Pulsatile FSH delivery for female fertility enhancement and delaying reproductive aging

Abstract

Provided herein are methods and compositions comprising pulsatile follicle stimulating hormone (FSH). The pulsatile FSH may be used in methods of treating infertility, restoring normal hormonal levels, stimulating secretion of a sex steroid, treating or preventing fatty tissue accumulation, treating or preventing loss of bone density, preventing ovarian inflammation, and / or promoting metabolic health.

Description

[0001] Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0002] PULSATILE FSH DELIVERY FOR FEMALE FERTILITY ENHANCEMENT AND DELAYING REPRODUCTIVE AGING

[0003] FIELD OF THE INVENTION

[0004] The present disclosure relates generally to methods of using pulsatile Follicle-Stimulating Hormone (FSH) therapies to treat infertility and improve reproductive lifespan.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 737,171, filed December 20, 2024, which is incorporated by reference herein in its entirety.

[0007] BACKGROUND

[0008] The two closely related hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are synthesized in pituitary gonadotropes. LH and FSH are heterodimers, and the beta subunit is hormone and receptor specific. Conventional FSH therapies to treat sub- or infertility or in assisted reproductive therapy (ART) protocols typically include multiple single dose daily injections of follicle-stimulating hormone (FSH). However, this often does not provide adequate efficacy. Accordingly, there is a need for novel and improved methods for ART / IVF protocols and therapies for subfertility and / or infertility.

[0009] SUMMARY

[0010] The present disclosure provides compositions and methods of using pulsatile Follicle-Stimulating Hormone (FSH) to treat infertility and improve reproductive lifespan.

[0011] In some aspects, the disclosure provide a method of treating infertility in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

[0012] In some aspects, the disclosure provides a method of improving egg quality, inducing egg growth or maturation, increasing number of eggs, or improving embryo quality, in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the number of eggs is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, or about 8-fold. In some embodiments, the eggs maintain developmental competence. In some embodiments, the developmental competence Attorney Docket No.: CUJ-00525 (CU 2025-135) is measured by percent (%) expanded blastocysts.

[0013] In some aspects, the disclosure provides a method of restoring normal hormonal levels in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

[0014] In some aspects, the disclosure provides a method of stimulating secretion of a sex steroid in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the sex steroid is estradiol, estrogen, or progesterone.

[0015] In some aspects, the disclosure provides a method of treating or preventing fatty tissue accumulation in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

[0016] In some aspects, the disclosure provides a method of treating or preventing loss of bone density in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the subject has osteoporosis. In some embodiments, the rFSH prevents loss of bone mineral density (BMD) or bone mineral content (BMC). In some embodiments, the rFSH increases bone mineral density (BMD), or bone mineral content (BMC).

[0017] In some aspects, the disclosure provides a method of treating or preventing ovarian inflammation and delaying reproductive aging in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

[0018] In some aspects, the disclosure provides a method of treating or preventing weight gain in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the rFSH decreases levels of lean fat, decreases levels of total fat, or decreases total body weight.

[0019] In some aspects, the disclosure provides a method of promoting metabolic health in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

[0020] In some embodiments, the rFSH increases serum levels of spermidine, decreases serum levels of asymmetric dimethylarginine (ADMA), or increases serum levels of D-glucono-1,5- lactone. In some embodiments, the levels of spermidine are increased by about two-fold. In some embodiments, the levels of ADMA are decreased by about two-fold. In some embodiments, the levels of D-glucono-l,5-lactone are increased by about two-fold. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0021] In some embodiments, the subject is obese. In some embodiments, the subject has been diagnosed with diminished ovarian reserve (DOI). In some embodiments, the subject is of reproductive age.

[0022] In some embodiments, the subject is administered about 20 to about 50 international units (IUS) of recombinant FSH (rFSH) every 60-90 minutes for about 8 to about 10 hours. In some embodiments, the subject is administered about 30 international units (IUs) of recombinant FSH (rFSH) every hour for about 10 hours. In some embodiments, the subject is administered a total dose of about 200 units, about 300 units, about 400 units, or about 500 units of recombinant FSH (rFSH).

[0023] In some embodiments, the pulsatile rFSH is administered intravenously. In some embodiments, a gonadotropin releasing hormone (GnRH) antagonist is administered to the subject. In some embodiments, the GnRH antagonist is Cetrorelix.

[0024] In some embodiments, pulsatile rFSH is formulated in a pharmaceutically acceptable carrier.

[0025] In some embodiments, the pulsatile rFSH induces differential expression of one or more genes in the ovary selected from Upklb, Cetn4, Hcst, Fam89a, Grik3, Abhdl, Slc6al4, Tecta, Ccl22, Coch, Olfrl034, Rprm, Fst, S100a8, Myll, Noval, Oit3, Stcl, Erdrl, Vtn, Myom2, Eya4, Trim34b, TmemlOO, Tacr2, Gbp2b, Defb25, Dpp6, Faml81b, Cyp26bl, Msmp, Cyp4fl4, Tmem72, Nebl, Trankl, Tbx3, Slc39a8, ApollOb, Ntng2, Angptl7, Rbm46, Slc28a2, Aim2, Atpl2a, Tdgfl, Adam23, Sstrl, Tmem267, Adhl, H33, Camkkl, Amd2, Cntfr, Slc52a3, Plac9a, Eif3j 1 , Jmjd7, Cdld2, Slcl4al, Drd4, Lrrn4, Tchh, Cilp, Hoxb3, Hoxb2, and Otof, compared to continuous rFSH.

[0026] In some embodiments, treatment with pulsatile rFSH increases expression of one or more genes in the ovary selected from Cypl9al, Egrl, Gas2, Inhbb, Kcnj8, Gata6, Dusp9, Trim5, and Rgsl4 compared to treatment with continuous rFSH. In some embodiments, treatment with pulsatile rFSH decreases expression of one or more genes in the ovary selected from Ereg, SlOOg, Tnfaip6, Itgav, Bmp7, Pdzkl compared to treatment with continuous rFSH.

[0027] In some embodiments, the subject maintains normal estrus cycles. In some embodiments, the subject maintains normal antral follicles and corpora lutea (CL) in the ovary. In some embodiments, the subject has an increased number of ovulations. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows that chronic overexpression of supra-pharmacological levels of FSH results in disruption of ovarian folliculogenesis with many follicles showing hemorrhage and cysts.

[0030] FIG. 2A-2E shows the effect of pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT) in Fshb / _(null) mice on ovulation efficiency. FIG. 2A: Summary of average number of eggs produced within each group. In contrast to control mice (Ctrl) and Fshb null mice expressing constitutive FSH (Fshb / _HFSHBWT), Fshb / _null mice expressing pulsatile FSH (Fshb / _HFSHBMut) showed significantly increased number of ovulations (55 vs. 8-9 eggs; n= 10 mice per genotype; P< 0.05 by ANOVA). FIG. 2B: Morphology of fertilized one-cell embryos retrieved from oviducts of naturally mated mice with vaginal plugs on embryonic day 3.5. FIG. 2C: Six times more one-cell embryos are present in oviducts of mutant FSH- expressing mice. FIG. 2D: In vitro culture of fertilized eggs results in viable and apparently healthy blastocysts in all genotypes. FIG. 2E: Quantification of fully expanded blastocysts reveals comparable efficiencies across all genotypes. All data are mean ± SEM; n = 10 mice per group.

