WO2025226902A1 - Inhibine et fsh en tant que biomarqueurs pour signalisation de temps de cycle dans le cycle menstruel - Google Patents

Inhibine et fsh en tant que biomarqueurs pour signalisation de temps de cycle dans le cycle menstruel

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Publication number
WO2025226902A1
WO2025226902A1 PCT/US2025/026119 US2025026119W WO2025226902A1 WO 2025226902 A1 WO2025226902 A1 WO 2025226902A1 US 2025026119 W US2025026119 W US 2025026119W WO 2025226902 A1 WO2025226902 A1 WO 2025226902A1
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inhibin
fsh
cycle
day
days
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Harvinder Singh Gill
Stephen USALA
Logan WILKS
Gaurav JOSHI
David VINEYARD
Adao Alexandre TRINDADE
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Texas Tech University TTU
Texas Tech University System
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Texas Tech University TTU
Texas Tech University System
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates in general to the field of biomarkers, and more particularly, to an assay, apparatus, kit, and methods for determining female fertile and infertile phases during the menstrual cycle and for assessing fertility and infertility in general.
  • Point of Care (POC) devices and home monitoring of reproductive hormones during a woman’s menstrual cycle and non-ovulatory times of life are now a reality (1 - 4).
  • Devices for measuring urinary luteinizing hormone (LH) and estrone-3 -glucuronide (E3G) have been available for decades (CLEARBLUE FERTILITY MONITORTM), and more recently urinary pregnanediol -3 -glucuronide (PDG) has been added to the home measurable hormone panel (INITO FERTILITY MONITORTM, and MIRA FERTILITY TRACKERTM) (5 - 13).
  • the present POC fertility monitoring devices are inadequate for birth control (18, 19). Ovulation occurs approximately 24 h after the urine LH (luteinizing hormone) peak (5, 6).
  • the fertile interval or ‘fertile window’ is generally accepted to be Day -4 to Day +1 (a 6-day interval) where Day 0 is the first day of positive urine LH or Day -5 to Day 0, if Day 0 is defined as the day of ovulation (20 - 23).
  • the ‘Fertile Start Day’ is Day -4 or Day -5 depending upon the basis for indexing ovulation.
  • an aspect of the present disclosure relates to an improved method and apparatus for assessing the fertile and infertile phases of the female menstrual cycle in humans and mammals.
  • an aspect of the present disclosure relates to an assay to measure an ovulation cycle in a mammalian female comprising the detection of inhibin A and inhibin B in one or more biological samples to determine a concentration of the inhibin A and the inhibin B in the biological sample to calculate a data output and the analysis of the data output to determine a timepoint in the ovulation cycle of the mammalian female.
  • the assay further comprises detecting follicle stimulating hormone (FSH) in the biological sample to determine a concentration of inhibin A and inhibin B and FSH in the biological sample to calculate a data output, and analyzing the data output to determine a timepoint in the ovulation cycle of the female.
  • FSH follicle stimulating hormone
  • the assay further comprises obtaining, of having obtained, one or more biological samples from the female; detecting the concentration in the one or more biological samples of inhibin A and inhibin B and FSH polypeptides or mRNA that encodes the inhibin A and inhibin B and FSH, or combinations thereof; and determining an individual and a relative expression of inhibin A and inhibin B and FSH polypeptides or mRNA in the one or more biological samples from the female at one or more timepoints, and collating the detection of the inhibin A and inhibin B and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.
  • A inhibin A concentration or level
  • B inhibin B concentration or level
  • FSH FSH concentration or level
  • the assay further comprises a point-of-care (POC) apparatus adapted for clinic or home use for precise signaling of fertile and infertile days of a human ovulation cycle.
  • the assay further comprises a point-of-care (POC) apparatus that is used to determine at least one of: a time-of-cycle during an ovulatory or menstrual cycle for which a female user can use for family planning, to enhance or to avoid conception; ovarian dysfunction, menopausal state, polycystic ovary syndrome (PCOS), or primary ovarian insufficiency (POI); assesses male fertility, and fertility impairment; or evaluate puberty status and abnormalities thereof.
  • POC point-of-care
  • PCOS polycystic ovary syndrome
  • POI primary ovarian insufficiency
  • the POC apparatus uses a lateral or vertical flow device with high- affinity inhibin A beta subunit, inhibin B beta subunit, alpha subunit antibodies to measure human inhibin A and inhibin B levels from serum, plasma, whole, or fingerstick blood, where the inhibin A and inhibin B levels are measured simultaneously, separately, or both, on a test substrate, strip, or filter in the POC apparatus.
  • the POC apparatus simultaneously with A and B assays measures with high-affinity monoclonal or polyclonal antibodies to FSH the serum, plasma, whole or fingerstick blood levels of FSH.
  • the Inhibin A, Inhibin B, and optionally FSH levels are measured one or more times at least daily or one or several days during an ovulatory or menstrual cycle to determine time-of-cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used to compute one or more of the following: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used in one or more diverse computational functions, formulations, algorithmic equations, using Inhibin A and Inhibin B, and optionally FSH levels, as one or more variables to quantify at least one of: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • the Inhibin A and Inhibin B levels with FSH levels are used with one or more diverse computational functions, formulations, algorithmic equations, using Inhibin A and Inhibin B levels with or without FSH levels as one or more variables to quantify at least one of: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • an aspect of the present disclosure relates to a kit comprising: a skin puncture device; and one or more containers comprising reagents to detect inhibin A and inhibin B or inhibin A and inhibin B and FSH in a biological sample obtained using the skin puncture device, and instructions or software to calculate a concentration of inhibin A and inhibin B or inhibin A and inhibin B and FSH in the biological sample, and using the concentration of inhibin A and inhibin B or inhibin A and inhibin B and FSH in the biological sample to determine at least one of: measure of a fertility of a female or a timepoint in the ovulation cycle.
  • the kit further comprises instructions for the steps of: providing one or more biological samples from a female; detecting in the one or more biological samples inhibin A and inhibin B or inhibin A and inhibin B and FSH polypeptides or mRNA that encodes inhibin A and inhibin B, and FSH, and determining an individual or relative expression of the inhibin A and inhibin B and FSH polypeptides or mRNA at one or more timepoints, and collating the detection of the inhibin A and inhibin B or inhibin A and inhibin B and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.
  • the kit comprises a point-of-care (POC) apparatus adapted for clinic or home use for precise signaling of fertile and infertile days of a human ovulation cycle.
  • POC point-of-care
  • the apparatus is used to determine at least one of: a time-of-cycle during an ovulatory or menstrual cycle for which a female user can use for family planning, to enhance or to avoid conception; ovarian dysfunction, menopausal state, polycystic ovary syndrome (PCOS), or primary ovarian insufficiency (POI); or assesses male fertility, and fertility impairment; or to evaluate puberty status and abnormalities thereof.
