WO2025226902A1 - Inhibins and fsh as biomarkers for signaling time-of-cycle in the female menstrual cycle - Google Patents

Abstract

Provided herein are methods, assays, kits, and systems for use of inhibins with or without FSH to calculate a data output and the analysis of the data output to determine a timepoint in the menstrual cycle and in particular in reference to ovulation, fertility, and infertility.

Description

INHIBINS AND FSH AS BIOMARKERS FOR SIGNALING TIME-OF-CYCLE IN THE FEMALE MENSTRUAL CYCLE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/638,491, filed April 25, 2024, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] 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.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0003] None.

BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with biomarkers of the ovulation cycle.

[0005] 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 MONITOR™), and more recently urinary pregnanediol -3 -glucuronide (PDG) has been added to the home measurable hormone panel (INITO FERTILITY MONITOR™, and MIRA FERTILITY TRACKER™) (5 - 13). These examples and related technology are broadly termed “FemTech”, which has been rapidly gaining research, financial, and commercial interest (14, 15). The specific applications of POC monitoring methods and devices span a wide spectrum: ‘understanding your hormones and tracking your symptoms’ (HORMONA™), addressing anxiety over possible reduced ovarian reserve, enhancing fertility, and as Fertility Awareness Methods (FAMs) for birth control (1, 16, 17).

[0006] However, 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). Thus, the ‘Fertile Start Day’ is Day -4 or Day -5 depending upon the basis for indexing ovulation. Urinary E3G levels during the fertile window cover a wide range; there is a considerable standard deviation from the mean day-specific levels and, therefore, absolute levels cannot be used to identify the start of the 6-day fertile window (24 - 27). Both the MIRA™ and INITO™ monitor systems, which quantify E3G levels, give fluctuating E3G levels preceding the start of and during the fertile window (9, 10, 24). Only 75% of women had adequate warning of the 6-day fertile interval to avoid pregnancy using the E3G-based CLEARBLUE FERTILITY MONITOR™ (24). Another fertility tracking monitor, the PROOV COMPLETE™, provided an average of 2.68 days between the E3G rise and the LH surge, which is unsuitable for birth control applications (2). Furthermore, these tracking systems lack precision in identifying the day of transition to the luteal phase, which shortens the ‘safe’ infertile luteal interval for sexual intercourse. Multiple studies set a PDG threshold of 5 mcg/mL for entry into this safe infertile luteal phase, which allows a corresponding length of 8.8 days on average (7, 8, 28 - 31). With the very precise means of establishing the 24 hour interval of ovulation by follicular sonography and serum hormone levels, the inventors themselves have confirmed the inadequacies of LH, E3G, and PDG for accurate predictions of the Fertile Start Day and day of ovulatory /luteal transition needed for family planning (19).

[0007] The markers targeted by commercial kits suffer from one or more of the following problems. The markers that look at a surge of LH appear too close to ovulation, and thus the user misses the most fertile period. Those that measure E3G show large fluctuations between individual cycles, leading to an imprecise determination of the start of the fertile window. Finally, those that measure a surge in PDG detect a biomarker that occurs sometime after ovulation and imprecisely marks the start of the luteal phase.

[0008] Thus, despite these advances, what is needed are methods, kits, and devices that provide the user with data along the entire cycle of ovulation, that is timely with regard to the start of fertility, and that is accurate even if there is a large range in levels for the biomarker(s) being measured.

SUMMARY OF THE INVENTION

[0009] As embodied and broadly described herein, 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.

[0010] As embodied and broadly described herein, 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. In one aspect, 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. In another aspect, 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. In another aspect, an output from the assay is analyzed by application of at least one algorithm selected from the following, where A = inhibin A concentration or level and B = inhibin B concentration or level, and FSH is FSH concentration or level: (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, loglO(abs(B-A/A)), loglOB/loglOA and/or alternative mathematical transformations, formulations, computations, or algorithms, wherein at least one algorithm quantifies one or more timepoints in the ovulation cycle. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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.

[0011] As embodied and broadly described herein, 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. In one aspect, 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. In another aspect, an output from the kit is analyzed by application of at least one algorithm selected from the following, where A = inhibin A level or concentration and B = inhibin B level or concentration, and 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, loglO(abs(B-A/A)), loglOB/loglOA and/or alternative mathematical transformations formulations, computations, or algorithms, wherein at least one algorithm quantifies one or more timepoints in the ovulation cycle. In another aspect, 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. In another aspect, 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. In another aspect, the POC apparatus uses a lateral or vertical flow device with antibodies to inhibin A beta subunit, inhibin B beta subunit and 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 but separately on a test substrate, strip, or filter in the POC apparatus. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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.

