)octadecyloxypropylamine, bis-(2-hydroxyethyl)tallowamine, poly(5)oxyethylenesoctadecylamine, poly(15)oxyethylenesoctadecylamine, poly(3)oxyethylene 1,3-diaminopropane, or bis(2-hydroxyethyl)cocoamine. In some embodiments, the surfactant includes surfactants derived from... Any surfactant from the surfactant family.

[0085] In some embodiments, the preservation solution includes at least one of the following: a preservative, a dissociating agent, or a combination thereof. In some embodiments, the preservative includes a zwitterionic compound, a permeabilizer, an apoptosis inhibitor, a non-reducing sugar or polyol, a disaccharide derivative, a chelating agent, a pH buffer, a phosphatase inhibitor, a protease inhibitor, or a combination thereof. In some embodiments, the dissociating agent includes a mucolytic agent, an expectorant, a surfactant, a nuclease, a protease, or a combination thereof. In some embodiments, the preservation solution further includes an incorporation. In some embodiments, the preservation solution is substantially composed of: a zwitterionic compound, a permeabilizer, an apoptosis inhibitor, a non-reducing sugar or polyol, a chelating agent, a pH buffer, a phosphatase inhibitor, a protease inhibitor, a mucolytic agent, an expectorant, a surfactant, a nuclease, a protease, an incorporation, or any combination thereof. In some embodiments, the preservation solution includes a reagent for selectively lysing non-target cells rather than target cells in the sample. In some embodiments, the preservation solution contains a reagent for selectively lysing cells that are not endometrial cells. In some embodiments, the reagent for selective lysis includes a dissociating agent. In some embodiments, the reagent for selective lysis includes a nuclease, a protease, or a combination thereof. In some embodiments, the preservation solution selectively lyses approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of non-target cells in the sample. In some embodiments, the preservation solution selectively lyses approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells in the sample that are not target cells. In some embodiments, the preservation solution also includes a binding agent.

[0086] In some embodiments, the preservation solution comprises a zwitterionic compound. In some embodiments, the zwitterionic compound comprises betaine or a betaine analogue. In some embodiments, the zwitterionic compound comprises trimethylamine N-oxide (TMAO). In some embodiments, the zwitterionic compound includes N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid; 3-(N-morpholino)propanesulfonic acid; 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; tris(hydroxymethyl)aminomethane; piperazine-N,N'-bis(2-ethanesulfonic acid); 2-(N-morpholino)ethanesulfonic acid hydrate; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; N-[tris(hydroxymethyl)methyl]glycine; 3-((3-acrylamidopropyl)-dimethylammonium)-propane-1-sulfonate; hydroxyectoine; ectoine; 4,5,6,7 -Tetrahydro-2-methyl-1H-(1,3)-diaza-4-carboxylic acid (homoectoine); L-carnitine; sarcosine; N,N-dimethylglycine triethylacetate ammonium; glycerol phosphate; tricine; pentaerythritol; N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butylsulfonate; 3-morpholino-2-hydroxypropanesulfonic acid; 4-(2-ethoxy-2-oxoethyl)-4-ethylmorpholino-4-onium bromide; N-(2-ethoxy-2-oxoethyl)-3-hydroxy-N,N-bis(2-hydroxyethyl)prop-1-amineonium bromide; 2-ethoxy-N,N,N-triethyl-2-oxoethylamineonium bromide Compounds; 2-((3-hydroxypropyl)dimethylammonium)acetate; 2-((2-hydroxypropyl)dimethylammonium)acetate; 2-(2-(hydroxymethyl)-1-methylpiperidinium-1-yl)acetate; 2-((2-hydroxyethyl)dimethylammonium)acetate; 2-((2,3-dihydroxypropyl)dimethylammonium)acetate; 1-(2-ethoxy-2-oxoethyl)-4-hydroxy-1-methylpiperidinium bromide; 2-(4-hydroxy-1-methylpiperidinium-1-yl)acetate; 2-ethoxy-N-(2-(2-hydroxyethoxy)ethyl)-N,N-dimethyl-2-oxoethylamineonium bromide; 2-((2-(2-hydroxyethoxy)ethyl)-N,N-dimethyl-2-oxoethylamineonium bromide; 2-(bis(2-hydroxyethyl)-(methyl)ammonium)acetate; 4-(2-hydroxyethyl)-4-methyl-2-oxomorpholin-4-onium bromide; 2-(bis(2-hydroxyethyl)-(methyl)ammonium)acetate; 2-(4-(2-hydroxyethyl)morpholin-4-onium)acetate; 4-(2-ethoxy-2-oxoethyl)-4-methylmorpholin-4-onium bromide; 1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidineonium bromide; 2-(benzyl(2-hydroxy-ethyl)(methyl)ammonium)acetate; 3-(2,3-dihydroxypropyl)-1-methyl-1H-imidazol-3-onium chloride;1,3-Dimethyl-1H-imidazol-3-onthium methyl sulfate; N-benzyl-2-ethoxy-N,N-dimethyl-2-oxoethylamineonium bromide; 1-(2-ethoxy-2-oxoethyl)-1-methylpiperidineonium bromide; N-(2-ethoxy-2-oxoethyl)-N,N-dimethylphenylammonium bromide; 1-(2-ethoxy-2-oxoethyl)-3-hydroxy-1-methylpiperidineonium bromide; 3-(2-(2-hydroxyethyl) (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-1-methyl-1H-imidazol-3-onium chloride; 1-methyl-3-tetradecyl-1H-imidazol-3-onium bromide; N-(2-ethoxy-2-oxoethyl)-N,N-dimethylcyclohexylammonium bromide; 3-((2-hydroxy-ethyl)dimethyl-ammonium)propionate; or any combination thereof. In some embodiments, the zwitterionic compound comprises a zwitterionic polymer. In some embodiments, the zwitterionic polymer includes carboxybetaine methacrylate-1; carboxybetaine methacrylate-1-tertiary amine; carboxybetaine methacrylate-2; carboxybetaine acrylamide-2; carboxybetaine acrylamide-2-ethyl ester; carboxybetaine acrylamide-2-RGD; carboxybetaine diacrylamide crosslinking agent; glycine betaine; polysulfonated betaine; or any combination thereof.

[0087] In some embodiments, the preservation solution includes an osmotic protectant. In some embodiments, the osmotic protectant includes trimethylammonium acetate; glycerol phosphate; diglyceride phosphate; N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine; 3-(N-morpholino)-2-hydroxypropanesulfonic acid; pentaerythritol; glyceric acid; malic acid; tartaric acid; lactic acid; glycolic acid; 2-hydroxybutyric acid; 3-hydroxybutyric acid; 4-amino-3-hydroxybutyric acid; 3-(1-azabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate; 1-(2-carboxyethyl)-1-azabicyclo[2.2.2]oct-1-onium; or any combination thereof.

[0088] In some embodiments, the preservation solution includes an apoptosis inhibitor. In some embodiments, the apoptosis inhibitor includes PERK-eIF2-α inhibitors, ASK1 inhibitors, NRF2-KEAP1 inhibitors, JNK inhibitors, p38 MAP kinase inhibitors, IRE1 inhibitors, GSK3 inhibitors, PIK3 pathway inhibitors, MEK inhibitors, calpain inhibitors, caspase-1 inhibitors, or any combination thereof.

[0089] In some embodiments, the preservation solution comprises non-reducing sugars or polyols. In some embodiments, non-reducing sugars or polyols include ethylene glycol, glycerol, erythritol, threitol, aritol, xylitol, ribitol, edetol, mannitol, sorbitol, galactitol, fucitol, idoteol, inositol, edetol, sucralfate, sucrose octasulfate, sucrose, trehalose, or any combination thereof. In some embodiments, the preservation solution comprises disaccharide derivatives. In some embodiments, disaccharide derivatives include sucralose, trichloromaltose, or combinations thereof.

[0090] In some embodiments, the preservation solution includes a chelating agent. In some embodiments, the chelating agent includes diethylenetriaminepentaacetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); ethylene glycol tetraacetic acid (EGTA); trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid; sodium gluconate; nitrotriacetic acid (NTA); or combinations thereof.

[0091] In some embodiments, the preservation solution includes a pH buffer. In some embodiments, the pH buffer includes citric acid; tartaric acid; malic acid; sulfosalicylic acid; sulfoisophthalic acid; oxalic acid; borate; CAPS (3-(cyclohexylamino)-1-propanesulfonic acid); CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid); EPPS (4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid); HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid); MES (2-(N-morpholino)ethanesulfonic acid); MOPS (3-(N-morpholino)propanesulfonic acid); MOPSO (3-morpholino-2-hydroxypropanesulfonic acid); PIPE S(1,4-piperazine diethanesulfonic acid); TAPS(N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid); TAPSO(2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid); TES(N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid); bicine(N,N-bis(2-hydroxyethyl)glycine); tricine(N-[tris(hydroxymethyl)methyl]glycine); tris(tris(hydroxymethyl)aminomethane); bis-tris(2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol); or combinations thereof.

[0092] In some embodiments, the preservative includes a phosphatase inhibitor. In some embodiments, the phosphatase inhibitor comprises β-glycerophosphate, aprotinin, phenbutastatin, EDTA, leucopeptidase, pepsin A, or combinations thereof.

[0093] In some embodiments, the preservative includes a protease inhibitor. In some embodiments, the protease inhibitor includes (2R)-2-mercaptomethyl-4-methylpentanoyl-β-(2-naphthyl)-Ala-Ala amide; 2-antifibrinolytic enzyme; 3,4-dichloroisocoumarin; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; 5-(R,S)-T-trans-cinnamamido-7-methyl-4-oxo-octanoyl-L-prolyl-L-proline; α1-antichymotrypsin; α1-antitrypsin; α 2-Antifibrinolytic enzyme; α2-macroglobulin; Antithrombin III; Aprotinin; Bromoenolactone; BTEE; C1 esterase inhibitor; Chymotrypsin inhibitor; Complement C1 esterase inhibitor; Disodium dichloromethylene bisphosphonate; Diisopropyl fluorophosphate; β-aminohexanoic acid; Escherichia coli; EDTA; Leech inhibitor C fragment 60-63 methyl ester; Gabexate mesylate; Histamine 5; Ile-Pro-Ile; Isopentylphosphonyl-Gly -L-Pro-L-Ala; Leucinogen; Na-p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride; N-acetyl-leech inhibitor C; N-toluenesulfonyl-L-phenylalanine chloromethyl ketone; p-chloromercuric benzoic acid free acid; benzyl sulfonyl fluoride; trypsin inhibitor; trypsin-chymotrypsin inhibitor; ZL-Phe chloromethyl ketone; Boc-Asp(OMe)-fluoromethyl ketone; Z-Ala-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone; antiprotease dihydrochloride derived from microbial protease inhibitors; CA-074 methyl ester; calpain inhibitor I; calpain inhibitor II; cysteine ​​protease inhibitor; E-64 protease inhibitor; leucinogen trifluoroacetate; α2-macroglobulin; cathepsinogen B; Z-Leu-Leu-Leu-fluoromethyl ketone; Z-Phe-Phe-fluoromethyl ketone; or combinations thereof.

[0094] Preparation of menstrual fingerprints

[0095] Methods for preparing menstrual fingerprint profiles are also described. In some embodiments, a menstrual genome comprises the entirety of molecules found in menstrual fluid, molecules separated from cells found in menstrual fluid, and cells found in menstrual fluid, as well as information determined from these molecules and cells. In some embodiments, a sample menstrual fingerprint profile includes differences in the levels and / or presence of multiple menstrual biomarkers from biological materials from a first sample and a second sample. In some embodiments, a menstrual fingerprint profile includes biological characteristics of biomarkers specific to a particular state in the menstrual cycle. In some embodiments, a menstrual fingerprint profile represents a specific genomic profile of menstrual fluid suitable for diagnostic development. In some embodiments, a menstrual fingerprint profile serves as a non-invasive biopsy for collecting endometrial tissue. In some embodiments, menstrual biomarkers comprise a biomarker matrix from endometrial tissue shed during menstruation.

[0096] In some aspects, the systems and methods described herein include methods for preparing menstrual fingerprints. In some embodiments, the method for preparing a menstrual fingerprint includes obtaining a sample using the methods and systems described herein. In some embodiments, the method for preparing a menstrual fingerprint includes obtaining a first sample and a second sample using the methods and systems described herein. In some embodiments, the method includes extracting biological material from one or more samples obtained herein into an aqueous buffer. In some embodiments, the method includes isolating biological material from one or more obtained samples. In some embodiments, the method includes constructing a menstrual fingerprint.

[0097] In some embodiments, the sample menstrual fingerprint includes differences in the levels and / or presence of multiple menstrual biomarkers in the biological material from a first sample and a second sample. In some embodiments, the sample menstrual fingerprint includes differences in the levels and / or presence of multiple menstrual biomarkers in the biological material from the first sample and / or the second sample compared to a reference menstrual fingerprint. In some embodiments, the sample menstrual fingerprint includes the levels and / or presence of multiple menstrual biomarkers in the biological material from the first sample. In some embodiments, the sample menstrual fingerprint is compared to a reference fingerprint. In some embodiments, the biomarkers described herein show different presence or levels in cervical vaginal fluid or menstrual fluid compared to those in peripheral blood or cervical vaginal tissue.

[0098] In some embodiments, the method includes obtaining a first sample and a second sample from a subject, wherein the first sample and the second sample comprise cervical vaginal fluid or menstrual fluid collected on a first absorbent sample collector and a second absorbent sample collector; eluting the first sample and the second sample from the first sample collector and the second sample collector, respectively, into an aqueous buffer; isolating biological material from each of the first sample and the second sample; and constructing a sample menstrual group fingerprint, wherein the sample menstrual group fingerprint includes the levels and / or differences in the presence of multiple menstrual group biomarkers in the biological material from the first sample and the second sample. In some embodiments, the method includes: obtaining a first sample and a second sample from a subject, wherein the first sample and the second sample comprise cervical vaginal fluid or menstrual fluid collected on a first absorbent sample collector and a second absorbent sample collector; eluting the first sample and the second sample from the first sample collector and the second sample collector, respectively, into an aqueous buffer; isolating biological material from each of the first sample and the second sample; constructing a sample menstrual group fingerprint, wherein the sample menstrual group fingerprint includes differences in the levels and / or presence of multiple menstrual group biomarkers in the biological material from the first sample and / or the second sample compared to a reference menstrual group fingerprint. In some embodiments, the method includes: obtaining a first sample from a subject, wherein the first sample comprises cervical vaginal fluid or menstrual fluid collected on an absorbent sample collector; eluting the first sample from the sample collector into an aqueous buffer; isolating biological material from the first sample; constructing a sample menstrual group fingerprint, wherein the sample menstrual group fingerprint includes the levels and / or presence of multiple menstrual group biomarkers in the biological material from the first sample; and comparing the sample menstrual group fingerprint with a reference fingerprint. In some embodiments, samples are collected and / or preserved using the methods and apparatus described herein. In some embodiments, the subject has or is suspected of having endometriosis.

[0099] In some embodiments, the subject is female. In some embodiments, the subject suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. In some embodiments, the subject is a mammal. In some embodiments, the mammal is human. In some embodiments, the subject is suspected of having endometriosis. In some embodiments, the subject has not yet received a surgical diagnosis of endometriosis. In some embodiments, the subject has a family history of endometriosis. In some embodiments, the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE).

[0100] In some embodiments, the sample includes any biological material or sample described herein, including menstrual fluid samples and cervical vaginal fluid samples. In some embodiments, the biological material includes one or more biological materials described herein. In some embodiments, the biological material includes, but is not limited to, RNA, DNA, miRNA, proteins, microorganisms, and mammalian cells. In some embodiments, the biological material is RNA, and multiple menstrual biomarkers include the expression levels of multiple genes. In some embodiments, the biological material is RNA, and multiple menstrual biomarkers include the presence and / or levels of multiple miRNAs. In some embodiments, the biological material is cells, and multiple menstrual biomarkers measure the presence and / or quantity of one or more cell types. In some embodiments, the biological material is DNA, and multiple menstrual biomarkers measure the presence and / or level of one or more microorganisms. In some embodiments, the biological material is DNA, and multiple menstrual biomarkers measure microbial diversity.

[0101] In some embodiments, the methods described herein include methods or assays for isolating biological material from one or more samples described herein or for analyzing extracted biological material. In some embodiments, the methods described herein include isolating biological material from a sample. In some embodiments, isolating biological material from a sample includes separating biological material from a sample using the methods or assays described herein. In some embodiments, the biological material includes nucleic acids, proteins, cells, or combinations thereof.

[0102] In some embodiments, the method or assay includes separating nucleic acids, proteins, or combinations thereof from the cervical-vaginal sample described herein. In various embodiments, aliquots of the sample are created. In some embodiments, the method or assay includes separating nucleic acids from a first aliquot of the sample and separating proteins from a second aliquot of the sample. Separating nucleic acids, proteins, or combinations thereof from the sample includes lysis of cells in the sample; extraction of nucleic acids, proteins, or combinations thereof from the sample; and / or purification of the extracted nucleic acids, extracted proteins, or combinations thereof.