[0031] FIG. 3A-3B shows number of implanted embryos in uterine horns by natural mating in Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT) at 10.5 days post conception (dpc). FIG. 3A: Gross morphological image of murine uterine horns across groups. FIG. 3B: Number of embryos at 10.5 days post conception (dpc) across groups. Compared to constitutive FSH-expressing mice, pulsatile FSH expressing mice showed significantly higher number of implantations (P< 0.05, T-test; n=6 mice).

[0032] FIG. 4A-4B shows ovaries from female mice at 6 months, 12 months, and 17 months of age in Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT). Embryos and blastocysts were immunostained to show spindles and blastocysts. FIG. 4A: Gross morphologic images and PAS-hemotoxylin stained ovaries at 6 months, 12 months or 17 months across groups. CL indicates corpora lutea. FIG. 4B: Quantification of number of embryos in 6 week old and 6 month old mice.

[0033] FIG. 5A-5C shows RNA sequencing (RNA-seq) of adult ovaries (3-4 months) in Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT, “continuous”). FIG. 5A: Differentially expressed genes (DEGs) between Fshb null mice versus Fshb null mice expressing continuous FSH (HFSHBWT). FIG. 5B: DEGs between Fshb null Attorney Docket No.: CUJ-00525 (CU 2025-135) mice versus Fshb null mice expressing pulsatile FSH (HFSHBMut). FIG. 5C: DEGs between Fshb null mice expressing pulsatile FSH (HFSHBMut) compared to continuous FSH (HFSHBWT). n=3 per group.

[0034] FIG. 6A-6B shows proteomics (FIG. 6A) and phospho-proteomics (FIG. 6B) of ovaries from adult 3-4 month old Fshb / _(null) mice compared to Fshb null mice expressing continuous FSH (FR), and Fshb null mice expressing pulsatile FSH. n=3 per group. KO= Fshb null, FSHc = continuous FSH (HFSHBWT), FSHp = pulsatile FSH (HFSHBMut).

[0035] FIG. 7A-7B shows effects of continuous and pulsatile FSH on metabolic health in mice fed a normal versus high-fat / high-sucrose diet (HF / HS). Mice were weaned at 3 weeks of age and fed on high-fat / high-sucrose (HF / HS) diet beginning at 4wks of age for 10 wks. FIG. 7A: Body weight (g) of mice over time across groups, measured once per week from 3 to 14 weeks of age. FIG. 7B: Live mice (n=6-10) expressing constitutive and pulsatile FSH were also non- invasively analyzed for total body fat content by echo MRI.

[0036] FIG. 8 shows gene expression in the ovary of Fshb null mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT) fed a normal or high-fat diet (HFD). Ovaries were collected from mice (n=6) fed on normal or HF / HS diet and RNA-Seq data were validated by qPCR analysis using cDNA samples in triplicate. Genes were normalized to the housekeeping gene Ppill. Both known and novel FSH-responsive genes were assayed.

[0037] FIG. 9A-9C shows serum metabolic profiles of Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut, FRP) compared to wild type FSH (HFSHBWT, FR) in young mice at 12 weeks of age. Mice were subjected to untargeted metabolomics analysis using mass spectrometry. FIG. 9A: Heatmap of differentially regulated metabolites in the serum of young mice across groups. FIG. 9B: Normalized levels of spermidine in the serum of young Fshb null mice compared to young FR and young FRP mice. FIG. 9C: Normalized levels of Asymmetric dimethylarginine (ADMA) in young Fshb null mice compared to constitutive FSH (FR) and pulsatile FSH (FRP) mice. n=6 mice per genotype.

[0038] FIG. 10A-10B shows serum metabolic profiles of Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut, FRP) compared to wild type FSH (HFSHBWT, FR) in aged mice at 14 months of age. Mice were subjected to untargeted metabolomics analysis using mass spectrometry. FIG. 10A: Heatmap of differentially regulated metabolites in the serum of aged mice across groups. FIG. 10B: Normalized levels of D-Glucono-l,5-lactone in the serum of aged Fshb null mice Attorney Docket No.: CUJ-00525 (CU 2025-135) compared to aged FR and aged FRP mice. n=6 mice per genotype.

[0039] FIG. 11A-11E shows metabolic health between Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT) in young mice (between 2-3 months old). FIG. 11A: Body weight (g) of mice across groups. FIG. 11B: Bone mineral density (BMD) across groups. FIG. 11C: Bone mineral content (BMC) across groups. FIG. 11D: Lean fat (g) across groups. FIG. HE: Total fat (g) across groups. * p<0.05. n=4-6 mice per genotype.

[0040] FIG. 12A-12F shows metabolic health between Fshb / _(null) mice expressing pulsatile FSH (HFSHBMut) compared to wild type FSH (HFSHBWT) in aged mice (between 12-14 months old). FIG. 12A: Body weight (g) of mice across groups, quantified by DEXA. FIG. 12B: Bone mineral density (BMD) across groups, quantified by DEXA. FIG. 12C: Bone mineral content (BMC) across groups, quantified by DEXA. FIG. 12D: Lean fat (g) across groups, quantified by DEXA. FIG. 12E: Total fat (g) across groups, quantified by DEXA. FIG. 12F: Total fat (g) across groups, quantified by Echo MRI. * p<0.05. n=4-6 mice per genotype.

[0041] FIG. 13A-13B shows comparative analysis of C’ terminus heptapeptide of LHb in mammalian species. Analysis was performed using GENBANK sequence alignment and a phylogenetic tree was generated. In collaboration with Mass General Brigham hospital investigators, 7,500 infertile patient DNA sample data was screened for any mutations in the heptapeptide-encoding sequence. FIG. 13A: Amino acid sequences of the LH heptapeptide across species, corresponding to SEQ ID NOs: 1-10. FIG. 13B: Phylogenetic tree of the LH heptapeptide across species.

[0042] DETAILED DESCRIPTION

[0043] In the following detailed description, embodiments of the present disclosure are described in detail to enable practice thereof. Although the invention is described with reference to these specific embodiments, it should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. All publications cited herein are incorporated by reference in their entireties for their teachings.

[0044] The invention includes numerous alternatives, modifications, and equivalents as will become apparent from consideration of the following detailed description. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0045] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the description and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] Definitions

[0047] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0048] As used herein, the term “Follicle Stimulating Hormone (FSH)” refers to a hormone secreted by the anterior pituitary gland which promotes the formation of ova or sperm.