[0012] As embodied and broadly described herein, 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. In one aspect, 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. In another aspect, the output from the assay is analyzed by application of at least one algorithm selected from the following, where A = inhibin A level or concentration and B = inhibin B level or concentration, and 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, loglO(abs(B- A/A)), loglOB/loglOA and/or alternative mathematical transformations formulations, computations, or algorithms, wherein at least one algorithm quantifies a timepoint in the ovulation cycle. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, the female is a human.

[0013] As embodied and broadly described herein, 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.

[0014] Other aspects of the application and various embodiments described herein will become apparent to those skilled in the art once the preferred embodiments have been shown and described. These objectives are not to be construed as limitations of applicant’s invention, but are merely aimed to suggest some of the many benefits that may be realized by the apparatus and methods of the present application and with its many embodiments.

[0015] As embodied and broadly described herein, 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. As a further embodiment and broadly described herein this novel physiology, the FSH<->B function, was discernable using hitherto unavailable day-specific levels of inhibin A, inhibin B, and FSH and advances in functional data analysis.

[0016] Furthermore, knowledge of the periodicity of the oscillatory inhibin B and FSH levels which precedes the timing of the dominant follicle (DF) enabled derivation of algorithms that signal the end of the infertile follicular phase, appearance of a DF, and entry into the fertile window culminating in ovulation. [0017] As embodied and broadly described herein, 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. In one aspect, 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. Furthermore, data output and analysis may use subsets of inhibin A, inhibin B, and FSH polypeptide or mRNA levels from biological samples.

[0018] 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. In one embodiment, 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: In another embodiment 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.

[0019] In another aspect, 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 phase; vi) Area Under the Curve (AUC) algorithm (27), where B/A is a variable to determine time-of-cycle; vii) Fertility Indicator Equation (73, 74), where B/A or A are variables to determine time of cycle; vii) and the following functions with the subset {B, A} as variables in algorithms to determine time-of-cycle: LoglO(abs((inhibin A- inhibin B)/ inhibin A)) = loglO(abs((inhibin B- inhibin A)/ inhibin A), LoglOB/LoglOA (although the skilled artisan will recognize that using base 10 is an example, as other logs such as log e, can also be used and are encompassed by the present invention), (inhibin A- inhibin B)/ inhibin B; (inhibin A x inhibin B); or (rate of change of inhibin A) x (rate of change of inhibin B); wherein the at least one algorithm quantifies a timepoint or interval or phase in the ovulation cycle.

[0020] In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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.

[0021] As embodied and broadly described herein, 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. In one aspect, 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. In another aspect, 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; vii) Fertility Indicator Equation (73, 74), where B/A or A are variables to determine time of cycle; vii) and the following functions with the subset {B, A} as variables in algorithms to determine time-of-cycle: Logio(abs((inhibin A- inhibin B)/ inhibin A)) = logio(abs((inhibin B- inhibin A)/ inhibin A), LogioB/LogioA (although the skilled artisan will recognize that using base 10 is an example, as other logs such as log e, can also be used and are encompassed by the present invention), (inhibin A- inhibin B)/ inhibin B; (inhibin A x inhibin B); or (rate of change of inhibin A) x (rate of change of inhibin B); wherein the at least one algorithm quantifies a timepoint or interval or phase in the ovulation cycle. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures. In the following figures, 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).

[0023] 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 MIRA™, 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]). Furthermore, reliable identification of the post-ovulatory/luteal phase by a significant PDG rise is seen with a delay of 2 - 3 days after ovulation. The analysis of MIRA™ hormone levels with this precision of the time of ovulation is novel and has been published in a peer-reviewed journal (19).

[0024] 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. The early infertile follicular phase levels of FSH and B indicated oscillatory behavior which was further examined by way of functional data analysis (FIG 3). The day-specific FSH, B, and A levels from these individual cycles were used to derive the algorithms to predict time-of-cycle.

[0025] 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. A major embodiment of this disclosure is that 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). This discovery of a differing FSH<->B function between infertile and fertile follicular phases of the menstrual cycle, along with the distinctive plot of A, enabled creation of the disclosed algorithms to map and signal the time of cycle. In this figure, Day 0 is the day of ovulation as detailed in FIG 1 and FIG 2 legends. The shaded interval [-5, 0] is the fertile window. The start of the fertile window is Day -5.

