Methods comprising ubiquitin c-terminal hydrolase l1 (UCHL1) for determining ovarian reserve

Abstract

The present disclosure generally relates to methods utilizing UCHL1 as a biomarker which indicates the quantity and quality of the ovarian reserve from the time of birth and thereafter in subjects having at least one ovary (e.g., female subjects).

Description

[0001] METHODS COMPRISING UBIQUITIN C-TERMINAL HYDROLASE LI (UCHL1) FOR DETERMINING OVARIAN RESERVE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 655,680, filed June 4, 2024, which is hereby incorporated herein by reference in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The present application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML file, created on April 29, 2025, is named 371288-3008WO1 Sequence Listing.xml and is 4,096 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Ovarian reserve has a significant impact on women’s fertility and overall health. For example, at least 1% of women worldwide experience primary ovarian insufficiency (POI) that is caused by a reduction in the formation or maintenance of the primordial follicle pool. In adult women, POI manifests as the early onset of a menopausal state including reduced estrogen levels, increased Follicle Stimulating Hormone (FSH) levels, and infertility.

[0008] Polycystic Ovary' Syndrome (PCOS) affects an estimated 8-13% of reproductive-aged women and up to 70% of affected women remain undiagnosed worldwide. PCOS can cause hormonal imbalances, irregular periods, excess androgen levels and cysts in the ovaries. Irregular periods, usually with a lack of ovulation, can make it difficult to become pregnant and PCOS is a leading cause of infertility. Because women affected with PCOS have higher ovarian reserve, a practical and accurate test for ovarian reserve could help to increase diagnosis rates (and thus treatment rates) for PCOS patients.

[0009] Currently, indirect measures of oocyte abundance include the "‘gold standard” Anti- Mullerian Hormone (AMH) which is produced by ovarian somatic cells, and follicle stimulating hormone (FSH) which is produced by the pituitary gland [Raeissi, et al., Int J Fertil Steril. 2015 Apr Jun;9(l):27-32], While serum AMH levels report the secreted levels from the somatic cells of maturing secondary ovarian follicles, the ovarian reserve is, instead, the stockpile of much earlier primordial follicles that steadily declines from birth until menopause in girls and women. Therefore, AMH can only assess the quantity of mature follicle stages which become abundant after puberty, and there is no reliable way to measure the ovarian reserve in girls from birth to puberty. Further, there is no oocyte-specific serum biomarker of ovarian reserve identified to date.

[0010] New tools are needed for directly monitoring the ovarian reserve in females at any life stage from the time of birth (e.g, in prepubescent females and after reproductive maturity in women), as it would directly reflect oocyte abundance (rather than current markers which provide an indirect measure of ovarian follicle maturation). Further, because the reduction of estrogen in peri-menopausal women significantly precipitates heart, bone, and cognitive decline, as well as overall shorter lifespan, a serum biomarker is needed to monitor ovarian reserve health and abundance, and to prevent the negative extra-ovarian health effects of perimenopause, POI, PCOS, and related conditions. The present invention addresses this unmet need.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, the invention provides a method for determining relative ovarian reserve level in a subject having at least one ovary, the method comprising:

[0013] (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; and

[0014] (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining the relative ovarian reserve level in the subject.

[0015] In various embodiments, the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

[0016] In various embodiments, the measuring comprises use of a UCHL1 concentration calibration curve.

[0017] In various embodiments, the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

[0018] In various embodiments, one or more of the following applies: (i) the UCHL1 concentration is measured directly from the at least one blood sample (z.e., from whole blood);

[0019] (ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or

[0020] (iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma.

[0021] In various embodiments, the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count and / or by measuring follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), and / or estradiol.

[0022] In various embodiments, the reference subjects comprise age-matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

[0023] In various embodiments, the reference subjects comprise age-matched individuals who do not have and / or did not develop a disease or condition related to abnormal ovarian reserve level.

[0024] In various embodiments, the reference subjects comprise age-matched individuals having a disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed a disease or condition related to abnormal ovarian reserve level, grouped at least by type of disease or condition related to abnormal ovarian reserve level.

[0025] In various embodiments, the method further comprises diagnosing the subject with the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects having the disease or condition related to abnormal ovarian reserve level.

[0026] In various embodiments, the method further comprises predicting that the subject is at risk of developing the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects who later developed the disease or condition related to abnormal ovarian reserve level.

[0027] In various embodiments, the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

[0028] In various embodiments, the subject is a human. In various embodiments, the subject ranges in age from about 1 day old to about 51 years old.

[0029] In one aspect, the invention provides a method of preventing or treating at least one symptom associated with a disease or condition related to abnormal ovarian reserve level in a subject having at least one ovary, the method comprising:

[0030] (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration;

[0031] (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining relative ovarian reserve level in the subject; and

[0032] (iii) administenng an effective amount of at least one therapy which prevents or treats the at least one symptom associated with the disease or condition related to abnormal ovarian reserve level when the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level.

[0033] In various embodiments, the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

[0034] In various embodiments, the measuring comprises use of a UCHL1 concentration calibration curve.

[0035] In various embodiments, the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

[0036] In various embodiments, one or more of the following applies:

[0037] (i) the UCHL1 concentration is measured directly from the at least one blood sample (z.e., from whole blood);

[0038] (ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or

[0039] (iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma. In various embodiments, the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count (AFC) and / or by measuring follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), and / or estradiol.

[0040] In various embodiments, the reference subjects comprise age-matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

[0041] In various embodiments, the reference subjects comprise age-matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0042] In various embodiments, the reference subjects further comprise age-matched individuals who do not have and / or did not develop the disease or condition related to abnormal ovarian reserve level.

[0043] In various embodiments, the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration and / or the relative ovarian reserve level in the subject correlates with that of the reference subjects comprising age- matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0044] In various embodiments, the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

[0045] In various embodiments, the at least one therapy comprises a therapy selected from the group consisting of hormone replacement therapy (HRT), a hormone-based contraceptive, an anti-depressant, exercise, metformin, gonadotropins, acne treatment, spironolactone, eflomithine, hair removal, and infertility treatment.

