Inhibition of telomere length increase and / or telomere shortening.

Abstract

This invention relates to a mitochondrial targeting agent and its usefulness in increasing telomere length and / or suppressing telomere shortening.

Description

Technical Field

[0001] The present invention relates to telomeres, the treatment of telomere-related disorders and / or fertilization.

Background Art

[0002] Telomeres are repetitive DNA elements that cap the ends of chromosomes and are important protective factors for genomic stability. Telomere length is an important determinant of healthy aging and lifespan in mammals. Telomeres shorten with each somatic cell division, and when they fall below a threshold length, cellular senescence is triggered, contributing to tissue dysfunction and pathology. Thus, long telomeres are associated with good health in old age and increased lifespan, while shorter telomere length is a biomarker for susceptibility to aging and a wide range of age-related comorbidities, particularly cardiovascular disease. Effective treatment of telomere-related disorders is needed.

[0003] Furthermore, telomere length at birth is a major determinant of lifetime telomere length and has a greater influence than environmental stresses that affect the rate of telomere shortening throughout the rest of life. However, despite being a major risk factor for chronic disease and early death, the understanding of the developmental mechanisms that establish neonatal telomere length is relatively limited.

[0004] Preimplantation embryo development is an exceptional situation where telomeric DNA elongates. Specifically, rapid telomere elongation occurs between fertilization and blastocyst formation. The telomere length achieved by the inner cell mass (ICM), which is a pluripotent precursor to all fetal tissues, is thought to be the maximum set point for its future individual, from which telomeres continuously shorten during fetal development, childhood, and adulthood. Thus, the initial elongation and resetting mechanisms that establish ICM telomere length are essential for genomic integrity and determine the progenitor cell replication capacity in the offspring.

[0005] The mechanisms regulating the resetting of ICM telomere length during embryonic development are unknown, and it is necessary to understand how maternal physiological factors affect the setpoint. Shorter telomeres are observed in children of women with obesity or metabolic syndrome, or in children born to women of higher reproductive age. Similarly, children conceived using in vitro embryo culture have shorter telomeres than their counterparts conceived in vivo. Short telomeres in such children are problematic and increase the risk of telomere-related disorders later in life, for example. [Overview of the project] [Means for solving the problem]

[0006] This invention overcomes one or more of the above-mentioned problems.

[0007] The inventors have found the usefulness of mitochondrial targeting agents in the treatment of telomere-related disorders.

[0008] The inventors have found that gametes and / or their fertilized products having elevated levels of mitochondrial reactive oxygen species are associated with telomere elongation defects during preimplantation embryogenesis. Surprisingly, contacting such gametes and / or their fertilized products with mitochondrial targeting agents can increase telomere length and (partially or completely) restore telomere elongation defects.

[0009] Advantageously, the inventors have shown for the first time that mitochondrial targeting agents increase telomere length (for example, by promoting telomere elongation). For example, telomere length can be increased by contacting a mitochondrial targeting agent in vitro with gametes (e.g., oocytes) and / or their fertilized products, or by administering a mitochondrial targeting agent to a mammal before ovulation and / or fertilization. In particular, telomere length can be increased by contacting a mitochondrial targeting agent in vitro with oocytes and / or their fertilized products, or by administering a mitochondrial targeting agent to a female mammal before ovulation and / or fertilization.

[0010] Therefore, the present invention may find particular utility in treating telomere-related disorders by increasing telomere length during embryonic development.

[0011] Additionally or alternatively, the present invention may enable the generation of improved embryos that have the ability to develop into healthier offspring.

[0012] The present invention may be particularly suitable for use in in vitro fertilization, where the use of mammalian gametes with reduced reproductive capacity and / or where mitochondrial reactive oxygen species have been shown to be elevated in the gametes (e.g., in oocytes).

[0013] In one aspect, the present invention provides a mitochondrial targeting agent for use in treating telomere-related disorders. In a related aspect, a method for treating telomere-related disorders is provided, comprising the administration of a mitochondrial targeting agent. In another related aspect, the use of a mitochondrial targeting agent in the manufacture of a pharmaceutical for treating telomere-related disorders is provided.

[0014] In one aspect, the present invention provides a mitochondrial targeting agent for use in increasing telomere length. In a related aspect, the present invention provides a method for increasing telomere length, comprising the administration of a mitochondrial targeting agent. In another related aspect, the present invention provides the use of a mitochondrial targeting agent in the manufacture of a pharmaceutical for increasing telomere length.