[0103] In some embodiments, the method or assay includes lysis of cells in the sample. In some embodiments, lysis is chemical lysis, mechanical lysis, or a combination thereof. In some embodiments, chemical lysis includes adding a lysin, a dissociative agent, a detergent, or a combination thereof to the sample. In some embodiments, mechanical lysis includes homogenization, sonication, shearing, or shocking the cells. In some embodiments, shocking includes osmotic shock. In some embodiments, lysis results in the release of nucleic acids and proteins from the cells. In some embodiments, the method or assay includes purifying nucleic acids, proteins, or a combination thereof from the sample.

[0104] In some embodiments, the method or assay includes extracting nucleic acids, proteins, or combinations thereof from a sample. In some embodiments, the nucleic acid is DNA, RNA, or a combination thereof. In some embodiments, the RNA includes mRNA, tRNA, rRNA, miRNA, siRNA, or a combination thereof. Extraction includes organic phase extraction. In some embodiments, the method or assay includes purification of the extracted nucleic acids, extracted proteins, or combinations thereof.

[0105] In some embodiments, the method or assay includes sequencing nucleic acids from a sample or an enriched sample. In some embodiments, sequencing is whole-genome sequencing or whole-exome sequencing. In some embodiments, sequencing is high-throughput sequencing. In some embodiments, nucleic acids are sequenced to a depth of at least 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, 300x, or more than 300x coverage. In some embodiments, sequencing is targeted sequencing, wherein one or more pre-selected nucleic acid targets are sequenced. In some embodiments, one or more pre-selected nucleic acid targets are one or more biomarkers specific to endometriosis. In some embodiments, sequencing includes sequencing 16S rRNA or 16S rDNA. In some embodiments, the method or assay includes bisulfite treatment prior to sequencing. In some embodiments, the method or assay described herein includes determining the methylation status (i.e., methylated or unmethylated) of nucleic acids in the nucleic acid sequence. In some embodiments, the nucleic acid is cytosine. In some embodiments, the methods or assays described herein include determining the methylation pattern of the nucleic acid sequence.

[0106] In some embodiments, the method or assay includes determining the expression level of one or more microRNAs (miRs) selected from the group consisting of miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p from an individual's biological sample. In some embodiments, the method or assay includes determining the expression level of one or more microRNAs (miRs) selected from the group consisting of miR-23b-3p, miR-30a-3p / 5p, and miR-34a-5p from an individual's biological sample. In some embodiments, the method or assay includes determining the expression levels of one or more miRs selected from the group consisting of: let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2-3p, miR-1 7-3p,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,miR-200 c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-222-3 p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409, and miR-98-5p. In some embodiments, the method or assay includes determining the expression level of one or more microRNAs from an individual's biological sample, which regulate or are expected to regulate the expression of at least one gene involved in at least one KEGG pathway. In some embodiments, the KEGG pathway is: "ECM-receptor", "adhesion junction", "proteoglycan in cancer", "TGF-β signaling", "Hippo signaling", "microRNA in cancer", "pathway in cancer", "hepatitis B", "glioma", "chronic myeloid leukemia", "bladder cancer", or a combination thereof. In some implementations, at least one KEGG pathway involves Wnt / JNK / VEGF signaling.

[0107] In some embodiments, the method or determination includes determining the methylation profile of one or more CpG sites selected from the CpG sites in Table 4.

[0108] In some embodiments, the menstrual footprint includes determining a measure of bacterial diversity in the biological sample. In some embodiments, the measure of bacterial diversity is the amount of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of bacteria.

[0109] In some embodiments, the biological sample comprises one or more bacterial cells. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the phyla Bacteroidetes, Proteobacteria, Actinobacteria, Cyanobacteria, Fusobacteria, Spirulina, Soft-walled Bacteria, Acidobacteria, TM7, or Mutualistic Bacteria. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the following genera: *Lactobacillus*, *Gardnerella*, *Fusobacterium*, *Staphylococcus*, *Streptococcus*, *Mageeibacillus*, *Mageeibacillus*, *Aegilops*, *Mycoplasma*, *Bacteroidetes*, *Prevotella*, *Porphyeromonas*, *Alisteria*, *Mageeibacillus*, *Macrococcus*, *Propionibacterium*, and *Porphyromonas*. The genera *Bacteria*, *Desmodium*, *Moraxella*, *Anaerobic Cocci*, *Peptostreptococcus*, *Campylobacter*, *Corynebacterium*, *Franklinella*, *Klebsiella*, *Peptone*, *Seneca*, *Ureaplasma*, *Fingoldii*, *Actinomyces*, *Clostridium*, *Veillonella*, *Peptone*, *Andekella*, *Femtobacter*, *Haemophilus*, *Sphingomonas*, *Bacillus*, *Wexneria*, *Bifidobacterium*, *Brutella*, or combinations thereof. In some embodiments, one or more bacteria include those from... Figure 4C , Figure 4D , Figure 4E Bacteria of the genera described therein, or combinations thereof. In some embodiments, the measure of bacterial diversity includes the ratio of at least one first bacterium to at least one second bacterium.

[0110] In some embodiments, the measure of bacterial diversity includes a diversity index. In some embodiments, the diversity index includes the Shannon diversity index, the Simpson diversity index, or the Berger-Parker diversity index. In some embodiments, bacterial diversity measures the diversity of bacterial species, genera, families, functional types, or haplotypes. In some embodiments, bacterial diversity is determined by sequencing. In some embodiments, sequencing includes Sanger sequencing or high-throughput sequencing. In some embodiments, sequencing identifies bacterial species in a biological sample. In some embodiments, sequencing identifies the abundance of bacterial species. In some embodiments, sequencing is sequencing of 16S rRNA or a portion thereof.

[0111] In some embodiments, the biomarker shows differences in levels in cervical vaginal fluid or menstrual fluid between one or more health conditions. In some embodiments, the biomarker shows different presence or levels in cervical vaginal fluid or menstrual fluid compared to peripheral blood or cervical vaginal tissue.

[0112] In some embodiments, the method further includes comparing a sample menstrual group fingerprint with a reference menstrual group fingerprint. In some embodiments, the reference menstrual group fingerprint includes threshold levels or presence of multiple menstrual group biomarkers associated with a health status. In some embodiments, the healthy reference menstrual group fingerprint includes principal component analysis; t-distributed random neighborhood embedding; heatmap; diversity index; classical, metric, and non-metric multidimensional scaling (MDS); diffusion plot; recipient operating characteristic curve; k-means clustering; discriminant model construction; multivariate logistic regression with stepwise feature selection; tree; random forest; and principal component analysis. In some embodiments, the reference status includes a health status before or after the procedure. In some embodiments, the reference status includes a patient without endometriosis. In some embodiments, the reference status includes a healthy subject. In some embodiments, the healthy subject is a subject without a family history of endometriosis. In some embodiments, the healthy subject is a subject who does not have or is not suspected of having a reproductive disorder, including but not limited to polycystic ovary syndrome (PCOS), endometriosis, or a combination thereof. In some embodiments, the healthy subject is a subject with a family history of a reproductive disorder.

[0113] In some embodiments, the first and second samples include any of the samples or biological samples described herein. In some embodiments, the first and second samples include biological materials collected from the subject at different time points. In some embodiments, the time points are separated by a time interval of approximately 15 minutes to approximately 30 days. In some embodiments, the time points are separated by a time interval of at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the time points are separated by a time interval of at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the time points are separated by a time interval of at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the time points are spaced no more than approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes apart. In some embodiments, the time points are spaced no more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days apart. In some embodiments, the time points are spaced no more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart. In some embodiments, the time points are spaced no more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years apart, or more than 10 years apart.

[0114] In some embodiments, time points include different days within the subject's menstrual cycle. In some embodiments, a normal menstrual cycle occurs approximately monthly, including the shedding of the uterine lining through the vagina. In some embodiments, a normal menstrual flow lasts approximately 4 or 5 days, up to 7 days, and occurs every 21 to 35 days. In some embodiments, time points are within a single menstrual cycle. In some embodiments, time points include days within different menstrual cycles. In some embodiments, time points are during one or more days of the subject's menstrual period. In some embodiments, one time point is during the subject's menstrual period while another time point is not. In some embodiments, at least one time point is during a day of heavy bleeding. In some embodiments, at least one time point is during a day of light bleeding. In some embodiments, at least one time point is not during a day of bleeding. In some embodiments, at least one time point is during ovulation.

[0115] In some embodiments, two or more health conditions are included before and after medical treatment. In some embodiments, the medical treatment is surgery, such as surgery to treat endometriosis or other menstrual disorders. In some embodiments, health conditions include health conditions before or after surgery. In some embodiments, a healthy state includes chronic pelvic pain, infertility, heavy menstrual bleeding, eating disorders; extreme weight loss; excessive exercise; polycystic ovary syndrome (PCOS); ovarian cysts; premature ovarian failure; breast cancer; ovarian cancer; infertility; decreased ovarian reserve; chronic or frequent urinary tract infections; ectopic pregnancy; heart disease; type 1 diabetes; type 2 diabetes; autoimmune diseases such as lupus, multiple sclerosis, or rheumatoid arthritis; pelvic inflammatory disease (PID); fibroids (e.g., uterine fibroids); adenomyosis; cervical cancer; endometrial cancer; uterine cancer; bacterial vaginosis, chlamydia, gonorrhea, genital herpes, hepatitis, human immunodeficiency virus, acquired immunodeficiency syndrome, human papillomavirus, syphilis, trichomoniasis, or cervical or endometrial infection, or a combination thereof. In some embodiments, a healthy state includes menstrual disorders. In some implementations, endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE).

[0116] In some implementations, the reference footprint includes the expression levels of one or more microRNAs, methylation profiles, measures of bacterial diversity, or combinations thereof, of individuals with a known endometriosis status.

[0117] In some embodiments, the method or assay further includes generating a report based on a biomarker or biomarker feature. In some embodiments, the biomarker or biomarker feature includes the expression level of one or more miRNAs relative to a reference expression level, the methylation profile of one or more genomic regions, a measure of bacterial diversity, or a combination thereof.

[0118] In some embodiments, methods or assays for classifying or detecting endometriosis in an individual include determining the expression levels of one or more microRNAs (miRs) from a biological sample (e.g., a menstrual fluid sample). In some cases, biological or menstrual fluid samples are collected on days 1, 2, 3, 4, 5, 6, and / or 7 of an individual's menstrual cycle. In other cases, biological or menstrual fluid samples are collected on day 2 of an individual's menstrual cycle.

[0119] In some embodiments, the method or assay further includes determining the expression levels of two, three, or more microRNAs (miRs) from an individual's biological sample. In some embodiments, the miRs are selected from miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and / or miR-410-3p. In some embodiments, miRs include intracellular miRs, extracellular miRs, or both intracellular and extracellular miRs. In some embodiments, miRs are separated from cells in the biological sample. In various embodiments, miRs are separated from the non-cellular portion of the biological sample. In some embodiments, miRs are separated from the total biological sample (e.g., from both the intracellular and extracellular portions of the biological sample). In some embodiments, miRs are assessed or detected by any suitable method. In some embodiments, sequencing is used to assess or detect miRs.

[0120] In some embodiments, the biological sample includes menstrual fluid, cervical-vaginal fluid, or both. In some embodiments, the biological sample is disposed in a sample collector as provided herein. In some embodiments, the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, interlabial pad, or a combination thereof.

[0121] Disease detection

[0122] In some embodiments, this document describes methods or assays for detecting a condition in an individual. In some embodiments, this document describes methods or assays for detecting endometriosis in an individual. In some embodiments, the individual is female. In some embodiments, the individual suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. In some embodiments, the individual is a mammal. In some embodiments, the mammal is human. In some embodiments, the individual is suspected of having endometriosis. In some embodiments, the individual has not yet received a surgical diagnosis of endometriosis. In some embodiments, the individual has a family history of endometriosis. In some embodiments, the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE). In some embodiments, the methods or assays described herein have a false detection rate of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the methods or assays described herein have a false detection rate of 5% or less. In some embodiments, this document provides methods or assays for classifying or detecting endometriosis in an individual. In some embodiments, the methods or assays have a specificity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0123] In some embodiments, the method or assay described herein includes removing the sample described herein from the system. In some embodiments, the sample is removed from the system through a port located on the system. In some embodiments, the sample is removed from the system via a syringe inserted through the port. In some embodiments, about 2 ml to about 4 ml of sample is removed from the system. In some embodiments, about 1 ml to about 5 ml of sample is removed from the system.

[0124] In some embodiments, the method or assay includes separating a biomarker from a sample, including but not limited to nucleic acids, proteins, or cells. In some embodiments, the method or assay includes separating nucleic acids, proteins, or combinations thereof from a sample. In various embodiments, aliquots of the sample are created. In some embodiments, the method or assay includes separating nucleic acids from a first aliquot of the sample and separating proteins from a second aliquot of the sample. Separating nucleic acids, proteins, or combinations thereof from a sample includes lysis of cells in the sample; extraction of nucleic acids, proteins, or combinations thereof from the sample; and / or purification of the extracted nucleic acids, extracted proteins, or combinations thereof. In some embodiments, the biomarker shows a different presence or level in cervical vaginal fluid or menstrual fluid compared to peripheral blood or cervical vaginal tissue.

[0125] In some embodiments, the method or assay includes lysis of cells in the sample. In some embodiments, lysis is chemical lysis, mechanical lysis, or a combination thereof. Chemical lysis includes adding a lysin, a dissociative agent, a detergent, or a combination thereof to the sample. Mechanical lysis includes homogenization, sonication, shearing, or shocking the cells. In some embodiments, shocking includes osmotic shock. In some embodiments, lysis results in the release of nucleic acids and proteins from the cells. In some embodiments, the method or assay includes purifying nucleic acids, proteins, or a combination thereof from the sample.

[0126] In some embodiments, the method or assay includes extracting nucleic acids, proteins, or combinations thereof from a sample. In some embodiments, the nucleic acid is DNA, RNA, or a combination thereof. In some embodiments, the RNA includes mRNA, tRNA, rRNA, miRNA, siRNA, or a combination thereof. Extraction includes organic phase extraction. In some embodiments, the method or assay includes purification of the extracted nucleic acids, extracted proteins, or combinations thereof.

[0127] In some embodiments, the method or assay includes sequencing nucleic acids from a sample or enriched sample. In some embodiments, sequencing is whole-genome sequencing or whole-exome sequencing. In some embodiments, sequencing is high-throughput sequencing. In some embodiments, nucleic acids are sequenced to a depth of at least 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, 300x, or more than 300x coverage. In some embodiments, sequencing is targeted sequencing, wherein sequencing is performed on one or more pre-selected nucleic acid targets. In some embodiments, one or more pre-selected nucleic acid targets are one or more biomarkers specific to endometriosis. In some embodiments, sequencing includes sequencing of 16S rRNA or 16S rDNA. In some embodiments, the method or assay includes bisulfite treatment prior to sequencing. In some embodiments, the method or assay described herein includes determining the methylation status (i.e., methylated or unmethylated) of nucleic acids in the nucleic acid sequence. In some embodiments, the nucleic acid is cytosine. In some embodiments, the methods or assays described herein include determining the methylation pattern of the nucleic acid sequence.

[0128] In some embodiments, the method or assay includes determining the expression level of one or more microRNAs (miRs) selected from the group consisting of miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p from an individual's biological sample. In some embodiments, the method or assay includes determining the expression level of one or more microRNAs (miRs) selected from the group consisting of miR-23b-3p, miR-30a-3p / 5p, and miR-34a-5p from an individual's biological sample. In some embodiments, the method or assay includes determining the expression levels of one or more miRs selected from the group consisting of: let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2-3p, miR-1 7-3p,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,miR-200 c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-222-3 p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409, and miR-98-5p. In some embodiments, the method or assay includes determining the expression level of one or more microRNAs from an individual's biological sample, which regulate or are expected to regulate the expression of at least one gene involved in at least one KEGG pathway. In some embodiments, the KEGG pathway is: "ECM-receptor", "adhesion junction", "proteoglycan in cancer", "TGF-β signaling", "Hippo signaling", "microRNA in cancer", "pathway in cancer", "hepatitis B", "glioma", "chronic myeloid leukemia", "bladder cancer", or a combination thereof. In some implementations, at least one KEGG pathway involves Wnt / JNK / VEGF signaling.

[0129] In some embodiments, the method or assay includes comparing the expression levels of one or more miRs to a reference expression level. In some embodiments, the comparison includes performing differential expression analysis. Machine learning algorithms are used for differential expression analysis. In some embodiments, the reference expression level is obtained from a healthy subject. In some embodiments, the healthy subject is a subject who does not have endometriosis or is not suspected of having endometriosis. In some embodiments, the healthy subject is a subject without a family history of endometriosis. In some embodiments, the healthy subject is a subject who does not have or is not suspected of having a reproductive disorder, including but not limited to polycystic ovary syndrome (PCOS), endometriosis, or a combination thereof. In some embodiments, the healthy subject is a subject with a family history of a reproductive disorder. In some embodiments, an increase or decrease in the expression level of one or more miRs relative to a reference expression level indicates that the subject has endometriosis.

[0130] In some embodiments, the method or determination includes determining the methylation profile of one or more CpG sites selected from the CpG sites in Table 4.

