Methods for collecting cervical-vaginal fluids and isolating exosome and microvesicles for molecular analysis

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.