[0026] 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. In addition, these algorithms have been tested against day-specific mean B, A, and FSH levels from 10 cycles of Sehested et al., ‘Seh’ (64) and against the day-specific mean levels from 10 cycles of Gilfillan et al., ‘Gilf (69); these levels were obtained by digitalization of published data and indexing the levels to the day after the LH peak, which also corresponded to the midcycle B peak. Day-specific values in the table are indexed to Day 0, day of dominant follicle rupture by sonography and mid-cycle peak inhibin B level and validated by serum progesterone levels. Bold ‘Day of Cycle’ and light shaded region is the interval, [-5, 0], the fertile window. 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. Underlined values are the uninterrupted string >2.0 that indicate days of potential fertility in the fertile window. (A/FSH)algo values are computed as (A/FSH)/(A/FSH)ave where inhibin A level is divided by the corresponding FSH level normalized to the average A/FSH for the days of menses. The start day for the fertile window, the Fertile Start Day, by (A/FSH)algo (value in bold) is identified by a value of > 2.0. Underlined values are the uninterrupted string >2.0 that indicates days of potential fertility in the fertile window. The FSH<->B preovulatory oscillatory function derived from the functional data analysis of the SI, S2, S3, S4 cycles was crucial in showing the distinctive change in behavior of FSH and B between the pre-DF and post- DF intervals of the follicular phase. Consequently, B/A is revealed in this invention to be particularly useful in defining the end of the early infertile follicular interval and expanse of the fertile window and the day(s) of highest probability of conception. There is an excellent correlation between Aalgo and (A/FSH)algo in determining the start of the fertile window. The start days of the fertile window, Fertile Start Day, computed by Aalgo and (A/FSH)algo, (Cycle, (Aalgo, (A/FSH)algo) ) are: SI (-5, -5) S2 (-6, -7), S3 (-5, -5), (S4 (-7, -6). For the 20 averaged test cycles the Aalgo and (A/FSH)algo fertile window start days were: Seh (-5, -5) and Gilf (-6, - 6). Thus an embodiment of this patent disclosure, the algorithms based upon a novel discovery, enables adequate advance notice of the fertile window for birth control purposes and minimizes the length of abstinence. In addition a potentially most or highly fertile day was indicated with the B/A algorithm, the first rise after a minimum during the fertile window: (Cycle (Day)), SI (-1), S2 (-1), S3 (0), S4 (-1), Seh (-1), Gilf (-1). Furthermore, highly fertile days could be signaled simply by counting 2 - 3 days after the start signal of the fertile window.

[0027] FIG. 5 shows plots of the Aalgo and (A/FSH)algo (top) and B/A (bottom) values for the combined cycles: SI, S2, S3, S4, and the 10 cycles for Sehested et al., ‘Seh’, (64) and 10 cycles for Gilfillan et al., ‘Gilf, (69). The values for these plots are given in FIG 4 (table). A 2-fold increase with the Aalgo or (A/FSH)algo, starting with daily measurements from the beginning of the cycle, is the signal for the start of the fertile window. The light shaded interval [-5, 0] is the fertile window and the darker shaded interval [-7, -5] is the immediate approach to the fertile window. Note that between Day -7 and Day -5, a signal for the start of the fertile window, has occurred for all 24 cycles. The B/A plot (here denoted ‘(B/A)algo’) for the combined cycles demonstrates a signature curve during the fertile window of: uninterrupted sequential decrease, a minimum, and ending with a maximum at Day 0 or occasionally at Day -1. An important embodiment of B/A as an algorithm is that the B/A value is always low (<1) in the infertile luteal region after Day +1 or Day +2 until the start of the next ovulatory cycle. B/A can be used to reliably distinguish the infertile follicular phase from the infertile luteal phase.