  • PCOS polycystic ovary syndrome
  • POI primary ovarian insufficiency
  • the POC apparatus uses a lateral or vertical flow device with high-affinity inhibin A beta subunit, inhibin B beta subunit, alpha subunit antibodies to measure human inhibin A and inhibin B levels from serum, plasma, whole, or fingerstick blood, where the inhibin A and inhibin B levels are measured simultaneously, separately, or both, on a test substrate, strip, or filter in the POC apparatus.
  • the POC apparatus simultaneously with A and B assays measures with high-affinity monoclonal or polyclonal antibodies to FSH the serum, plasma, whole or fingerstick blood levels of FSH.
  • the Inhibin A, Inhibin B, and optionally FSH levels are measured one or more times at least daily or one or several days during an ovulatory or menstrual cycle to determine time-of-cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used to compute one or more of the following: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used in diverse computational functions, formulations, algorithmic equations, using Inhibin A and Inhibin B and optionally FSH levels as one or more variables to quantify at least one of: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • an aspect of the present disclosure relates to a method for determining an ovulation cycle in a female, the method comprising: obtaining or having obtained one or more biological samples from the female: assaying the one or more biological samples to detect a level of inhibin A and inhibin B or inhibin A and inhibin B and follicle stimulating hormone (FSH) in the one or more biological samples; using an algorithm to parse or map the ovulation cycle in a female based on the level of inhibin A and inhibin B with or without FSH in the one or more biological samples, to provide a data output and an analysis of the data output that measures the ovulation cycle in the female.
  • FSH follicle stimulating hormone
  • the method further comprises the steps of: providing an isolated biological sample from the female; detecting in the biological sample inhibin A and inhibin B and FSH polypeptides or mRNA that encodes the inhibin A and inhibin B and FSH, determining individual and relative expression of inhibin A and inhibin B and FSH polypeptides or mRNA at one or more timepoints, and collating the detection of each of the inhibin A and inhibin B and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.
  • A inhibin A level or concentration
  • B inhibin B level or concentration
  • FSH FSH level or concentration: (B/A) with application of various identifying sequences for phase of cycle; Aalgo; (A/FSH)algo; Slope (B/A)/FSH; Slope A; AUC(B/A) or its first derivative,
  • the method further comprises a point-of-care (POC) apparatus adapted for clinic or home use for precise and real-time signaling of fertile and infertile days of a human ovulation cycle.
  • POC point-of-care
  • the POC apparatus is used to determine at least one of: a time- of-cycle during an ovulatory or menstrual cycle for which a female user can use for family planning, to enhance or to avoid conception; ovarian dysfunction, menopausal state, polycystic ovary syndrome (PCOS), or primary ovarian insufficiency (POI); assesses male fertility, and fertility impairment; or to evaluate puberty status and abnormalities thereof.
  • PCOS polycystic ovary syndrome
  • POI primary ovarian insufficiency
  • the POC apparatus uses a lateral or vertical flow device with high-affinity inhibin A beta subunit, inhibin B beta subunit, alpha subunit antibodies to measure human inhibin A and inhibin B levels from serum, plasma, whole, or fingerstick blood, where the inhibin A and inhibin B levels are measured simultaneously, separately, or both, on a test substrate, strip, or filter in the POC apparatus.
  • the POC apparatus simultaneously with A and B assays measures with high-affinity monoclonal or polyclonal antibodies to FSH the serum, plasma, whole or fingerstick blood levels of FSH.
  • the Inhibin A, Inhibin B, and optionally FSH levels are measured one or more times at least daily or one or several days during an ovulatory or menstrual cycle to determine time-of-cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used to compute one or more of the following: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • the Inhibin A, Inhibin B, and optionally FSH levels are used in one or more diverse computational functions, formulations, algorithmic equations, using Inhibin A and Inhibin B and optionally FSH levels as one or more variables to quantify at least one of: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle.
  • the female is a human.
  • an aspect of the present disclosure relates to an system for determining an ovulation cycle in a female, the system comprising: one or more biological samples obtained from the female: assaying the one or more biological samples to detect a level of inhibin A and inhibin B with or without FSH in the one or more biological samples; and using a processor to calculate a timepoint in the ovulation cycle in the female using an algorithm that uses the level of inhibin A and inhibin B with or without FSH in the one or more biological samples, to provide a data output and an analysis of the data output to determine in real-time the timepoint of the ovulation cycle in the female.
  • an aspect of the present disclosure relates to the discovery of oscillatory behavior between inhibin B and FSH levels in the follicular phase before the fertile window where a positive-negative feedback loop, ‘FSH ⁇ ->B’, is discernable on a daily basis or after a few days of measurements, hitherto unrealized in the state of the art and unknown.
  • FSH ⁇ ->B positive-negative feedback loop
  • an aspect of the present disclosure relates to an assay to measure an ovulation cycle in a mammalian female comprising the detection of inhibin A, inhibin B, and FSH in a biological sample to determine a concentration of inhibin A, inhibin B and FSH in the biological sample to calculate a data output and the analysis of the data output to determine a timepoint in the ovulation cycle of the female.
  • assay further comprises one or more biological samples from the female; detecting the concentration in the one or more biological samples of inhibin A, inhibin B, and FSH polypeptides or mRNA that encodes inhibin A, inhibin B, and FSH and determining a relative expression of inhibin A, inhibin B, and FSH polypeptides or mRNA in the one or more biological samples from the female at one or more timepoints, and collating the detection of inhibin A, inhibin B, and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.
  • data output and analysis may use subsets of inhibin A, inhibin B, and FSH polypeptide or mRNA levels from biological samples.
  • a further embodiment of this invention is the measurement of what can be construed as the set of hormone levels, ⁇ inhibin B, inhibin A, FSH ⁇ denoted as ⁇ B, A, FSH ⁇ hereafter, on a single day, 3 - 4 sequential days, or more days.
  • the measurement of ⁇ B, A, FSH ⁇ starts on the first day of the cycle (denoted calendar day 1 (CD1)) and progresses with daily measurements to signal the crucial and characteristic features, timing, and events of the ovulatory cycle: preovulatory infertile days, a Fertile Start Day, a fertile interval, one or more days of increased or maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a day of transition to a luteal phase, one or more days of luteal phase, or days of immediate approach to the next cycle:
  • a single measurement of ⁇ B, A, FSH ⁇ or 3 or 4 sequential measurements at any time or interval in the cycle serve as the basis in an algorithm to identify a phase, interval, or day of the cycle.
  • the output from the assay of ⁇ B, A, FSH ⁇ measured for one or more days, starting from CD1 or from another point in the cycle is analyzed by application of at least one algorithm selected from: i) (B/A), as an identifying sequence signaling infertile follicular, fertile follicular, ovulatory, and luteal phases, that is, the crucial and characteristic features, timing, and events of the ovulatory cycle; ii) ‘A algo’, where a computed increase in A signals the Fertile Start Day and fertile window; iii) ‘(A/FSH)/algo’, where a computed increase in A/FSH signals the Fertile Start Day, iv) ‘slope (B/A)/FSH’, as an identifying sequence marking the fertile window; v) slope A (daily change in A), which is used in conjunction with slope (B/A)/FSH and (B/A) to distinguish the fertile window from the luteal
  • the assay further comprises a point-of-care (POC) apparatus adapted for clinic or home use for precise signaling of fertile and infertile days of a human ovulation cycle.