[0046] In various embodiments, the infertility treatment comprises egg retrieval, egg freezing, ovulation induction, intrauterine insemination (IUI), donor egg(s), donor embryo(s), and / or in vitro fertilization (IVF).

[0047] In various embodiments, the subject is a human.

[0048] In various embodiments, the subject ranges in age from about 1 day old to about 51 years old. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0050] FIG. 1 is a chart showing that UCHL1 is detected in the serum of mice (n=4 of 21 day old male and female mice, n=3 of 6 month old female mice) and humans (n=2 adult females, n=4 adult males), with much higher levels observed in females compared to that in age-matched males, indicating a female-specific source of the additional UCHL1.

[0051] FIG. 2 is a chart showing that measurement of UCHL1 in ' / b / 4h-heterozygous and homozygous mutant mice (a mouse model of oocyte-depletion and primary' ovarian insufficiency7(POI); n=l per genotype) demonstrates a significant reduction of serum UCHL1 in heterozygous animals and a loss of serum UCHL1 in homozy gous animals which possess very few oocytes, indicating an oocyte-specific source for UCHL1 in serum.

[0052] FIG. 3 is a chart showing a significant reduction of serum UCHL1 concentration in female conditional UCHL1 knockout (cKO) mice (n=3) compared to female wild-type (WT) mice (n=3), indicating an oocyte-specific source for serum UCHL1. The cKO mice are deficient in UCHL1 expression specifically in oocytes, whereas UCHL1 expression is preserved at normal levels in all other cell types and tissues. As a negative control, female global UCHL1 knockout mice (n=3) have no detectable serum UCHL1.

[0053] FIG. 4 is a chart showing that measurement of UCHL1 in human follicular fluid demonstrates that, whereas the follicular fluid UCHL1 level varies among patients, the UCHL1 concentration is approximately 10-fold higher in follicular fluid compared to that in serum, indicating a local source (z.e., the oocyte) of UCHL1 secretion.

[0054] DETAILED DESCRIPTION

[0055] Ubiquitin C-Terminal Hydrolase LI (UCHL1) is a deubiquitinating enzyme known to be secreted by neurons [Konya, et al., Neurochem Int. 2011 Aug;59(2):251-258], and is reportedly useful as a biomarker for diagnosing traumatic brain injury (WO2018 / 067468 Al). The regulation of protein turnover by UCHL1 is only seen in neurons, spermatogonia, and oocytes. In females, UCHL1 is required for proper folliculogenesis in mice and demonstrates similar expression dynamics in the human fetus [Woodman, et al., Biology of Reproduction 2022 May 3; ioac086; Ozcan, et al., Reprod Sei. 2023 Jun 5],

[0056] The present disclosure relates to the discovery7that, as demonstrated herein in Example 1. UCHL1 is detected in the serum of mouse and human females at much higher levels than age-matched males, that serum UCHL1 is undetectable in a mouse model of POI, and that UCHL1 is detected at very high levels in human follicular fluid, indicating that UCHL1 in this fluid is derived from the oocyte. Thus the present disclosure is directed primarily to methods utilizing UCHL1 as a biomarker (e.g. a serum biomarker) which indicates the quantity and quality of the ovarian reserve from the time of birth and thereafter in subjects having at least one ovary.

[0057] In one aspect, the present disclosure provides a method for determining relative ovarian reserve level in a subject having at least one ovary, the method comprising: (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; and (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining the relative ovarian reserve level in the subject.

[0058] In one aspect, the present disclosure provides a method of preventing or treating at least one symptom associated with a disease or condition related to abnormal ovarian reserve level in a subject having at least one ovary, the method comprising: (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining relative ovarian reserve level in the subject; and (iii) administering an effective amount of at least one therapy which prevents or treats the at least one symptom associated with the disease or condition related to abnormal ovarian reserve level when the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level.

[0059] It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well know n to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by Green and Sambrook, and Harlow et al.. Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0060] Definitions

[0061] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as w ell as other forms, such as “includes” and “included,” is not limiting. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0062] Generally, nomenclature used in connection with cell and tissue culture, molecular biology7, immunology7, microbiology7, genetics and protein and nucleic acid chemistry7and hybridization described herein is w ell-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzy matic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of. analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry7described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0063] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. That the disclosure may be more readily understood, select terms are defined below.

[0064] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0065] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0,1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0066] As used herein, “antibody’' (Ab) is used in the broadest sense and specifically may include any immunoglobulin, whether natural or partly or wholly synthetically produced, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), and antibody fragments. Thus, the term “antibody” as used in any context within this specification is meant to include, but not be limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgGl. IgG2a, IgG2b, IgG3. IgG4, IgM. IgAl, IgA2, IgD, and IgE) and biologically relevant fragment or specific binding member thereof, including but not limited to Fab, F(ab')2, scFv (single chain or related entity ) and (scFv)2. The term “antibody fragments” as used herein may include those antibody fragments obtained using techniques readily known and available to those of ordinary skill in the art. as reviewed herein. Therefore, in addition to the definition for “antibody” presented supra, the term “antibody” may further encompass any polypeptide or protein comprising a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibody fragments include, but are not limited to. Fab, Fab', F(ab')2, and Fv fragments; diabodies, and linear antibodies.

[0067] As used herein, the term "antigen" refers to a molecule to which an antibody can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. Preferably, the target antigen is a protein or fragment thereof or a complex of proteins.