DETAILED DESCRIPTION

[0028] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0029] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0030] The limitations in the existing art and science of biomarkers for time-of-cycle prediction, and the novel features of the method herein, can be further understood after a detailed description of pituitary -ovarian physiology. It is known that there can be preovulatory LH surges and unsustained estrogen rises throughout a normal ovulatory cycle complicating the analysis of normal and abnormal cycles using standard hormone levels (19, 32 - 34). It appears that contrary to past conventional wisdom, which asserted single recruitment of ovarian follicles occurs during the menstrual cycle in women, there are multiple cohorts or waves of antral follicle recruitment (35 - 39). A wave of follicular development is defined as a group of antral follicles that progresses in size and hormonal attributes during the menstrual cycle; follicles in each wave are of similar, but not identical, diameters, and display differing levels of potential biomarker molecules (36, 37, 40 - 43). The development of primordial follicles from the resting to the growing state has not been completely elucidated but can be generally considered as the resultant of factors (endocrine, paracrine, and autocrine) that prompt follicular cell proliferation, growth, and differentiation and those promoting apoptosis (36, 37). Pre-antral follicles (~ 0.1 - 0.2 mm) develop independently of LH and follicle stimulating hormone (FSH). At a diameter of ~ 0.2 - 0.4 mm, a fluid-filled cavity (called an antrum) begins to form and a follicular cohort responsive to FSH grows that can be visualized by ultrasonography. This follicular process continues and leads to the selection of a ‘dominant follicle’ (DF), a follicle of - 10 mm diameter, that grows independently from FSH, unlike the subordinate follicles of the cohort which become atretic. The mean follicular growth rate of the cohort is 1.42 ± 0.05(SEM) mm/day prior to the point of selection. After selection, 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. Once a DF is ‘chosen’ during the follicular phase, there are - 7 days until the ovulatory event (36). Thus, assessment of the Fertile Start Day and the 6-day fertile window with suitable biomarkers is dependent upon biological and endocrinological knowledge of the developing follicles and in particular the DF.

[0031] The present model for dominant follicle (DF) selection asserts that there is an FSH threshold above which FSH levels must be maintained in order to stimulate growth and development of the cohort of antral follicles to a mid-point of the follicular phase (38, 44). After the start of menses, the days to reach this approximate mid-point can be variable, and this fact is one of the greatest challenges for any fertility tracking method and device. At the time point where the DF has reached suitable maturation in the follicular phase interval, the FSH level falls below a threshold and the DF progresses until ovulation in approximately seven days. Put another way a DF is established about seven days before ovulation in the context of diminishing FSH levels. The uniqueness of the DF and its role in: influencing levels of pituitary gonadotropins, the length of the follicular phase and the timing of ovulation was shown in experiments where the DF was excised (i.e., follicle-ectomy) at various times prior to ovulation (45, 46).

[0032] 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). FSH is the central regulator of ovarian follicular development, and activins are dimers of 0A and 0B which enhance FSH transcription and pituitary secretion (47 - 55). The present paradigm is that inhibins are competitive antagonists to activin action at the level of their cognate pituitary receptors, which thereby reduce FSH transcription (47 - 55). Inhibin A suppresses FSH levels by way of a ternary complex with activin type II receptor and betaglycan, whereas inhibin B has this capability, and in addition, binds to a gonadotrope-restricted transmembrane protein, TGFBR3L (47). Importantly it is thought that inhibin B rather than inhibin A has the primary role in regulating FSH and is instrumental in dominant follicle DF selection (44, 56 - 62). However, the mechanistic details of how FSH and inhibin B levels interact to process folliculogenesis and selection of the DF have not been elucidated.

[0033] Multiple studies have published the mean day-specific serum inhibin B and inhibin A levels indexed to the LH peak during the ovulatory cycle; in these studies, the presumptive day of ovulation, Day 0, would be the day after the LH peak (42, 63 - 69). 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). In contrast, the mean day-specific serum inhibin A profile is characterized by: a) low levels in the early follicular phase, b) a rise beginning at the time of DF selection (~ Day -7), and c) high levels throughout the early and midpostovulatory phase (42, 63 - 69). What is problematic here for fertility tracking with inhibin levels is that only the mean day-specific profiles for inhibin B and inhibin A levels are known, and a woman’s day-specific hormone levels and even the time-function can vary widely from the mean. Another technical issue is that inhibins are not detectable in urine and therefore fertility tracking based upon inhibins requires blood measurements (70).

[0034] Indeed, the state of present knowledge lacks the information necessary to use inhibins as biomarkers for fertility tracking technology. It is anticipated that the amount of 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.

[0035] In summary, critical to a sensitive and accurate use of inhibins for time-of-cycle mapping and a POC device is knowledge of inhibin levels as a function of time for individual cycles and these data are unknown.