  • POC point-of-care
  • this is used to determine at least one of: a time-of-cycle during an ovulatory or menstrual cycle for which a female user can use for family planning, to enhance or to avoid conception; ovarian dysfunction, polycystic ovary syndrome (PCOS), or primary ovarian insufficiency (POI); assesses male fertility, and fertility impairment; or to evaluate puberty status and abnormalities thereof.
  • PCOS polycystic ovary syndrome
  • POI primary ovarian insufficiency
  • the POC apparatus uses an inhibin and FSH VFA (vertical flow assay) apparatus or alternative lateral flow assay (LFA) apparatus to measure human inhibin A, inhibin B, and FSH levels in serum, plasma, whole blood, or fingerstick blood.
  • the inhibin A, inhibin B, and FSH levels are measured one or more times during at least daily or one or several days, or during an ovulatory or menstrual cycle to determine time- of-cycle.
  • the inhibin A, inhibin B, and FSH levels are used to compute one or more of the following: an infertile follicular phase, a Fertile Start Day, a fertile interval, days of maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a transition to a luteal phase, the infertile luteal days, and the luteal days immediately prior to the next cycle.
  • the inhibin A, inhibin B, and FSH levels are variables in mathematical transformations using one or more diverse computational functions, formulation functions, or algorithmic functions to quantify at least one of: an infertile follicular interval or phase, a Fertile Start Day, a fertile interval, days of maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a transition to a luteal phase, an infertile luteal phase, and the luteal days immediately prior to the next cycle.
  • an aspect of the present disclosure relates to a kit comprising: a skin puncture device; and one or more containers comprising reagents to detect inhibin A, inhibin B, and FSH in a biological sample obtained using the skin puncture device, and instructions or software to calculate a concentration of inhibin A, inhibin B, and FSH in the biological sample, and using the concentration of inhibin A, inhibin B and FSH in the biological sample to determine at least one of: measure of a fertility or an infertility of a female or a timepoint in the ovulation cycle.
  • assay further comprises one or more biological samples from the female; detecting the concentration in the one or more biological samples of inhibin A, inhibin B, and FSH polypeptides or mRNA that encodes inhibin A, inhibin B, and FSH and determining a relative expression of inhibin A, inhibin B, and FSH polypeptides or mRNA in the one or more biological samples from the female at one or more timepoints, and collating the detection of the inhibin A, inhibin B, and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.
  • the output from the assay is analyzed by application of at least one algorithm selected from: i) (B/A), as an identifying sequence signaling infertile follicular, fertile follicular, ovulatory, and luteal phases, that is, the crucial and characteristic features, timing, and events of the ovulatory cycle; ii) ‘A algo’, where a computed increase in A signals the Fertile Start Day and fertile window; iii) ‘(A/FSH)/algo’, where a computed increase in A/FSH signals the Fertile Start Day, iv) ‘slope (B/A)/FSH’, as an identifying sequence marking the fertile window; v) slope A (daily change in A), which is used in conjunction with slope (B/A)/FSH and (B/A) to distinguish the fertile window from the luteal phase; vi) Area Under the Curve (AUC) algorithm (27), where B/A is a variable to determine time-of-cycle;
  • the assay with method further comprises a point-of-care (POC) apparatus adapted for clinic or home use for precise signaling of fertile and infertile days of a human ovulation cycle.
  • POC point-of-care
  • the assay is used to determine at least one of: a time-of-cycle during an ovulatory or menstrual cycle for which a female user can use for family planning, to enhance or to avoid conception; ovarian dysfunction, polycystic ovary syndrome (PCOS), or primary ovarian insufficiency (POI); assesses male fertility, and fertility impairment; or to evaluate puberty status and abnormalities thereof.
  • PCOS polycystic ovary syndrome
  • POI primary ovarian insufficiency
  • the POC apparatus uses an Inhibin and FSH VFA (vertical flow assay) apparatus or alternative lateral flow assay (LFA) apparatus to measure human inhibin A, inhibin B, and FSH levels in serum, plasma, whole blood, or fingerstick blood.
  • inhibin A, inhibin B, and FSH levels are measured one or more times during, at least daily or one or several days, an ovulatory or menstrual cycle to determine time-of-cycle.
  • the inhibin A, inhibin B, and FSH levels are used to compute one or more of the following: days of an infertile follicular phase, a Fertile Start Day, a fertile interval, days of maximum fertility, a peri-ovulatory interval, a day of ovulation/follicular rupture, a transition to a luteal phase, days of an infertile luteal phase, and luteal days approaching the next cycle.
  • the inhibin A, inhibin B, and FSH levels are variables in mathematical transformations using one or more diverse computational functions, formulation functions, or algorithmic functions to quantify at least one of: the following: days of an infertile follicular phase, a Fertile Start Day, a fertile interval, days of maximum fertility, a peri -ovulatory interval, a day of ovulation/follicular rupture, a transition to a luteal phase, days of an infertile luteal phase, and luteal days approaching the next cycle.
  • the method further comprises updating at least one of the algorithms, formulations, and transformations to increased precision and information for a single subject or multiple subjects after measuring the inhibin A and inhibin B and/or FSH levels from multiple cycles.
  • a and B refer to inhibin A level and inhibin B level, respectively.
  • B/A or B/A refers to the function or variable (inhibin B level/inhibin A level).
  • FSH refers to FSH (follicle stimulating hormone).
  • FIG. 1 shows plots of day-specific urinary E3G (estrone-3 -glucuronide) and PDG (pregnanediol -3 -glucuronide) levels from three subjects, S2, S3, S4. After menses daily E3G and PDG levels were obtained by these subjects with the MIRATM, a popular fertility tracking home device. The day-specific levels are indexed to Day 0, the day of ovulation determined by dominant follicle (DF) rupture and confirmed by serum progesterone levels. These data corroborate that a urinary E3G rise is an inadequate signal to reliably identify the start of the fertile window (shaded region, [Day -5, Day 0]).
  • DF dominant follicle
  • FIG. 2 shows plots of day-specific serum FSH, B, and A levels from subjects SI, S2, S3, and S4. These subjects provided daily blood by venipuncture the entire cycle, starting the first day of the cycle (CD1 (calendar day 1) the start of menses) until the beginning of the subsequent menses. FSH levels were measured by batch with a commercial platform (Abbott Architect ci4100) and the inhibins measured by batch with sensitive ELISA kits. The day-specific levels are indexed to Day 0, the day of ovulation determined by dominant follicle (DF) rupture and serum progesterone levels, which corresponded to the B mid-cycle peak.