[0049] As used herein, the term “Constitutive FSH” refers to wild-type FSH which is secreted constitutively in many species. The mouse model representing constitutive FSH is Fshb / _HFSHBWTor “FR”.

[0050] As used herein, the term “pulsatile FSH” (e.g., mutant FSH, or rerouted FSH) refers to a form of FSH that is secreted in pulses. The pulsatile secretion behavior of leutinizing hormone (LH) is due to a carboxyterminal heptapeptide which is a gonadotropin-sorting determinant. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice and is released in response to gonadotropin-releasing hormone, similar to LH. Pulsatile FSH can either be engineered to express the C’ terminal heptapeptide of LH in mice or delivered in “pulses” in humans.

[0051] Also as used herein, the terms "treat," "treating" or "treatment" may refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial and / or therapeutic effect, to a subject afflicted with a condition, disorder, disease or illness, including, for example, improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disorder, disease or illness, delay of the onset of the disease, disorder, or illness, and / or change in clinical parameters of the condition, disorder, disease or illness, etc., as would be well known in the art.

[0052] As used herein, the terms "prevent," "preventing" or "prevention of (and grammatical variations thereof) may refer to prevention and / or delay of the onset and / or progression of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset and / or progression of the disease, disorder and / or clinical symptom(s) relative to what Attorney Docket No.: CUJ-00525 (CU 2025-135) would occur in the absence of the methods of the invention. In representative embodiments, the term "prevent," "preventing," or "prevention of (and grammatical variations thereof) refer to prevention and / or delay of the onset and / or progression of a metabolic disease in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset and / or the progression is less than what would occur in the absence of the present invention.

[0053] An "effective amount" or "therapeutically effective amount" may refer to an amount of a compound or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic and / or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, during the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount or therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and / or by using routine experimentation. (See, for example, REMINGTON, THE SCIENCE AND PRACTICE OF PHARMACY (latest edition)).

[0054] A "nucleic acid" may include single-stranded and double-stranded nucleic acids and includes ribonucleic acids as well as deoxyribonucleic acids. It may include naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified.

[0055] A "vector" or "expression vector" may refer to and include any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and / or eukaryotic host cells and, where appropriate, may be integrated into a genome of and / or expressed in the host cell. Vectors may thus be replicated and / or expressed in the host cells. A vector may include one or more selection markers for selecting host cells comprising the vector. Vectors enabling the expression of recombinant proteins including FSH of the inventive concept as described herein, as well as suitable expression cassettes and expression elements which enable the expression of a recombinant protein with high yield in a host cell are well known and are commercially available, and may include any that may be appreciated by one of skill in the art. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0056] A "cell," "cells," and a "cell line" may be used interchangeably, and may refer to one or more cells and, in some embodiments, refer to mammalian cells, such as human cells. The term includes progeny of a cell or cell population. One of skill in the art will appreciate that "cells" include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or change. A "cell" may refer to isolated cells and / or cultivated cells which are not incorporated in a living human, non-human or animal body.

[0057] Compositions

[0058] Follicle-stimulating hormone (FSH) is a gonadotropin. FSH is synthesized and secreted by the gonadotropic cells of the anterior pituitary gland and regulates the development, growth, pubertal maturation, and reproductive processes of the body.

[0059] FSH is a 35.5 kDa glycoprotein heterodimer, consisting of two polypeptide units, alpha and beta. Its structure is similar to those of luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The alpha subunits of the glycoproteins LH, FSH, TSH, and hCG are identical and consist of 96 amino acids, while the beta subunits vary. The alpha subunit of the glycoprotein hormones are encoded by the CGA gene (Gene ID: 1081). Both the alpha and beta subunits are required for biological activity. FSH has a beta subunit of 111 amino acids (FSH 0), which confers its specific biologic action, and is responsible for interaction with the follicle-stimulating hormone receptor.

[0060] In some embodiments, the present disclosure provides a formulation or composition comprising recombinant human FSH0 (rFSH) for use in any one of the conditions described herein. In some embodiments, the rFSH is set forth in SEQ ID NO: 11. In some embodiments, the rFSH has an amino acid sequence comprising about 80%, about 95%, about 90%, about 91%, about 92%, about 93% about 94% about 95%, about 96% about 97% about 98%, about 99% or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0061] Methods of Treatment

[0062] The association between obesity and reduced reproductive fitness is well documented. Obese women have low gonadotropin and ovarian hormone levels, including lower follicular serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B levels Attorney Docket No.: CUJ-00525 (CU 2025-135) compared to women of normal weight. In women with infertility and obesity, a higher dose of exogenous gonadotropin is needed to achieve adequate ovarian stimulation and an increase in body mass index (BMI) negatively impacts outcomes (oocyte retrieval, clinical pregnancy, live birth and miscarriage) in women undergoing in vitro fertilization (IVF). These findings suggest that both pituitary and ovarian dysfunction may be implicated in the evident adverse effects of obesity on fertility and fecundity.

[0063] In some embodiments, the present disclosure provides methods of treating infertility in an obese female subject comprising administering pulsatile recombinant FSH (rFSH).

[0064] In some embodiments, the present disclosure provides methods of improving egg quality, inducing egg growth and / or maturation, increasing number of eggs, and / or improving embryo quality in an obese female subject comprising administering pulsatile recombinant FSH (rFSH). In some embodiments, the present disclosure provides methods of restoring normal hormonal levels in an obese female subject comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the number of eggs is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, or about 8-fold. In some embodiments, the eggs maintain developmental competence. In some embodiments, the developmental competence is measured by percent (%) expanded blastocysts.

[0065] In some embodiments, the present disclosure provides methods of treating a non-obese female human subject. In some embodiments, the human subject is a female human subject. In some embodiments, the female human subject may be premenopausal. In some embodiments, the female human subject may be perimenopausal. In some embodiments, the female subject may be postmenopausal, or in menopause. In some embodiments, the female subject has been diagnosed with diminished ovarian reserve (DOI).

[0066] In some embodiments, the present disclosure provides methods of stimulating secretion of a sex steroid in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the sex steroid is estradiol, estrogen, or progesterone.

[0067] In some embodiments, the present disclosure provides methods of treating or preventing fatty tissue accumulation in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject. In some embodiments, the fatty tissue accumulation is measured by total body weight, amount of lean fat, and / or amount of total fat. In some Attorney Docket No.: CUJ-00525 (CU 2025-135) embodiments, the fatty tissue accumulation is measured by vivo dual-energy X-ray absorptiometry (DEX A) or Echo MRI. Treating and / or preventing fatty tissue accumulation according to embodiments of the inventive concept may include administration of compositions and / or formulations of the inventive concept as described herein to treat and / or prevent fatty tissue accumulation that may result from, for example, a female subject, such as a female human subject approaching or entering menopause, e.g., a perimenopausal female human subject, or treating and / or preventing fatty tissue accumulation in a female human subject that is in menopause, but is not limited thereto.