[0036] The present inventors recognized that, in order to accurately know the time-of-cycle, ‘follicular metrics’ are necessary, especially to signal the day of selection of the dominant follicle and the Fertile Start Day. Follicle stimulating hormone (FSH) is the crucial pituitary hormone that controls early follicular development to the selection of the dominant follicle (DF). Inhibins are the follicular proteins that signal to the pituitary and other local regulatory ovarian cells (i.e., hormone, autocrine, and paracrine effects) the state of follicle development. Inhibins are multifunctional molecules involved in not only the control of pituitary FSH secretion vis-a-vis negative feedback but also paracrine regulation of ovarian and testicular function. 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.

[0037] Profound changes occur in the follicles in terms of hormonal microenvironment at follicular diameters of 8 - 11mm corresponding to follicular selection (40 - 44). 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. Another recent study concurred with these findings in a different way; Lawrenz et al., (80), showed that 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.

[0038] There are multiple studies where serum day-specific inhibin A and inhibin B levels were measured during the ovulatory cycle using different antibodies and indexed to the day of putative ovulation mainly by LH peak (42, 63 - 69). They are all in agreement and reveal a reproducible signature. For the day-specific inhibin A curve: very low inhibin A levels occur in the early follicular phase, an increase starts approximately 5 days before ovulation and always before the LH surge, and high levels continue through the mid-luteal phase. For the day-specific inhibin B curve: 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).

[0039] However, it is clear from these published data, that because the absolute levels of inhibin A and inhibin B show such large ranges of values, the absolute levels - as is the case for urinary E3G and PDG - are not suitable as reliable indicators for time-of-cycle. These large ranges of A and B levels are one reason why there is no prior teaching of the use of inhibins to assess the time- of-cycle for conception planning for the ovulatory female.

[0040] There are other reasons why the prior art and science say virtually nothing about inhibins as a tool for such applications, either through a clinical lab or POC device. The first, and apparently only, reference regarding 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). Sheth, et al., suggested this “inhibin-like” protein could serve as a better signal for the fertile period prior to ovulation and noted ‘the use of E3G as a marker for identifying the fertile period has been debated’ (82). However, the “inhibin-like” molecule was never identified, and as such there are, to date, no reports of the use of inhibins as biomarkers for predicting or calculating the onset of the fertile phase or ovulation or time-of-cycle in general. Importantly, the result of Sheth, et al. has never been repeated, and furthermore, sensitive assays have failed to reliably measure inhibin A or inhibin B in urine (70).

[0041] The inventors recognized that 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.

[0042] As used herein, a “subject” refers to a mammal, whether female or male. In certain embodiments, the subject is a female, and a specific case a female human. In other embodiments, the female is a non-human mammal, such as domesticated animals, domestic pets, rodents, and primates. In certain examples, the mammals are selected from cats, dogs, equines, bovines/cattle, pigs, goats, buffalo, sheep, goats, camels, or deer.

[0043] As used herein, 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. In one example, the substance measured is inhibin A and inhibin B and FSH. In another aspect, subsets of these levels: 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.

[0044] As used herein, the term “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). 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. The term “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. However, 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 (BIAcore™), scintillation proximity assays, ORIGEN immunoassay (IGEN), fluorescence quenching, and/or fluorescence transfer. Binding affinity may also be screened using a suitable bioassay.

[0045] 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.

[0046] 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.

[0047] A control sample or reference sample can contain a known amount of the biomarker to be assessed. In some embodiments, 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.

[0048] Thus, 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 Fertility™ (https://modemfertility.com/; ZRT labs™ (www.zrtlab.com)(88, 89). However, these developments are not POC and require mail-in of blood spots on filters to commercial laboratories. [0049] 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). [0050] 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. The 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. However, 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. [0051] 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).

[0052] ACE2, the receptor for entry into target cells by SARS-CoV-2/COVID-19, is found on Sertoli and germ cells (96). There is evidence to suggest that COVID-19 in the acute infection stage can have deleterious effects on male gonadal function (97-103). In a hamster model, 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). In addition, 86% of these CO VID men were not hypoxic and did not have severe symptomatology, radiological findings, or require intervention.

[0053] There is a paucity of data regarding the long-term effects following COVID-19 infection on male gonadal function, specifically fertility. The best study on chronic effects of COVID-19 and male fertility is from the Spanish COVID19-ALC Research Group (104). The aim of this study was to determine the prevalence of low serum testosterone and impaired fertility potential (Leydig and Sertoli cell dysfunction, respectively) in COVID-19 male survivors. Sertoli cell dysfunction was defined as inhibin B < 89 pg/ml. For 77 days, 143 patients were evaluated, and low levels of inhibin B were detected in 25 patients (18.1%; 95% CI: 12.5 - 25.3%).