  • DF dominant follicle
  • FIG. 3 shows the scaled cross-sectional mean day-specific FSH, B, and A levels (top and bottom plots) and B/A values (middle plot) computed for SI, S2, S3, S4 cycles. These plots were derived using functional data analysis (75). There is an oscillatory time-function for FSH, B, and B/A, and this novel discovery was used to derive the algorithms of this disclosure that signal and predict the key phases of the ovulatory cycle.
  • FSH and B levels have a periodic relationship driven by positive-negative feedback mechanism, denoted as ‘FSH ⁇ ->B’, that changes and therefore distinguishes the infertile follicular phase from the fertile window.
  • the FSH ⁇ ->B function has a period of approximately 4 - 5 days prior to approximately Day -7, the day of DF appearance, whereas this period is increased to approximately 7 days during the fertile window until ovulation. Furthermore, whereas there is a lag of approximately one day in the induction of B by FSH and the suppression of FSH by B during the early infertile follicular phase, this effect is abrogated during the fertile window. In contrast, A levels do not display such a periodic function, but rather, begin to rise at the time of the DF, Day -7. There is oscillatory behavior with the B/A function (middle plot).
  • FIG. 4 is a table which shows the day-specific values for algorithms using B, A, and FSH levels from cycles SI, S2, S3, S4. These algorithms predict: the Fertile Start Day, the days of the fertile window, day(s) of potential maximum fertility, and the transition point from ovulation to luteal infertile phase, the infertile luteal phase, and the luteal days just prior to the next cycle.
  • the algorithms displayed here are: ‘B/A’, ‘Aalgo’, and ‘(A/FSH)algo’; their embodiments are herein described.
  • Interval [-7, -6], the dark shaded region, is the immediate approach to the fertile window and approximate time of dominant follicle selection.
  • the B/A columns show sequences of B/A used in an algorithmic form to: i) confirm the days of the fertile window, ii) identify the potentially most fertile day, and ii) identify the transition to the luteal phase after ovulation.
  • Underlined values are the uninterrupted decreasing sequence of B/A, an identifying metric, which begins with the start signal for the fertile window identified by Aalgo or (A/FSH)algo (below).
  • This B/A sequence increases before Day 0, and the bold B/A value (mostly on Day -1) is the first increase after the nadir which signals a day of high or highest probable fertility.
  • the italicized values after Day 0 are ⁇ 1.0 and identify the luteal phase. Days of late luteal phase B/A values >1 signal the approach to the next cycle.
  • Aalgo values are computed as A/Aave, where the inhibin A level is normalized by Aave, the average inhibin A level over the menses which were 4 - 7 days in the cycles.
  • the start day for the fertile window, the Fertile Start Day, by Aalgo (value in bold) is identified by a value of > 2.0.
  • the start days of the fertile window are: SI (-5, -5) S2 (-6, -7), S3 (-5, -5), (S4 (-7, -6).
  • SI 5, -5)
  • S2 -6, -7
  • S3 5, -5
  • S4 -7, -6
  • Seh -5, -5
  • Gilf -6, - 6
  • the DF growth rate is 1.62 ⁇ 0.05(SEM) mm/day with a maximum size prior to ovulation of 21.2 ⁇ 0.3(SEM) mm.
  • a key hormonal regulator of DF selection is an inhibin (44, 47).
  • Inhibins are a member of the transforming growth factor-0 superfamily and as ovarian hormones serve in autocrine, paracrine, and endocrine regulatory roles (48 - 55).
  • Inhibin A and inhibin B are each formed by a common a-subunit which is linked by a disulfide bridge to one of two highly homologous 0- subunits, 0A and 0B, resulting in OC-0A (inhibin A) and OC-0B (inhibin B), respectively (48 - 55).
  • the mean day-specific inhibin B profile is characterized by: a) an increase starting the first day of menses and peaking ⁇ Day -9 - Day -7, b) a nadir at Day -2, c) a second peak at Day 0, and d) post-ovulatory low levels with a rise before the start of the next menses (42, 63 - 69).
  • inhibin B secreted from follicles of the early pre-ovulatory phase is dependent on the distribution of follicle number and size, which is variable in ovulatory cycles (40 - 44, 71, 72).
  • the early and mid-follicular phase antral follicle counts for follicle sizes 2 - 6 mm and >6 - 10mm have been report at ⁇ 18 ⁇ 7(SD) and ⁇ 5 to 7 ⁇ 3(SD), respectively (72). Consequently, it is not clear how inhibin B could be used in a fertility tracking method and device.
  • Inhibins are glycoproteins produced by the granulosa and thecal cells of the ovary. There are at least two active hormonal forms, inhibin A and inhibin B. Inhibins are heterodimeric and consist of an alphasubunit (a subunit) covalently linked to a 0 subunit (0 A) of inhibin A and a 0 subunit (0B) of inhibin B. There appear to be several isoforms of inhibin A and inhibin B in human serum.
  • Kristensen et al., (40) measured the concentrations of inhibin A and inhibin B in 958 follicles from 286 women during the follicular phase of the ovulatory cycle up to the time of dominant follicle selection (max diameter tested, 13mm). Most notably, there was a dramatic fall in follicular inhibin B with dominant selection in contrast to a dramatic rise in follicular inhibin A with this transformation.
  • inhibin A has strong predictive value as a metric for oocyte/follicular maturation. This work indicated that only follicles with a size of 12mm or larger contribute to the serum inhibin A level, making it a potential signal for the start of the fertile interval of the ovulatory cycle.
  • inhibin B levels increase during the early follicular phase and reach a peak, fall about the time of the inhibin A increase, reach an ovulatory peak, and are low throughout the early and middle luteal phase (42, 63-69).
  • inhibins as a Fertility Assessment Method (FAM) was by A. R. Sheth who first observed an immunoreactive “inhibin-like” substance in the urine of ovulatory women that surged 3 - 4 days before the urinary LH peak (81). This urinary immunoreactive “inhibin- like” protein was an approximately 19,000 Molecular Weight protein (82).
  • inhibins are an important indicator of the follicular state and could be used as variables in mathematical transformations to predict the phase of the ovulatory cycle. Furthermore, these predictive formulations and algorithms could be extended by considering the set of hormone levels ⁇ B, A, FSH ⁇ . Significantly, the inventors recognized FSH- inhibin B positive-negative oscillations, that is, FSH ⁇ ->B, prior to DF selection and a change in this infertile follicular phase signature curve with progression through the fertile window to ovulation.
  • a key concept introduced with the herein invention is to use the measurements simultaneously and distinctly of ⁇ B, A, FSH ⁇ from bodily fluids as variables in novel mathematical formulations to determine time-of-cycle and diagnose abnormal ovarian function.
  • a “subject” refers to a mammal, whether female or male.
  • the subject is a female, and a specific case a female human.
  • the female is a non-human mammal, such as domesticated animals, domestic pets, rodents, and primates.