[0068] Treating and / or preventing loss of bone density according to embodiments of the inventive concept may include treating and / or preventing conditions and diseases including administration of compositions and / or formulations of the inventive concept as described herein to a subject in which the inhibition of bone loss and / or the promotion of bone formation is desirable. Such conditions and diseases include, for example, osteoporosis, osteomyelitis, Paget's disease, periodontitis, hypercalcemia, osteonecrosis, osteosarcoma, osteolyic metastases, familial expansile osteolysis, prosthetic loosening, periprostetic osteolysis, cleiodocranial dysplasia (CCD), osteoporosis from arthritides, and bone loss due to metastatic disease and humoral hypercalcemia. In some embodiments, treating and / or preventing loss of bone density includes methods of treating and / or preventing osteoporosis in a subject. In some embodiments, treating and / or preventing loss of bone density includes treating a human subject. In some embodiments, the human subject is a female human subject. In some embodiments, the female human subject. In some embodiments, the female human subject may be premenopausal. In some embodiments, the female human subject may be perimenopausal. In some embodiments, the female subject may be postmenopausal, or in menopause.

[0069] Treating and / or preventing fatty tissue accumulation according to embodiments of the inventive concept may include administration of compositions and / or formulations of the inventive concept as described herein to treat and / or prevent fatty tissue accumulation that may result from, for example, a female subject, such as a female human subject approaching or entering menopause, e.g., a perimenopausal female human subject, or treating and / or preventing fatty tissue accumulation in a female human subject that is in menopause, but is not limited thereto. In some embodiments, the present disclosure provides a method of treating or preventing weight gain in a female subject in need thereof. In some embodiments, the pulsatile rFSH Attorney Docket No.: CUJ-00525 (CU 2025-135) decreases levels of lean fat, total fat, or total body weight.

[0070] Treating infertility, or infertility treatment, according to embodiments of the inventive concept include treatment of a dysfunction, disorder, or disease related to reproduction or fertility in a human or non-human (animal) subject including administration of compositions and / or formulations of the inventive concept as described herein. In some embodiments, treating infertility includes assisted reproductive technologies (ART), including but not limited to in vitro fertilization, in vitro fertilization with intracytoplasmic sperm injection, ovulation induction, and ovulation induction with intrauterine insemination. In some embodiments, treating infertility includes treatment of ovulatory disorders, treatment of disorders related to egg maturation, as well as inducing, enabling and / or improving folliculogenesis and / or spermatogenesis, inducing, enabling and / or improving Sertoli cell proliferation, and treating hypogonadotropic hypogonadism in the subject.

[0071] In some embodiments, treating infertility according to the inventive concept may include methods of inducing and / or stimulating secretion / production of sex steroids in the subject including administration of compositions and / or formulations of the inventive concept as described herein. Sex steroids, or sex hormones, refer to steroid hormones that interact with androgen or estrogen receptors. Sex steroids include: androgens, e.g., anabolic steroids, androstenedione, dehydroepiandrosterone, dihydrotestosterone and testosterone; estrogens, e.g., estradiol, estriol and estrone; and progesterone. In some embodiments, inducing and / or stimulating secretion / production of sex steroids include inducing and / or stimulating secretion / production of estrogens, for example, estradiol, and / or inducing and / or stimulating secretion / production progesterone.

[0072] In some embodiments, treating infertility according to the inventive concept may include induction of follicle growth and / or maturation including administration of compositions and / or formulations of the inventive concept as described herein. In some embodiments, treating infertility may include induction of oocyte / egg growth and / or maturation including administration of compositions and / or formulations of the inventive concept as described herein.

[0073] In some embodiments, treating infertility according to the inventive concept may include inducing, enabling and / or improving Sertoli cell proliferation including administration of compositions and / or formulations of the inventive concept as described herein. In some embodiments, treating infertility may include treating hypogonadotropic hypogonadism Attorney Docket No.: CUJ-00525 (CU 2025-135) including administration of compositions and / or formulations of the inventive concept as described herein.

[0074] In some embodiments, the disclosure provides methods of treating or preventing ovarian inflammation and delaying reproductive aging in a female subject in need thereof.

[0075] In some embodiments, the disclosure provides methods of promoting metabolic health in a female subject in need thereof. In some embodiments, the rFSH increases serum levels of spermidine, decreases serum levels of asymmetric dimethylarginine (ADMA), or increases serum levels of D-glucono-l,5-lactone. In some embodiments, the levels of spermidine are increased by about two-fold. In some embodiments, the levels of ADMA are decreased by about two-fold. In some embodiments, the levels of D-glucono-l,5-lactone are increased by about twofold.

[0076] In some embodiments, the pulsatile rFSH increases induces differential expression of one or more genes in the ovary selected from Upklb, Cetn4, Hcst, Fam89a, Grik3, Abhdl, Slc6al4, Tecta, Ccl22, Coch, Olfrl034, Rprm, Fst, S100a8, Myll, Noval, Oit3, Stcl, Erdrl, Vtn, Myom2, Eya4, Trim34b, TmemlOO, Tacr2, Gbp2b, Defb25, Dpp6, Faml81b, Cyp26bl, Msmp, Cyp4fl4, Tmem72, Nebl, Trankl, Tbx3, Slc39a8, ApollOb, Ntng2, Angptl7, Rbm46, Slc28a2, Aim2, Atpl2a, Tdgfl, Adam23, Sstrl, Tmem267, Adhl, H33, Camkkl, Amd2, Cntfr, Slc52a3, Plac9a, Eif3j 1 , Jmjd7, Cdld2, Slcl4al, Drd4, Lrrn4, Tchh, Cilp, Hoxb3, Hoxb2, and Otof, compared to continuous rFSH.

[0077] Methods of Making

[0078] "FSH" refers to follicle-stimulating hormone, a gonadotropin. In embodiments, the FSH is human FSH, in particular human FSH, composed of an alpha-subunit and a beta-subunit. "Recombinant FSH," as used herein, may refer to FSH that is not naturally produced by a living, for example, human or animal body / subject, and then obtained from a sample derived therefrom, such as urine, blood or any other body fluid, waste, e.g., fecal matter, or tissue derived from the human or animal body / subject. In some embodiments, recombinant FSH may be obtained from cells which have been biotechnologically engineered, for example, cells which have been transformed or transfected with a nucleic acid encoding FSH or the alpha- or beta-subunits of FSH. In some embodiments, the recombinant FSH comprises the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the rFSH comprises at least 80%, at least 85%, at Attorney Docket No.: CUJ-00525 (CU 2025-135) least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12.

[0079] According to some embodiments, recombinant FSH is obtained from human cells, mammalian cells, or non mammalian, e.g., insect cells, comprising an exogenous nucleic and encoding for an FSH of the disclosure. Respective exogenous nucleic acids can be introduced e.g. by using one or more expression vectors, for example, an expression vector including a nucleic acid encoding an FSH as described herein, operably linked to a heterologous promoter, which can be introduced into the host cell e.g. via transfection. Methods for recombinantly producing expression vectors, cell lines including expression vectors, proteins and FSH are well known, and any recombinant method as would be appreciated by one of skill in the art may be used for producing expression vectors, cell lines including expression vectors, proteins and FSH of the present disclosure. In some embodiments, the recombinant FSH may be introduced into a host cell by genetic engineering using any method that would be appreciated by one of skill in the art.