[0054] Thus, the pandemic of COVID-19 can potentially have profound effects on global male fertility. 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. [0055] A significant increase in precocious puberty, rapidly progressive puberty, and precocious menarche has recently been reported (105). In addition, 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.

[0056] 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).

[0057] 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.

[0058] Another application of measuring inhibins is in cancer detection and treatment (115, 116). 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).

[0059] Throughout the description of the invention and its embodiments the term ‘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.

[0060] Generally disclosed is a method interfaceable with apparatus for POC, in clinic and home, for assessment of the time-of-cycle for the ovulatory woman and for fertility status in general for adult males and females, and for adolescents approaching or demonstrating puberty, via measurement of A and B in serum, plasma, whole blood, and fingerstick blood or A and B and FSH in serum, plasma, whole blood, and fingerstick blood or a subset of such hormones in serum, plasma, whole blood, and fingerstick blood.

[0061] According to a preferred embodiment of the disclosed method, 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.

[0062] Application of the following mathematical transformations, formulations, or algorithms:

• (B/A) with application of various identifying sequences for phase or point of cycle

• Aalgo

• (AZFSH)algo

• Slope (B/A)/FSH

• Slope A

• AUC(B/A) and/or its first derivative, logio(abs(B-A/A)), logioB/logioA and/or alternative formulations, computations, or algorithms to determine the time-of-cycle known as: o Infertile preovulatory phase days, o Fertile Start Day, o Fertile interval or fertile window days, o Days or Day of high or maximum fertility, o Days of peri-ovulatory interval, o Day of ovulation/follicular rupture, o Day of transition to the luteal phase. o Infertile luteal phase days o Days near or at the end of the luteal phase prior to the start of the next cycle.

[0063] The significance, relevance, importance, usefulness, and application of these days for avoiding or enhancing conception during an ovulatory cycle are familiar to those with knowledge of science and art of the field. [0064] According to one method, 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(B-A/A)), loglOB/loglOA and/or alternative formulations, computations, or algorithms; and interpretation corresponding to male or female or adolescent age.

[0065] 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.

[0066] 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. These markers of the ovulatory cycle are well established and known to those familiar with the science and art of the fertility field.

[0067] 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.

[0068] Another embodiment of the disclosed methodology is that (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.

[0069] FIG. 1 shows tracking of the ovulatory cycle with urinary hormones, E3G and PDG, with the MIRA™ 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. [0070] 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. These day-specific data do not exist in the literature and were analyzed with functional data analysis (75) rendering a novel finding - an interpretable FSH-B feed-back mechanism, ‘FSH<->B’, the curve of which changes pre- and post-DF appearance at approximately Day -8 to Day -7. This novel result from special analysis of these data is a key point of this disclosure leading to the development of the herein mathematical transformations, formulations, algorithms to predict especially the timing of the Fertile Start Day and the fertile window ending with ovulation.

[0071] 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). However, inhibin A does not exhibit such an oscillatory behavior. These novel results were revealed by analysis of the SI, S2, S3, S4 individual cycles with day-specific B and A levels. The FSH, B, A functions of time-of- cycle herein disclosed are used for prediction of time-of-cycle.

[0072] 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. This information is thus also used to identify the day of ovulation. 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.

[0073] FIG. 5 shows plots of the results from FIG. 3. Here, 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. The Fertile Start Day signals, values of Aalgo and (A/FSH)algo >2.0, all fall within Day -7 and Day -5. By Day -7 all B/A values are decreasing until Day -2, and in one case, Day -1; this identifying B/A string reveals the fertile window and the peri-ovulatory events (bottom panel). B/A is <1 during the luteal phase after Day 0 or Day +1 and rises prior to the next cycle. These results are computed from the individual cycles SI, S2, S3, S4, and for the 10 averaged Seh cycles and the 10 averaged Gilf cycles. The disclosed method is used in a POC fertility tracking device to inform on time-of- cycle.

[0074] 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.

[0075] Table 1. B/A pattern in early follicular phase and transition to fertile

[0076] For a further explication of Table 1 : B/A values provided in FIG. 4 (table) 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. Starting on the day after menses, 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. Importantly, 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). 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).

[0077] Table 2. Slope (B/A)/FSH and B/A as state functions for time-of-cycle for cycles S2 and Seh.

[0078] 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. (d) 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, (e) 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.

[0079] 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.

