# US 2017/0283789 A1 · Methods for collecting cervical-vaginal fluids and isolating exosome and microvesicles for molecular analysis

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- Number: US 2017/0283789 A1
- Document ID: US20170283789A1
- Status: Published application
- Kind: A1
- Application number: 15/507,218
- Filed: 2015-08-27
- Published: 2017-10-05
- Earliest claimed: 2014-08-29
- Assignees: Hitachi Chemical Company, Ltd.; Hitachi Chemical Company America, Ltd.
- Applicants: Hitachi Chemical Co., Ltd.; Hitachi Chemical Co. America, Ltd.
- Inventors: Cindy M. Yamamoto; Vivian Tovar; Taku Murakami; Mieko Ogura; Melanie Oakes; Masato Mitsuhashi
- Web page: https://patentgrep.com/patent/US20170283789A1

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- A61P 15/02: for disorders of the vagina
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6872: involving mass spectrometry
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/68: involving nucleic acids
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present disclosure relates to methods of collecting cervical-vaginal fluid exosomes and microvesicles and isolating corresponding mRNA. In particular, certain embodiments relate to the method of collecting the cervical-vaginal fluids with a tampon and releasing the cells, exosomes and microvesicles using excess buffer and a syringe or syringe-like device. The resulting expunged fluid can be applied to a filter device that is capable of capturing exosomes and microvesicles. Nucleic acids such as mRNA can be isolated from the cervical-vaginal fluid exosome and microvesicles using an oligo(dT)-coated plate designed to accommodate the filter device and then used for further molecular analysis. Quantification of the collected nucleic acids may then be used in the diagnosis and/or treatment of gynecological diseases or conditions.

## Claims

### Claim 1

A method of collecting cervical-vaginal fluids and identifying biomarkers for gynecological diseases and conditions, comprising:
(I) obtaining cervical-vaginal fluids by tampon usage;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon containing cervical-vaginal fluids; and
(III) detecting expression of a biomarker composed of one or more of the following:
IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from the isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting the liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for the biomarker of the gynecological disease or condition and a DNA polymerase in order to generate amplified DNA.

### Claim 2 (depends on 1)

The method of claim 1, wherein said isolating cervical-vaginal fluids comprises wearing a tampon for up to 8 hours during non-menstrual periods.

### Claim 3 (depends on 2)

The method of claim 2, wherein said tampon is composed of a combination of cotton and rayon fibers that can absorb up to 6 g.

### Claim 4 (depends on 1)

The method of claim 1, wherein said isolating cervical-vaginal fluids comprises adding excess buffer to the tampon to release one or more membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles.

### Claim 5 (depends on 4)

The method of claim 4 wherein excess buffer is between 10 to 30 mL.

### Claim 6 (depends on 4)

The method of claim 4, wherein said excess buffer is composed of pH greater than 4 and less than 10 and not containing any detergent.

### Claim 7 (depends on 1)

The method of claim 1, wherein said isolating cervical-vaginal fluids comprises using physical forces including a syringe or syringe-like device, centrifugation, shaking, air or liquid pressure to expunge fluids and/or membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from the tampon.

### Claim 8 (depends on 1)

The method of claim 1, wherein filtration traps said one or more of membrane particles, exosomes, exosome-like vesicles, and microvesicles on a filter device.

### Claim 9 (depends on 1)

The method of claim 1, wherein centrifugation isolates one or more of membrane particles, exosomes, exosome-like vesicles, and microvesicles.

### Claim 10

A method of collecting cervical-vaginal fluids and identifying biomarkers for gynecological diseases and conditions, comprising:
(I) obtaining cervical-vaginal fluids by tampon usage;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon containing cervical-vaginal fluids; and
(III) identifying mRNA expression profile or mutations by a method comprising:
(a) liberating RNA from the isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) preparing double-stranded cDNA library for RNA sequencing; and
(c) performing RNA sequencing.

### Claim 11 (depends on 10)

The method of claim 10, wherein said isolating cervical-vaginal fluids comprises wearing a tampon for up to 8 hours during non-menstrual periods.

### Claim 12 (depends on 11)

The method of claim 11, wherein said tampon is composed of a combination of cotton and rayon fibers that can absorb up to 6 g.