[0080] Pharmaceutical Compositions

[0081] According to further embodiments, provided are pharmaceutical compositions including a therapeutically effective amount of the pulsatile rFSH. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to any substance, not itself a therapeutic agent, used as at least in part a vehicle for delivery of a therapeutic agent to a subject. Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, and various types of wetting agents. Further, in preparing such pharmaceutical compositions comprising the active ingredient or ingredients in admixture with components necessary for the formulation of the compositions, other conventional pharmacologically acceptable additives may be incorporated, for example, excipients, stabilizers, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity agents, buffering agents, antioxidants and the like. Additives may include, for example, starch, mannitol, sorbitol, precipitated calcium Attorney Docket No.: CUJ-00525 (CU 2025-135) carbonate, crystalline cellulose, carboxymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, sodium metabi sulfite, and the like.

[0082] Formulations suitable for administering the composition of the present concept may be suitable for any route of administration as would be appreciated by one of skill in the art. Routes of administration may include, but are not limited to intravenous, oral, parenteral, subcutaneous, topical and / or vaginal methods of administration.

[0083] Methods of Administration

[0084] Also provided herein are methods for administering to a subject in need thereof a compound or pharmaceutical composition as described herein. For administration, either the compound or pharmaceutical composition is understood as being the active ingredient and capable of administration to a subject, and thus, in some instances, the terms are interchangeable.

[0085] The method of administration of the compound or pharmaceutical composition as described herein is not particularly limited, and any method that would be appreciated by one of skill in the art for the compound or pharmaceutical composition in a particular formulation as described herein. Routes of administration may include, but are not limited to intravenous, oral, parenteral, subcutaneous, topical and / or vaginal methods of administration. In some embodiments, the composition is further administered a gonadotropin releasing hormone (GnRH) antagonist. In some embodiments, the GnRH antagonist is Cetrorelix. In some embodiments, the GnRH antagonist is selected from one or more of Ganirelix, Cetrorelix, Elagolix, Relugolix, and Degarelix.

[0086] Subjects suitable to be treated with the composition, compositions and formulations include, but are not limited to, mammalian subjects. Mammals include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, humans and the like, and mammals in utero. Any mammalian subject in need of being treated or desiring treatment according to the present invention is suitable. In some embodiments, the subject is a human subject. Although the human subject treated according to methods described herein may be of any gender (for example, male, female or transgender) and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult, elderly), in some embodiments, the human subject is a premenopausal, menopausal, or postmenopausal female human subject. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0087] Dosages of compositions and formulations may depend on route of administration and intended use. It will be appreciated by one of skill in the art that international units (IU) for FSH may refer to the fourth International Standard for human urinary FSH (Stoning and Gaines Das 2001, J Endocrinol. 171, 119-129), determined as described by Steelman and Pohley 1953, Endocrinol. 53, 604-616. For example, 450 IU of FSH, corresponds to 33 mcg of FSH (41 IU corresponds to 3 mcg, -13.67 lU / mcg). Typical dosages available of commercially available FSH, such as GONAL-F®, GONAL-F RFF®, FOLLISTIM AQ®, and PUREGON®, may be in a range of about 1-10,000 IU, 10-3,600 IU and / or 37.5-1,200 IU, and include dosages of, for example, 37.5 IU, 75 IU, 150 IU, 300 IU, 450 IU, 600 IU, 750 IU, 900 IU, 1,050 IU, and 1,200 IU, which may be provided as a solid for dissolving in a carrier for administration, such as subcutaneous administration, or provided as a solution in, for example, 0.75 mL or 1.0 mL volume of a suitable carrier for subcutaneous administration / inj ection. Dosages and dosing regimens of FSH compositions are known, and any dosage and dosing regimens for FSH compositions and formulations may be used for the FSH compositions and formulations of the present disclosure. Known dosing regimens include, for example, daily subcutaneous administration, initially for 5 days, 7 days or 14 days, with duration of therapy running until response is achieved.

[0088] In some embodiments, the subject is administered about 20 to about 50 international units (IU) of rFSH every 60 to 90 minutes for about 8 to about 10 hours. In some embodiments, the subject is administered about 30 IU of rFSH every hour for about 10 hours. In some embodiments, the subject is administered a total dose of about 200 units, about 300 units, about 400 units, or about 500 units of recombinant FSH (rFSH).

[0089] EXAMPLES

[0090] Example 1. Pulsatile FSH transgenic mouse model

[0091] During vertebrate evolution, the female reproductive pattern underwent a remarkable transition from spawning of large number of eggs in primitive species under favorable conditions to more tightly controlled ovarian cycles in higher vertebrates, such that only a limited number (rodents) or a single (human and nonhuman primates) egg is released per cycle. Coincident with this event, the single pituitary gonadotropic hormone that exists in primitive vertebrates has given rise to two gonadotropins, FSH and luteinizing hormone (LH), which coordinate Attorney Docket No.: CUJ-00525 (CU 2025-135) gametogenesis and steroidogenesis. FSH and LH are heterodimeric glycoproteins that contain a common a-subunit and a hormone specific P-subunit. Although synthesized in the same cell, the gonadotrope FSH is mostly constitutively released in many species, whereas LH is stored in dense core granules (DCGs) and secreted in pulses via the regulated pathway in response to gonadotropin releasing hormone (GnRH). Thus, LH is secreted in pulses whereas FSH is secreted constitutively. The pulsatile secretion behavior of LH is due to a carboxyterminal heptapeptide which is a gonadotropin-sorting determinant. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice and is released in response to gonadotropin-releasing hormone, similar to LH. The present disclosure demonstrates that FSHP can be modified to contain a carboxyterminal (C')-heptapeptide from the human LHP (LSGLLFL), which results in secretion of the modified FSHP in a pulsatile manner (e.g., pulsatile FSH).

[0092] Human transgenes encoding wild-type or mutant FSHP containing the C'-heptapeptide were engineered for expression in mice. Multiple lines of transgenic mice were generated and the different transgenes were introduced separately onto an Fshb-null background, resulting in distinct expression and secretion patterns of murine and human FSH. Control mice expressed murine FSH a and b, FSHb / _(Fshb null) mice expressed murine FSHa but lacked expression of FSHb, FSHb / _HFSHBWTmice expressed mouse FSHa and human wild type FSHb (continuous FSH, Fshb HFSHBWTor FR), and FSHb / _HFSHBMutmice expressed mouse FSHa and human mutant FSHb (pulsatile FSH, Fshb HFSHBMutor FRP). HFSHBWTmice express continuous human FSH and HFSHBMutmice express pulsatile FSH.