[0131] In some embodiments, the method or assay includes determining a measure of bacterial diversity in a biological sample. In some embodiments, the measure of bacterial diversity is the quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of bacteria. In some embodiments, the biological sample comprises one or more bacterial cells. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the phyla Bacteroidetes, Proteobacteria, Actinobacteria, Cyanobacteria, Fusobacteria, Spirulina, Soft-walled Bacteria, Acidobacteria, TM7, or Mutual Atrophy. In some embodiments, the one or more bacterial cells comprise one or more bacteria from the genera *Lactobacillus*, *Gardnerella*, *Fusobacterium*, *Staphylococcus*, *Streptococcus*, *Mageeibacillus*, *Mageeibacillus*, *Aegilops*, *Mycoplasma*, *Bacteroidetes*, *Prevotella*, *Porphyeromonas*, *Alistella*, *Mageeibacillus*, *Mageeibacillus*, *Mageeibacillus*, *Propionibacterium*, *Porphyromonas*. Genus, *Desmodium*, *Moraxella*, *Anaerobic Cocci*, *Peptostreptococcus*, *Campylobacter*, *Corynebacterium*, *Franklinella*, *Klebsiella*, *Peptobacter*, *Seneca*, *Ureaplasma*, *Fingoldii*, *Actinomyces*, *Clostridium*, *Veillonella*, *Peptobacter*, *Andekella*, *Femtobacter*, *Haemophilus*, *Sphingomonas*, *Bacillus*, *Wexneria*, *Bifidobacterium*, *Brutella*, or combinations thereof. In some embodiments, one or more bacteria include those from... Figure 4C , Figure 4D , Figure 4EBacteria of the genera described in the description, or combinations thereof, *Lactobacillus leucocephala* subsp. *leucocephala*, species 3 of the genus *Lactobacillus*, species 9 of the genus *Lactobacillus*, or combinations thereof. In some embodiments, bacterial diversity is measured as the ratio of at least one first bacterium to at least one second bacterium.

[0132] In some embodiments, bacterial diversity is measured as a diversity index. In some embodiments, the diversity index is the Shannon diversity index, the Simpson diversity index, or the Berger-Parker diversity index. In some embodiments, bacterial diversity measures the diversity of bacterial species, genera, families, functional types, or haplotypes. In some embodiments, bacterial diversity is determined by sequencing. In some embodiments, sequencing is Sanger sequencing or high-throughput sequencing. In some embodiments, sequencing identifies bacterial species in a biological sample. In some embodiments, sequencing identifies the abundance of bacterial species. In some embodiments, sequencing is sequencing of 16S rRNA or a portion thereof. In some embodiments, an increase in bacterial diversity in a biological sample relative to a reference bacterial diversity indicates that the subject has endometriosis. In some embodiments, the reference bacterial diversity level is the bacterial diversity in a healthy individual. In some embodiments, a healthy subject is a subject who does not have endometriosis or is not suspected of having endometriosis. In some embodiments, a healthy subject is a subject who does not have or is not suspected of having a reproductive disorder, including but not limited to polycystic ovary syndrome (PCOS), endometriosis, or a combination thereof. In some embodiments, a healthy subject is a subject with a family history of a reproductive disorder.

[0133] In one example, the bacteria is *Propionibacterium acnes*. In some embodiments, an increase in *Propionibacterium acnes* abundance relative to a reference *Propionibacterium acnes* abundance indicates that the subject has endometriosis. In some embodiments, an increase in *Propionibacterium acnes* abundance of at least 5, at least 10, or at least 15 times compared to a reference *Propionibacterium acnes* abundance indicates that the subject has endometriosis. In some embodiments, the reference *Propionibacterium acnes* abundance is the abundance of *Propionibacterium acnes* in a healthy subject. In some embodiments, the healthy subject is a subject who does not have endometriosis or is not suspected of having endometriosis. In some embodiments, the healthy subject is a subject who does not have or is not suspected of having a reproductive disorder, including but not limited to polycystic ovary syndrome (PCOS), endometriosis, or a combination thereof. In some embodiments, the healthy subject is a subject with a family history of reproductive disorders.

[0134] In some embodiments, the method or assay includes applying a classifier algorithm (or classifier) ​​to the expression levels of one or more microRNAs, methylation profiles, measures of bacterial diversity, or combinations thereof, of a biological sample from an individual to generate a classification of the individual. In some embodiments, the classification is selected from: possible endometriosis and unlikely endometriosis. In some embodiments, the classification of possible endometriosis is selected from: high probability of endometriosis, moderate probability of endometriosis, and low probability of endometriosis. In some embodiments, the classification is a numerical score quantifying the likelihood that an individual has endometriosis. In some embodiments, the method or assay includes using a machine learning model to generate a classifier algorithm. Generating the classifier algorithm includes using training data from individuals with a known endometriosis status (e.g., individuals diagnosed with endometriosis or individuals diagnosed without endometriosis). In some embodiments, the training data includes the expression levels of one or more microRNAs, methylation profiles, measures of bacterial diversity, or combinations thereof, of individuals with a known endometriosis status. In some implementations, the classifier algorithm includes decision trees, random forests, Bayesian networks, support vector machines, neural networks, or logistic regression algorithms. In some implementations, the classifier is a random forest classifier. In some implementations, the random forest classifier includes at least 10, 20, 50, 100, 1000, or 5000 decision trees. The machine learning model is used for differential expression analysis.

[0135] In some embodiments, the method or assay further includes generating a report based on a biomarker or biomarker signature. In some embodiments, the biomarker or biomarker signature includes the expression level of one or more miRNAs relative to a reference expression level, methylation profiles of one or more genomic regions, a measure of bacterial diversity, or a combination thereof. In some embodiments, the method or assay further includes transmitting the report to a healthcare professional. In some embodiments, the report includes a recommendation for administering an intervention to an individual. In some embodiments, the intervention includes a surgical intervention, a therapeutic intervention, or a combination thereof. In some embodiments, the surgical intervention includes surgical removal of at least a portion of an endometriotic lesion, hysterectomy, salpingo-oophorectomy, presacral neurectomy, or laparoscopic uterine nerve ablation. In some embodiments, the therapeutic intervention includes the administration of a therapeutic agent. In some embodiments, the therapeutic agent is a hormone, a hormone agonist, a hormone antagonist, an aromatase inhibitor, an anti-inflammatory therapy, an acetyltransferase, a histone deacetylase inhibitor, a phosphodiesterase inhibitor, or a combination thereof. In some embodiments, the hormone is a synthetic hormone. In some embodiments, the hormone is an estrogen, progesterone, progesterone, androgen, gonadotropin-releasing hormone (Gn-RH), or a combination thereof. In some embodiments, the hormone agonist is a gonadotropin-releasing hormone (Gn-RH) agonist. In some embodiments, the hormone antagonist is a gonadotropin-releasing hormone (Gn-RH) antagonist. In some embodiments, the therapy is a contraceptive containing a hormone. In some embodiments, the anti-inflammatory therapy is an NSAID, a JNK inhibitor, a TNF inhibitor, an interleukin (IL) inhibitor, or a combination thereof.

[0136] In some embodiments, the method or assay further includes administering an intervention to an individual. In some embodiments, the intervention is determined from a report. In some embodiments, the intervention is determined without a report. In some embodiments, the intervention includes a surgical intervention, a therapeutic intervention, or a combination thereof. In some embodiments, the surgical intervention includes surgical removal of at least a portion of an endometriotic lesion, hysterectomy, salpingo-oophorectomy, presacral neurectomy, or laparoscopic uterine nerve ablation. In some embodiments, the therapeutic intervention includes administering a therapeutic agent. In some embodiments, the therapeutic agent is a hormone, a hormone agonist, a hormone antagonist, an aromatase inhibitor, an anti-inflammatory therapy, an acetyltransferase, a histone deacetylase inhibitor, a phosphodiesterase inhibitor, or a combination thereof. In some embodiments, the hormone is a synthetic hormone. In some embodiments, the hormone is an estrogen, progesterone, progesterone, androgen, gonadotropin-releasing hormone (Gn-RH), or a combination thereof. In some embodiments, the hormone agonist is a gonadotropin-releasing hormone (Gn-RH) agonist. In some embodiments, the hormone antagonist is a gonadotropin-releasing hormone (Gn-RH) antagonist. In some embodiments, the therapy is a contraceptive containing hormones. In some embodiments, the anti-inflammatory therapy is an NSAID, a JNK inhibitor, a TNF inhibitor, an interleukin (IL) inhibitor, or a combination thereof. In some embodiments, the method or assay further includes assessing the success of the intervention, the likelihood of success, incomplete success or failure, and the likelihood of failure.

[0137] In some embodiments, methods or assays for classifying or detecting endometriosis in an individual include determining the expression levels of one or more microRNAs (miRs) from a biological sample (e.g., a menstrual fluid sample). In some cases, biological or menstrual fluid samples are collected on days 1, 2, 3, 4, 5, 6, and / or 7 of an individual's menstrual cycle. In other cases, biological or menstrual fluid samples are collected on day 2 of an individual's menstrual cycle.

[0138] In some embodiments, the method or assay further includes determining the expression levels of two, three, or more microRNAs (miRs) from an individual's biological sample. In some embodiments, the miRs are selected from miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and / or miR-410-3p. In some embodiments, miRs include intracellular miRs, extracellular miRs, or both intracellular and extracellular miRs. In some embodiments, miRs are separated from cells in the biological sample. In various embodiments, miRs are separated from the non-cellular portion of the biological sample. In some embodiments, miRs are separated from the total biological sample (e.g., from both the intracellular and extracellular portions of the biological sample). In some embodiments, miRs are assessed or detected by any suitable method. In some embodiments, sequencing is used to assess or detect miRs.

[0139] In some embodiments, the method or assay further includes comparing the expression levels of one or more miRs to a reference expression level. In various cases, an increase or decrease in the expression level of one or more miRs relative to a reference expression level indicates that an individual has endometriosis. In some embodiments, the biological sample includes menstrual fluid, cervical-vaginal fluid, or both. In some embodiments, the biological sample is disposed in a sample collector as provided herein. In some embodiments, the sample collector includes a pad, tampons, vaginal cups, cervical caps, menstrual discs, cervical discs, sponges, interlabial pads, or combinations thereof.

[0140] Treatment of endometriosis

[0141] In some embodiments, this document describes methods and systems for treating subjects suspected of having endometriosis. In some embodiments, the method includes obtaining, or having obtained from the subject, a biological sample as described herein; performing, or having performed, measurements on the biological sample to determine whether the subject possesses a biomarker indicative of endometriosis; administering an intervention to the subject if the subject possesses a biomarker indicative of endometriosis, and not administering an intervention if the subject does not possess a biomarker indicative of endometriosis. In some embodiments, the biomarker indicative of endometriosis is a microRNA expression signature indicative of endometriosis. In some embodiments, the microRNA expression signature indicating endometriosis includes significantly different expression of one or more microRNAs (miRs, miRNAs) relative to the expression of one or more microRNAs selected from the group consisting of individuals without endometriosis: miR-1271-5p, miR-4485-3p, miR-125b-2-3p, miR-410-3p, let-7c-5p, miR-10 0-5p,miR-149-5p,miR-193b-3p,miR-221-5p,miR-363-3p,miR-99a-5p,let-7e-5p,miR-10a-5 p,miR-10b-5p,miR-125b-5p,miR-127-3p,miR-132-3p,miR-141-3p,miR-142-5p,miR-143-3p,m iR-144-5p,miR-145-5p,miR-152-3p,miR-16-2-3p,miR-17-3p,miR-195-5p,miR-196b-5p,miR -199a-3p / 199b-3p,miR-200a-3p,miR-200c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21- 5p, miR-22-3p, miR-222-3p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409 and miR-98-5p and combinations thereof.In some embodiments, the microRNA expression characteristics indicative of endometriosis include significantly different expression of one or more microRNAs (miRs) relative to the expression of one or more microRNAs selected from the group consisting of miR-1271-5p, miR-4485-3p, miR-125b-2-3p, miR-410-3p, and combinations thereof in individuals without endometriosis.

[0142] In some embodiments, the methods and systems described herein include measuring, or having measured, a biological sample to determine whether a subject possesses a biomarker indicative of endometriosis, including: extracting nucleic acids from, or having extracted from, the biological sample, and sequencing, or having sequenced, one or more biomarkers derived from the extracted nucleic acids. In some embodiments, one or more biomarkers are one or more microRNAs. In some embodiments, the biomarker shows a different presence or level in cervical vaginal fluid or menstrual fluid compared to its presence in peripheral blood or cervical vaginal tissue.

[0143] In some embodiments, the intervention includes surgical intervention, therapeutic intervention, or a combination thereof. In some embodiments, the surgical intervention includes surgical removal of at least a portion of the endometriotic lesion, hysterectomy, salpingo-oophorectomy, presacral neurectomy, or laparoscopic uterine nerve ablation. In some embodiments, the therapeutic intervention includes administration of a therapeutic agent. In some embodiments, the therapeutic agent is a hormone, a hormone agonist, a hormone antagonist, an aromatase inhibitor, an anti-inflammatory therapy, an acetyltransferase, a histone deacetylase inhibitor, a phosphodiesterase inhibitor, or a combination thereof. In some embodiments, the hormone is a synthetic hormone. In some embodiments, the hormone is estrogen, progesterone, progesterone, androgen, gonadotropin-releasing hormone (Gn-RH), or a combination thereof. In some embodiments, the hormone agonist is a gonadotropin-releasing hormone (Gn-RH) agonist. In some embodiments, the hormone antagonist is a gonadotropin-releasing hormone (Gn-RH) antagonist. In some embodiments, the therapeutic agent is a contraceptive containing a hormone. In some implementations, the anti-inflammatory therapy is an NSAID, a JNK inhibitor, a TNF inhibitor, an interleukin (IL) inhibitor, or a combination thereof.

[0144] In some embodiments, this document describes a method for predicting the success of an intervention for endometriosis in an individual. In some embodiments, the method includes obtaining a biological sample from the individual prior to an intervention such as surgery or administration of a therapeutic composition. In some embodiments, the intervention is a treatment for endometriosis. In some embodiments, the method includes measuring the obtained biological sample prior to the intervention to determine biomarkers or biomarker characteristics of the individual. In some embodiments, the method includes obtaining a biological sample from the individual after the intervention. In some embodiments, the method includes measuring the obtained biological sample after the intervention to determine biomarkers or biomarker characteristics of the individual. In some embodiments, the method includes comparing biomarkers or biomarker characteristics in a biological sample obtained prior to the intervention with biomarkers or biomarker characteristics in a biological sample obtained after the intervention. Predicting the success of an intervention is based on a comparison of biomarkers or biomarker characteristics in a biological sample obtained prior to the intervention, biomarkers or biomarker characteristics in a biological sample obtained after the intervention, biomarkers or biomarker characteristics in a biological sample obtained prior to the intervention, and biomarkers or biomarker characteristics in a biological sample obtained after the intervention, or a combination thereof. Predicting the success of an intervention involves comparing a biomarker or biomarker characteristic with a known biomarker or biomarker characteristic from an individual receiving the intervention, where the individual's outcome is known, such as intervention success or intervention failure.

[0145] Sample collection system

[0146] In some embodiments, this document describes a system or apparatus for collecting cells and other biological materials (nucleic acids, proteins, metabolites) from biological samples such as menstrual fluid. In some embodiments, the system or apparatus is used in conjunction with the methods described herein. In some embodiments, the system includes an upper portion; a lower portion; a central portion including a first end configured to be operatively coupled to the upper portion and a second end configured to be operatively coupled to the lower portion; and a compression member including: a first compression end disposed in the central portion, the first compression end forming a compression base in contact with an inner surface of the central portion, the compression base and the central portion forming a first central cavity configured to receive a sample collector; and a second compression end coupled to the lower portion; wherein the compression member is configured to compress the sample collector upon activation of the lower portion. In some embodiments, the upper portion includes an upper cavity configured to retain a preservation solution. In some embodiments, the upper cavity is accessible via a destructible member. In some embodiments, the central portion is configured to retain a preservation solution. In some embodiments, the preservation solution (e.g., disposed within the destructible member) is disposed at or adjacent to the compression base.

[0147] In some embodiments, the sample collection device includes a system for collecting biological samples from a subject, the system including a sample collector that collects the biological samples from the subject in a non-invasive manner. In some embodiments, the sample collector is inserted into the vaginal cavity of the subject to collect the biological sample. In some embodiments, the system described herein collects a volume of biological sample, including menstrual fluid, cervical vaginal fluid, secreted mucus, exfoliated uterine cells, exfoliated ovarian cells or other cells, tissues, or fluids. In some embodiments, the sample collector is made of a material capable of collecting and / or retaining biological samples. In some embodiments, the sample collector is made of a highly absorbent material that rapidly absorbs liquid samples. In some embodiments, the sample collector is made of a material that rapidly releases the absorbed liquid sample, such as when a compression mechanism (e.g., pressure, force) is applied to the sample collector. In some embodiments, the system includes an extractor for retrieving the biological sample from the sample collector. In some embodiments, the extractor includes components for applying a compression mechanism to the sample collector. In some embodiments, the components for applying the compression mechanism include, but are not limited to, springs, threaded screws, levers, airtight plungers, or roller-based compression. In some embodiments, the liquid sample absorbed on the sample collector is extracted by applying a compression mechanism to the sample collector. In some embodiments, the system includes a compression mechanism. In some embodiments, the system does not include a compression mechanism. In some embodiments, the compression mechanism is compressed externally to the system. In some embodiments, closing or sealing the system activates the compression mechanism. In some embodiments, closing or sealing the system does not activate the compression mechanism. In some embodiments, activation of the compression mechanism is separate from closing or sealing the system. In some embodiments, the liquid sample absorbed on the sample collector is extracted without a compression mechanism. In some embodiments, the liquid sample absorbed on the sample collector is eluted into a buffer solution described herein. In some embodiments, the extractor includes a sample container that receives the sample collector via an opening, and a reservoir in fluid communication with the sample container to receive biological samples released from the sample collector. In some embodiments, the reservoir and / or container contains a solution containing one or more reagents for analyzing, preserving, storing, or transporting the collected biological sample. In some embodiments, one or more reagents are necessary for hydrolyzing, diffusing, or releasing the biological sample. In some embodiments, one or more reagents are essential for analyzing, preserving, or extracting deoxyribonucleic acid, ribonucleic acid, or proteins from biological samples. In some embodiments, one or more reagents are essential for reducing analytical background noise. In some embodiments, one or more reagents are essential for precipitating or removing contaminants from biological samples. In some embodiments, one or more reagents are essential for testing biological samples for the presence or absence of an analyte.In some embodiments, the container contains reagents necessary to dissolve the sample collector upon contact with it. Therefore, in some embodiments, the sample collector is made of a material that dissolves upon contact with reagents stored in the container, thereby releasing the biological sample into the container. In some embodiments, the system also includes a cartridge containing a chamber, wherein the cartridge and / or chamber are connected to the reservoir via a docking unit such that, upon contact with the reservoir, the released biological sample flows into the cartridge and / or chamber. In some embodiments, the docking unit includes a one-way pressure valve. In some embodiments, the docking unit includes a resealable slit. In some embodiments, the cartridge containing the collected biological sample is covered or sealed. In some embodiments, the cartridge containing the collected biological sample is transported without damage or degradation of the collected biological sample.