[0068] As used herein, the term “binding” refers to the adherence of molecules to one another, such as, but not limited to, enzy mes to substrates, antibodies to antigens, DNA strands to their complementary’ strands. Binding occurs because the shape and chemical nature of parts of the molecule surfaces are complementary. A common metaphor is the “lock-and-key” used to describe how enzy mes fit around their substrate. As used herein, the term "biological sample" refers to a sample obtained from an individual and used in a diagnostic or monitoring assay. Biological samples encompass, e.g., a clinical sample, cells in culture, cell supernatants, cell lysates, plasma, serum, biological fluid (e.g., urine), and tissue samples. The source of the biological sample may be solid tissue (e.g.. from a fresh, frozen, and / or preserved organ, tissue sample, biopsy, or aspirate), blood or any blood constituents such as serum or plasma, bodily fluids (such as, e.g.. urine, lymph, cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid, semen), or cells from any time in gestation or development of the individual. The biological sample may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, or antibiotics. In certain embodiments, the biological sample is a blood sample. In certain embodiments, the biological sample is whole blood. In certain embodiments, the biological sample is serum. In certain embodiments, the biological sample is plasma.

[0069] As used herein, the terms ' biomarker' ‘ or "‘marker” generally refers to a nucleic acid molecule, clinical indicator, protein, or other analyte that is associated with a disease. In various embodiments, a biomarker is differentially present in a biological sample obtained from a subject having or at risk of developing a disease or disorder (e.g., an disease or disorder related to abnormal ovarian reserve level) relative to a reference. A marker is differentially present if the mean or median level of the biomarker present in the sample is statistically different from the level present in a reference. A reference level may be, for example, the level present in a sample obtained from a healthy control subject or the level obtained from the subject at an earlier timepoint, z.e., prior to treatment. Common tests for statistical significance include, among others, t-test. ANOVA, Kruskal -Wallis, Wilcoxon, Mann- Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest. The differential presence of a marker of the invention in a subj ect sample can be useful in characterizing the subject as having or at risk of developing a disease, for determining the stage or type of a disease (e.g.. acute disease or chronic disease), for determining the prognosis of the subject, for evaluating therapeutic efficacy, or for selecting a treatment regimen.

[0070] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like have the meaning ascribed to them in U.S. Patent law and mean “includes,” “including,” and the like; “consisting essentially of?or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0071] As used herein, the term '‘disease” refers to a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. A “disease subtype” is a state of health of an animal wherein animals with the disease manifest different clinical features or symptoms.

[0072] A “disorder” as used herein, is used interchangeably with '‘condition,” and refers to a state of health in an animal, w herein the animal is able to maintain homeostasis, but in w hich the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0073] “Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte in the sample being analyzed. The dynamic range can be the range of linearity of the standard curve.

[0074] As used herein, the term "effective amount” refers to the amount required to reduce or improve at least one symptom of a condition, disease, or disorder relative to an untreated patient or to practice the methods of the invention w ith a successful or enhanced outcome. The effective amount of active compound(s) used to practice the present invention varies depending upon the manner of administration, the age, body weight, and general health of the subject as well as requirements of the methods of the invention.

[0075] The term “elevated” as used herein when applied to a gene, protein or chemical reaction means that the expression, activity or concentration of the gene, protein or reaction is higher compared to an appropriate control.

[0076] The phrase '‘inhibit,” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e , antagonists.

[0077] As used herein, the term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0078] As used herein the term "immobilized" refers to bound directly or indirectly to a surface of, e.g., a device, including attachment by covalent binding or noncovalent binding (e.g., hydrogen bonding, ionic interactions, van der Waals forces, or hydrophobic interactions).

[0079] As used herein “instructional material” includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the device in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the device of the invention or be shipped together with a container that contains the device. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the device cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

[0080] “Label” and “detectable label” as used herein refer to a moiety' attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include signalproducing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g.. acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling. Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as3H,14C,32P,33P,33S,90Y, "Tc,mIn,123I,1311,177Lu,166HO, and153Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein ( e.g., 5-fluorescein. 6-carboxy- fluorescein, 3’6-carboxy-fluorescein, 5(6)-carboxy-fluorescein, 6-hexachloro-fluorescein, 6- tetrachloro-fluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon.

[0081] As used herein the term ‘'level” is intended to refer to amount of a unit of a compound being measured, for example a protein. It is also intended to encompass “concentration” expressed as amount per volume or weight per volume and any other depiction of concentration as known in the art.

[0082] “Linking sequence” or “linking peptide sequence” refers to a natural or artificial polypeptide sequence that is connected to one or more polypeptide sequences of interest (e.g., full-length, fragments, etc.). The term “connected” refers to the joining of the linking sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. Linking sequences can have a length of from about 4 to about 50 amino acids. Preferably, the length of the linking sequence is from about 6 to about 30 amino acids. Natural linking sequences can be modified by amino acid substitutions, additions, or deletions to create artificial linking sequences. Linking sequences can be used for many purposes, including in recombinant Fabs. Exemplary linking sequences include, but are not limited to: (i) Histidine (His) tags, such as a 6X His tag (aka His tag), are useful as linking sequences to facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) Enterokinase cleavage sites, like 6X His tags, are used in the isolation and purification of proteins and antibodies of interest. Often, enterokinase cleavage sites are used together with 6X His tags in the isolation and purification of proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. (iii) Miscellaneous sequences can be used to link or connect the light and / or heavy chain variable regions of single chain variable region fragments. Examples of other linking sequences can be found in Bird et al.. Science 242:423-426 (1988); Huston et al., PNAS USA 85: 5879-5883 (1988); and McCafferty et al., Nature 348: 552-554 (1990). Linking sequences also can be modified for additional functions, such as attachment of detectable labels or drugs or attachment to solid supports. In the context of the present disclosure, the monoclonal antibody, for example, can contain a linking sequence, such as a His tag, an enterokinase cleavage site, or both.

[0083] As used herein, the terms “prevent,” “preventing,” “prevention,” and the like refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

[0084] As used herein the terms “purified” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0085] “Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity7panels. A “calibrator” or “standard” typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a predetermined positive / negative cutoff, reference level or control level (e.g., “low,” “medium,” or “high” levels), can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of calibrators )) can be used in conjunction to comprise a “sensitivity7panel.”