[0015] In one aspect, the present invention provides a mitochondrial targeting agent for use in inhibiting telomere shortening. In a related aspect, the present invention provides a method for inhibiting telomere shortening, comprising the administration of a mitochondrial targeting agent. In another related aspect, the present invention provides the use of a mitochondrial targeting agent in the manufacture of a pharmaceutical for inhibiting telomere shortening. By increasing telomere length (e.g., of a subject) and / or inhibiting telomere shortening, mitochondrial targeting agents may promote healthy aging and / or lifespan. Healthy aging may be aging unrelated to disorders caused by short telomeres, such as telomere-related disorders described herein. For example, a mitochondrial targeting agent may be prophylactically administered to a subject (e.g., as a supplement) to increase telomere length and / or inhibit telomere shortening. Such administration may promote healthy aging and / or lifespan.

[0016] In one embodiment, the present invention provides a mitochondrial targeting agent for use in increasing telomere length during embryonic development, wherein the use comprises contacting gametes and / or their fertilized products with the mitochondrial targeting agent. In a related embodiment, a method for increasing telomere length during embryonic development is provided, comprising contacting gametes and / or their fertilized products with the mitochondrial targeting agent. In another related embodiment, a use of a mitochondrial targeting agent in the manufacture of a pharmaceutical for increasing telomere length during embryonic development is provided, comprising contacting gametes and / or their fertilized products with the mitochondrial targeting agent. The contact may be in vivo. For example, gametes of a male or female mammal may be in contact with the mitochondrial targeting agent by administration of the mitochondrial targeting agent to a male or female mammal that is producing, will produce, and / or has produced gametes. In the first example, a male or female mammal administered with the mitochondrial targeting agent may optionally mate with a mammal of the opposite sex that may also be administered with the mitochondrial targeting agent. Telomere length may increase during subsequent in vivo embryonic development. In a second example, the gametes may then be used in a reproductive aid method. In this method, embryonic development occurs, and therefore telomere length increase may occur.

[0017] Preferably, at least the fertilized product of the gametes is brought into contact with the female subject containing the fertilized product by administering a mitochondrial targeting agent.

[0018] In one embodiment, the present invention provides a method (preferably in vitro) for increasing telomere length during embryonic development, comprising contacting gametes and / or their fertilized products with a mitochondrial targeting agent in vitro.

[0019] In one embodiment, the present invention provides a use (preferably in vitro) of a mitochondrial targeting agent for increasing telomere length during embryonic development, comprising contacting gametes and / or their fertilization products with the mitochondrial targeting agent in vitro.

[0020] In one aspect, the present invention provides a method (preferably in vitro) for generating an embryo (e.g., having an increased telomere length), the method comprising contacting a gamete and / or its fertilization product with a mitochondrial targeting agent in vitro.

[0021] The gamete and / or its fertilization product contacted with the mitochondrial targeting agent in vitro can be used in an assisted reproduction method.

[0022] For example, the method or use may (a) contacting a gamete with a mitochondrial targeting agent in vitro; and (b) fertilizing the gamete with a gamete obtainable from a mammalian of the opposite sex to produce a fertilization product; and (c) preferably culturing the fertilization product (e.g., to generate an embryo). and may include.

[0023] Steps (b) and (c) can also be performed in vitro.

[0024] For example, the method or use may (a) contacting a fertilization product of a gamete with a mitochondrial targeting agent in vitro; and (b) culturing the fertilization product (e.g., to generate an embryo). and may include.

[0025] Step (b) can also be performed in vitro.

[0026] When the fertilization product is cultured, an embryo with an increased telomere length can be generated.

[0027] In one aspect, the present invention provides a method (preferably in vitro) for increasing the telomere length during embryogenesis, the method comprising using a gamete contacted with a mitochondrial targeting agent in an assisted reproduction method, preferably an in vitro fertilization method.

[0028] In one embodiment, the present invention provides a use (preferably in vitro) of a mitochondrial targeting agent for increasing telomere length during embryonic development, which includes using a gamete that has been in contact with the mitochondrial targeting agent in an assisted reproductive method, preferably an in vitro fertilization method.

[0029] In one embodiment, the present invention provides a method for generating an embryo (preferably in vitro), comprising using gametes that have been contacted with a mitochondrial targeting agent in an assisted reproductive method, preferably an in vitro fertilization method.

[0030] In one embodiment, the present invention provides a method (preferably in vitro) for increasing telomere length during embryonic development, comprising using a fertilized product of gametes in an assisted reproductive method, wherein the fertilized product is in contact with a mitochondrial targeting agent.