[0148] Figure 5 and Figures 6A-6C An embodiment of a menstrual fluid cell collection system 400 for collecting samples as described herein is illustrated. In some embodiments, system 400 includes an upper portion 401, a central portion 402, a lower portion 403, and a compression member 404.

[0149] In some embodiments, the upper portion 401 includes an upper cavity 405, a destructible member 406, an inner surface 425 of the upper portion 401, and a destructive element 407. In some embodiments, the upper portion 401 is coupled to a first end 414 of the central portion 402. In some embodiments, the upper portion 401 is threadedly coupled to the first end 414. In some embodiments, the upper portion 401 cannot be removably coupled to the central portion 402, for example, by a patient. In some embodiments, the upper portion 401 is removably coupled to the central portion 402 by removing a screw or other suitable coupling member from the upper portion 401, for example, by a medical practitioner or technician. In some embodiments, the upper portion 401 is sealed or configured to seal the first central cavity 411. In some embodiments, one of the upper portion 401 or the first end 414 of the central portion 402 includes a seal such that fluid communication from the first central cavity 411 to the outside of the system is prohibited (e.g., fluid cannot flow out of the first central cavity 411).

[0150] In some embodiments, system 400 further includes a connector that flexibly couples upper portion 401 to central portion 402. In some embodiments, the connector flexibly couples upper portion 401 to central portion 402 such that the connector bends to allow upper portion 401 to couple to a first end 414 of central portion 402. In some embodiments, the connector comprises polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, rubber, silicone, thermoplastic elastomer (TPE), or combinations thereof, or formed therefrom.

[0151] In some embodiments, the destructible component 406 surrounds the preservation solution (e.g., Biomatrica). In some embodiments, the amount of preservation solution surrounded by the destructible member 406 is in the range of about 3 ml to about 12 ml, about 5 ml to about 10 ml, about 7.5 ml to about 10 ml, or about 7 ml to about 8 ml. In some embodiments, the destructible member 406 comprises polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, aluminum, or combinations thereof, or formed therefrom. In some embodiments, the aluminum is a heat-sealable aluminum foil.

[0152] In some embodiments, the rupture element 407 includes a first surface 408, a second surface 409, and an opening 410. In some embodiments, the first surface 408 of the rupture element 407 is adjacent to the destructible member 406. In some embodiments, the second surface 409 of the rupture element 407 is adjacent to a first central cavity 411. In some embodiments, the rupture element 409 is configured to rupture the destructible member 406 upon activation of the upper portion 403. In some embodiments, the rupture element 409 is displaced toward the destructible member 406 such that a force is applied to the destructible member 406. In some embodiments, this force causes the destructible member 406 to break, break, fail, or open. In some embodiments, a perforator (e.g., a floating perforator) is disposed within the destructible member 406 (e.g., within a preservation solution). In some embodiments, the perforator causes the destructible member 406 to break, break, or open upon compression of the destructible member 406.

[0153] In some embodiments, the disruptive element 409 includes one or more protrusions (e.g., on the first surface 408) configured to cut or pierce the disruptive member 406, for example, when the disruptive element 407 is pressed against or displaced toward the disruptive member 406. In some embodiments, the one or more protrusions include blades, tips, points, or other suitable protrusions configured to disrupt the disruptive member 406. In some embodiments, the upper portion 401 is activated by a first end 414 coupling the upper portion 401 to the central portion 402. In some embodiments, upon activation of the upper portion 401 and / or subsequent disruption of the disruptive member 406, the opening 409 of the disruptive element 410 allows or permits fluid communication between the upper cavity 405 and the first central cavity 411. In some embodiments, alternatively, a preservation solution flows out of the disrupted disruptive member 406, through the opening 409, and into at least a portion of the first central cavity 411. In some embodiments, the preservation solution is configured to flow into or enter the first central cavity 411 when the destructible component is damaged.

[0154] In some embodiments, when the sample collector is positioned in the first central cavity 411, the disrupting element 407 is configured to apply a first force to the sample collector upon activation of the lower portion 403. In some embodiments, activation of the lower portion 403 (e.g., rotation of the lower portion 403 relative to the central portion 402) causes the compression first end 418 to displace or move toward the upper cavity 405. In some embodiments, activation of the lower portion 403 causes the compression base 417 to displace toward the upper cavity 405. Displacement of the compression base 417 is configured to apply a second force to the sample collector, for example, when the sample collector is positioned in the first central cavity 411. In some embodiments, the lower portion 403 provides a mechanical advantage that allows a patient to compress the sample collector using the system 400. Figure 5 As shown, in some embodiments, the lower portion 403 is coupled (e.g., threadedly coupled) to the central portion 402. In some embodiments, the interaction between the lower portion 403 and the central portion 402 provides a mechanical advantage when activated by a user, such that sufficient force is applied to at least a portion of the sample collector to compress or crush it. In some embodiments, loads exceeding 20 lbs, 30 lbs, 40 lbs, 50 lbs, 60 lbs, 70 lbs, 80 lbs, 90 lbs, or 100 lbs are applied to the sample collector by the system 400. In some embodiments, loads less than 200 lbs, 180 lbs, 160 lbs, 140 lbs, 120 lbs, 100 lbs, or 80 lbs are applied to the sample collector by the system 400.

[0155] In some embodiments, the central portion 402 includes a first central cavity 411, a second central cavity 412, an inner surface 413, a first end 414, and a second end 415. In some embodiments, the central portion 402 also includes a stop 424. In some embodiments, the central portion 402 is coupled to a lower portion 403. In some embodiments, the central portion 402 is threadedly coupled to the lower portion 403. In some embodiments, the lower portion 403 is rotatable relative to the central portion 402 in a first direction. In some embodiments, the first direction relative to the central portion 402 is clockwise rotation. In some embodiments, the lower portion 403 is not rotatable relative to the central portion 402 in a second direction. In some embodiments, the second direction relative to the central portion 402 is counterclockwise rotation. In some embodiments, the first central cavity 411 is disposed between the compression base 417 and the first end 414 of the central portion. In some embodiments, the compression base 417 and the central portion 402 further form a second central cavity 412. In some embodiments, the second central cavity 412 is configured to receive the preservation solution and biological samples from the sample collector.

[0156] In some embodiments, the central portion 402 includes a port 416. In some embodiments, the port 416 is disposed through at least a portion of the second end 415 of the central portion 415. In some embodiments, the port 416 allows access to the second central cavity 412. In some embodiments, the port 416 is a valve. In some embodiments, the valve is a self-sealing valve, a safety valve, a sampling valve, a one-way valve, a check valve, a duckbill valve, a baffle valve, an umbrella valve, a diaphragm valve, or other suitable valve. In some embodiments, the port 416 is accessed via a syringe (e.g., the syringe can be displaced through at least a portion of the port 416). In some embodiments, the central portion 402 includes one, two, three, four, five, or more than five ports. In some embodiments, the port 416 is accessed through an external opening 423 on the base 422.

[0157] In some embodiments, the compression member 404 includes a compression base 417, a first compression end 418, and a second compression end 419. In some embodiments, the compression base 417 includes a compression base seal 420 and an outer surface 421 of the compression base 417. In some embodiments, the compression base seal 420 includes, or is formed from, nitrile rubber, ethylene propylene rubber, perfluoroelastomer (FFKM), fluorosilicone rubber, neoprene rubber, chloroprene, polyurethane, silicone rubber, fluorocarbons, or combinations thereof. In some embodiments, the ethylene-propylene rubber is an ethylene-propylene copolymer (EPR) or an ethylene-propylene-diene terpolymer (EPDM).

[0158] In some embodiments, a portion of the compression member (e.g., an elongated member) extends through at least a portion of the second end 415 of the central portion 402. In some embodiments, the compression base 417 includes a compression base seal 420 such that fluid communication is permitted in a first direction around at least a portion of the compression base 417 and prohibited or restricted in a second direction around at least a portion of the compression base 417. In some embodiments, the first direction is from the first central cavity 411 to the second central cavity 412. In some embodiments, the second direction is from the second central cavity 412 to the first central cavity 411. In some embodiments, the compression base seal 420 extends around the outer surface 421 of the compression base 417. In some embodiments, the compression base seal 420 is disposed between the compression base 417 and the inner surface 413 of the central portion 402. In some embodiments, when a portion of the compression member 404 extends through an orifice or opening in the second end 415 of the central portion 402, the orifice includes a seal such that fluid communication between the second central cavity and the outside of the central portion is prohibited.

[0159] In some embodiments, a first central cavity 411 located near the first end 414 of the central portion 402 has a first diameter. In some embodiments, a second central cavity 412 located near the second end 415 of the central portion 402 has a second diameter. In some embodiments, the first central cavity 411 and the second central cavity 412 located at one or more locations between the first end 414 and the second end 415 have a third diameter. In some embodiments, the diameters of the first central cavity 411 and the second central cavity 412 located at one or more locations between the first end 414 and the second end 415 increase or decrease. In some embodiments, the diameters gradually increase or decrease (e.g., the inner surface 413 is inclined).

[0160] In some embodiments, the first diameter and the second diameter are substantially equal. In various embodiments, the first and second diameters are smaller than the third diameter. In some embodiments, when the compression base 417 is installed near the second end 415 of the central portion 402, the compression base seal 420 forms a seal between the compression base 417 and the inner surface 413 of the central portion 402. In some embodiments, when the compression base 417 is installed near the first end 414 of the central portion 402, the compression base seal 420 forms a seal between the compression base 417 and the inner surface 413 of the central portion 402. In some embodiments, when the compression base 417 is installed between the first end 414 and the second end 415 of the central portion 402, the compression base seal 420 does not form a seal between the compression base 417 and the inner surface 413 of the central portion 402. In some embodiments, when the compression base 417 is displaced between the first end 414 and the second end 415 of the central portion 402, no seal is formed between the compression base 417 and the inner surface 413 of the central portion 402. In some cases, if a seal is formed at one or more locations between the first end 414 and the second end 415 of the central portion 402, excessive pressure can accumulate in the system 400 (e.g., in one or more of the first central cavity 411 or the second central cavity 412). In some embodiments, the compression base seal 420 is a pressure seal. In some embodiments, if the pressure within at least a portion of the system 400 increases above a threshold level, the pressure seal is released or configured to release the pressure in the system 400 (e.g., by allowing fluid or air to pass through).

[0161] refer to Figure 5In some embodiments, system 400 further includes one or more ridges or ribs 427. As illustrated, a ridge 427 extends along a portion of the inner surface 413 of the central portion 402. In some embodiments, one or more recesses extend along a portion of the inner surface 413 of the central portion 402. In some embodiments, the ridge 427 or recesses interrupt the formation of a seal between the compression base 417 and the inner surface 413 of the central portion 402 at one or more locations between a first end 414 and a second end 415 of the central portion 402, such that the pressure within at least a portion of system 400 does not increase above a threshold level (e.g., to avoid or suppress the development of excessive pressure within system 400).

[0162] In some embodiments, the lower portion 403 includes a base 422. In some embodiments, the base 422 includes an external opening 423. In some embodiments, the base 422 includes one, two, three, four, five, or more than five openings. In some embodiments, the external opening 423 allows, permits, or provides access to the port 416.

[0163] Figures 7A-7C and Figures 8A-8C The illustration shows the use of an embodiment of the system 400 described herein. In some embodiments, although the upper portion 401 is configured to be operatively connected to the central portion 402, it is not connected to the central portion 402 until a sample collector is inserted via the first end of the central portion 414. Figure 7A In some embodiments, the lower portion 403 is operatively coupled to the central portion 402 before being inserted into the sample collector via the first end 414 of the central portion 402. Figure 7A ).

[0164] In some embodiments, the sample collector 426 is placed in the first central cavity 411 of the system 400. Figure 8A In some embodiments, after the sample collector 426 is inserted into the first central cavity 411 via the first end 414 of the central portion 402, the upper portion 401 is operatively coupled to the central portion 402. Figure 7B Operable coupling of the upper portion 401 to the central portion 402 includes threading the upper portion 401 to the central portion 402 and rotating the upper portion 401 in a first direction to activate the upper portion 401. In some embodiments, the upper portion 401 is rotated until it engages or contacts the stop 424. Figure 7B ).

[0165] In some embodiments, the upper portion 401 is rotated until the system provides a signal (e.g., a tactile signal). In some embodiments, the signal is sound. In some embodiments, the sound is a clicking sound. Activating the upper portion 401 by rotating it includes reducing the distance between the disruptive element 407 and the inner surface of the upper portion 425. Figure 8B In some embodiments, activation of the upper portion 401 includes rotation of the upper portion 401 relative to the central portion 402 in a first direction. In some embodiments, the first direction is clockwise. In some embodiments, the upper portion 401 cannot rotate relative to the central portion 402 in a second direction. In some embodiments, the second direction is counterclockwise. In some embodiments, the space between the destructive element 407 and the inner surface of the upper portion 425 includes an upper cavity 405 accommodating the destructible member 406.

[0166] In some embodiments, activation of the upper portion 401 is completed when the upper portion 401 connects to or contacts the stop member 424, or when a signal is given by the system. In some embodiments, activation of the upper portion 401 is completed when no additional rotation of the upper portion 401 in the first direction is achieved or performed. In some embodiments, activation of the upper portion 401 is completed upon the breakage of the destructible member 406. In some embodiments, activation of the upper portion 401 results in compression of the sample collector 426. Figure 8C ).

[0167] In some embodiments, the lower portion 403 is activated after the upper portion 401 is activated. In some embodiments, activation of the lower portion 403 includes rotation of the lower portion 403 relative to the central portion 402 in a first direction. In some embodiments, the lower portion 403 is rotated until it engages with or contacts the stop member 424. Figure 7C In some embodiments, the lower portion 403 is rotated until the system provides a signal (e.g., a tactile signal). In some embodiments, the signal is sound. In some embodiments, the sound is a clicking sound. In some embodiments, activation of the lower portion 403 is completed when no additional rotation of the lower portion 403 in the first direction is achieved or performed. In some embodiments, activation of the lower portion 403 causes the compression first end 418 of the compression base 417 to shift toward the breaking element 407. Figure 8B and Figure 8CIn some embodiments, the destructive element 407 is configured to apply a first force to the sample collector upon activation of the lower portion 403. In some embodiments, the first force compresses the sample collector between the first compression end 418 and the destructive element 407. In some embodiments, the compression of the sample collector causes the preservation solution to mix with the biological sample entering the second central cavity 412. In various embodiments, the upper portion 401 is coupled to the central portion 402. In such a configuration, the upper portion 401 is sealed to the central portion 402. In some embodiments, the formation of the seal is indicated by a tactile signal (e.g., a click). In some embodiments, after the upper portion 401 is coupled to the central portion 402 and a seal is formed, the lower portion 403 is activated, causing the preservation solution to be released from the destructible member 406. In some embodiments, the preservation solution does not leak or flow out of the system 400 (e.g., around the seal) and come into contact with the user.

[0168] In some embodiments, the compression base seal 420 allows fluid communication between the first central cavity 411 and the second central cavity 412 before the lower portion 403 is fully activated. In some embodiments, the fluid communication between the first central cavity 411 and the second central cavity 412 allows a preservation solution mixed with the biological sample to enter the second central cavity 412. In some embodiments, the completion of the activation of the lower portion 403 inhibits or prevents fluid communication between the first central cavity 411 and the second central cavity 412 through or around the compression base seal 420. In some embodiments, the activation of the lower portion 403 is completed when the lower portion 403 engages or contacts the stop 424, when the system gives a signal, when no additional rotation of the lower portion 403 in a first direction is achieved or performed, or a combination thereof.