[0080] 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. In addition, it is shown herein the recognition of the change in oscillatory function of FSH related to B during the follicular phase is crucial for algorithmic creation to identify time-of-cycle. The skilled artisan will recognize that other formulations and algorithms that utilize both inhibin A and inhibin B levels together, with or without FSH, including differences in the levels, can be applied to determine the different phases of the ovulation cycle.

[0081] The specific days and rate of changes signaled by the embodiments, i.e., mathematical transformations, computations, formulations, and algorithms such as (B/A) and its functions, Aalgo, (A/FSH)algo, slope (B/A)/FSH, and slope A as disclosed herein, are not absolute and may change depending upon calibration parameters for measurement of A and B.

[0082] It naturally follows for those skilled in the art of fertility assessment, that from the signatures of the disclosed embodiments as formulations and algorithms for normal ovulatory women, that disturbances of ovarian function found in PCOS and other conditions can be identified and diagnosed. Furthermore, the disclosed embodiments herein would be quite useful in signaling the return of the dominant follicle (DF) during breastfeeding. It would also offer diagnostic applications in the identification of primary ovarian insufficiency (POI) and perimenopause and menopause. [0083] The mathematical transformations, formulations, and algorithms, with B/A, etc. and further ones that follow, should not be construed as limiting the disclosed invention in terms of other such equations with A and/or B, and A and/or B, and/or FSH, as variables to predict and diagnosis fertility and infertility.

[0084] A person of skill in the art will readily recognize that a lateral flow assay (LFA) or vertical flow assay (VFA) can be utilized to measure whole blood, serum or plasma samples levels of B, A, and FSH either for a single day, several days or on a daily basis for application of the disclosed invention.

[0085] A person of skill in the art will readily recognize that steps of various above-described methods can be performed by programmed computers. Herein, some embodiments are also intended to cover 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.

[0086] The functions of the various elements shown in the figures, including any functional blocks labeled as "modules", may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with the appropriate software. When provided by a processor, 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. Moreover, explicit use of the term "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. Other hardware, conventional and/or custom, may also be included.

[0087] In summary, 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.

[0088] The present application and its various include as a method and device for POC, clinic and home, identification and diagnosis of ovarian dysfunction such as polycystic ovary syndrome (PCOS) and primary ovarian insufficiency (POI). [0089] It is a further aspect of the present application and various embodiments to enable POC, at home and in the clinic, determinations of male fertility status and identification and diagnosis of gonadal dysfunction which may occur in such conditions as COVID-19.

[0090] It is another aspect of the present application and various embodiments to enable POC clinic evaluation and diagnosis of disorders of puberty during adolescence in males and females.

[0091] It is another aspect of this disclosure to describe the use of 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.

[0092] It is another aspect of this disclosure to describe the use of 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.

[0093] It is another aspect of this disclosure to describe measurement daily, or for a few consecutive days, or throughout an ovulatory cycle 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, and to subsequently process A and B with or without FSH as variables in diverse disclosed formulations to monitor the ovulatory cycle for use to time sexual relations for family planning.

[0094] 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.

[0095] Another aspect of the disclosure is the use of diverse computations, formulations and algorithms to accomplish objectives.

[0096] 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. [0097] A further aspect of the disclosure is the function, (B/A), as a method of quantifying certain metrics for the ovulatory cycle.

[0098] A further aspect of the disclosure is the function, Aalgo, as a method of quantifying certain metrics for the ovulatory cycle.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] It is a further aspect of the disclosure to describe the use of fingerstick blood, serum, plasma, or other bodily fluids to determine A and B with or without FSH concentrations and by analysis of these levels to quantify certain metrics of the ovulatory cycle.

[0105] It is a further aspect of the disclosure that 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.

[0106] A person of skill in the art would readily recognize that steps of various above-described methods can be performed by one or more programmed computers, each having one or more computer processors. Herein, some embodiments are also intended to cover 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.

[0107] The functions of the various elements shown in the figures, including any functional blocks labeled as “modules”, may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with the appropriate software. When provided by a processor, 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. Moreover, explicit use of the term “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. Other hardware, conventional and/or custom, may also be included.

[0108] Other aspects of the disclosure and embodiments described herein will become apparent to those skilled in the art once the preferred embodiments have been shown and described. These objectives are not to be construed as limitations of the applicant’s invention, but are merely aimed to suggest some of the many benefits that may be realized by the methods and apparatus of the present application and with its many embodiments.

[0109] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0110] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0111] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0112] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0113] As used in this specification and claim(s), 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. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, 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. As used herein, 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.