  • the mammals are selected from cats, dogs, equines, bovines/cattle, pigs, goats, buffalo, sheep, goats, camels, or deer.
  • the terms “determining” or “measuring,” or alternatively “detecting,” may include assessing the presence, absence, quantity and/or amount (which can be an effective amount) of a substance or biomarker within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values and/or categorization of such substances in a sample from a subject.
  • the substance measured is inhibin A and inhibin B and FSH.
  • inhibin B and inhibin A which is ⁇ B, A ⁇ or ⁇ A, B ⁇
  • inhibin A and FSH which is ⁇ A, FSH ⁇ or ⁇ FSH, A ⁇
  • inhibin A which is ⁇ A ⁇ , etc.
  • binding affinity refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its cognate binding partner (e.g., an antigen).
  • a molecule e.g., an antibody
  • its cognate binding partner e.g., an antigen
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • high affinity antibody refers to those antibodies having a binding affinity to their target of at least about 10' 8 M, at least about 10' 9 M, at least about 10' 10 M; at least about 10' 11 M; or at least about 10' 12 M.
  • antibodies for use with the present invention can include those with a binding affinity of at least about 10' 6 M, or at least about 10' 7 M.
  • a variety of methods of measuring binding affinity are known in the art, e.g., ELISA, Kinexa Biosensor, surface plasmon resonance (BIAcoreTM), scintillation proximity assays, ORIGEN immunoassay (IGEN), fluorescence quenching, and/or fluorescence transfer. Binding affinity may also be screened using a suitable bioassay.
  • the levels of one or more of the biomarkers in a sample obtained from a subject may be measured by the assay methods described herein, or equivalents thereof, and used for various clinical purposes. These clinical purposes may include but are not limited to identifying a subject having infertility, detecting or diagnosing the opening and/or closing of fertility in a subject trying to, or not trying to, become pregnant.
  • the level of a biomarker in a sample as determined by assay methods described herein may be normalized with an internal control in the same sample or with a standard sample (having a predetermined amount of the biomarker) to obtain a normalized value.
  • An important aspect of the present invention is that inhibins vary widely in overall expression, however, the present invention works regardless of whether a raw value or a normalized value of the biomarker is used.
  • a deviated (e.g., increased or reduced) value of one inhibin in a sample obtained from a subject relative to the value of the other inhibin is indicative of the point in the ovulation cycle.
  • a control sample or reference sample can contain a known amount of the biomarker to be assessed.
  • the control sample or reference sample is a sample obtained from a control subject.
  • a control level can be a predetermined level or threshold. Such a predetermined level can represent the level of the protein in a population of subjects throughout the ovulation cycle. It can also represent the level of the protein in a population of subjects that do not ovulate or no longer has ovaries.
  • a method and apparatus based on inhibins A and B with or without FSH would need to use serum or plasma or whole blood for fertility tracking.
  • Whole blood assays for molecules - fingerstick sampling for hormones, in particular protein hormones - are receiving increasing research attention and commercial product development (83-87).
  • Concordance of measured hormone levels between fingerstick and venipuncture samples for fertility protein hormones such as anti-mullerian hormone (AMH), FSH, LH, and prolactin, has been proven, and blood spots on filters obtained by women at home can now be sent into various companies for results (Modern FertilityTM (https://modemfertility.com/; ZRT labsTM (www.zrtlab.com)(88, 89).
  • inhibin A Serum levels of inhibin A have been studied as an indicator for oocyte maturation in ovarian stimulation for assisted reproductive techniques, but no method or apparatus is mentioned or discussed, or envisioned for fertility tracking for home use by women (80).
  • inhibin B In contrast to the female, in the male, inhibin B is in the afferent arm of the feedback loop from the testis that regulates FSH secretion; inhibin A is undetectable in the peripheral serum of adult men (90-92). Inhibin B is produced mainly from the Sertoli cells, which line the convoluted seminiferous tubules and are controlled by FSH.
  • Sertoli cells secrete regulatory and nutrient molecules which are crucial for spermatogenesis.
  • the classical definition of male hypogonadism in the past has referred to testicular failure associated with androgen deficiency, without considering potential deficiencies in germ and Sertoli cells.
  • Sertoli-cell-only syndrome in male infertility is relatively common and the patients present with no sexual abnormality.
  • causes of Sertoli-cell-only syndrome include: some genetic mutations in the AZF region (azoospermia factor region), toxins including chemotherapy, radiation therapy, and viral infection.
  • Apical secretion of inhibin B appears to have considerable value in aiding the assessment of the status of the seminiferous epithelium/Sertoli cell health (90-92). Serum inhibin B levels in various male age cohorts is being established as a maker of Sertoli cell function, that is, for spermatogenesis and male infertility (93-95).
  • ACE2 the receptor for entry into target cells by SARS-CoV-2/COVID-19, is found on Sertoli and germ cells (96).
  • COVID-19 in the acute infection stage can have deleterious effects on male gonadal function (97-103).
  • SARS- CoV-2 via intranasal infection has been shown to cause acute testicular damage with subsequent chronic asymmetric testicular atrophy and associated hormonal changes despite resolution of the pneumonic phase (97, 98).
  • Another initial study from China showed the ratios of testosterone/LH and FSH/LH were abnormal in 81 reproductively aged men with COVID-19 compared to 100 age-matched healthy men (99).
  • 86% of these CO VID men were not hypoxic and did not have severe symptomatology, radiological findings, or require intervention.
  • the present invention includes an apparatus, kit, method, and system for determining inhibin B levels of male control subjects and patients after COVID-19 infection. And a practical way to investigate this question, and the hypothesis of reduced male global fertility from CO VID- 19, would be the development of a POC inhibin B measuring device.
  • a significant increase in precocious puberty, rapidly progressive puberty, and precocious menarche has recently been reported (105).
  • inhibin B levels have been advanced as a way for differentiating between congenital hypogonadotropic hypogonadism and constitutional delay of growth and puberty (106).
  • the disclosed invention as a POC method and device for measuring inhibin B, would have a significant impact on understanding the effect of COVID-19 on pubertal development and disorders of puberty.
  • inhibin A and inhibin B Given the periodic change in inhibin A and inhibin B in the normal ovulatory cycle, it would be anticipated that knowledge of these levels would aid in the diagnosis of such conditions as primary ovarian insufficiency, menopausal state, and polycystic ovary syndrome. POC measurement of inhibin A and/or inhibin B levels would be potentially clinically important in diagnosis and treatment of these common conditions (107-112). Also, a suitable POC method and device for inhibins could be very helpful in signaling to breastfeeding women the return of fertility after lactational amenorrhea (113,114). Breastfeeding is used in many parts of the world as a form of family planning, spacing children, and an inhibin POC method and device would offer a significant improvement (113,114).
  • the invention described herein solves the various problems discussed above.
  • the present invention provides methods related to kits or devices for the ovulatory woman to know where she is in her cycle, especially for identifying with precision the start of fertility prior to ovulation, a capability lacking in the present state of the art.