[0086] As used herein the term “reference” is meant a standard or control value often used as a basis for comparison. The terms “reference” and “control” are used interchangeably herein. A “reference” or “control” means a value or level measured in a sample obtained from a suitable subject (a “reference subject”). A “reference” or “control” also means a value or level that is a composite of values or levels obtained from a group of reference subjects as described herein, or as generated by an algorithm using multiple measured values or levels.

[0087] A “reference level” as used herein refers to an assay cutoff value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.) This disclosure provides exemplary7reference levels. However, it is well-known that reference levels may vary depending on the nature of the immunoassay (e.g., antibodies employed, reaction conditions, sample purity7, etc.) and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific reference levels for those other immunoassays based on the description provided by this disclosure. Whereas the precise value of the reference level may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays.

[0088] "Risk assessment," "risk classification," "risk identification," or "risk stratification" of subjects (e.g., patients) as used herein refers to the evaluation of factors including biomarkers, to predict the risk of occurrence of future events including disease onset or disease progression, so that treatment decisions regarding the subject may be made on a more informed basis.

[0089] "Sample," "test sample," "specimen," "sample from a subject," and "patient sample" as used herein may be used interchangeable and may be a sample of blood such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. As used herein the term “blood sample” refers to whole blood or blood that has been pre-treated and / or processed to obtain serum or plasma.

[0090] "Sensitivity" of an assay as used herein refers to the proportion of subjects for whom the outcome is positive that are correctly identified as positive.

[0091] "Specificity" of an assay as used herein refers to the proportion of subjects for whom the outcome is negative that are correctly identified as negative.

[0092] As used herein the term “signal” refers to light intensity (e.g., light generated by fluorescence, bioluminescence, or phosphorescence), ionizing radiation, particle emission, magnetism, staining, or a product of a reaction involving an enzyme. Diffraction, absorbance, polarization, reflection, deflection, increases, decreases, or amplification of a signal may be indicative of an event (e.g., binding of a biomarker or biomarker complex to an antibody immobilized on the surface of a diffraction-based device).

[0093] As used herein the term "single molecule detection" refers to the detection and / or measurement of a single molecule of an analyte in a test sample at very low levels of concentration (such as pg / mL or femtogram / mL levels). A number of different single molecule analyzers or devices are known in the art and include nanopore and nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in International Patent Publication No. WO 2016 / 161400. which is hereby incorporated by reference in its entirety.

[0094] As used herein the term ‘'specifically binds” refers to an antibody or fragment thereof that recognizes and binds an antigen, but that does not substantially recognize or bind to other molecules in a biological sample. Specific recognition of an antigen by an antibody may be assayed by using, e.g.. light diffraction devices with an immobilized capture surface or using standard techniques known to one of skill in the art, such as immunoprecipitation, Western blotting, and ELISA.

[0095] "Solid phase" or "solid support" as used interchangeably herein, refers to any material that can be used to attach and / or attract and immobilize (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. The solid phase can be chosen for its intrinsic ability to attract and immobilize a capture agent. Alternatively, the solid phase can have affixed thereto a linking agent that has the ability to attract and immobilize the (1) capture agent or capture specific binding partner, or (2) detection agent or detection specific binding partner. For example, the linking agent can include a charged substance that is oppositely charged with respect to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself or to a charged substance conjugated to the (1) capture agent or capture specific binding partner or (2) detection agent or detection specific binding partner. In general, the linking agent can be any binding partner (preferably specific) that is immobilized on (attached to) the solid phase and that has the ability to immobilize the (1) capture agent or capture specific binding partner, or (2) detection agent or detection specific binding partner through a binding reaction. The linking agent enables the indirect binding of the capture agent to a solid phase material before the performance of the assay or during the performance of the assay. For examples, the solid phase can be plastic, derivatized plastic, magnetic, or non-magnetic metal, glass or silicon, including, for example, a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.

[0096] As used herein the terms “subject,” “individual,” “patient,” and the like are used interchangeably herein, and refer to any vertebrate, including, but not limited to, a mammal (e.g., a human, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig. cat, dog, rat, mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.)) or cells thereof, whether in vitro or in situ, amenable to the methods described herein. In some embodiments, the term “subject” refers to a mammal, including a human or non-human mammal, having at least one ovary, e.g., a female. The term ■‘subject” may refer to a human or other animal which is the object of treatment, observation, or experiment (e.g., a patient).

[0097] As used herein the terms “therapeutic” and “therapy” refers to a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0098] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or improving a disease or condition and / or symptom associated therewith. It will be appreciated that, although not precluded, treating a disease or condition does not require that the disease, condition or symptoms associated therewith be completely ameliorated or eliminated. The term also refers to reducing the severity of a disease or condition or symptoms associated with such disease or condition prior to affliction with the disease or condition. Such prevention or reduction of the severity7of a disease or condition prior to affliction refers to administration of a pharmaceutical composition to a subject that is not at the time of administration afflicted with the disease. “Preventing” and “prevent” also refer to preventing the recurrence of a disease or condition or of one or more symptoms associated with such disease or condition.

[0099] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0100] The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0101] Methods

[0102] In one aspect, the present disclosure provides a method for determining relative ovarian reserve level in a subject having at least one ovary, the method comprising: (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; and (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby- determining the relative ovarian reserve level in the subject.

[0103] In one aspect, the present disclosure provides a method of preventing or treating at least one symptom associated with a disease or condition related to abnormal ovarian reserve level in a subject having at least one ovary , the method comprising: (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining relative ovarian reserve level in the subject; and (iii) administering an effective amount of at least one therapy which prevents or treats the at least one symptom associated with the disease or condition related to abnormal ovarian reserve level when the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level.

[0104] In certain embodiments, the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

[0105] In certain embodiments, the measuring comprises use of a UCHL1 concentration calibration curve.

[0106] In certain embodiments, the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof. Suitable immunoassays for measuring or assessing UCHL1 at low levels in a biological sample over a wide range of concentrations are known in the art (see, e.g., WO 2018 / 067468 which is incorporated by reference in its entirety- herein). Such immunoassays have a wide dynamic range, e.g., a dynamic range of about five logs (e.g., 5 pg / mL - 50,000 pg / mL).