[0169] In some embodiments, the kit described herein includes the system described herein and a sample collector (e.g., a tampon). In some embodiments, the kit includes identification descriptions, labels, and / or packaging inserts. In some embodiments, the kit also includes a transport package. In some embodiments, the transport package is used for transporting the system after use. In some embodiments, the transport package includes a hydrophilic material. In some embodiments, the hydrophilic material includes cotton, cellulose, hydrogel, absorbent polymer, or combinations thereof. In some embodiments, the transport package includes 1, 2, 3, 4, 5, or more layers of hydrophilic material. If the transport package includes more than two layers of hydrophilic material, at least one of the more than two layers of hydrophilic material is different from the remaining layers. In some embodiments, the hydrophilic material is contained in a pouch. In some embodiments, the pouch is formed of polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, or combinations thereof. In some embodiments, the transport package includes adhesive strips, glue, or a waterproof zipper for sealing the transport package after the system including the sample collector is placed inside. In some embodiments, the transport package is pre-labeled. In some implementations, the transport packaging also includes at least one layer of absorbent material.

[0170] In some embodiments, the kit also includes a label or packaging insert. In some embodiments, the label or packaging insert includes a list of kit contents, instructions relating to the use of the kit in the methods described herein, or a combination thereof. In some embodiments, the label is on or associated with the system. In some embodiments, the label is on the system when the letters, numbers, or other characters forming the label are attached, molded, or etched into the system itself. In some embodiments, the label is associated with the system when it is present, for example, as a packaging insert within a container or carrier that also holds the system. In some cases, the label is used to indicate that the contents will be used for a specific application, such as collecting a sample from menstrual fluid.

[0171] Implementation of numbering

[0172] The disclosure herein is further defined by the following numbered embodiments: 1. An assay for classifying or detecting endometriosis in a subject, comprising determining the expression levels of one or more microRNAs (miRs) from a menstrual fluid sample of the subject, wherein the menstrual fluid sample is collected on the first, second, third, fourth, fifth, sixth, and / or seventh day of the subject's menstrual cycle. 2. The assay according to embodiment 0, wherein the menstrual fluid sample is collected on the second day of the subject's menstrual cycle. 3. The assay according to embodiment 0 or embodiment

[0163] , further comprising determining the expression levels of two or more microRNAs (miRs) from the menstrual fluid sample of the subject. 4. The assay according to embodiment 0 or embodiment

[0163] , further comprising determining the expression levels of three or more microRNAs (miRs) from the menstrual fluid sample of the subject. 5. The assay method according to any one of Embodiments 1-

[0163] , wherein the miRNA is selected from miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p. 6. The assay method according to any one of Embodiments 1-

[0163] , further comprising comparing the expression level with a reference expression level of the one or more miRs, wherein an increase or decrease in the expression level of the one or more miRs relative to the reference expression level indicates that the subject has endometriosis. 7. The assay method according to any one of Embodiments 1-

[0163] , wherein the miR is an intracellular miR. 8. The assay method according to any one of Embodiments 1-

[0163] , wherein the miR is an extracellular miR. 9. The assay method according to any one of Embodiments 1-

[0163] , wherein the miR is both intracellular and extracellular miR. 10. The assay method according to any one of Embodiments 1-

[0163] , wherein the menstrual fluid further includes cervical vaginal fluid. 11. The determination method according to any one of embodiments

[0163] -

[0163] , wherein the menstrual fluid sample is placed in a sample collector. 12. The determination method according to embodiment

[0163] , wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad.13. A system for collecting biological samples from a sample collector, the system comprising an upper portion; a lower portion; a central portion including a first end configured to be operatively coupled to the upper portion and a second end configured to be operatively coupled to the lower portion; a destructible member for retaining a preservation solution; and a compression member comprising: a compression first end disposed in the central portion, the compression first end forming a compression base in contact with an inner surface of the central portion, the compression base and the central portion forming a first central cavity configured to receive the sample collector; and a compression second end coupled to the lower portion; wherein the compression member is configured to compress the sample collector upon activation of the lower portion, such that the destructible member is destructed and the preservation solution is released. 14. The system according to embodiment 13, wherein the destructible member is disposed in the upper cavity of the upper portion. 15. The system according to embodiment 13 or

[0163] , further comprising a destructive element configured to destruct the destructible member upon activation of the upper portion. 16. The system according to embodiment

[0163] , wherein the destructive element includes an opening that allows fluid communication between the upper cavity and the first central cavity. 17. The system according to embodiment

[0163] or embodiment

[0163] , wherein the destructive element includes a first surface adjacent to the destructible member and a second surface adjacent to the first central cavity. 18. The system according to any one of embodiments

[0163] -

[0163] , wherein when the sample collector is disposed in the first central cavity, the destructive element is configured to apply a first force to the sample collector upon activation of the lower portion. 19. The system according to any one of embodiments

[0163] -

[0163] , wherein when the sample collector is disposed in the first central cavity, activation of the lower portion displaces the destructive element toward the upper cavity. 20. The system according to any one of embodiments 1-

[0163] , wherein the destructible member surrounds the preservation solution. 21. The system according to any one of Embodiments 1-

[0163] , wherein the destructible member comprises polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, aluminum foil, or a combination thereof. 22. The system according to Embodiment

[0163] , wherein the aluminum foil is heat-sealable. 23. The system according to any one of Embodiments 1-

[0163] , wherein the preservation solution is configured to flow into the first central cavity when the destructible member is destroyed. 24. The system according to Embodiment 13, wherein the destructible member is disposed adjacent to the compression base. 25. The system according to Embodiment

[0163] , wherein the destructible member surrounds the preservation solution.26. The system according to embodiment

[0163] or embodiment

[0163] , wherein the destructible component comprises polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, aluminum foil, or a combination thereof. 27. The system according to embodiment

[0163] , wherein the aluminum foil is heat-sealable. 28. The system according to any one of embodiments

[0163] -

[0163] , wherein the preservation solution is configured to flow into the first central cavity when the destructible component is destructed. 29. The system according to any one of embodiments 13-

[0163] , wherein the upper portion is configured to seal the central cavity. 30. The system according to any one of embodiments 13-

[0163] , wherein one of the upper portion or the first end of the central portion includes a seal such that fluid communication from the first central cavity to the outside of the system is prohibited. 31. The system according to any one of embodiments

[0163] -

[0163] , wherein the upper portion is threadably coupled to the first end of the central portion. 32. The system according to embodiment

[0163] , wherein the upper portion cannot be removably coupled to the central portion by the patient. 33. The system according to embodiment

[0163] or embodiment

[0163] , wherein the upper portion is rotatable by the patient relative to the central portion in a first direction, and wherein the lower portion is not rotatable by the patient relative to the center in a second direction. 34. The system according to any one of embodiments

[0163] -

[0163] , wherein the first cavity is disposed between the compression base and the first end of the central portion. 35. The system according to any one of embodiments

[0163] -

[0163] , wherein the compression base and the central portion further form a second central cavity, the second central cavity being configured to receive the preservation solution and biological samples from a sample collector. 36. The system according to any one of embodiments

[0163] -

[0163] , wherein activation of the lower portion causes the compression base to shift toward the upper cavity. 37. The system according to embodiment

[0163] , wherein the displacement of the compression base is configured to apply a second force on the sample collector. 38. The system according to any one of embodiments

[0163] -

[0163] , wherein the central portion is threadedly coupled to the lower portion. 39. The system according to embodiment

[0163] , wherein the threaded coupling of the central portion to the lower portion provides a mechanical advantage. 40. The system according to embodiment

[0163] or embodiment

[0163] , wherein the lower portion is rotatable by the patient relative to the central portion in a first direction, and wherein the lower portion is not rotatable by the patient relative to the central portion in a second direction.41. The system according to any one of embodiments 1-