  • the invention includes novel algorithms with inhibin A, inhibin B, and FSH levels that reveals day-specific fertility information.
  • the invention can include a point-of-care (POC) method and device, for home and clinic, offering wide-ranging diagnostic possibilities for fertility disorders.
  • POC point-of-care
  • Inhibin is a heterodimeric TGF-P family ligand that is expressed in many cancers and is a selective biomarker for ovarian cancers, however, its tumor-specific functions remain unknown.
  • the a subunit of inhibin A and B which is critical for the functionality of their dimeric structures, correlates with microvessel density in human ovarian tissues and xenografts and is predictive of poor clinical outcomes in multiple cancers (116).
  • the a subunit of the inhibins has been found to be elevated in patient ascites and supports the idea that inhibins may promote ascites formation, likely through increased vascular permeability.
  • the effectiveness of anti-angiogenic therapies is attributed to increased vascular normalization resulting in reduced intra-tumoral hypoxia, perfused and functional vessels that improve the delivery of other chemotherapeutics, and enhanced immune response (115).
  • A refers to inhibin A concentration or level and ‘B’ refers to inhibin B concentration or level as measured in serum, plasma, whole blood, or fingerstick blood.
  • FSH refers to follicle stimulating hormone concentration or level as measured in serum, plasma, whole blood, or fingerstick blood.
  • the fertile or infertile state for any given days of the ovulatory cycle is assessed via: (i) Collecting a sample of serum, plasma, whole blood, or fingerstick blood for at least one day, or more typically for several consecutive days, or daily throughout much or all of a menstrual cycle; (ii) Application of this sample to a suitable test strip, filter, device or apparatus; and/or (iii) Determination of A and B or A and B and FSH. This may be denoted below as the measured set of hormones: ⁇ B,A ⁇ or ⁇ B,A,FSH ⁇ or subsets thereof.
  • male fertility, pathology of ovarian function such as PCOS, or adolescent pubertal status are assessed via: (i) collecting a sample of serum, plasma, whole blood, or fingerstick blood for at least one to three days or several days; (ii) application of this sample to a suitable test strip, filter, device or apparatus; (iii) determination of the inhibin A and inhibin B and FSH concentrations (levels); and/or (iv) application of the mathematical transformations, formulations, computations, or algorithms described which include: (B/A) with application of various identifying sequences for phase or point of cycle; Aalgo; (A/FSH)algo; slope (B/A)/FSH; slope A; AUC(B/A) and/or its first derivative, loglO(abs(
  • the disclosed methodology is novel and provides unique insight into analyzing the time- of-cycle in ovulatory cycles.
  • the methodology provided creates and reveals the need for an inhibin A and inhibin B test strip or filter or device or other detection apparatus, with or without an FSH measurement component.
  • the fertile interval is generally taken as Day -5 to Day 0, with Day 0 defined as the day of ovulation generally taken as the day of follicular rupture and hours close to this rupture; that is, the fertile interval is the 6-day interval counting back from the day of ovulation (5,6, 20-23).
  • the Fertile Start Day critical for enhancement of conception, or correspondingly for natural methods of birth control (i.e., avoidance of sexual relations from the Fertile Start Day to the luteal infertile phase), is therefore Day -5.
  • One embodiment of the disclosed method is the capability to distinguish (B/A) ratios and other computations with (B,A) between normal women and women with PCOS and for diagnosis of disturbances in puberty.
  • (B/A) and other diverse functions of (B,A), with or without FSH as a transformative variable can be used during the ovulatory cycle to determine in real-time precise points in the preovulatory, periovulatory, and luteal phases in general, and more precisely: (1) the appearance of the DF, (2) the Fertile Start Day, (3) the days of probable maximal fertility, (4) the day of ovulation/follicle rupture, and/or (5) the day of transition from the peri -ovulatory interval to the luteal phase, (6) the days of the infertile luteal phase, and/or (7) luteal days immediately prior to the start of the next cycle.
  • FIG. 1 shows tracking of the ovulatory cycle with urinary hormones, E3G and PDG, with the MIRATM device and demonstrates the inadequacy of this present technology in accurately and reliably signaling the Fertile Start Day, the fertile window, and the precise day of ovulation and transition to the luteal phase.
  • FIG. 2 shows the unique day-specific B and A levels for four ovulatory cycles SI, S2, S3, and S4 with the day of ovulation determined by follicular rupture and the adjunct data of serum progesterone and mid-cycle inhibin B peak.
  • FIG. 3 shows the FSH ⁇ ->B oscillatory behavior in the infertile follicular phase (approximately Day -14 to Day-8 or Day -7), by way of scaled cross-sectional mean levels (75), and the change in the FSH, B relationship from ⁇ Day -8 to Day 0 (top plot).
  • the change in oscillatory behavior is also detectable with (B/A) (middle plot).
  • inhibin A does not exhibit such an oscillatory behavior.
  • FIG. 4 shows application of B/A, Aalgo, and (A/FSH) algorithms to determine time-of- cycle. The results are for cycles SI, S2, S3, S4. In addition, the algorithms have been tested with 10 averaged cycles from the literature, ‘Seh’ (64), and another 10 averaged cycles, ‘Gilf (69). In these applications fingerstick blood testing begins on CD1 (menses start) and is performed daily. B/A values determine an identifying sequence for the approach to the fertile window and the days of the fertile window, an uninterrupted decreasing string which ends with a minimum generally at Day -2. Day -2 is identified by a subsequent increase in this B/A string, which occurs mostly on Day -1, and less frequently on Day 0.
  • the luteal phase is consistently signaled by a B/A >1 after Day 0 or Day +1.
  • B/A is always >1 on Day 0, the day of ovulation.
  • Aalgo uses a normalized A level, from the mean of the days of the menses.
  • the Fertile Start Day is signaled by an Aalgo level >2.0.
  • the (A/FSH)algo corroborates the (B/A) and Aalgo signals.
  • (A/FSH) is a normalized level from the days of menses.
  • the Fertile Start Day is signaled by an (A/FSH)algo level >2.0.
  • the disclosed method is used in a POC fertility tracking device to inform on time-of-cycle.
  • FIG. 5 shows plots of the results from FIG. 3.
  • the values for Aalgo, and (A/FSH)algo as a function of time-of-cycle, which were used to determine the Fertile Start Day are provided (top panel).
  • Day 0 is the day of ovulation; the fertile window, Day -5 to Day 0, is in light shade; the immediate approach to the fertile window, Day -7 to Day -5, is in dark shade.
  • Table 1 shows the application of the B/A function to signal infertile preovulatory or infertile follicular days and the approach or time of fertile days which is particularly useful in cases of a protracted infertile preovulatory or infertile follicular phase.