[0107] In certain embodiments, the methods described herein use an isolated antibody that specifically binds to ubiquitin carboxy-terminal hydrolase LI ("UCHL1") (or fragments thereof), referred to as an “anti-UCHLl antibody" or "UCHL1 antibody." The UCHL1 antibodies can be used to assess the UCHL1 status as a measure of relative ovarian reserve level, detect the presence of UCHL1 in a biological sample, quantify the amount (e.g., concentration) of UCHL1 present in a biological sample, or detect the presence of and quantify the amount of UCHL1 in a biological sample (e.g., a blood sample, a serum sample, and / or a plasma sample). In certain embodiments, the UCHL1 concentration is measured directly from the at least one blood sample (z.e. , from whole blood). In certain embodiments, the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum. In certain embodiments, the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma. In certain embodiments, the UCHL1 concentration is measured from at least one serum sample. In certain embodiments, the UCHL1 concentration is measured from at least one plasma sample.

[0108] In certain embodiments, the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count and / or by measuring follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), and / or estradiol.

[0109] In certain embodiments, the reference subjects comprise age-matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

[0110] In certain embodiments, the reference subjects comprise age-matched individuals who do not have and / or did not develop a disease or condition related to abnormal ovarian reserve level.

[0111] In certain embodiments, the reference subjects comprise age-matched individuals having a disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed a disease or condition related to abnormal ovarian reserve level, grouped at least by ty pe of disease or condition related to abnormal ovarian reserve level.

[0112] In certain embodiments, the method further comprises diagnosing the subject with the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects having the disease or condition related to abnormal ovarian reserve level.

[0113] In certain embodiments, the method further comprises predicting that the subject is at risk of developing the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects who later developed the disease or condition related to abnormal ovarian reserve level.

[0114] In certain embodiments, the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause. In certain embodiments, the subject is a human.

[0115] In certain embodiments, the subject is a human and ranges in age from about 1 day old to about 51 years old.

[0116] In other embodiments, the subject may be a non-human mammal selected from cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog. rat. mouse, and a non-human primate.

[0117] In certain embodiments, the reference subjects comprise age-matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0118] In certain embodiments, the reference subjects further comprise age-matched individuals who do not have and / or did not develop the disease or condition related to abnormal ovarian reserve level.

[0119] In certain embodiments, the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration and / or the relative ovarian reserve level in the subject correlates with that of the reference subjects comprising age- matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0120] In certain embodiments, the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

[0121] In certain embodiments, the at least one therapy comprises a therapy selected from the group consisting of hormone replacement therapy (HRT), a hormone-based contraceptive, an anti-depressant, exercise, metformin, gonadotropins, acne treatment, spironolactone, eflomithine, hair removal, and infertility treatment.

[0122] In certain embodiments, the infertility treatment comprises egg retrieval, egg freezing, ovulation induction, intrauterine insemination (IUI), donor egg(s), donor embryo(s), and / or in vitro fertilization (IVF).

[0123] Although it has been previously reported (WO 2018 / 067468) that the plasma UCHL1 concentration from 50 apparently healthy human donors ranged from approximately 5 pg / mL to approximately 175 pg / mL, the report did not specify the age or gender of the donors or make any comments, hypotheses, or conclusions regarding any differences in plasma UCHL1 concentration related to age and / or gender of the healthy donors. In contrast, as demonstrated herein, the average serum UCHL1 concentration from healthy adult human females, each having two ovaries, is about 50 pg / mL, ranging from about 20 pg / mL to about 80 pg / mL, whereas UCHL1 concentration was significantly lower (i.e., UCHL1 was not detected) in serum of healthy adult human males.

[0124] In particular, with respect to a predetermined level as employed for monitoring disease progression and / or treatment or for determining the risk of a subject of developing a disease or condition related to abnormal ovarian reserve level, the amount or concentration of UCH-L1 or UCH-L1 fragment may be "unchanged," "favorable" ( or "favorably altered"), or "unfavorable" (or "unfavorably altered"). "Elevated" or "increased" refers to an amount or a concentration in a test sample that is higher or greater than a typical or normal level or range (e.g., predetermined level), or is higher or greater than another reference level or range ( e.g., earlier or baseline sample). The term "lowered" or "reduced" refers to an amount or a concentration in a test sample that is lower or less than a typical or normal level or range (e.g.. predetermined level), or is lower or less than another reference level or range ( e.g., earlier or baseline sample). The term "altered" refers to an amount or a concentration in a sample that is altered (increased or decreased) over a ty pical or normal level or range (e.g., predetermined level), or over another reference level or range (e.g., earlier or baseline sample).

[0125] Ubiquitin carboxy -terminal hydrolase LI ("UCHL1"), which is also known as "ubiquitin C-terminal hydrolase," is a deubiquitinating enzy me. UCHL1 is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCHL1 is highly specific to neurons and to cells of the diffuse neuroendocrine system and their tumors. It is abundantly present in all neurons (accounts for 1-2% of total brain protein), and is expressed specifically' in neurons and testis / ovary. The catalytic triad of human UCHL1 contains a cysteine at position 90, an aspartate at position 176, and a histidine at position 161 that are responsible for its hydrolase activity.

[0126] In some embodiments, human UCHL1 has the following amino acid sequence:

[0127] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLL LFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGF EDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFIL FNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALC KAA (SEQ ID NO: 1). In some embodiments, the human UCHL1 is a fragment or variant of SEQ ID NO: 1. The fragment of UCHL1 may be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 175 amino acids, or between 175 and 225 amino acids in length. The fragment may comprise a contiguous number of amino acids from SEQ ID NO: 1.