[0163] , wherein a portion of the compression member extends through the second end of the central portion. 42. The system according to embodiment 41, wherein the portion of the compression member extends through an orifice in the second end of the central portion, and wherein the orifice includes a seal such that fluid communication between the second central cavity and the outside of the central portion is prohibited. 43. The system according to any one of embodiments 1-42, wherein the compression base includes a compression base seal such that fluid communication is permitted in a first direction and prohibited in a second direction. 44. The system according to embodiment 43, wherein the compression base seal extends around an outer surface of the compression base. 45. The system according to embodiment 43 or embodiment 44, wherein the compression base seal is disposed between the compression base and the inner surface of the central portion. 46. The system according to any one of embodiments 43-45, wherein the first direction is from the first central cavity to the second central cavity, and wherein the second direction is from the second central cavity to the first central cavity. 47. The system according to any one of embodiments 1-46, wherein the central portion includes a port. 48. The system according to embodiment 47, wherein the port is disposed through the second end of the central portion. 49. The system according to embodiment 47 or embodiment 48, wherein the port allows access to the second central cavity. 50. The system according to any one of embodiments 47-49, wherein the port is a valve. 51. The system according to embodiment 50, wherein the valve is a self-sealing valve, a diaphragm, a check valve, a safety valve, or a sampling valve. 52. The system according to any one of embodiments 1-50, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. 53. The system according to any one of embodiments 1-52, wherein the volume of the preservation solution is 5 ml to 10 ml. 54. The system according to embodiment 53, wherein the volume of the preservation solution is about 7.5 mL. 55. The system according to any one of embodiments 1-54, wherein the osmolality of the preservation solution is about 310 to about 410 mOsmkg. -1 56. The system according to any one of embodiments 1-54, wherein the osmolality of the preservation solution is about 95 to about 210 mOsm kg. -157. A kit comprising: a system of any one of embodiments 1-56; and a sample collector. 58. The kit according to embodiment 57, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad. 59. The kit according to embodiment 57 or embodiment 58, further comprising a transport package. 60. The kit according to embodiment 59, wherein the transport package comprises a hydrophilic material. 61. The kit according to embodiment 60, wherein the hydrophilic material comprises cotton, cellulose, hydrogel, absorbent polymer, or a combination thereof. 62. The kit according to embodiment 60 or embodiment 61, wherein the transport package comprises at least one layer of the hydrophilic material. 63. The kit according to embodiment 60 or embodiment 61, wherein the hydrophilic material is contained in a pouch. 64. The kit according to embodiment 63, wherein the pouch is formed of polyethylene, polypropylene, polyester, nylon, polyvinyl chloride, polystyrene, poly(methyl methacrylate), polyetheretherketone, or a combination thereof. 65. The kit according to embodiment 59, wherein the transport package includes means for sealing the transport package. 66. The kit according to embodiment 65, wherein the means for sealing the transport package includes adhesive strips, glue, a waterproof zipper, or a combination thereof. 67. The kit according to any one of embodiments 59-66, wherein the transport package includes a label. 68. The kit according to any one of embodiments 57-67, further comprising instructions for use of the system. 69. A biological sample collected using the system according to any one of embodiments 1-56 or the kit according to any one of embodiments 57-68. 70. A method for collecting a biological sample from a sample collector, the method comprising: a. an apparatus comprising: an upper portion including an upper cavity configured to retain a preservation solution, wherein the upper cavity is accessible via a destructible member; a lower portion; a central portion including a first end configured to be operatively coupled to the upper portion and a second end operatively coupled to the lower portion; and a compression member comprising: a first compression end disposed in the central portion, the first compression end forming a compression base in contact with an inner surface of the central portion, the compression base and the central portion forming a first central cavity configured to receive a sample collector; and a second compression end coupled to the lower portion; wherein the compression member is configured to compress the sample collector upon activation of the lower portion; and b. placing the sample collector into the first central cavity; c. activating the lower portion to compress the sample collector to release the biological sample from the sample collector; and d. collecting the biological sample. 71. The method according to embodiment 70, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad.72. The method according to embodiment 70 or embodiment 71, wherein activating the lower portion includes rotating the lower portion relative to the central portion in a first direction. 73. The method according to any one of embodiments 70-72, wherein the compression base and the central portion further form a second central cavity, the second central cavity being configured to receive the preservation solution and a biological sample from a sample collector. 74. The method according to any one of embodiments 70-73, wherein the collection includes extracting the biological sample from the second central cavity. 75. The method according to embodiment 74, wherein the extraction of the biological sample is through a port allowing access to the second central cavity on the device. 76. The method according to embodiment 75, wherein the port is a valve. 77. The method according to embodiment 76, wherein the valve is a self-sealing valve, a diaphragm, a check valve, a safety valve, or a sampling valve. 78. The method according to any one of embodiments 75-77, wherein the port is disposed through the second end of the central portion. 79. The method according to embodiment 78, wherein the collection includes extracting the biological sample from the second central cavity using a syringe inserted into the second central cavity through the port. 80. A method for detecting endometriosis in an individual, comprising determining the expression level of one or more microRNAs (miRs) selected from a biological sample of the individual: miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p. 81. The method of embodiment 80, further comprising comparing the expression level to a reference expression level of the one or more miRs, wherein an increase or decrease in the expression level of the one or more miRs relative to the reference expression level indicates that the subject has endometriosis. 82. The method of embodiment 80, wherein the reference expression level is obtained from a subject who has never had a reproductive disorder or is not suspected of having a reproductive disorder. 83. The method of embodiment 82, wherein the reproductive disorder is endometriosis. 84. The method of any one of embodiments 80-83, wherein the individual suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. 85. The method according to any one of embodiments 80-84, wherein the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE).86. The method according to any one of embodiments 80-85 further comprises determining the expression level of one or more miRs selected from the group consisting of: let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2- 3p,miR-17-3p,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,m iR-200c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-2 22-3p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409, and miR-98-5p. 87. The method according to any one of embodiments 80-86 further includes determining the methylation profile of one or more CpG sites selected from the CpG sites in Table 4. 88. The method according to any one of embodiments 80-87 further includes determining a measure of bacterial diversity in the biological sample. 89. The method according to embodiment 88, wherein the measure of bacterial diversity is the amount of at least one type of bacteria. 90. The method according to embodiment 89, wherein the at least one bacterium is selected from the genera *Propionibacterium*, *Alternaria*, *Porphyromonas*, *Streptococcus*, *Desmodium*, *Moraxella*, *Anaerobes*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Franklinella*, and *Klebsiella*. 91. The method according to any one of embodiments 88-90, further comprising comparing the measure of bacterial diversity with a reference measure of bacterial diversity. 92. The method according to any one of embodiments 88-91, wherein the measure of bacterial diversity is the ratio of at least one first bacterium to at least one second bacterium. 93. The method according to any one of embodiments 80-92, further comprising determining the amount of *Propionibacterium acnes*.94. The method according to embodiment 93 further includes comparing the amount of *Propionibacterium acnes* with a reference amount of *Propionibacterium acnes*. 95. The method according to any one of embodiments 80-94, wherein the biological sample is menstrual fluid. 96. The method according to embodiment 95, wherein the menstrual fluid further includes cervical vaginal fluid. 97. The method according to embodiment 95 or embodiment 96, wherein the biological sample is collected on the second day of the individual's menstrual cycle. 98. The method according to any one of embodiments 95-97, wherein the biological sample is collected on a day in the individual's menstrual cycle when the individual experiences heavy menstrual flow. 99. The method according to any one of embodiments 80-98, wherein the biological sample is collected before treatment is administered to the individual. 100. The method according to any one of embodiments 80-98, wherein the biological sample is collected after treatment is administered to the individual. 101. The method according to any one of embodiments 80-100, wherein the biological sample is disposed in a sample collector. 102. The method of embodiment 101, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. 103. The method of any one of embodiments 80-102, further comprising administering treatment for endometriosis to the individual. 104. The method of embodiment 103, wherein the treatment is selected from surgical intervention, administration of therapeutic agents, and combinations thereof. 105. The method of embodiment 104, wherein the therapeutic agent is selected from hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 106. The method of any one of embodiments 80-105, further comprising generating a report based on the expression level of the one or more miRs relative to the reference expression level. 107. The method of embodiment 106, further comprising transmitting the report to a healthcare professional. 108. The method of embodiment 106 or embodiment 107, wherein the report contains a recommendation for administering a therapeutic agent to the individual. 109. The method according to any one of embodiments 106-108, wherein the report includes a recommendation for surgical intervention. 110. The method according to any one of embodiments 80-109, wherein the method has a false detection rate of 5% or less. 111. A method for detecting endometriosis in an individual, comprising determining from a biological sample of the individual a methylation profile of one or more CpG sites selected from CpG sites in Table 4; b. a measure of bacterial diversity in the biological sample; and c. a combination thereof. 112. The method according to embodiment 111, wherein the measure of bacterial diversity is the amount of at least one bacterium.113. The method according to embodiment 112, wherein the at least one bacterium is selected from the following genera: *Propionibacterium*, *Propionibacterium*, *Dialectae*, *Porphyromonas*, *Streptococcus*, *Desmodium*, *Moraxella*, *Anaerobic Cocci*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Franklinella*, and *Klebsiella*. 114. The method according to embodiment 112 or embodiment 113 further includes comparing the measure of bacterial diversity with a reference measure of bacterial diversity. 115. The method according to any one of embodiments 112-114, wherein the measure of bacterial diversity is the ratio of at least one first bacterium to at least one second bacterium. 116. The method according to any one of embodiments 111-115 further includes determining the amount of *Propionibacterium acnes*. 117. The method according to embodiment 116 further includes comparing the amount of *Propionibacterium acnes* with a reference amount of *Propionibacterium acnes*. 118. The method according to any one of embodiments 111-117, wherein the biological sample is menstrual fluid. 119. The method according to embodiment 118, wherein the menstrual fluid further includes cervical vaginal fluid. 120. The method according to embodiment 118 or embodiment 119, wherein the biological sample is collected on the second day of the individual's menstrual cycle. 121. The method according to any one of embodiments 118-120, wherein the biological sample is collected on a day in the individual's menstrual cycle when the individual experiences heavy menstrual flow. 122. The method according to any one of embodiments 111-121, wherein the biological sample is collected before treatment is administered to the individual. 123. The method according to any one of embodiments 111-121, wherein the biological sample is collected after treatment is administered to the individual. 124. The method according to any one of embodiments 111-123, wherein the biological sample is disposed in a sample collector. 125. The method of embodiment 124, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. 126. The method of any one of embodiments 111-125, further comprising administering treatment for endometriosis to the individual. 127. The method of embodiment 126, wherein the treatment is selected from surgical intervention, administration of a therapeutic agent, and combinations thereof. 128. The method of embodiment 127, wherein the therapeutic agent is selected from hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 129. The method of any one of embodiments 111-128, further comprising generating a report based on the expression level of the one or more miRs relative to the reference expression level.130. The method of embodiment 129, further comprising transmitting the report to a healthcare professional. 131. The method of embodiment 129 or embodiment 107, wherein the report contains recommendations for the administration of a therapeutic agent. 132. The method of any one of embodiments 129-131, wherein the report contains recommendations for surgical intervention. 133. The method of any one of embodiments 111-132, wherein the method has a false detection rate of 5% or less. 134. A method for detecting endometriosis in an individual, comprising: a. determining the expression level of one or more microRNAs selected from the group consisting of miR-1271-5p, miR-4485-3p, miR-125b-2-3p, and miR-410-3p from a biological sample of the individual; and b. applying a classifier algorithm to the expression level of the one or more microRNAs to generate a classification of the individual. 135. The method of embodiment 134, wherein the individual suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. 136. The method according to embodiment 134 or embodiment 135, wherein the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE).137. The method according to any one of embodiments 134-136 further includes determining the expression level of one or more miRs selected from the group consisting of: let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2- 3p,miR-17-3p,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,m iR-200c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-2 22-3p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409, and miR-98-5p. 138. The method according to any one of embodiments 134-137, further comprising determining a methylation profile of one or more CpG sites selected from the CpG sites in Table 4. 139. The method according to embodiment 138, further comprising applying the classifier algorithm to the methylation profile. 140. The method according to any one of embodiments 134-139, further comprising determining a measure of bacterial diversity in the biological sample. 141. The method according to embodiment 140, wherein the measure of bacterial diversity is the amount of at least one type of bacteria. 142. The method according to embodiment 141, wherein the at least one bacterium is selected from the following genera: *Propionibacterium*, *Propionibacterium*, *Dialectae*, *Porphyromonas*, *Streptococcus*, *Dermatobacterium*, *Moraxella*, *Anaerobic Cocci*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Franklinella*, and *Klebsiella*. 143. The method according to any one of embodiments 140-142, wherein the measure of bacterial diversity is the ratio of at least one first bacterium to at least one second bacterium. 144. The method according to any one of embodiments 140-143, further comprising determining the amount of *Propionibacterium acnes*.145. The method according to any one of embodiments 140-144, further comprising applying the classifier algorithm to the measure of bacterial diversity. 146. The method according to any one of embodiments 134-145, wherein the classification is selected from: possible endometriosis and unlikely endometriosis. 147. The method according to embodiment 146, wherein the classification of possible endometriosis is selected from: high probability of endometriosis, moderate probability of endometriosis, and low probability of endometriosis. 148. The method according to any one of embodiments 134-147, wherein the classifier algorithm includes a decision tree, random forest, Bayesian network, support vector machine, neural network, or logistic regression algorithm. 149. The method according to any one of embodiments 134-148, wherein the biological sample is menstrual fluid. 150. The method according to embodiment 149, wherein the menstrual fluid further includes cervical vaginal fluid. 151. The method according to embodiment 149 or embodiment 150, wherein the biological sample is collected on the second day of the individual's menstrual cycle. 152. The method according to any one of embodiments 149-151, wherein the biological sample is collected on a day during the individual's menstrual cycle when the individual experiences heavy menstrual flow. 153. The method according to any one of embodiments 134-152, wherein the biological sample is collected before treatment is administered to the individual. 154. The method according to any one of embodiments 134-152, wherein the biological sample is collected after treatment is administered to the individual. 155. The method according to any one of embodiments 134-154, wherein the biological sample is disposed in a sample collector. 156. The method according to embodiment 155, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad. 157. The method according to any one of embodiments 134-156, further comprising administering treatment for endometriosis to the individual. 158. The method according to embodiment 157, wherein the treatment is selected from surgical intervention, administration of a therapeutic agent, and combinations thereof. 159. The method of embodiment 158, wherein the therapeutic agent is selected from hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 160. The method of any one of embodiments 134-159, further comprising generating a report based on the disease state. 161. The method of embodiment 160, further comprising transmitting the report to a healthcare professional. 162. The method of embodiment 160 or embodiment 161, wherein the report contains recommendations for the administration of a therapeutic agent.163. The method according to any one of embodiments 160-162, wherein the report includes a recommendation for surgical intervention. 164. The method according to any one of embodiments 134-163, wherein the method has a false detection rate of 5% or less. 165. A method for detecting endometriosis in an individual, comprising: a. determining, from one of a cervical vaginal fluid sample or a menstrual fluid sample of the individual, the expression level of one or more microRNAs (miRs) selected from: miR-1271-5p, miR-4485-3p, miR-125b-2-3p, miR-410-3p, let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-2 21-5p,miR-363-3p,miR-99a-5p,let-7e-5p,miR-10a-5p,miR-10b-5p,miR-125b-5p,miR-127-3p,miR- 132-3p,miR-141-3p,miR-142-5p,miR-143-3p,miR-144-5p,miR-145-5p,miR-152-3p,miR-16-2-3p,miR -17-3p,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,miR-200c-3p,miR-203a-3p,m iR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-222-3p,miR-224-5p,miR-23b-3p,miR-27b-3p,miR- 28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409, and miR-98-5p; and b. comparing the expression level with a reference expression level of one or more miRs; wherein an increase or decrease in the expression level of the one or more miRs relative to the reference expression level indicates that the subject has endometriosis. 166. The method according to embodiment 165, wherein the one or more miRNAs include miR-23b-3p, miR-30a-3p / 5p, and miR-34a-5p. 167. The method according to embodiment 165 or embodiment 166, wherein the reference expression level is obtained from a subject who has never had a reproductive disorder or is not suspected of having a reproductive disorder. 168. The method according to embodiment 166, wherein the reproductive disorder is endometriosis.169. The method according to any one of embodiments 165-168, wherein the individual suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. 170. The method according to any one of embodiments 165-169, wherein the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE). 171. The method according to any one of embodiments 165-170, further comprising determining the methylation profile of one or more CpG sites selected from the CpG sites in Table 4. 172. The method according to any one of embodiments 165-171, further comprising determining a measure of bacterial diversity in the biological sample. 173. The method according to embodiment 172, wherein the measure of bacterial diversity is the amount of at least one type of bacteria. 174. The method according to embodiment 173, wherein the at least one bacterium is selected from the following genera: *Propionibacterium*, *Propionibacterium*, *Dialectae*, *Porphyromonas*, *Streptococcus*, *Desmodium*, *Moraxella*, *Anaerobic Cocci*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Franklinella*, and *Klebsiella*. 175. The method according to any one of embodiments 172-174, further comprising comparing the measure of bacterial diversity with a reference measure of bacterial diversity. 176. The method according to any one of embodiments 172-175, wherein the measure of bacterial diversity is the ratio of at least one first bacterium to at least one second bacterium. 177. The method according to any one of embodiments 165-176, further comprising determining the amount of *Propionibacterium acnes*. 178. The method according to embodiment 177, further comprising comparing the amount of *Propionibacterium acnes* with a reference amount of *Propionibacterium acnes*. 179. The method according to any one of embodiments 165-178, wherein the biological sample is disposed in a sample collector. 180. The method according to embodiment 179, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. 181. The method according to any one of embodiments 165-180, further comprising administering treatment for endometriosis to the individual. 182. The method according to embodiment 181, wherein the treatment is selected from surgical intervention, administration of a therapeutic agent, and combinations thereof. 183. The method according to embodiment 182, wherein the therapeutic agent is selected from hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 184. The method according to any one of embodiments 165-183, further comprising generating a report based on the expression level of the one or more miRs relative to the reference expression level.185. The method according to embodiment 184, further comprising transmitting the report to a healthcare professional. 186. The method according to embodiment 184 or embodiment 185, wherein the report contains recommendations for the administration of a therapeutic agent. 187. The method according to any one of embodiments 184-186, wherein the report contains recommendations for surgical intervention. 188. The method according to any one of embodiments 165-187, wherein the method has a false detection rate of 5% or lower. 189. A method for detecting endometriosis in an individual, comprising: a. determining the expression level of one or more microRNAs from a biological sample of the individual, said microRNAs regulating the expression of at least one gene selected from at least one KEGG pathway: ECM-receptor, adhesion junctions, proteoglycans in cancer, TGF-β signaling, Hippo signaling, microRNAs in cancer, pathways in cancer, hepatitis B, glioma, chronic myeloid leukemia, bladder cancer, and combinations thereof; and b. comparing said expression level with a reference expression level, wherein an increase or decrease in the expression level of said one or more microRNAs or one or more genes relative to said reference expression level indicates that the subject has endometriosis. 190. The method according to embodiment 189, wherein said one or more microRNAs are selected from miR-23b-3p, miR-30a-3p / 5p, miR-34a-5p, and combinations thereof.191. The method according to embodiment 189, wherein the one or more microRNAs are selected from let-7c-5p, miR-100-5p, miR-149-5p, miR-193b-3p, miR-221-5p, miR-363-3p, miR-99a-5p, let-7e-5p, miR-10a-5p, miR-10b-5p, miR-125b-5p, miR-127-3p, miR-132-3p, miR-141-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-152-3p, miR-16-2-3p, miR-17-3p. ,miR-195-5p,miR-196b-5p,miR-199a-3p / 199b-3p,miR-200a-3p,miR-200c-3 p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-21-5p,miR-22-3p,miR-222-3p, miR-224-5p,miR-23b-3p,miR-27b-3p,miR-28-3p,miR-30a-3p,miR-30a-5p,m iR-34a-5p,miR-34c-5p,miR-365a-3p / 365b-3p,miR-375,miR-409 and miR-98-5p. 192. The method according to any one of embodiments 189-191, wherein the one or more genes are selected from TGF-α, TGF-β, progesterone receptor A, progesterone receptor B, estrogen receptor A, E-cadherin, N-cadherin, and combinations thereof. 193. The method according to any one of embodiments 189-192, wherein the at least one KEGG pathway involves Wnt / JNK / VEGF signaling. 194. The method according to any one of embodiments 189-193, wherein the reference expression level is obtained from a subject who has never had a reproductive disorder or is not suspected of having a reproductive disorder. 195. The method according to embodiment 194, wherein the reproductive disorder is endometriosis. 196. The method according to any one of embodiments 189-195, wherein the individual suffers from chronic pelvic pain, infertility, heavy menstrual bleeding, or a combination thereof. 197. The method according to any one of embodiments 189-196, wherein the endometriosis is deep invasive endometriosis (DIE), superficial peritoneal endometriosis (SPE), or ovarian endometrioma (OE). 198. The method according to any one of embodiments 189-197, further comprising determining the methylation profile of one or more CpG sites selected from the CpG sites in Table 4.199. The method according to any one of embodiments 189-198 further includes determining a measure of bacterial diversity in the biological sample. 200. The method according to embodiment 199, wherein the measure of bacterial diversity is the amount of at least one bacterium. 201. The method according to embodiment 200, wherein the at least one bacterium is selected from the genera *Streptococcus*, *Propionibacterium*, *Dialectae*, *Porphyromonas*, *Streptococcus*, *Desmodium*, *Moraxella*, *Anaerobes*, *Peptostreptococcus*, *Lactobacillus*, *Prevotella*, *Campylobacter*, *Corynebacterium*, *Franklinella*, and *Klebsiella*. 202. The method according to any one of embodiments 199-201 further includes comparing the measure of bacterial diversity with a reference measure of bacterial diversity. 203. The method according to any one of embodiments 199-202, wherein the measure of bacterial diversity is the ratio of at least one first bacterium to at least one second bacterium. 204. The method according to any one of embodiments 189-203, further comprising determining the amount of *Propionibacterium acnes*. 205. The method according to embodiment 204, further comprising comparing the amount of *Propionibacterium acnes* with a reference amount of *Propionibacterium acnes*. 206. The method according to any one of embodiments 189-205, wherein the biological sample is menstrual fluid. 207. The method according to embodiment 206, wherein the menstrual fluid further includes cervical vaginal fluid. 208. The method according to embodiment 206 or embodiment 207, wherein the biological sample is collected on the second day of the individual's menstrual cycle. 209. The method according to any one of embodiments 206-208, wherein the biological sample is collected on a day in the individual's menstrual cycle when the individual experiences heavy menstrual flow. 210. The method according to any one of embodiments 189-209, wherein the biological sample is collected before treatment is administered to the individual. 211. The method according to any one of embodiments 189-209, wherein the biological sample is collected after treatment is administered to the individual. 212. The method according to any one of embodiments 189-211, wherein the biological sample is disposed in a sample collector. 213. The method according to embodiment 212, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual tray, cervical tray, sponge, or interlabial pad. 214. The method according to any one of embodiments 189-213, further comprising administering treatment for endometriosis to the individual. 215. The method according to embodiment 214, wherein the treatment is selected from surgical intervention, administration of a therapeutic agent, and combinations thereof.216. The method of embodiment 215, wherein the therapeutic agent is selected from hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 217. The method of any one of embodiments 189-216, further comprising generating a report based on the expression level of the one or more miRs relative to the reference expression level. 218. The method of embodiment 217, further comprising transmitting the report to a healthcare professional. 219. The method of embodiment 217 or embodiment 218, wherein the report contains recommendations for administering the therapeutic agent. 220. The method of any one of embodiments 217-219, wherein the report contains recommendations for surgical intervention. 221. The method of any one of embodiments 217-220, wherein the method has a false detection rate of 5% or less. 222. A method of treating a subject suspected of having endometriosis, comprising: obtaining a biological sample from or having obtained the subject; measuring or having measured the biological sample to determine whether the subject has a microRNA expression signature indicative of endometriosis; administering an intervention to the subject if the subject has the microRNA expression signature indicative of endometriosis, and not administering an intervention if the subject does not have a methylation signature indicative of endometriosis.223. The method according to embodiment 222, wherein the microRNA expression characteristics indicative of endometriosis include significantly different expression of one or more microRNAs (miRs) relative to the expression of one or more microRNAs selected from the group consisting of individuals without endometriosis: miR-1271-5p, miR-4485-3p, miR-125b-2-3p, miR-410-3p, let-7c-5p, miR-100-5p,miR-149-5p,miR-193b-3p,miR-221-5p,miR-363-3p,miR-99a-5p,let-7e-5p,miR- 10a-5p,miR-10b-5p,miR-125b-5p,miR-127-3p,miR-132-3p,miR-141-3p,miR-142-5p,miR-143- 3p,miR-144-5p,miR-145-5p,miR-152-3p,miR-16-2-3p,miR-17-3p,miR-195-5p,miR-196b-5p, miR-199a-3p / 199b-3p,miR-200a-3p,miR-200c-3p,miR-203a-3p,miR-205-5p,miR-21-3p,miR-2 1-5p, miR-22-3p, miR-222-3p, miR-224-5p, miR-23b-3p, miR-27b-3p, miR-28-3p, miR-30a-3p, miR-30a-5p, miR-34a-5p, miR-34c-5p, miR-365a-3p / 365b-3p, miR-375, miR-409 and miR-98-5p and combinations thereof. 224. The method of embodiment 222, wherein the microRNA expression signature indicating endometriosis includes significantly different expression of one or more microRNAs (miRs) relative to the expression of one or more microRNAs selected from the group consisting of miR-1271-5p, miR-4485-3p, miR-125b-2-3p, miR-410-3p, and combinations thereof in an individual without endometriosis. 225. The method of any one of embodiments 222-224, wherein performing the assay on or on the biological sample to determine whether the subject has a microRNA expression signature indicating endometriosis comprises: extracting nucleic acid from or on the biological sample, and sequencing one or more microRNAs from the extracted nucleic acid. 226. The method of embodiment 225, wherein the nucleic acid is RNA.227. The method according to any one of embodiments 222-226, wherein the intervention is selected from surgical intervention, therapeutic intervention, and combinations thereof. 228. The method according to embodiment 227, wherein the surgical intervention is selected from: surgical removal of at least a portion of an endometriotic lesion, hysterectomy, salpingo-oophorectomy, presacral nerve resection, and laparoscopic uterine nerve ablation. 229. The method according to embodiment 227, wherein the therapeutic intervention comprises administering a therapeutic agent selected from: hormones, hormone agonists, hormone antagonists, aromatase inhibitors, anti-inflammatory therapies, acetyltransferases, histone deacetylase inhibitors, phosphodiesterase inhibitors, and combinations thereof. 230. The method according to embodiment 229, wherein the hormone is selected from estrogen, progesterone, androgens, and gonadotropin-releasing hormone (Gn-RH). 231. The method according to embodiment 229 or embodiment 230, wherein the hormone is a synthetic hormone. 232. The method of embodiment 229, wherein the hormone agonist or antagonist is a gonadotropin-releasing hormone (Gn-RH) agonist or a Gn-RH antagonist. 233. A method for preserving cells from a menstrual fluid sample, the method comprising placing the menstrual fluid sample containing the cells in a preservation solution to form a mixture of the menstrual fluid sample containing the cells and the preservation solution. 234. The method of embodiment 233, further comprising contacting the cells in the menstrual fluid sample with an antibody that binds to a cell surface antigen of a target cell in the cells of the menstrual fluid sample. 235. The method of embodiment 234, wherein the antibody is attached to a solid support. 236. The method of embodiment 235, wherein the solid support is a bead. 237. The method of embodiment 236, wherein the bead is a magnetic bead. 238. The method of any one of embodiments 234-237, wherein the antibody is conjugated to a detectable marker. 239. The method according to embodiment 238, wherein the detectable marker is a fluorophore. 240. The method according to any one of embodiments 234-239, wherein the target cell is selected from endothelial cells, epithelial cells, leukocytes, mesenchymal cells, and combinations thereof. 241. The method according to any one of embodiments 234-240, wherein the target cell is an endothelial cell.242. The method according to embodiment 241, wherein the cell surface antigen is selected from: CD31 / PECAM-1, CD34, CD36 / SR-B3, CD39, CD44, CD47, CD54 / ICAM-1, CD61, CD62E, CD62P, CD80, CD86, CD93, CD102, CD105, CD106, CD112, CD117, ESAM, endothelial mucin, CXCL16, CD121a, CD141, CD142, CD143, CD144, CD146, CD147, CD151, CD160, CD201, CD213a, CD248, CD309, ADAM 8, ADAM 9, ADAM 10, ADAM 11, ADAM 12, ADAM 13, ADAM 14, ADAM 15, ADAM 16, ADAM 17. ADAM 33, ADAMTS-13, ADAMTS-18, VWF, TEM8, NOTCH, and KLF4. 243. The method according to any one of embodiments 234-240, wherein the target cell is an epithelial cell. 244. The method according to embodiment 243, wherein the cell surface antigen is selected from: epithelial cell adhesion molecule (EpCAM), E-cadherin, and CD326. 245. The method according to any one of embodiments 234-240, wherein the target cell is a leukocyte. 246. The method according to embodiment 245, wherein the cell surface antigen is CD45. 247. The method according to any one of embodiments 234-240, wherein the target cell is a mesenchymal cell. 248. The method of embodiment 247, wherein the cell surface antigen is selected from N-cadherin, OB-cadherin, α-5β-1 integrin, α-Vβ-6 integrin, and multiligand proteoglycan-1. 249. The method of any one of embodiments 234-248, further comprising separating the target cells from the menstrual fluid sample. 250. The method of embodiment 249, wherein the separation comprises fluorescence-activated cell sorting (FACS), magnetically activated cell sorting, or a combination thereof. 251. The method of any one of embodiments 234-248, further comprising removing the target cells from the menstrual fluid sample. 252. The method of any one of embodiments 233-251, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the menstrual fluid sample are intact.253. The method according to any one of embodiments 233-252, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the menstrual fluid sample are viable. 254. The method according to any one of embodiments 233-253, wherein the osmolality of the preservation solution is about 310 to about 410 mOsm kg. -1 255. The method according to any one of embodiments 233-252, wherein the osmolality of the preservation solution is about 95 to about 210 mOsmkg. -1256. The method according to any one of embodiments 233-255, wherein the volume of the preservation solution is about 5 ml to about 10 ml. 257. The method according to any one of embodiments 233-255, wherein the volume of the preservation solution is about 7.5 ml. 258. A menstrual fluid cell sample comprising: one or more cells from a menstrual fluid sample; and a preservation solution. 259. The sample according to embodiment 258, wherein the one or more cells are selected from endothelial cells, epithelial cells, leukocytes, mesenchymal cells, and combinations thereof. 260. The sample according to embodiment 259, wherein the epithelial cells are endometrial epithelial cells. 261. The sample according to any one of embodiments 258-260, wherein the preservation solution comprises a precipitant. 262. The sample according to embodiment 261, wherein the precipitant is selected from: 5-(4-dimethyl)aminobenzylmethylrhodanine, sulfosalicylic acid, lithium chloride, and lithium hydroxide. 263. The sample according to any one of embodiments 258-262, wherein the preservation solution comprises a lower alcohol. 264. The sample according to Embodiment 263, wherein the lower alcohol is selected from: methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol (2-methylprop-1-ol). 265. The sample according to any one of Embodiments 258-264, wherein the preservation solution includes a clinker. 266. The sample according to Embodiment 265, wherein the clinker is selected from: guanidine hydrochloride, guanidine thiocyanate, potassium thiocyanate, sodium thiocyanate, and urea. 267. The sample according to any one of Embodiments 258-266, wherein the preservation solution includes a chelating agent. 268. The sample according to embodiment 267, wherein the chelating agent is selected from: diethylenetriaminepentaacetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); ethylene glycol tetraacetic acid (EGTA); trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid; and nitrilotriacetic acid (NTA). 269. The sample according to any one of embodiments 258-268, wherein the preservation solution includes a reducing agent. 270. The sample according to Embodiment 269, wherein the reducing agent is selected from: 2-mercaptoethanol, thiosulfate, TCEP (tris-(2-carboxyethyl)phosphine), dithiothreitol, and dithioerythritol. 271. The sample according to any one of Embodiments 258-270, wherein the preservation solution contains a pH buffer.272. The sample according to Embodiment 271, wherein the pH buffer is selected from: citric acid; tartaric acid; malic acid; sulfosalicylic acid; sulfoisophthalic acid; oxalic acid; borate; CAPS (3-(cyclohexylamino)-1-propanesulfonic acid); CAPS (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid); EPPS (4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid); HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid); MES (2-(N-morpholino)ethanesulfonic acid); MOPS (3-(N-morpholino)propanesulfonic acid); MOPSO (3-morpholino-2-hydroxy Propanesulfonic acid); PIPES (1-4-piperazine diethanesulfonic acid); TAPS (N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid); TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid); TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid); bicine (N,N-bis(2-hydroxyethyl)glycine); tricine (N-[tris(hydroxymethyl)methyl]glycine); tris (tris(hydroxymethyl)aminomethane); and bis-tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol). 273. A sample according to any one of embodiments 258-272, wherein the preservation solution comprises a surfactant. 274. A sample according to any one of embodiments 258-273, wherein the osmolality of the preservation solution is about 310 to about 410 mOsm kg. -1 275. The sample according to any one of embodiments 258-273, wherein the osmolality of the preservation solution is about 95 to about 210 mOsm kg. -1276. The sample according to any one of embodiments 258-275, wherein the preservation solution does not include a fixative. 277. The sample according to any one of embodiments 258-276, further comprising menstrual fluid. 278. The sample according to any one of embodiments 258-277, further comprising cervical vaginal fluid. 279. The sample according to any one of embodiments 258-278, wherein the volume of the menstrual fluid sample is about 100 μl to about 1 ml. 280. The sample according to any one of embodiments 258-279, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the sample are intact. 281. The sample according to any one of embodiments 258-280, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the sample are viable. 282. The sample according to any one of embodiments 258-281 further includes bacterial cells, yeast cells, sperm, or combinations thereof. 283. The sample according to any one of embodiments 258-282 further includes a portion of a sample collector. 284. The sample according to embodiment 283, wherein the sample collector is a pad, tampon, vaginal cup, cervical cap, menstrual disc, cervical disc, sponge, or interlabial pad.