  • a prolonged infertile follicular or infertile preovulatory phase - that is a phase before DF selection - can occur sporadically during normal cycles and in PCOS. This derivation is based upon the novel discovery of the oscillatory FSH ⁇ ->B function prior to DF selection. It does not require measurements starting from CD1, but measurements can commence during any prolonged cycle interval of unknown phase. This particularly useful for family planning, to avoid or enhance pregnancy.
  • An infertile day is signaled as an increase in B/A from the prior day. But any decrease in B/A from the prior day is a potentially fertile day, and two sequentially decreasing B/A values (i.e., a string of three decreasing B/A values) would mark the approach to or entry into the fertile window. Shown are applications of the algorithm to individual cycles S1,S2,S3,S4, and averaged 10 cycles, Seh, and averaged 10 cycles, Gilf. These cycles were all ovulatory, so there was a limited interval of infertile follicular phase days. The up arrows darkly shaded show the days that would be considered infertile.
  • the down arrows are days of potential fertility, but the days following the lightly shaded down arrows are part of the identifying string of decreasing B/A values for the fertile window.
  • the darkly shaded up arrows would be considered infertile and ‘safe’ for sexual intercourse to avoid pregnancy. This algorithm should provide such ‘up arrow safe days’ for prolonged infertile follicular phase situations and during such prolonged infertile stretches in PCOS.
  • B/A values provided in FIG. 4 were used in this algorithmic form to differentiate the infertile follicular phase days from the fertile follicular phase days - that is, to predict the ‘safe’ infertile follicular phase days and the fertile follicular phase days.
  • B/A values are determined as an increase from the preceding day (up arrow) or as a decrease from the preceding day (down arrow). The first day starting the sequence is indicated as a blank, since an up/down value cannot be assigned.
  • Up arrows (increased B/A) are shown as dark shading, and the first down arrow (decreased B/A) initiating the extended follicular phase interval of uninterrupted decreasing B/A is shown in the lighter shading.
  • this analysis involves B/A values that are >1 distinguishing the domain for this analysis from the luteal phase; thus, it could be performed during extended infertile phases such as occur in polycystic ovary syndrome (PCOS) (76 - 79).
  • PCOS polycystic ovary syndrome
  • Infertile days during the follicular phase before DF selection, useful for birth control purposes, would be any ‘up arrow’ (increased B/A) days.
  • One algorithm with this method for determining the start of a fertile interval after an extended infertile follicular phase would be the 2nd or 3rd day of a ‘down arrow’ (decreased B/A) after an ‘up arrow’.
  • Individual cycles SI, S2, S3, and S4 were analyzed. Results from 10 averaged cycles, ‘Seh’, from Sehested et al. (64) and 10 averaged cycles, ‘Gilf from Gilf et al. (69) are also shown.
  • the first day of the extended negative B/A sequence (light shaded down arrow) following an ‘up arrow’ (increased B/A) is: SI (Day -9), S2 (Day -8), S3 (Day -6), S4 (Day -6), Seh (Day -8), Gilf (Day -7).
  • Table 2 shows the computation of day-specific slope of (B/A)/FSH alongside B/A which serve as state functions to determine time-of-cycle for cycle S2 and the average cycles, ‘Seh’, from Sehested et al., (64).
  • Slope (B/A)/FSH is computed as the difference, (B/A)/FSH on day, D, minus (B/A)/FSH on day, D-l. SI and ‘Seh’ cycles are provided as examples for this algorithm.
  • Slope (B/A)/FSH and B/A serve as state functions to uniquely define time-of-cycle.
  • Two to four daily blood measurements are used to determine the point of a cycle: (a) During the early follicular phase, before the fertile window, positive slope (B/A)/FSH values interrupt negative values until Day -6 to Day -9: (SI (Day -9), S2 (Day -5), S3 (Day -5), S4 (Day -6), Seh (Day -6), Gilf (Day - 7)). (b) Slope (B/A)/FSH during fertile window [-5, -2] is always negative; that is, there are always four uninterrupted sequential negative values in this interval, (c) Slope (B/A)/FSH is always positive on Day 0; it may be positive or negative on Day -1.
  • B/A is: always >1 on Day 0, followed by a >1 or ⁇ 1 value on Day +1 and always ⁇ 1 on Day +2 to approximately Day +10 of the luteal phase,
  • An increasing B/A, > or ⁇ 1 occurs in the late luteal phase to signal the impending next cycle.
  • Bold Day -5 and Day 0 in the Day of Cycle column with the shaded rows delineates the fertile window.
  • the bold slope (B/A)/FSH values is the identifying uninterrupted negative string that signals the approach to and includes the fertile window.
  • the bold B/A values is the identifying string, the uninterrupted sequential decreasing B/A ending with a minimum B/A on Day -2 (arrow) followed by two increasing B/A values, that signals the approach to the fertile window, the fertile window, day of ovulation, and the transition point to the luteal phase.
  • the present invention does not preclude the use of the urine LH to help identify the time close to ovulation or the use of a urine PDG stick to double-check for the luteal phase.
  • the invention disclosed herein may be combined with existing fertility tracker technologies, including, e.g., luteinizing hormone (LH), estrone-3 -glucuronide (E3G), pregnanedi ol-3 -glucuronide (PDG), and/or follicle stimulating hormone (FSH), detected using urine or other samples.
  • LH luteinizing hormone
  • E3G estrone-3 -glucuronide
  • PDG pregnanedi ol-3 -glucuronide
  • FSH follicle stimulating hormone
  • the present invention is not constrained by the above mathematical transformations, formulations, or algorithms with ⁇ B, A, FSH ⁇ . It is shown herein that the signature of inhibin B and inhibin A levels during the ovulatory cycle can be applied to diverse analytic functions; this signature is a) in the early part of the cycle, inhibin B is rising whereas inhibin A is virtually undetectable, b) then the dominant follicle appears and inhibin B is falling whereas inhibin A is rising, and c) then ovulation with rupture of the follicle producing a brief burst of inhibin B with subsequent falling and low levels whereas inhibin A levels remain high, through much of the luteal phase.
  • LFA lateral flow assay
  • VFA vertical flow assay
  • program storage devices e.g., digital data storage media, which are machine or computer-readable and encode machine-executable or computer-executable programs of instructions, wherein said instructions perform some or all of the steps of said above-described methods.
  • the program storage devices may be, e.g., digital memories, magnetic storage media such as magnetic disks and magnetic tapes, hard drives, or optically readable digital data storage media.
  • the embodiments are also intended to cover computers programmed to perform said steps of the above-described methods.
  • modules may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with the appropriate software.
  • the functions may be provided by a single dedicated processor, by a single shared processor, or by a plurality of individual processors, some of which may be shared.
  • module should not be construed to refer exclusively to hardware capable of executing software, and may implicitly include, without limitation, digital signal processor (DSP) hardware, network processor, application-specific integrated circuit (ASIC), field- programmable gate array (FPGA), read-only memory (ROM) for storing software, random access memory (RAM), and nonvolatile storage.