### Claim 13 (depends on 10)

The method of claim 10, wherein said isolating cervical-vaginal fluids comprises adding excess buffer to the tampon to release one or more membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles.

### Claim 14 (depends on 13)

The method of claim 13 wherein excess buffer is between 10 to 30 mL.

### Claim 15 (depends on 13)

The method of claim 13, wherein said excess buffer is composed of pH greater than 4 and less than 10 and not containing any detergent.

### Claim 16 (depends on 10)

The method of claim 10, wherein said isolating cervical-vaginal fluids comprises using physical forces including a syringe or syringe-like device, centrifugation, shaking, air or liquid pressure to expunge fluids and/or membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from the tampon.

### Claim 17 (depends on 10)

The method of claim 10, wherein filtration traps said one or more of membrane particles, exosomes, exosome-like vesicles, and microvesicles on a filter device.

### Claim 18 (depends on 10)

The method of claim 10, wherein centrifugation isolates one or more of membrane particles, exosomes, exosome-like vesicles, and microvesicles.

### Claim 19

A method of collecting vaginal fluids and analyzing biomarkers to determine whether a subject is suffering from a gynecological disease or condition, the method comprising:
(I) obtaining a tampon comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon;
(III) detecting expression of a biomarker associated with a gynecological disease or condition, wherein said biomarker is selected from the group consisting of: IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for said biomarker and a DNA polymerase in order to generate amplified DNA;
(IV) comparing said expression of said biomarker to expression of said biomarker in a control sample; and
(V) determining that said subject is suffering from a gynecological disease or condition when expression of said biomarker is greater when compared to said expression in said control sample.

### Claim 20 (depends on 1)

The method of claim 1, wherein said tampon was used by said subject for up to 8 hours during a non-menstrual time-period prior to said obtaining step.

### Claim 21 (depends on 1)

The method of claim 1, wherein said tampon is composed of a combination of cotton and rayon fibers that can absorb up to 6 g.

### Claim 22 (depends on 1)

The method of claim 1, wherein said isolation of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises adding an excess of a buffer to the tampon to release said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles.

### Claim 23 (depends on 4)

The method of claim 4, wherein between 10 to 30 mL of said excess buffer is added.

### Claim 24 (depends on 4)

The method of claim 4, wherein said excess buffer is composed of pH greater than 4 and less than 10, and wherein said excess buffer does not comprise a detergent.

### Claim 25 (depends on 1)

The method of claim 1, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon comprising cervical-vaginal fluids comprises using one or more types of physical force selected from the group consisting of using a syringe or syringe-like device, centrifugation, shaking, air pressure, and liquid pressure.

### Claim 26 (depends on 1)

The method of claim 1, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon comprising cervical-vaginal fluids comprises using a filter device.

### Claim 27 (depends on 1)

The method of claim 1, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises using centrifugation.

### Claim 28 (depends on 1)

The method of claim 1, wherein said biomarker is selected from the group consisting of IL-8, FTL, B2M, S100A8, S100A9, SAT1, IFITM2, SPRR3, SOD2, FTH1, CXCL8, GOS2, SRGN, IL-1B, and CXCL1.

### Claim 29

A method of collecting cervical-vaginal fluids from a subject to determine whether a subject has a gynecological disease or condition, the method comprising:
(I) obtaining a tampon comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon;
(III) identifying an mRNA expression profile, wherein said mRNA expression profile is generated for one or more mRNA associated with a gynecological disease or condition, by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles,
(b) preparing a double-stranded cDNA library for RNA sequencing from said RNA,
(c) performing sequencing of said cDNA,
(d) identifying said mRNA expression profile based on the results of said cDNA sequencing; and
(IV) determining whether a subject has a gynecological disease or condition based on comparing said mRNA expression profile of said subject to a control mRNA expression profile, wherein said expression of said biomarker is higher in said subject as compared to said control mRNA expression profile when said subject is suffering from a gynecological disease or condition.

### Claim 30 (depends on 29)

The method of claim 29, wherein said tampon is used by said subject for up to 8 hours during a non-menstrual time-period.

### Claim 31 (depends on 29)

The method of claim 29, wherein said tampon is composed of a combination of cotton and rayon fibers that can absorb up to 6 g.

### Claim 32 (depends on 29)

The method of claim 29, wherein said isolation of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises adding an excess of a buffer to the tampon to release said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles.