[0173] Example

[0174] Example 1: RNA-Seq time-course data

[0175] The quality metrics of RNA-seq libraries were assessed by examining the number of overall reads aligned with the human transcriptome. Continuous time-course samples (both menstrual samples (MB) and cervical-vaginal samples (CV)) from women were analyzed by collecting tampon-based samples every day of a 28-day cycle, including peripheral whole blood (WB) collection on days of heavy flow. Figure 5 The apparatus shown was used for sample collection. RNAgard was used as a sample preservation solution. RNA was sequenced from 244 samples (171 CVs, 46 MBs, and 27 WBs) from 27 participants. Typically, for high-quality genomic analysis, at least 70% of the sequencing reads should be aligned with the reference transcriptome. This is an industry standard and is independent of the sequencing platform used. Both menstrual blood and whole blood showed similar robustness in the percentage of reads aligned with the transcriptome (mean for menstrual fluid = 93.8%; StDev = 4.6%, and mean for whole blood = 95.5%; StDev = 1.1%). These percentages well exceed the 70% threshold for genomic analysis, indicating high-quality capture of nucleic acids from menstrual fluid.

[0176] To assess the inter-sample and intra-sample variability among participants, unsupervised principal component analysis (PCA) was performed, followed by t-SNE (a nonlinear dimensionality reduction technique that preferably preserves local and global structure) to visualize all variations within both dimensions. Figures 1C-1D Compared to CV, which exhibits high variability in the menstrual cycle, and WB, menstrual samples showed tight clustering, indicating high reproducibility of this sample type, and unique clusters representing approximately 800 differentially expressed genes were observed in the menstrual samples. Figure 1A-Figure 1B ).

[0177] To better understand the value of the menstrual group compared to whole blood samples, total RNA transcription in whole blood and menstrual fluid collected from the same patient at the same time during the three days prior to menstruation was compared. Similarly, menstrual fluid was a tampon-based sample, in which... Figure 5 The apparatus shown was used for sample collection. Whole blood samples were collected using venipuncture. By performing differential analysis on each matched sample for each day of menstruation, genes that were differentially expressed in menstrual fluid but not in whole blood were identified. Interestingly, on the first day of menstruation, little difference was observed in the relative abundance of gene transcripts between menstrual fluid and whole blood. However, on the second day of menstruation, often referred to as the "heavy flow day"—when most of the uterine lining has shed—more than 800 differentially expressed genes were identified, representing a unique genomic profile of menstrual fluid. Figure 1A-Figure 1B ).

[0178] In time-course analysis, several genes expressed in various reproductive tissues (expression of tissue-specific genes in cervical / vaginal cells, ovarian / fallopian tube cells, and endometrial cells) were identified. The relative abundance of each of these markers was compared throughout the female menstrual cycle. On the second day of menstruation, a significant overexpression of gene signals was found in endometrial tissue in menstrual samples. Figure 2B () Among samples collected during ovulation on non-menstrual days, cervical and vaginal samples showed the highest RNA and DNA yields, and enrichment of ovarian and fallopian tube-specific genes was found during ovulation.

[0179] Normalized gene expression values ​​of patient C000 across time-series samples spanning April 18 to May 17 were used for hierarchical clustering using the K-means clustering algorithm in Morpheus, an open-source software program developed by the Broad Institute. Figure 9The time-series data depicted shows five main clusters, with overexpression and underexpression observed as samples moved chronologically within the time series. In cluster 2, gene overexpression was detected in menstrual samples, along with underexpression of certain genes. Overexpression of genes was detected in cluster 5 during the postmenstrual phase, while overexpression of genes was detected in cluster 1 and underexpression of genes was detected in cluster 4 during the pre-ovulatory phase. Finally, overexpression of genes was detected in cluster 5 during the ovulation phase.

[0180] Perform Kegg pathway analysis on the gene lists from each cluster, such as... Figures 10A-10E The described cluster 1 contains genes downregulated in menstrual bleeding and shows that regulated KEGG pathways include lipid metabolism, arginine and proline metabolism, estrogen signaling pathways, FcγR-mediated phagocytosis, histidine metabolism, drug metabolism, α-linolenic acid metabolism, Staphylococcus aureus infection, linoleic acid metabolism, and circadian rhythms. Regulated KEGG pathways in cluster 2 are associated with transcriptional dysregulation in systemic lupus erythematosus, alcoholism, viral carcinogenesis, Alzheimer's disease, spliceosomes, Huntington's disease, oxidative phosphorylation, human T-cell leukemia virus 1 infection, prion diseases, and cancer. The regulated KEGG pathways in cluster 3 are associated with ribosome biosynthesis, ribosomes, mineral uptake in eukaryotes, microRNAs in cancer, epithelial cell signaling and endocytosis in Helicobacter pylori infection, pancreatic cancer, chronic myeloid leukemia, sulfur relay systems, and hepatocellular carcinoma. The regulated KEGG pathways in cluster 4 are associated with measles, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, Epstein-Barr virus (EBV) infection, Salmonella infection, NF-κB signaling pathways, p53 signaling pathways, cytokine-cytokine receptor interactions, transcriptional dysregulation in cancer, and human cytomegalovirus (CMV) infection. The regulated KEGG pathways in cluster 5 are associated with osteoclast differentiation, Staphylococcus aureus infection, tuberculosis, cytokine-cytokine receptor interactions, leishmaniasis, hematopoietic lineages, NOD-like receptor signaling pathways, chemokine signaling pathways, CMV infection, and TNF signaling pathways.

[0181] Example 2: Collection of whole blood samples, cervical and vaginal samples, and menstrual samples

[0182] Whole blood, cervical, vaginal, and menstrual samples were collected from women with suspected endometriosis (n=19), healthy women (n=55), and women with polycystic ovary syndrome (PCOS) (n=5). Epigenetic regulation (small RNA and DNA methylation), as well as RNA transcription sequencing and bacterial 16S sequencing, were analyzed. [The text then abruptly shifts to a different topic:] ...using... Figure 5 , Figures 6A-6D , Figures 7A-7C Menstrual and cervical vaginal samples were collected using the sample collection system shown in 8A-8C, and whole blood samples were collected via routine venipuncture. Nucleic acids were extracted, and sequencing libraries were prepared using Illumina reagents and sequenced on Illumina MiSeq, NextSeq 550, and HiSeq 4000 sequencers to compare the performance of each sample type in detecting endometriosis. Patient classification is as described in Table 1A. Tampons were collected from 5 patients before surgery and from 14 patients after surgery (these were not paired samples; pre- and post-operative tampons were collected from different participants). Menstrual cycle samples were collected on day 2 of the menstrual cycle. This allowed for evaluation of data from the perspective of pre- and post-operative genomic signals in the population of interest. The stage, classification, and anatomical location of the patients' disease were also recorded. Three post-operative samples did not meet quality control criteria and were therefore not included in the analysis. For the healthy population, women were recruited from the community and had never been diagnosed with reproductive disorders. These women were further subdivided into “truly healthy” and “suspected unhealthy” groups based on measurements of reproductive hormones (anti-Müllerian hormone (AMH), estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH)), recorded typical endometriosis symptoms, and vaginal infection symptoms (Table 1A). In summary, tampons collected from 5 “truly healthy” women were compared with those collected preoperatively from 5 endometriosis patients and postoperatively from 11 endometriosis patients.

[0183] Table 1A: Criteria for the "Truly Healthy" or "Suspected Unhealthy" Classification

[0184]

[0185] Table 1B: How to recruit healthy patients

[0186]

[0187]

[0188] Detection of miRNA biomarkers

[0189] To explore local and intracellular miRNA signaling, miRNAs in menstrual fluid were segmented and sequenced. A small subcohort of patients (five women with endometriosis and five truly healthy women) was analyzed to examine differential miRNA expression. Samples were collected from women with endometriosis prior to surgery, and all suspected endometriosis patients underwent surgical confirmation. Differential expression analysis was performed on normalized miRNAs from menstrual fluid. Forty-nine significantly dysregulated miRNAs (p < 0.05 at FDR < 0.05) were detected in preoperative endometriosis patients compared to menstrual fluid from truly healthy women (Table 2). Ten of these 49 markers had an area under the curve (AUC) of 0.95 or greater, with a false discovery rate of 5%. When the miRNA profiles of preoperative patients were compared with those collected postoperatively from tampons, no difference in miRNA expression was found between postoperative endometriosis subjects and healthy individuals. This is not to say that these patients are cured, as there is currently no cure for endometriosis, but the data demonstrate the ability to detect disease activity. Post-operative tampon samples were collected 3 months to 2 years after the procedure, and these patients did not experience disease recurrence for up to two years post-surgery. The results demonstrate that miRNAs can be used to monitor the efficacy of surgery or other interventions, including the efficacy of therapeutics that reduce disease activity.

[0190] Table 2: Compared with menstrual fluid from truly healthy women, miRNAs in patients with pre-operative endometriosis were significantly dysregulated.

[0191]

[0192] The development of methods to stratify patients based on DIE, SPE, OE, aromatase expression, retinoic acid imbalance, and estrogen and progesterone receptor bias can predict patient responses to interventions. To understand the relevant pathways involved in endometriosis in the data presented in this paper, pathway analysis was performed on 49 miRNAs using mirPath v.3, and the results were compared with 377 genes involved in epithelial-mesenchymal transition (EMT) and their respective Kegg pathways (referred to as the EMT database (dbEMT)). These candidate biomarkers for endometriosis and EMT share a total of 11 pathways (…). Figure 3 These pathways include the Hippo signaling pathway, TGF-β, and pathways involved in Wnt / JNK / VGEF signaling, key biological pathways involved in the pathology of endometriosis.

[0193] One hundred and four samples were collected from healthy or endometriosis patients, including 53 menstrual blood samples and 51 whole blood samples. As previously described, the samples were segregated and sequenced. Dimensional analysis using PCA or tSNE plots revealed that menstrual blood and whole blood samples clustered together. Figures 13A-13B This shows a clear difference in organizational type.

[0194] When the transcriptional patterns of menstrual blood from all healthy patients and all patients with endometriosis were compared together, there were no significantly different regulated miRNAs, such as Figure 13C The description is as follows. However, when menstrual blood samples were divided into pre- and post-operative samples, 49 genes showed significant differential expression between patients.