[0093] Example 2. Pulsatile FSH enhances ovulation efficiency and developmental competence

[0094] Chronic overexpression of supra-pharmacological levels of FSH results in disruption of ovarian folliculogenesis with many follicles showing hemorrhage and cysts (FIG. 1), thus alternative approaches to delivering FSH are needed to avoid toxicities.

[0095] Because the mouse uterus can have only limited implantations, and counting the pups will not give an accurate estimate of ovulations, natural matings were established with proven fertile males and the number of fertilized eggs retrieved from oviducts were counted. Adult female mice at the age of 42 days were set up for mating with proven fertile males. Vaginal plugs were monitored the next morning and eggs / l-cell embryos were collected from oviducts of Attorney Docket No.: CUJ-00525 (CU 2025-135) plugged females following incubation with hyaluronidase enzyme at 37C. The released eggs were counted in each case. In contrast to control mice (Ctrl) and Fshb null mice expressing constitutive FSH (Fshb / _HFSHBWT), Fshb / _null mice expressing pulsatile FSH (Fshb HFSHBMut) showed significantly increased number of ovulations (55 vs. 8-9 eggs; n= 10 mice per genotype; P< 0.05 by ANOVA). (FIG. 2A). Thus, the pattern of FSH release is critical for ovulation efficiency.

[0096] Next, fertilized one-cell embryos were cultured in vitro in M2- culture medium and on embryonic day 3.5, the percent (%) of fully expanded blastocysts were counted. The number of ovulations was dramatically increased (by six times) in Fshb-null mice expressing pulsatile FSH compared with WT FSH or normal control mice (FIG. 2B-2C). These fertilized one-cell embryos, when cultured in vitro, fully expanded into blastocysts as efficiently as controls (FIG. 2D-2E).

[0097] The number of natural implantations of embryos in uterine horns were scored from plugged females around mid-gestation stage (at embryonic day 10.5). Gross morphological examination of the uterus in mice expressing pulsatile FSH compared to mice expressing wildtype human FSH demonstrated an increased number of embryos in mice expressing pulsatile FSH on day 10.5 (FIG. 3A-3B). This indicates better quality and quantity of embryos produced by pulsatile FSH-expressing mice.

[0098] When transferred to pseudopregnant female mice, the embryos derived from pulsatile FSH mice produced higher percentages of viable pups. Control mice were treated with exogenous hormones (eCG +11CG) (e.g., the standard superovulation protocol) and compared to Fshb-null mice expressing pulsatile FSH. Table 1 shows comparison of embryo efficiency with standard superovulation achieved with eCG+hCG vs. pulsatile FSH mice. Moreover, not only did pulsatile FSH mice produce significantly higher number of oocytes, their quality in terms of their blastocyst formation and hatching was comparable to that of controls. The efficiency was almost 5 times more than the standard superovulation protocol. Thus, pulsatile FSH mice produce good quantity as well as good quality eggs.

[0099] Table 1. In vivo transfer efficiency of pulsatile FSH mice Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0100] Example 3. Pulsatile FSH delays reproductive aging

[0101] Ovaries from female mice at 6m of age and beyond (12m and 17m) were collected and gross images were taken. Histological analysis was done by PAS-hematoxylin staining to score follicles and observe corpora lutea (CL). Natural ovulations were counted in constitutive and pulsatile FSH expressing mice at 6 months of age. Embryos and blastocysts were immunostained to show spindles and blastocysts. Ovarian and uterine morphology was indistinguishable in controls, or Fshb null mice that express WT or mutant FSH. At 17 months, morphological examination of the ovary was consistent across groups and CL were visible. The presence of antral follicles and abundant corpora lutea (CL) in ovarian sections indicated normal estrus cycles in control and Fshb null mice expressing WT or mutant FSH (FIG. 4A). Female mice expressing pulsatile FSH show increased number of ovulations and corpora lutea in ovaries at 6 and 12 months (FIG. 4B). Eggs and embryos at 12 months appear healthy and have no indication of any abnormalities. The follicle pool remains the same and there is no rapid follicle activation leading to premature ovarian failure. Because there are no oogonial stem cells in the mouse ovary, the enhanced ovulations in pulsatile FSH mice are attributed and experimentally verified due to suppression of atresia, which randomly eliminates about 37- 40 follicles every cycle in the mouse. These data demonstrate that the manner in which FSH is delivered has a significant effect on fertility.

[0102] Example 4. Pulsatile FSH induces expression of distinct ovarian genes and proteins

[0103] To better understand the mechanistic effects of pulsatile versus continuous FSH, RNA- sequencing (RNA-seq) was conducted on the ovaries of mice across groups. Ovaries from adult (3m-4m) female mice (n=3) were collected and total RNA was isolated using Qiagen mini- RNeasy columns. The isolated RNA was used to generate RNA-Seq libraries and high- throughput sequencing was conducted. After the appropriate normalization process, bioinformatics and statistical analyses were performed to generate 3 different comparisons to identify differentially regulated genes. The heatmaps of top 100 differentially regulated genes were indicated, and the hierarchical clusters are indicated. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0104] Analysis was conducted between Fshb null mice versus Fshb null mice expressing continuous FSH (HFSHBWT) (FIG. 5A), Fshb null mice versus Fshb null mice expressing pulsatile FSH (HFSHBMut) (FIG. 5B), and Fshb null mice expressing pulsatile FSH (HFSHBMut) compared to Fshb null mice expressing continuous FSH (HFSHBWT) (FIG. 5C). It was shown that pulsatile FSH induces differential expression of several genes compared to continuous rFSH (Table 2). The human homologs from Gene Cards database and their IDs are listed in Table 2 below, corresponding with the heatmap in FIG. 5C. Un-annotated and pseudogenes were removed. Overall, several differences in pulsatile FSH regulated ovarian genes were identified.

[0105] Table 2. List of Differentially regulated (annotated) genes in ovaries of mice expressing continuous vs. pulsatile FSH Attorney Docket No.: CUJ-00525 (CU 2025-135) Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0106] Proteomics (FIG. 6A) and phospho-proteomics (FIG. 6B) in the ovary was next examined. Ovaries from adult (3m-4m) female mice (n=3) were collected and protein was isolated and subjected to proteomics and phospho-proteomic analysis using tandem mass spectrometry. After the appropriate normalization process, bioinformatics and statistical analyses were performed to generate 3 different comparisons to identify differentially regulated proteins. The heatmaps of top 100 differentially regulated proteins and phospho-proteins were indicated, and the hierarchical clusters were indicated. The comparisons shown are KO vs. constitutive FSH; KO vs. pulsatile FSH and constitutive vs. pulsatile FSH. Several differences in pulsatile FSH regulated ovarian proteins and phospho-proteins were identified.