### Claim 33 (depends on 32)

The method of claim 32, wherein between 10 to 30 mL of said excess buffer is added.

### Claim 34 (depends on 32)

The method of claim 32, wherein said excess buffer is composed of pH greater than 4 and less than 10, and wherein said excess buffer does not comprise a detergent.

### Claim 35 (depends on 29)

The method of claim 29, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises using one or more types of physical force selected from the group of using a syringe or syringe-like device, centrifugation, shaking, air pressure, or liquid pressure.

### Claim 36 (depends on 29)

The method of claim 29, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises using a filter device.

### Claim 37 (depends on 29)

The method of claim 29, wherein said isolating of said membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles from said tampon containing cervical-vaginal fluids comprises using centrifugation.

### Claim 38 (depends on 29)

The method of claim 29, wherein said biomarker is selected from the group consisting of IL-8, FTL, B2M, S100A8, S100A9, SAT1, IFITM2, SPRR3, SOD2, FTH1, CXCL8, GOS2, SRGN, IL-1B, and CXCL1.

### Claim 39

A method of treating a subject suffering from a gynecological disease or condition, comprising:
(I) ordering that a tampon with cervical-vaginal fluid that has been used by a subject is collected;
(II) ordering a test of said cervical-vaginal fluid of said tampon by a method comprising:
isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said cervical-vaginal fluid of said tampon;
liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
contacting said cDNA with sense and antisense primers that are specific for one or more markers of gynecological disease or condition and a DNA polymerase in order to generate amplified DNA, wherein said one or more markers of gynecological disease or condition is chosen from:
IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RP S11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN;
detecting the amount of expression of said one or more markers of gynecological disease or condition;
(III) obtaining the results of the test, wherein the test results indicate that said subject is suffering from a gynecological disease or condition when said expression of said marker is different than a control mRNA expression profile; and
(IV) treating the subject when said test results indicate that the subject is suffering from a gynecological disease or condition.

### Claim 40

A method of directing treatment of a subject suffering from a gynecological condition or disease, comprising:
(I) receiving cervical-vaginal fluid collected from a tampon that has been used by a subject;
(II) detecting expression of at least one marker of gynecological disease or condition from the group consisting of:
IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said cervical-vaginal fluid of said tampon;
liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
contacting said cDNA with sense and antisense primers that are specific for said marker of gynecological disease in order to generate amplified DNA;
(III) identifying said subject as suffering from a gynecological disease or condition when said expression of said marker is different as compared to a control mRNA expression profile;
(IV) informing a physician that it would be appropriate to treat said subject if said expression indicates that said subject is suffering from a gynecological disease or condition.

### Claim 41

A method of collecting vaginal fluids and analyzing biomarkers to determine whether a subject is suffering from a gynecological disease or condition, the method comprising:
(I) obtaining a tampon comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon;
(III) detecting expression of a biomarker associated with a gynecological disease or condition, wherein said biomarker is selected from the group consisting of: IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for said biomarker and a DNA polymerase in order to generate amplified DNA; and
(IV) using a computer configured to receive data from step (III) and programmed to determine whether said expression of said biomarker indicates that said subject is suffering from a gynecological disease or condition.

### Claim 42

A method of treating a subject suffering from a gynecological disease or condition, the method comprising:
(I) obtaining a tampon comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon;
(III) detecting expression of a biomarker associated with a gynecological disease or condition, wherein said biomarker is selected from the group consisting of: IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for said biomarker and a DNA polymerase in order to generate amplified DNA;
(IV) comparing said expression of said biomarker to expression of said biomarker in a control sample;
(V) treating said subject for a gynecological disease or condition when expression of said biomarker is different when compared to said expression in said control sample.

### Claim 43

A method of treating a subject suffering from a gynecological disease or condition, the method comprising:
(I) obtaining a tampon comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said tampon;
(III) identifying an mRNA expression profile, wherein said mRNA expression profile is generated for one or more mRNA associated with a gynecological disease or condition, by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles,
(b) preparing a double-stranded cDNA library for RNA sequencing from said RNA,
(c) performing sequencing of said cDNA,
(d) identifying said mRNA expression profile based on the results of said cDNA sequencing; and
(IV) treating said subject for a gynecological disease or condition when said mRNA expression profile of said subject is different when compared to a control mRNA expression profile.