[0195] The potential of these candidate biomarkers for molecular / genomic classification of the disease was also assessed by observing specific miRNA targets that coordinate epigenetic signaling with specific genes associated with key pathways involved in endometriosis. Using TarBase v.867, experimentally supported gene interactions were queried for 49 candidate biomarkers and compared with known cellular biomarkers for endometriosis. Three miRNAs (miR-23b-3p, miR-30a-3p / 5p, and miR-34a-5p) were identified with strong experimental support for many genes involved in endometriosis, including JNK1-3 and LATS1 (Hippo signaling). These miRNAs showed evidence of interactions with TGF-α, TGF-β, progesterone receptors A and B, estrogen receptors A and B, and E-cadherin and N-cadherin (Table 3). Figure 3 This study showed that endometriosis and EMT share 11 Kegg pathways. Figure 14 This paper details these 11 pathways, indicating their association with endometriosis, the significance (p-value) of these pathways among the 49 miRNAs provided in this paper, and the number of miRNAs supporting each pathway from the data provided here. These candidate biomarkers may be helpful in stratifying patients through molecular / genomic classification, identifying aromatase activity, the ratio of progesterone receptor A to progesterone receptor B, the ratio of N-cadherin expression to E-cadherin expression, and the ratio of estrogen receptor A to estrogen receptor B.

[0196] Furthermore, the miRNA levels differed from those described in previous studies and tissue types, as depicted in Table 3. These data indicate differential expression in menstrual fluid between patients with endometriosis and healthy individuals, contrasting with values ​​found in previous comparisons of ectopic endometrial tissue from patients with endometriosis versus eutopic endometrial tissue from healthy individuals, ectopic endometrial tissue from patients with endometriosis versus eutopic endometrial tissue from patients with endometriosis, and eutopic endometrial tissue from patients with endometriosis versus eutopic endometrial tissue from healthy individuals. The differential expressions are shown as log10 fold changes. This suggests that menstrual blood exhibits expression different from other tissues.

[0197] Table 3: Comparison of miRNA levels in menstrual blood with previous studies in other tissues

[0198]

[0199]

[0200]

[0201] *No previous association with endometriosis

[0202] Detection of methylation markers

[0203] Using the same patients from the miRNA analysis described above, DNA methylation patterns in endometriosis were also examined using Illumina's EPIC850k methylation array. Methylation features were mapped to the genome, and normalized intensity values ​​were compared between preoperative menstrual fluid collected from endometriosis patients and menstrual fluid from truly healthy individuals. In the initial sample set, over one thousand CpG methylation sites were identified, which were either undermethylated or overmethylated compared to menstrual fluid from healthy participants. Table 4 shows 370 CpG sites that exhibited the most significantly different methylation status between endometriosis and healthy individuals. Interestingly, in endometriosis patients, a higher percentage of undermethylated sites fell on the shore—the flanking regions of CpG islands in the genome, regions that are highly dynamic and associated with many downstream regulatory functions and diseases.

[0204] Table 4: CpG sites showing under-methylation or hypermethylation in individuals with endometriosis compared to healthy individuals.

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Referring to Figure 4, the following 13 sites are overmethylated: cg02858642, ch.20.53118117F, cg20768326, ch.17.48901549F, cg15202115, cg01372113, cg10296715, cg19430489, cg03356461, cg09669049, cg21846877, ch.5.2763962F, and cg07029980. The remaining sites listed in Table 4 are undermethylated.

[0213] Detection of bacterial markers

[0214] The human microbiome also provides a potential source of novel biomarkers for the detection of endometriosis. The microbiome is a collection of microorganisms in the body that exist in a mutually beneficial relationship with the host. Microbial metagenomics of cervical vaginal fluid and menstrual fluid were analyzed to understand bacterial diversity present in endometriosis compared to healthy controls (both truly healthy and suspected unhealthy – Table 1A). The analyzed population included 5 patients with polycystic ovary syndrome (PCOS), 19 patients with endometriosis (tampon samples collected pre- and post-operatively), 5 healthy individuals, and 50 “suspected unhealthy” individuals. 16S microbial sequencing was performed, in which regions of the ribosomal RNA genomic code were amplified and sequenced, achieving species-level resolution of bacterial composition. This information was used to compare the relative abundance of bacterial species between healthy individuals (divided into truly healthy and suspected unhealthy – Table 1A), PCOS, and endometriosis. Diversity present in each sample (α-diversity) and diversity between samples within the same cohort (β-diversity) were then examined. The Shannon diversity index was used, which considers the abundance of each bacterial species and how evenly the species is distributed across a sample or population. Increased bacterial species diversity was found in patients with endometriosis compared to healthy patients. Figure 4A and Figure 4BThis study was able to identify specific bacterial species associated with endometriosis, most notably *Propionibacterium acnes*, which was present at levels 15 times higher in patients with endometriosis than in healthy individuals. *Propionibacterium acnes* produces high levels of prostaglandin-like substances and porphyrins, both of which are associated with inflammation and dysmenorrhea.

[0215] Furthermore, by comparing the bacterial abundance of menstrual samples and cervical-vaginal samples in healthy cohort 1, it was observed that the abundance of a large number of bacterial genera was higher in menstrual fluid than in cervical-vaginal samples. Figure 4C This is unique to this cohort, producing a distinctive bacterial profile. Abundance was also compared in healthy cohort three, providing a more detailed list of bacteria overexpressed in menstrual fluid. This increase in the number of bacterial genera in healthy cohort three demonstrates a correlation between the number of overabundant species and the degree of "health" in the patient cohort. Figure 4D By examining menstrual blood alone, numerous bacterial genera were observed, and their abundance was either higher or lower than that in healthy cohort 1. Figure 4E ).

[0216] Furthermore, when comparing menstrual fluid samples from patients with preoperative endometriosis and healthy controls, another unique bacterial genus characteristic was observed, such as... Figure 15A The bacterial abundance in menstrual fluid was described. Post-operatively, the difference in bacterial abundance between endometriosis patients and healthy patients was as follows: Figure 15B As shown.

[0217] Example 4: Differential methylation patterns between menstrual fluid and whole blood

[0218] The raw IDAT files were provided by the Illumina Human Methylation EPIC array. 311 sample files were available: 50 menstrual blood samples, 26 whole blood samples, and 253 other samples. Data processing was performed using the minfiR package (Bioconductor).

[0219] When methylation patterns of whole blood and menstrual blood were analyzed by tissue type using principal component analysis and tSNE dimensions, they showed significant tissue type differences, such as... Figures 11A-11B The description. Figure 12A The location of differentially methylated CpGs is shown when whole blood is compared with menstrual blood. Figure 12B The differentially methylated regions between whole blood and menstrual blood were shown.

[0220] Example 5: Using menstrual fingerprinting on patient data

[0221] For many patients, there may be no phenotypic data to group them, or if data is analyzed solely based on phenotypic and clinical parameters, there may be undiagnosed health conditions that could hinder the analysis. Therefore, data-driven approaches are crucial when there is no prior knowledge of patient data. Figures 16A-16B In the image, we can see that the methylation data of menstrual fluid in our patient cohort exhibits three distinct clusters. These clusters can then be used to begin grouping patients into cohorts for additional genomic analysis. In this example, the methylation clusters are used to set up the patient cohort for differential expression analysis of miRNAs collected from the same patients. This, in turn, generates a list of significantly dysregulated miRNAs for each cluster.

[0222] Patients were identified from clusters 1 and 2 of methylation data and compared with the abundance of *Lactobacillus* in menstrual samples (a decrease in *Lactobacillus* often indicates certain unhealthy conditions). (As in...) Figure 16C As we can see, cluster 1 shows low abundance of Lactobacillus, while cluster 2 shows high abundance. Therefore, each of these clusters can be categorized into a generally healthy state and an unhealthy state. Cluster 1 represents unhealthy, while cluster 2 represents healthy.

[0223] Biological relevance can be determined by acquiring miRNA targets and examining gene expression (RNA-seq) data to determine whether expression changes indicating true relevance have occurred in these genes. For each gene targeted by the five miRNAs dysregulated between cluster 1 and cluster 2, the average expression values ​​for cluster 1 and cluster 2 are presented in Table 5. The log2 fold change between cluster 1 and 2 is presented to illustrate the expression changes between clusters.

[0224] Table 5: Changes in expression by clustering

[0225] Clustering Average expression value Log2 fold change between cluster 1 and cluster 2 CYP26A1 cluster 1 0.22606087 1.286420439 CYP26A1 cluster 2 0.09267749 BPIFB1 cluster 1 0.30686597 0.343109732 BPIFB1 clustering 2 0.24191478 SCGB2A2 cluster 1 1.22321014 -0.711181248 SCGB2A2 clustering 2 2.00257117 CDC42BPA Cluster 1 1.03676009 0.293218741 CDC42BPA Cluster 2 0.84607845

[0226] Cluster 1 enriched patients with confirmed endometriosis. Differentially regulated miRNAs (miR-1270, miR-204-5p, miR-574-3p, miR-203a-3p, and miR-99a-3p) based on methylation clustering showed overexpression of five key microRNAs involved in regulating CYP26A1, BPIFB1, SCGB2A2, and CDC42BPA. Among these genes, CYP26A1 is a retinoic acid regulator that is progesterone-dependent and highly dysregulated in endometriosis; BPIFB1 is a molecular signature of orthotopic endometrium in endometriosis patients and is significantly downregulated in these patients; SCGB2A2 is significantly downregulated in endometriosis patients; and CDC42BPA is a key cell cycle regulator in the menstrual cycle and is significantly dysregulated in endometriosis patients. These miRNAs showed biological relevance compared to RNA-seq data. Similarly, endometriosis is associated with lower abundance of Lactobacillus species, similar to patients in cluster 1.

[0227] References

[0228] 1 Laudanski,P.,Charkiewicz,R.,Kuzmicki,M., et al.MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarianendometriosis.Reprod Biol Endocrinol 11,78(2013).https: / / doi.org / 10.1186 / 1477-7827-11-78

[0229] 2 Wright KR, Mitchell B, Santanam N. Redox regulation of microRNAs inendometriosis-associated pain. Redox Biol. 2017; 12:956-966.doi:10.1016 / j.redox.2017.04.037

[0230] 3 Filigheddu N, Gregnanin I, Porporato PE, et al. Differential expression of microRNAs between eutopic and ectopic endometrium in ovarian endometriosis. J Biomed Biotechnol. 2010;2010:369549. doi:10.1155 / 2010 / 369549

[0231] 4 Hawkins SM, Creighton CJ, Han DY, et al. Functional microRNA involved in endometriosis. Mol Endocrinol. 2011;25(5):821 - 832. doi:10.1210 / me.2010 - 0371

[0232] 5 Nisenblat V, Circulating miRNAs in endometriosis, The Robinson Institute, 2013; https: / / digital.library.adelaide.edu.au / dspace / bitstream / 2440 / 91226 / 5 / 01front.pdf

[0233] 6 Vanhie A, O D, Peterse D, et al. Plasma miRNAs as biomarkers for endometriosis. Hum Reprod. 2019;34(9):1650 - 1660. doi:10.1093 / humrep / dez116

[0234] 7 Cosar E, Mamillapalli R, Ersoy GS, Cho SY, Seifer B, Taylor HS, Serum microRNAs as diagnostic markers of endometriosis: a comprehensive array - based analysis, Fert Sterility. 2016. DOI: https: / / doi.org / 10.1016 / j.fertnstert.2016.04.013

[0235] 8 WO2015148919A3; Circulating micrornas as biomarkers for endometriosis

[0236] 9 Ohlsson Teague EM, Van der Hoek KH, Van der Hoek MB, et al. MicroRNA-regulated pathways associated with endometriosis. Mol Endocrinol. 2009;23(2):265-275. doi:10.1210 / me.2008-0387

[0237] 10 Zhuo, Z.; Wang, C.; Li, G.; Yu, H. Plasma MicroRNAs Can be a Potential Diagnostic Biomarker for Endometriosis. Preprints 2019, 2019070108 (doi:10.20944 / preprints201907.0108.v1).

[0238] 11 Zhao L, Gu C, Ye M, et al. Integration analysis of microRNA and mRNA paired expression profiling identifies deregulated microRNA-transcription factor-gene regulatory networks in ovarian endometriosis. Reprod Biol Endocrinol. 2018;16(1):4. Published January 22, 2018. doi:10.1186 / s12958-017-0319-5

[0239] 12 Suryawanshi, S and Vlad, AM. Lin, HM et al. Plasma MicroRNAs as NovelBiomarkers for Endometriosis and Endometriosis-Associated Ovarian Cancer, American Association for Cancer Research, 2013. https: / / doi.org / 10.1158 / 1078-0432.CCR-12-2726

[0240] 13 Zhou CF, Liu MJ, Wang W, et al. miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells [Published correction: Stem Cell Res Ther. 2020 May 20; 11(1):188]. Stem Cell Res Ther. 2019; 10(1):287. Published on September 23, 2019. doi:10.1186 / s13287-019-1388-5

[0241] 14 Kim, MK, Lee, SK, Park, J. et al. Ginsenoside Rg3 Decreases Fibrotic and Invasive Nature of Endometriosis by Modulating miRNA-27b: In Vitro and InVivo Studies. Sci Rep 7, 17670 (2017). https: / / doi.org / 10.1038 / s41598-017-17956-0

[0242] While preferred embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, alterations, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of this disclosure described herein may be employed in practice. The following claims are intended to define the scope of this disclosure and therefore cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for preparing a menstrual fingerprint, comprising: (a) Preparation of the first menstrual group fingerprint spectrum, including: i. Obtain a first sample from the subject at the first time point of the subject's menstrual cycle, wherein the first sample comprises cervical vaginal fluid or menstrual fluid collected onto a first absorbent sample collector; ii. Elute the first sample from the first sample collector into an aqueous buffer solution; iii. Isolate a first biological material from the first sample, wherein the first biological material comprises an RNA sequence; iv. Identify a gene from the RNA sequence of the first sample, wherein the gene shows a different presence or level in the cervical vaginal fluid or menstrual fluid compared to the peripheral blood of the subject; v. Constructing a menstrual fingerprint of a first sample, wherein the menstrual fingerprint of the first sample includes the levels and / or presence of multiple menstrual biomarkers in the first biological material from the first sample. (b) Preparation of the second menstrual group fingerprint spectrum, including: i. Obtain a second sample from the subject at a second time point in the subject's menstrual cycle, wherein the second sample comprises cervical vaginal fluid or menstrual fluid collected onto a second absorbent sample collector; ii. Elute the second sample from the second sample collector into an additional aqueous buffer; iii. Isolate a second biological material from the second sample, wherein the second biological material contains an RNA sequence; iv. Identify a gene from the RNA sequence of the second sample, wherein the gene shows a different presence or level in the cervical vaginal fluid or menstrual fluid compared to the peripheral blood of the subject; v. Constructing a second sample menstrual fingerprint, wherein the second sample menstrual fingerprint includes the levels and / or presence of the plurality of menstrual biomarkers in the second biological material from the second sample. (c) Compare the fingerprint chromatograms of the first and second samples with the fingerprint chromatogram of a reference menstrual group. The menstrual biomarkers mentioned therein include biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid compared to peripheral blood.

2. The method according to claim 1, wherein the first biological material and the second biological material comprise one or more biological materials selected from the group consisting of RNA, DNA, methylated nucleic acids, miRNA, proteins, protein-nucleic acid complexes, microorganisms, and mammalian cell types.

3. The method of claim 1, wherein the plurality of menstrual group biomarkers include at least one biomarker listed in Tables 2, 3, and 4, or any combination thereof.

4. The method of claim 1, wherein the plurality of menstrual group biomarkers include biomarkers that show different presence or levels in cervical vaginal fluid or menstrual fluid between two or more health states.

5. The method of claim 4, wherein the two or more health conditions include those prior to and after medical treatment.

6. The method of claim 4, wherein the two or more health conditions include preoperative and postoperative conditions.

7. The method of claim 4, wherein one of the two or more health states includes menstrual disorders.

8. The method of claim 4, wherein one of the two or more health conditions includes endometriosis.

9. The method of claim 1, wherein the plurality of menstrual biomarkers include the presence, absence, or amount of a certain cell type.

10. The method of claim 1, wherein the reference menstrual group fingerprint includes the levels and / or presence of multiple menstrual group biomarkers in the subject reference group.

11. The method of claim 1, wherein the reference menstrual fingerprint includes threshold levels or presence of the plurality of menstrual biomarkers associated with health status.

12. The method of claim 1, wherein the first time point and the second time point comprise different numbers of days within a single menstrual cycle of the subject.

13. The method of claim 1, wherein the first time point and the second time point are within a single menstrual cycle of the subject.

14. The method of claim 1, wherein the first time point and the second time point occur during one or more days of the subject's menstrual cycle.

15. The method of claim 1, wherein the first time point is during the subject's menstrual period while the second time point is not during the subject's menstrual period.

16. The method of claim 1, wherein the first sample collector and the second sample collector are intravaginal sample collectors.

17. The method of claim 1, wherein the first sample collector and the second sample collector preserve the biological material in an intact state.

18. The method of claim 1, wherein the first sample collector and the second sample collector are capable of absorbing at least 3 ml of fluid.

19. The method of claim 1, wherein after collecting the first sample and the second sample, the first sample collector and the second sample collector are placed in a buffer solution.

Images