[0107] Example 5. Pulsatile FSH prevents weight gain in mice fed a high-fat / high-sucrose (HF / HS) diet

[0108] The association between obesity and reduced reproductive fitness is well documented. However, much less attention has been paid to infertility and poor response to treatment in women with obesity and regular, ovulatory cycles, compared to those women with obesity and PCOS. To identify the effects of FSH on obesity, Fshb null mice, Fshb null mice expressing continuous FSH (HFSHBWT), and Fshb null mice expressing pulsatile FSH (HFSHBMut) were fed a normal or high fat high sucrose (HF / HS) diet. Mice (n=6-10) were weaned at 3 weeks of age and were fed on high-fat / high-sucrose diet (HF / HS) beginning at 4 weeks of age for 10 weeks. All mice fed a normal diet maintained normal body weight over time, while Fshb null mice, Fshb null mice expressing continuous FSH, and wild-type mice fed a high-fat diet all gained weight as expected. However, the Fshb null mice expressing pulsatile FSH fed a high-fat diet were rescued from increased weight gain and maintained weight on a similar trend to mice fed a normal diet (FIG. 7A). In addition, Fshb null mice expressing pulsatile FSH had a decreased amount of fat compared to Fshb null mice expressing continuous FSH (FIG. 7B). Overall, these experiments show that even under adverse metabolic conditions, pulsatile FSH expressing mice do not gain body weight or total body fat when compared to constitutive FSH expressing mice or other control groups (Ctrl or Fshb null mice on HF / HS diet). Thus, pulsatile FSH mice are resistant to adverse condition such as metabolic dysfunction and maintain good Attorney Docket No.: CUJ-00525 (CU 2025-135) overall metabolic health.

[0109] Example 6. Pulsatile FSH modulates FSH-responsive genes in mice fed a high-fat / high sucrose (HF / HS) diet

[0110] Because of the effects of pulsatile FSH on prevention of weight gain in mice fed a high- fat diet, the mechanistic effects on gene expression were investigated. Ovaries were collected from mice (n=6) fed on normal or HF / HS diet and the RNA-Seq data were validated by qPCR analysis using cDNA samples in triplicate. The four groups tested were continuous FSH mice fed on normal vs. HF / HS diet and similarly, pulsatile FSH mice fed on normal vs. HF / HS diet. Both known and novel FSH-responsive genes were assayed.

[0111] Expression of several known FSH-responsive genes and novel FSH-responsive genes were assayed for changes in expression in mice expressing either constitutive or pulsatile FSH and fed either a high fat diet or normal diet (FIG. 8). It was shown that treatment with pulsatile rFSH increased expression of genes in the ovary including Cypl9al, Egrl, Gas2, Inhbb, Kcnj8, Gata6, Dusp9, Trim5, and Rgsl4 compared to treatment with continuous rFSH. Treatment with pulsatile rFSH decreased expression of genes in the ovary including Ereg, SI 00g, Tnfaip6, Itgav, Bmp7, Pdzkl compared to treatment with continuous rFSH. Genes were normalized to the housekeeping gene Ppill. Multiple differences were evident between normal chow vs. HFD among FR (constitutive FSH) or FRP (pulsatile FSH) genotypes. Bioinformatics analyses indicates that even under adverse metabolic conditions, FRP mice do not show ovarian inflammation. Thus, FSH not only alters the general metabolic health but preserves ovarian microenvironment .

[0112] Example 7. Pulsatile FSH improves serum metabolic health in young and aged mice

[0113] To better understand the effects of pulsatile FSH on overall metabolism, metabolomics was conducted on the serum of Fshb null mice, Fshb null mice expressing continuous FSH (HFSHBWT), and Fshb null mice expressing pulsatile FSH (HFSHBMut). Serum samples collected from either young mice (n=6 per genotype) at 12 weeks or aged mice (n=6 per genotype) at 12-14 months were subjected to untargeted metabolomics analysis using mass spectrometry. Heat maps of differentially regulated metabolites were generated, and the top 25 candidates were identified (FIG. 9A, 10A) Overall, Fshb null mice expressing continuous and Attorney Docket No.: CUJ-00525 (CU 2025-135) pulsatile FSH had distinct serum metabolic profiles compared to Fshb null mice (FIG. 9A-10A).

[0114] Serum metabolomic analyses indicates that pulsatile FSH maintains better metabolic health. Spermidine (indicator of better oocyte quality), asymmetrical dimethyl arginine (ADMA) and glutamine (indicators of poor oocyte quality) are the prominent metabolites. In pulsatile FSH mice, spermidine was increased (FIG. 9B), and ADMA (FIG. 9C) and glutamine (data not shown) were decreased compared to constitutive FSH expressing mice. This suggests that these metabolites favor better oocyte health and could be one reason for delayed female reproductive aging in pulsatile FSH expressing mice. Without being bound by theory, in aged mice, free fatty acids are reduced in pulsatile FSH expressing mice, which is an indicator of better liver metabolism and general health. Moreover, in pulsatile FSH expressing mice, pentose phosphate pathway instead of glycolysis is upregulated (FIG. 10B) also indicating better metabolic health and energy homeostasis. These data show that the pattern of FSH delivery affects systemic metabolism in both young and aged mice.

[0115] Example 8. Pulsatile rFSH promotes bone health in young and aged mice

[0116] Bone mineral density and content of female Fshb null mice expressing pulsatile or continuous FSH was examined in young mice at 2-3 months of age using in vivo dual-energy X- ray absorptiometry (DEXA). In addition to showing significantly decreased body weight (FIG. HA), Fshb null mice expressing pulsatile FSH had significantly increased bone mineral density (FIG. 11B) and bone mineral content (FIG. 11C). Young Fsh null mice expressing pulsatile FSH also showed significantly decreased lean fat (FIG. 11D) and total fat (FIG. HE). Next, bone mineral density and content of female Fshb null mice expressing pulsatile or continuous FSH was examined in aged mice at 12-14 months of age using DEXA. Aged Fshb null mice expressing pulsatile FSH had moderately decreased body weight (FIG. 12A) and significantly increased bone mineral density (FIG. 12B) and bone mineral content (FIG. 12C). Aged Fshb null mice expressing pulsatile FSH also showed significantly decreased lean fat (FIG. 12D) and total fat quantified by DEXA (FIG. 12E) and MRI (FIG. 12F). These data show that pulsatile FSH significantly improves bone health and reduces weight gain in both young and aged mice. Young and aged mice expressing pulsatile FSH do not lose body weight, gain bone mineral density, and content and accumulate less fat compared to those seen in mice expressing constitutive FSH. Thus, FSH regulates its own microenvironment within the follicle. Attorney Docket No.: CUJ-00525 (CU 2025-135)

[0117] Example 9. Phylogenetic modeling of LH

[0118] The C-terminus heptapeptide of human LHP subunit is a gonadotrope sorting determinant which promotes secretion of LH in a pulsatile manner. Comparative analysis of C’ terminus heptapeptide of LHP in mammalian species was performed using GENBANK sequence alignment and a phylogenetic tree was generated. In collaboration with Mass General Brigham hospital investigators, 7,500 infertile patient DNA sample data was screened for any mutations in the heptapeptide-encoding sequence. The LH heptapeptide has been conserved across species (FIG. 13A-13B). Horse, pig and zebra fish do not contain the heptapeptide-encoding sequence. It is not known if these 3 species exhibit a broad LH peak. Whether there are benefits of pulsatile FSH to large animal species is also worth exploring. If LL residues are modified by PCR site- directed mutagenesis, the gonadotrope-specific routing will be lost in pituitaries of mice. The fact that this specific peptide motif is conserved across various species (e.g., humans, rodents, bovines, and even avian species) indicates that this mechanism of differential sorting and regulated secretion is critical for reproductive functions and has been maintained through evolution.