[0128] Other names (aliases) for Ubiquitin C-terminal hydrolase LI (UCHL1) include EC 3.4.19.12; HEL-117; HEL-S-53; NDGOA; Neuron cytoplasmic protein 9.5; PARK5; PGP 9.5; PGP9.5; PGP9.5Uch-Ll; PGP95; SPG79, ubiquitin carboxyl-terminal esterase LI (ubiquitin thiolesterase); ubiquitin carboxy -terminal hydrolase isozyme LI; ubiquitin C- terminal hydrolase; Ubiquitin thioesterase LI; UCHL1; UCH-L1; Uch-Ll; and UCHL-1.

[0129] In certain embodiments, the analyte of interest, and / or peptides of fragments thereof ( e.g., UCH-L 1, and / or peptides or fragments thereof, i.e., UCH-L1 fragments), may be analyzed using UCH-L1 antibodies in an immunoassay. The presence or amount of analyte (e.g.. UCH-L1) can be determined using antibodies and detecting specific binding to the analyte (e.g., UCH-L1). For example, the antibody, or antibody fragment thereof, may specifically bind to the analyte (e.g., UCH-L1). If desired, one or more of the antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies are available from companies such as R&D Systems. Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0130] It should be understood that the method, the compositions and the kits that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, protein purification methods, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventor regards as his invention.

[0131] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel. 1987); “PCR: The Polymerase Chain Reaction”, (Mullis. 1994); "Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0132] It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0133] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0134] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0135] The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

[0136] EXAMPLES

[0137] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

[0138] Example 1

[0139] Materials and Methods

[0140] Animals

[0141] C57bl / 6 (wild-type) mice were obtained from the Jackson Laboratory' (strain # 000664). 7b / 4 / ?-heterozygous and homozy gous mutant animals (a mouse model of postnatal oocyte loss) were gifted by Dr. Richard Freiman (Lovasco, et al.. Biology of Reproduction. 2010 Jan;81(2):23-34). Global UCHL1 knockout mice were obtained from the Jackson Laboratory' (strain # 024355), while conditional knockout mice were generated at Brown University. To create these conditional animals, the Brown Transgenic and Genome Editing Facility generated a floxed Uchll allele, and these animals were then bred to GDF9-iCre animals obtained from Jackon Laboratory (strain # 01 1062) to create oocyte-specific knockouts. All animal protocols were reviewed and approved by Brown University- Institutional Animal Care and Use Committee and w ere performed in accordance w ith the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0142] Human Subjects

[0143] Reproductive-age human subjects were enrolled in a prospective study to detect UCHL1 in serum or follicular fluid. All experiments were performed in accordance with the relevant guidelines and regulations of the Women and Infants Hospital Institutional Review Board committee. Serum Analysis

[0144] For UCHL1 analysis, whole blood was collected from wild-type mice at 21 days and at 6 months of age, or from POI and UCHL1 mouse models at 3 months of age by cardiac puncture. Whole blood from adult human subjects was obtained by venous draw. Blood was collected into serum separator tubes, allowed to clot for 30 min, then spun at 3000 g for 15 min at 4°C. The supernatant was collected as serum for analysis. Serum was analyzed via enzyme-linked immunosorbent assay (ELISA) via manufacturer’s instructions to determine UCHL1 concentration (LSBio #LS-F33472-1 for mouse serum; R&D Systems #DY6007-05 for human serum). Standards were used to generate a standard curve and Graphpad Prism was used to interpolate the standard curve and determine UCHL1 concentrations from the given specimens.

[0145] Results

[0146] In addition to producing oocytes, the ovary’ produces steroid hormones such as estrogen and progesterone that are required for promoting healthy egg production and successful pregnancy, and which are also required for extra-ovarian health including heart, bone and brain health. Interestingly, these critical functions are both dependent on functional units of the ovary' called ovarian follicles. Ovarian follicles each contain a single oocyte that matures into the egg cell surrounded by somatic granulosa cells which nurture the oocyte and also produce estrogen and other secreted factors into the bloodstream for systemic delivery’ and use outside the ovary.

[0147] Strikingly, all the ovarian follicles that survive and mature in the adult ovary were first bom in the fetal ovary as a population of primordial follicles that establish the finite ovarian reserve. These fetal oocytes complete the first few steps of meiosis I and then remain arrested in meiotic prophase I until they prepare to be ovulated from the post-pubertal adult ovary’. The successful arrest of oocytes in meiosis I and envelopment in somatic cells is seen in an average of 1-2 million oocytes in human newborn girls. This initial endowment of follicles is known as the ovarian reserve and determines a large part of a woman’s reproductive potential and, subsequently, her reproductive lifespan. These adult oocytes remain quiescent until they are either reawakened for ovulation or undergo atresia (a specialized form of cell death). Ovarian follicles are therefore a non-renewing resource in the adult ovary that are matured from the finite pool of primordial follicles, and menopause marks the ultimate depletion of the primordial follicle pool and reproductive senescence in women. The experiments described herein were designed to evaluate whether UCHL1 is a useful serum biomarker of ovarian reserve. First, serum UCHL1 concentration was measured via ELISA assay for adult female and adult male human subjects, and for 21 day female mice, 21 day male mice, and 6 month female mice. The results show that UCHL1 is detectable in the serum of mouse and humans, with much higher levels observed in females compared to that in age-matched males, indicating a female-specific source for the additional UCHL1 (FIG. 1).

[0148] Next, serum UCHL1 concentration was measured via ELISA assay for female Taf4b- heterozygous and homozygous mutant mice (a mouse model of oocyte-depletion and primary ovarian insufficiency (POI)) compared to that in female wild-type control mice. The results show a significant reduction of serum UCHL1 in heterozygous animals and a loss of serum UCHL1 in homozygous animals which lack oocytes, indicating an oocyte-specific source for UCHL1 in serum (FIG. 2).

[0149] To further explore the source of serum UCHL1, concentration of serum UCHL1 was measured via ELISA assay for female mice of two genetic mouse models that perturb UCHL1 expression. The conditional UCHL1 knockout (cKO) mice are deficient in UCHL1 expression specifically in oocytes, whereas UCHL1 expression is preserved at normal levels in all other cell types and tissues. The global UCHL1 knockout (KO) mice lack UCHL1 expression in all cells and tissues. A dramatic reduction in circulating UCHL1 was observed in the UCHL1 cKO mice compared to the level in female wild-type (WT) animals, demonstrating that much of the serum UCHL1 observed in wild- type animals is derived from an oocyte-specific source (FIG. 3). As expected, the control UCHL1 KO mice have no detectable UCHL1 in the serum.