### Claim 44 (depends on 43)

The method of claim 43, wherein the difference in the expression profile of the subject as compared to a control mRNA expression profile is an increase in the expression of a biomarker associated with a gynecological disease or condition.

### Claim 45 (depends on 43)

The method of claim 43, wherein the difference in the expression profile of the subject as compared to a control mRNA expression profile is a decrease in the expression of a biomarker associated with a gynecological disease or condition.

### Claim 46

A method of treating a subject suffering from a gynecological disease or condition, the method comprising:
(I) obtaining a female hygiene product comprising cervical-vaginal fluids that has been used by a subject;
(II) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said female hygiene product;
(III) detecting expression of a biomarker associated with a gynecological disease or condition, wherein said biomarker is selected from the group consisting of: IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for said biomarker and a DNA polymerase in order to generate amplified DNA;
(IV) comparing said expression of said biomarker to expression of said biomarker in a control sample;
(V) treating said subject for a gynecological disease or condition when expression of said biomarker is different when compared to said expression in said control sample.

### Claim 47

A method of collecting vaginal fluids and analyzing biomarkers to determine whether a subject is suffering from a gynecological disease or condition, the method comprising:
(A) having a female hygiene product comprising cervical-vaginal fluids that has been used by a subject placed into a fully automated machine for the machine to perform an assay comprising the following steps:
(1) isolating one or more of membrane particles, cells, exosomes, exosome-like vesicles, and microvesicles from said female hygiene product;
(2) detecting the mRNA expression profile of a biomarker associated with a gynecological disease or condition, wherein said biomarker is selected from the group consisting of: IL8, FTL, B2M, S100A8, SAT1, IFITM2, S100A9, SPRR3, SOD2, FTH1, IFI30, H3F3B, BCL2A1, LITAF, FCER1G, ACTB, S100A1, GOS2, SRGN, LCE3D, GLUL, PI3, IL1B, IFITM3, IL1RN, CCL4, CYSTM1, SDCBP, PLEK, EIF1, CNFN, ANXA1, MYL6, GAPDH, C15orf48, KRT13, RGS2, SPRR1B, NOP10, GABARAP, TYROBP, PLAUR, SPRR2D, FPR1, SPRR2A, TMSB4X, TIMP1, FAM25A, CRCT1, GABARAPL2, RHOA, SLPI, ACTG1, ALOX5AP, LAPTM5, IFITM1, CXCL1, CSTB, CARD16, S100A12, NINJ1, AIF1, S100A7, AQP9, ARHGDIB, CCL3, IGSF6, NAMPT, CASP4, MNDA, LCP1, SAMSN1, ALDOA, CLIC1, SH3BGRL3, PNRC1, SPRR1A, TPI1, SERPINA1, TALDO1, LST1, LINC01272, GMFG, CRNN, CD53, TAGLN2, LY96, RAC2, IVNS1ABP, ISG20, PLSCR1, TPT1, MYL12A, LDHA, LCN2, S100A6, MXD1, SPINK7, RPLP1, UBE2B, CXCL8, DUSP1, RPL23, RPS11, PROK2, RPL27, CXCL2, ZFP36L1, BASP1, CSTA, FOX, PCBP1, RPL38, BRI3, SDCBP, CCL20, RPS12, RPL37A, CEBPB, SPRR2E, NFKBIA, RPL30, RPL24, CYSTM1, RGS2, RPS25, CXCR4, C4orf3, PABPC1, S100P, RPL26, GCA, MARCKS, RPS27A, SELK, ITM2B, MAL, HSPA1A, RPS29, PPP1CB, RPS20, IVNS1ABP, ZFP36, and TXN by a method comprising:
(a) liberating RNA from said isolated membrane particles, cells, exosomes, exosome-like vesicles, and/or microvesicles;
(b) contacting said liberated RNA with a reverse transcriptase to generate complementary DNA (cDNA); and
(c) contacting said cDNA with sense and antisense primers that are specific for said biomarker and a DNA polymerase in order to generate amplified DNA; and
(B) determining whether a subject has a gynecological disease or condition based on comparing said mRNA expression profile of said subject to a control mRNA expression profile, wherein said expression of said biomarker is higher in said subject as compared to said control mRNA expression profile when said subject is suffering from a gynecological disease or condition.