[0119] INCORPORATION BY REFERENCE

[0120] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. The FSH-beta subunit that is used in the embodiments of the inventive concept set forth herein may include, e.g., mutations to one or more glycosylation sites on the FSH-beta subunits, e.g., as described in PCT Application Publication Nos. WO 2021 / 092358 and WO 2023 / 215914, and U.S. Application Publication No. 2022 / 0281940, in addition to the seven amino acid hydrophobic peptide sequence (e.g., LSGLLFL) from LH that may be genetically fused onto the FSH-beta subunit at the C-terminal.

[0121] EQUIVALENTS

[0122] Those skilled in the art will recognize, or be able to ascertain using no more than routine Attorney Docket No.: CUJ-00525 (CU 2025-135) experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0123] SEQUENCE LISTING Attorney Docket No.: CUJ-00525 (CU 2025-135) Attorney Docket No.: CUJ-00525 (CU 2025-135)

Claims

Attorney Docket No.: CUJ-00525 (CU 2025-135)WHAT IS CLAIMED IS:

1. A method of treating infertility in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

2. A method of improving egg quality, inducing egg growth or maturation, increasing number of eggs, or improving embryo quality, in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

3. The method of claim 2, wherein the number of eggs is increased by about 2-fold, about 3- fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, or about 8-fold.

4. The method of claim 2, wherein the eggs maintain developmental competence.

5. The method of any one of claims 2-4, wherein the developmental competence is measured by percent (%) expanded blastocysts.

6. A method of restoring normal hormonal levels in an obese female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

7. A method of stimulating secretion of a sex steroid in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

8. The method of claim 7, wherein the sex steroid is estradiol, estrogen, or progesterone.

9. A method of treating or preventing fatty tissue accumulation in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

10. A method of treating or preventing loss of bone density in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

11. The method of claim 10, wherein the subject has osteoporosis.

12. The method of any one of claims 10-11, wherein the rFSH prevents loss of bone mineral density (BMD) or bone mineral content (BMC).

13. The method of any one of claims 10-12, wherein the rFSH increases bone mineral density (BMD), or bone mineral content (BMC).

14. A method of treating or preventing ovarian inflammation and delaying reproductiveAttorney Docket No.: CUJ-00525 (CU 2025-135) aging in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

15. A method of treating or preventing weight gain in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

16. The method of claim 15, wherein the rFSH decreases levels of lean fat, decreases levels of total fat, or decreases total body weight.

17. A method of promoting metabolic health in a female subject in need thereof comprising administering pulsatile recombinant FSH (rFSH) to the subject.

18. The method of any one of claims 1-17, wherein the rFSH increases serum levels of spermidine, decreases serum levels of asymmetric dimethylarginine (ADMA), or increases serum levels of D-glucono-l,5-lactone.

19. The method of claim 18, wherein the levels of spermidine are increased by about twofold.

20. The method of claim 18, wherein the levels of ADMA are decreased by about two-fold.

21. The method of claim 18, wherein the levels of D-glucono-l,5-lactone are increased by about two-fold.

22. The method of any one of claims 6-21, wherein the subject is obese.

23. The method of any one of claims 6-22, wherein the subject has been diagnosed with diminished ovarian reserve (DOI).

24. The method of any one of claims 1-23, wherein the subject is of reproductive age.

25. The method of any one of claims 1-24, wherein the subject is administered about 20 to about 50 international units (IUs) of recombinant FSH (rFSH) every 60-90 minutes for about 8 to about 10 hours.

26. The method of any one of claims 1-25, wherein the subject is administered about 30 international units (IUs) of recombinant FSH (rFSH) every hour for about 10 hours.

27. The method of any one of claim 1-26, wherein the subject is administered a total dose of about 200 units, about 300 units, about 400 units, or about 500 units of recombinant FSH (rFSH).Attorney Docket No.: CUJ-00525 (CU 2025-135)28. The method of any one of claims 1-27, wherein the pulsatile rFSH is administered intravenously.

29. The method of any one of claims 1-28, wherein a gonadotropin releasing hormone (GnRH) antagonist is administered to the subject.

30. The method of claim 29, wherein the GnRH antagonist is Cetrorelix.

31. The method of any one of claims 1-30, wherein the pulsatile rFSH is formulated in a pharmaceutically acceptable carrier.

32. The method of any one of claims 1-31, wherein the pulsatile rFSH induces differential expression of one or more genes in the ovary selected from Upklb, Cetn4, Hcst, Fam89a, Grik3, Abhdl, Slc6al4, Tecta, Ccl22, Coch, Olfrl034, Rprm, Fst, S100a8, Myll, Noval, Oit3, Stcl, Erdrl, Vtn, Myom2, Eya4, Trim34b, TmemlOO, Tacr2, Gbp2b, Defb25, Dpp6, Faml81b, Cyp26bl, Msmp, Cyp4fl4, Tmem72, Nebl, Trankl, Tbx3, Slc39a8, ApollOb, Ntng2, Angptl7, Rbm46, Slc28a2, Aim2, Atpl2a, Tdgfl, Adam23, Sstrl, Tmem267, Adhl, 1133, Camkkl, Amd2, Cntfr, Slc52a3, Plac9a, Eif3j 1 , Jmjd7, Cdld2, Slcl4al, Drd4, Lrrn4, Tchh, Cilp, Hoxb3, Hoxb2, and Otof, compared to continuous rFSH.

33. The method of any one of claims 1-32, wherein treatment with pulsatile rFSH increases expression of one or more genes in the ovary selected from Cypl9al, Egrl, Gas2, Inhbb, Kcnj8, Gata6, Dusp9, Trim5, and Rgsl4 compared to treatment with continuous rFSH.

34. The method of any one of claims 1-33, wherein treatment with pulsatile rFSH decreases expression of one or more genes in the ovary selected from Ereg, SI 00g, Tnfaip6, Itgav, Bmp7, Pdzkl compared to treatment with continuous rFSH.

35. The method of any one of claims 1-34, wherein the subject maintains normal estrus cycles.

36. The method of any one of claims 1-35, wherein the subject maintains normal antral follicles and corpora lutea (CL) in the ovary.

37. The method of any one of claims 1-36, wherein the subject has an increased number of ovulations.

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