  • DSP digital signal processor
  • ASIC application-specific integrated circuit
  • FPGA field- programmable gate array
  • ROM read-only memory
  • RAM random access memory
  • nonvolatile storage nonvolatile storage.
  • Other hardware conventional and/or custom, may also be included.
  • the various embodiments described herein provide an improved method and device for POC, at home and in the clinic, real-time determinations of the fertility status during days of the ovulatory cycle (that is, the process of ‘timing-of-cycle’) for both enhancement and avoidance of conception.
  • inhibin A and inhibin B levels (‘A’ and ‘B’, respectively) with or without FSH levels, from plasma, serum, whole blood, and fingerstick blood, for POC, at home and in the clinic, as the analytes to determine the objectives, namely, determine one or more of: preovulatory infertile days, the Fertile Start Day, fertile interval, days of high or maximum fertility, periovulatory interval, day of ovulation/follicular rupture, day of transition to the luteal phase, further days of the luteal phase, and luteal days immediately prior to the start of the next cycle.
  • inhibin A and inhibin B with or without FSH levels, from plasma, serum, whole blood, and fingerstick blood, for POC, at home and in the clinic, in computations, formulations, and algorithms to determine in an ovulatory cycle: preovulatory infertile days, the Fertile Start Day, fertile interval, days of high or maximum fertility, periovulatory interval, day of ovulation/follicular rupture, day of transition to the luteal phase, further days of the luteal phase, and luteal days immediately prior to the start of the next cycle.
  • Another aspect of the disclosure is to use sensitive and accurate measurements of inhibin A and inhibin B levels with or without FSH levels, from plasma, serum, whole blood, and fingerstick blood, for POC, at home and in the clinic, for the diagnosis and for treatment of gonadal dysfunction such as occurs in polycystic ovary syndrome (PCOS) in women, after COVID-19 infections in men, and may occur during puberty in adolescence.
  • PCOS polycystic ovary syndrome
  • Another aspect of the disclosure is the use of diverse computations, formulations and algorithms to accomplish objectives.
  • a further objective of the disclosure is the use of the FSH ⁇ ->B function, that is the change in the FSH, B relationship, that is a change in the oscillatory behavior of B and FSH and B/A, as a function of time-of-cycle as a method of quantifying certain metrics for the ovulatory cycle.
  • a further aspect of the disclosure is the function, (B/A), as a method of quantifying certain metrics for the ovulatory cycle.
  • a further aspect of the disclosure is the function, Aalgo, as a method of quantifying certain metrics for the ovulatory cycle.
  • a further aspect of the disclosure is the function, (A/FSH)algo, as a method of quantifying certain of the metrics of for the ovulatory cycle.
  • a further aspect of the disclosure is the function, slope (B/A)/FSH, as a method of quantifying certain of the metrics of for the ovulatory cycle.
  • a further aspect of the disclosure is the function, slope A, as a method of quantifying certain of the metrics of for the ovulatory cycle.
  • a further aspect of the disclosure is the function or first derivative of AUC(B/A) as a method of quantifying certain of the metrics of for the ovulatory cycle.
  • a further aspect of the disclosure is to describe a method using A,B or (B/A) or ⁇ B, A, FSH) or other variations of these variables interfaced to a point of care device where A and B with or without FSH are measured by an antibody -based test strip or filter and lateral or vertical flow assay or other detection apparatus to quantify certain metrics of the ovulatory cycle.
  • a and B with or without FSH are automatically fed to a computing system, and (B/A), Aalgo, and functions such as disclosed are processed, and information provided by number and/or text for: time-of-cycle, disturbances in the ovulatory or menstrual cycle, and dispositions relative to male and female infertility and pubertal status.
  • program storage devices e.g., digital data storage media, which are machine or computer-readable and encode machine-executable or computer-executable programs of instructions, wherein the instructions perform some or all of the steps of said above-described methods.
  • the program storage devices may be, e.g., digital memories, magnetic storage media such as magnetic disks and magnetic tapes, hard drives, or optically readable digital data storage media.
  • the embodiments are also intended to cover computers programmed to perform said steps of the above-described methods.
  • modules may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with the appropriate software.
  • the functions may be provided by a single dedicated processor, by a single shared processor, or by a plurality of individual processors, some of which may be shared.
  • module should not be construed to refer exclusively to hardware capable of executing software, and may implicitly include, without limitation, digital signal processor (DSP) hardware, network processor, application-specific integrated circuit (ASIC), field- programmable gate array (FPGA), read-only memory (ROM) for storing software, random access memory (RAM), and nonvolatile storage.
  • DSP digital signal processor
  • ASIC application-specific integrated circuit
  • FPGA field- programmable gate array
  • ROM read-only memory
  • RAM random access memory
  • nonvolatile storage nonvolatile storage.
  • Other hardware conventional and/or custom, may also be included.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.
  • “comprising” may be replaced with “consisting essentially of’ or “consisting of’.
  • the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
  • TGFBR3L is an inhibin B co-receptor that regulates female fertility. Sci Adv. 2021 Dec 17;7(51):eabl4391. doi: 10.1126/sciadv.abl4391. Epub 2021 Dec 15. PMID: 34910520; PMCID: PMC8673766. [0167] 48) Mathews LS. Activin receptors and cellular signaling by the receptor serine kinase family. Endocr Rev. 1994 Jun;15(3):310-25. doi: 10.1210/edrv-15-3-310. PMID: 8076584.

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Abstract

L'invention concerne des méthodes, des dosages, des trousses et des systèmes pour l'utilisation d'inhibines avec ou sans FSH afin de calculer une sortie de données et d'analyser cette sortie de données pour déterminer un moment précis du cycle menstruel, en particulier par rapport à l'ovulation, à la fertilité et à l'infertilité.
PCT/US2025/026119 2024-04-25 2025-04-24 Inhibine et fsh en tant que biomarqueurs pour signalisation de temps de cycle dans le cycle menstruel Pending WO2025226902A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210264604A1 (en) * 2018-06-22 2021-08-26 Oova, Inc. Methods, devices, and systems for detecting analyte levels
US20220187325A1 (en) * 2019-04-10 2022-06-16 Spd Swiss Precision Diagnostics Gmbh Assay device

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210264604A1 (en) * 2018-06-22 2021-08-26 Oova, Inc. Methods, devices, and systems for detecting analyte levels
US20220187325A1 (en) * 2019-04-10 2022-06-16 Spd Swiss Precision Diagnostics Gmbh Assay device

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FEMKE P HOHMANN, JOOP S E LAVEN, FRANK H DE JONG, BART C J M FAUSER: "Relationship between inhibin A and B, estradiol and follicle growth dynamics during ovarian stimulation in normo-ovulatory women", EUROPEAN JOURNAL OF ENDOCRINOLOGY, BIOSCIENTIFICA LTD., GB, vol. 152, no. 3, 1 March 2005 (2005-03-01), GB , pages 395 - 401, XP009565402, ISSN: 0804-4643, DOI: 10.1530/eje.1.01871 *

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