[0114] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “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. Continuing with this example, 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.

[0115] As used herein, 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. In general, but subject to the preceding discussion, 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%.

[0116] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0117] For each of the claims, 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.

[0118] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0119] All of the 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.

REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. 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.

2. The assay of claim 1, further comprising 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.

3. The assay of claim 1, further comprising: 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.

4. The assay of claim 1, wherein an output from the assay is analyzed by application of at least one algorithm selected from the following, where A = inhibin A concentration or level and B = inhibin B concentration or level, and FSH is FSH concentration or level:

(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, loglO(abs(B-A/A)), loglOB/loglOA and/or alternative mathematical transformations, formulations, computations, or algorithms, wherein at least one algorithm quantifies one or more timepoints in the ovulation cycle.

5. The assay of claim 1, further comprising 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.

6. The assay of claim 5, wherein 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.

7. The assay of claim 5, wherein the POC apparatus uses a lateral or vertical flow device with antibodies to inhibin A 0-subunit, inhibin B 0-subunit, a-subunit 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, and optionally the antibodies are medium to high-affinity antibodies.

8. The assay of claim 5, wherein the POC apparatus simultaneously with A and B assays measures with monoclonal or polyclonal antibodies to FSH the serum, plasma, whole or fingerstick blood levels of FSH.

9. The assay of claim 2, wherein 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.

10. The assay of claim 2, wherein 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.

11. The assay of claim 2, wherein the inhibin A, inhibin B, and optionally FSH levels are computed using one or more diverse computational functions, formulations, algorithmic equations, using inhibin A and inhibin B 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.

12. The assay of claim 2, wherein 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.

13. A kit compri sing : 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.

14. The kit of claim 13, comprising 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.

15. The kit of claim 13, wherein an output from the kit is analyzed by application of at least one algorithm selected from the following, where A = inhibin A level or concentration and B = inhibin B level or concentration, and 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, loglO(abs(B-A/A)), loglOB/loglOA and/or alternative mathematical transformations formulations, computations, or algorithms, wherein at least one algorithm quantifies one or more timepoints in the ovulation cycle.

16. The kit of claim 13, wherein 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.

17. The kit of claim 13, wherein 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.

18. The kit of claim 13, wherein the POC apparatus uses a lateral or vertical flow device with antibodies to inhibin A 0-subunit, inhibin B 0-subunit and a-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 but separately on a test substate, strip, or filter in the POC apparatus.

19. The kit of claim 13, wherein the POC apparatus uses a lateral or vertical flow device with antibodies to inhibin A P-subunit, inhibin B 0-subunit and a-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, and optionally the antibodies are medium to high- affinity antibodies.

20. The kit of claim 13, wherein the POC apparatus simultaneously with A and B assays measures with monoclonal or polyclonal antibodies to FSH the serum, plasma, whole or fingerstick blood levels of FSH.

21. The kit of claim 13, wherein 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.

22. The kit of claim 13, wherein 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.

23. The kit of claim 13, wherein the inhibin A, inhibin B, and optionally FSH levels are computed using one or more diverse computational functions, formulations, algorithmic equations, using inhibin A and inhibin B levels, 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.

24. 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.

25. The method of claim 24, further comprising 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, 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, inhibin B, and FSH polypeptides or nucleic acids into a data analyzable form; and optionally providing an output for the analyzed data.

26. The method of claim 24, wherein the output from the assay is analyzed by application of at least one algorithm selected from the following, where A = inhibin A level or concentration and B = inhibin B level or concentration, and 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, loglO(abs(B-A/A)), loglOB/loglOA and/or alternative mathematical transformations formulations, computations, or algorithms, wherein at least one algorithm quantifies a timepoint in the ovulation cycle.

27. The method of claim 24, further comprising 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.

28. The method of claim 24, wherein 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.

29. The method of claim 24, wherein the POC apparatus uses a lateral or vertical flow device with high-affinity inhibin A P-subunit, inhibin B P-subunit, a-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, and optionally the antibodies are medium to high- affinity antibodies.

30. The method of claim 24, wherein 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.

31. The method of claim 24, wherein 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.

32. The method of claim 24, wherein 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.

33. The method of claim 24, wherein the inhibin A, inhibin B, and optionally FSH levels are computed using one or more diverse computational functions, formulations, algorithmic equations, using inhibin A and inhibin B levels, 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.

34. The method of claim 24, wherein the female is a human.

35. The method of claim 24, further comprising 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.

36. A 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.