[0150] Next, UCHL1 concentration in follicular fluid of female human subjects was measured via ELISA assay. The results show that, whereas the follicular fluid UCHL1 level varies among patients, the UCHL1 concentration is approximately 10-fold higher in follicular fluid compared to that in serum, indicating a local source (z.e., the oocyte) of the UCHL1 secretion in the follicle (FIG. 4).

[0151] Collectively, the results disclosed herein indicate that UCHL1 is a novel, oocyte- secreted biomarker which can be used to determine and monitor the quantity and quality of the ovarian reserve from the time of birth and thereafter. It is contemplated herein that the UCHL1 biomarker is useful for determining which female babies and children are beginning life with lower-than-normal ovarian reserv e abundance, and who may suffer from diminished ovarian reserve and its consequences (including early menopause, increased infertility, cardiovascular risks, and osteopenia) later in life. With this knowledge in hand, these individuals can employ preventative care earlier in life to mitigate the premature onset of cardiovascular disease, osteoporosis, and cognitive decline associated with this ovarian dysfunction. It is further contemplated herein that the UCHL1 biomarker is also a powerful tool for monitoring the ovarian reserve after reproductive maturity, as it will directly reflect oocyte abundance in contrast to current methods that utilize an indirect measure of ovarian follicle maturation.

[0152] Enumerated embodiments:

[0153] The following exemplary’ embodiments are provided, the numbering of which is not to be construed as designating levels.

[0154] Embodiment 1 : A method for determining relative ovarian reserve level in a subject having at least one ovary, the method comprising:

[0155] (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; and

[0156] (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining the relative ovarian reserve level in the subject.

[0157] Embodiment 2: The method of embodiment 1, wherein the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

[0158] Embodiment 3 : The method of embodiment 1 or embodiment 2, wherein the measuring comprises use of a UCHLl concentration calibration curve.

[0159] Embodiment 4: The method of any one of embodiments 1-3, wherein the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

[0160] Embodiment 5: The method of any one of embodiments 1 -4, wherein: (i) the UCHL1 concentration is measured directly from the at least one blood sample ( / .e., from whole blood);

[0161] (ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or

[0162] (iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma.

[0163] Embodiment 6: The method of any one of embodiments 1-5, wherein the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count and / or by measuring follicle stimulating hormone (FSH), anti- mullerian hormone (AMH), and / or estradiol.

[0164] Embodiment 7: The method of any one of embodiments 1-6, wherein the reference subjects comprise age-matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

[0165] Embodiment 8: The method of any one of embodiments 1-7, wherein the reference subjects comprise age-matched individuals who do not have and / or did not develop a disease or condition related to abnormal ovarian reserve level.

[0166] Embodiment 9: The method of any one of embodiments 1-8, wherein the reference subjects comprise age-matched individuals having a disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed a disease or condition related to abnormal ovarian reserve level, grouped at least by type of disease or condition related to abnormal ovarian reserve level.

[0167] Embodiment 10: The method of embodiment 9, further comprising diagnosing the subject with the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects having the disease or condition related to abnormal ovarian reserve level.

[0168] Embodiment 11: The method of embodiment 9, further comprising predicting that the subject is at risk of developing the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects who later developed the disease or condition related to abnormal ovarian reserve level.

[0169] Embodiment 12: The method of any one of embodiments 9-11, wherein the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

[0170] Embodiment 13: The method of any one of embodiments 1-12, wherein the subject is a human.

[0171] Embodiment 14: The method of embodiment 13, wherein the subject ranges in age from about 1 day old to about 51 years old.

[0172] Embodiment 15: A method of preventing or treating at least one symptom associated with a disease or condition related to abnormal ovarian reserve level in a subject having at least one ovary, the method comprising:

[0173] (i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration;

[0174] (ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining relative ovarian reserve level in the subject; and

[0175] (iii) administering an effective amount of at least one therapy which prevents or treats the at least one symptom associated with the disease or condition related to abnormal ovarian reserve level when the relative ovarian reserv e level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level.

[0176] Embodiment 16: The method of embodiment 15, wherein the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject. Embodiment 17: The method of embodiment 15 or embodiment 16, wherein the measuring comprises use of a UCHLl concentration calibration curve.

[0177] Embodiment 18: The method of any one of embodiments 15-17, wherein the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

[0178] Embodiment 19: The method of any one of embodiments 15-18, wherein:

[0179] (i) the UCHL1 concentration is measured directly from the at least one blood sample (i.e., from whole blood);

[0180] (ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or

[0181] (iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma.

[0182] Embodiment 20: The method of any one of embodiments 15-19, wherein the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count (AFC) and / or by measuring follicle stimulating hormone (FSH), anti-mullerian hormone (AMH). and / or estradiol.

[0183] Embodiment 21: The method of any one of embodiments 15-20, wherein the reference subjects comprise age-matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

[0184] Embodiment 22: The method of any one of embodiments 15-21, wherein the reference subjects comprise age-matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0185] Embodiment 23: The method of embodiment 22, wherein the reference subjects further comprise age-matched individuals who do not have and / or did not develop the disease or condition related to abnormal ovarian reserve level. Embodiment 24: The method of embodiment 22 or embodiment 23, wherein the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration and / or the relative ovarian reserve level in the subject correlates with that of the reference subjects comprising age-matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

[0186] Embodiment 25: The method of any one of embodiments 15-24, wherein the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

[0187] Embodiment 26: The method of any one of embodiments 15-25, wherein the at least one therapy comprises a therapy selected from the group consisting of hormone replacement therapy (HRT), a hormone-based contraceptive, an anti-depressant, exercise, metformin, gonadotropins, acne treatment, spironolactone, eflomithine, hair removal, and infertility treatment.

[0188] Embodiment 27: The method of embodiment 26, wherein the infertility treatment comprises egg retrieval, egg freezing, ovulation induction, intrauterine insemination (IUI), donor egg(s), donor embryo(s), and / or in vitro fertilization (IVF).

[0189] Embodiment 28: The method of any one of embodiments 15-27, wherein the subject is a human.

[0190] Embodiment 29: The method of embodiment 28, wherein the subject ranges in age from about 1 day old to about 51 years old.

[0191] Other Embodiments

[0192] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method for determining relative ovarian reserve level in a subject having at least one ovary, the method comprising:(i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration; and(ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining the relative ovarian reserve level in the subject.

2. The method of claim 1, wherein the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

3. The method of claim 1 or claim 2, wherein the measuring comprises use of a UCHL1 concentration calibration curve.

4. The method of any one of claims 1-3, wherein the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

5. The method of any one of claims 1-4, wherein:(i) the UCHL1 concentration is measured directly from the at least one blood sample ( / .e., from whole blood);(ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or(iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma.

6. The method of any one of claims 1-5, wherein the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count and / or by measuring follicle stimulating hormone (FSH), anti- mullerian hormone (AMH), and / or estradiol.

7. The method of any one of claims 1-6, wherein the reference subjects comprise age- matched individuals having two ovaries if the subject has two ovaries or having only one ovary' if the subject has only one ovary.

8. The method of any one of claims 1-7, wherein the reference subjects comprise age- matched individuals who do not have and / or did not develop a disease or condition related to abnormal ovarian reserve level.

9. The method of any one of claims 1-8, wherein the reference subjects comprise age- matched individuals having a disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed a disease or condition related to abnormal ovarian reserve level, grouped at least by ty pe of disease or condition related to abnormal ovarian reserve level.

10. The method of claim 9, further comprising diagnosing the subject with the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects having the disease or condition related to abnormal ovarian reserve level.

11. The method of claim 9, further comprising predicting that the subject is at risk of developing the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration of the subject correlates with that of the reference subjects who later developed the disease or condition related to abnormal ovarian reserve level.

12. The method of any one of claims 9-11, wherein the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovary syndrome (POS), early menopause, and premature menopause.

13. The method of any one of claims 1-12, wherein the subject is a human.

14. The method of claim 13, wherein the subject ranges in age from about 1 day old to about 51 years old.

15. A method of preventing or treating at least one symptom associated with a disease or condition related to abnormal ovarian reserve level in a subject having at least one ovary', the method comprising:(i) measuring ubiquitin C-Terminal Hydrolase LI (UCHL1) concentration in at least one blood sample obtained from the subject to determine a measured UCHL1 concentration;(ii) comparing the measured UCHL1 concentration to at least one reference UCHL1 concentration, wherein the reference UCHL1 concentration comprises an average measured UCHL1 concentration obtained from blood samples of reference subjects, further wherein the relative ovarian reserve level of the reference subjects is known and / or determined, thereby determining relative ovarian reserve level in the subject; and(iii) administering an effective amount of at least one therapy which prevents or treats the at least one symptom associated with the disease or condition related to abnormal ovarian reserve level when the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level.

16. The method of claim 15, wherein the measuring comprises averaging the UCHL1 concentration from two or more blood samples obtained from the subject.

17. The method of claim 15 or claim 16. wherein the measuring comprises use of a UCHL1 concentration calibration curve.

18. The method of any one of claims 15-17, wherein the measuring comprises an immunoassay comprising an anti-UCHLl antibody or fragment thereof.

19. The method of any one of claims 15-18, wherein:(i) the UCHL1 concentration is measured directly from the at least one blood sample (z.e., from whole blood);(ii) the method further comprises preparing serum from the at least one blood sample and the UCHL1 concentration is measured from the serum; or(iii) the method further comprises preparing plasma from the at least one blood sample and the UCHL1 concentration is measured from the plasma.

20. The method of any one of claims 15-19, wherein the relative ovarian reserve level of the reference subjects is known and / or determined by a vaginal ultrasound measuring antral follicle count (AFC) and / or by measuring follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), and / or estradiol.

21. The method of any one of claims 15-20, wherein the reference subjects comprise age- matched individuals having two ovaries if the subject has two ovaries or having only one ovary if the subject has only one ovary.

22. The method of any one of claims 15-21, wherein the reference subjects comprise age- matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

23. The method of claim 22, wherein the reference subjects further comprise age-matched individuals who do not have and / or did not develop the disease or condition related to abnormal ovarian reserve level.

24. The method of claim 22 or claim 23, wherein the relative ovarian reserve level in the subject indicates that the subject is at risk of developing or already has the disease or condition related to abnormal ovarian reserve level when the measured UCHL1 concentration and / or the relative ovarian reserve level in the subject correlates with that of the reference subjects comprising age-matched individuals having the disease or condition related to abnormal ovarian reserve level, or age matched individuals who later developed the disease or condition related to abnormal ovarian reserve level.

25. The method of any one of claims 15-24, wherein the disease or condition related to abnormal ovarian reserve level is selected from primary ovarian insufficiency (POI), polycystic ovaiy syndrome (POS), early menopause, and premature menopause.

26. The method of any one of claims 15-25, wherein the at least one therapy comprises a therapy selected from the group consisting of hormone replacement therapy (HRT), a hormone-based contraceptive, an anti-depressant, exercise, metformin, gonadotropins, acne treatment, spironolactone, eflomithine, hair removal, and in ferti 1 i ty treatment.

27. The method of claim 26, wherein the infertility treatment comprises egg retrieval, egg freezing, ovulation induction, intrauterine insemination (IUI), donor egg(s), donor embryo(s), and / or in vitro fertilization (IVF).

28. The method of any one of claims 15-27, wherein the subject is a human.

29. The method of claim 28, wherein the subject ranges in age from about 1 day old to about 51 years old.

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