[0031] In one embodiment, the present invention provides a use (preferably in vitro) of a mitochondrial targeting agent for increasing telomere length during embryonic development, comprising using a fertilized product of gametes in an assisted reproductive method, wherein the fertilized product is in contact with the mitochondrial targeting agent.

[0032] In one embodiment, the present invention provides a method for generating an embryo (preferably in vitro), comprising using a fertilized product of gametes in an assisted reproductive method, wherein the fertilized product is in contact with a mitochondrial targeting agent.

[0033] Preferably, the methods or uses described herein are in vitro methods or uses.

[0034] In assisted reproductive methods, the methods or uses described herein, which include contacting gametes and / or their fertilized products with a mitochondrial targeting agent in vitro, or using gametes and / or their fertilized products that have been contacted with a mitochondrial targeting agent, preferably, embryogenesis and telomere lengthening occur in vitro. However, such embryogenesis and telomere lengthening may occur later in vivo. In some cases, fertilization is preferably performed in vitro. However, such fertilization may occur later in vivo.

[0035] In one embodiment, the present invention provides an embryo that can be obtained by the method described herein.

[0036] As used herein, the term "can be obtained" also includes the term "obtainable".

[0037] As used herein, the term "disorder" also includes "disease."

[0038] Mitochondrial reactive oxygen species (MiTOs) are representative of mitochondrial superoxide production. Gametes and / or their fertilized products may preferably contain elevated levels of MiTOs, for example, before contact with the Mitochondrial Targeting Agent of the present invention. The gametes and / or their fertilized products may preferably additionally contain elevated levels of MiTOs during, but not after, contact with the Mitochondrial Targeting Agent. Gametes and / or their fertilized products containing elevated levels of MiTOs may have levels of MiTOs at least 10%, 20%, 30%, 40%, 50%, 75%, or 100% higher than the levels of MiTOs in the reference gametes and / or their fertilized products.

[0039] Preferably, the gametes contain elevated levels of mitochondrial reactive oxygen species, for example, prior to contact with the mitochondrial targeting agent of the present invention.

[0040] The reference gamete may be a gamete obtained from a mammal with normal or increased reproductive capacity. In other words, in one embodiment, the mammal is not suffering from or susceptible to a condition associated with reduced reproductive capacity and / or reduced gamete quality. The reference gamete may be a gamete obtained from a mammal having normal gamete production levels and / or rates. The reference gamete may be a gamete obtained from a mammal having normal blood glucose and insulin levels and / or not being exposed to high-fat diets. The reference gamete may be a gamete obtained from a healthy mammal. The reference gamete may be a gamete obtained from a mammal that has never smoked and / or does not smoke. Preferably, the reference gamete may be a gamete obtained from a mammal that is neither overweight nor obese. Most preferably, the reference gamete may be a gamete obtained from a mammal, which is neither overweight nor obese, does not have abnormal levels and / or rates of gamete production, does not have polycystic ovary syndrome, does not have metabolic syndrome, does not have reduced fertility, does not have low fertility, does not have infertility, does not have ovarian dysfunction, does not have anovulation, does not have a reduced ovulation rate, does not have prediabetes, does not have diabetes, does not have hyperandrogenism, does not have insulin resistance, and does not have glucose tolerance. The ovarian function is not impaired, blood glucose levels are not elevated, there is no hyperinsulinemia, there is no dyslipidemia, ovarian reserve is not low, there is no premature ovarian failure, there is no ovarian aging, sperm quality is not low (e.g., sperm motility, viability and / or acquisition of fertilizing ability are not low, sperm morphology and / or DNA integrity are not insufficient), sperm count is not low, there is no exposure to high-fat diet, there is no aging, there is no history of recurrent miscarriage, and / or there is no history of smoking and / or there is no smoking.

[0041] The reference fertilization product may be produced from any of the reference gametes mentioned above.

[0042] Suitable methods for measuring mitochondrial reactive oxygen species levels are described in the examples herein, such as MitoSOX Red staining.

[0043] Gametes and / or their fertilized products containing elevated levels of mitochondrial reactive oxygen species may, for example, be gametes and / or their fertilized products that were exposed to high levels of oxygen during the in vitro fertilization process. The term "high levels of oxygen" used in this context refers to oxygen levels that embryos would not normally be exposed to in vivo, such as standard air containing approximately 20% oxygen. In other words, the term "high levels of oxygen" may refer to non-physiological oxygen levels. Therefore, gametes and / or their fertilized products containing elevated levels of mitochondrial reactive oxygen species may be gametes and / or their fertilized products that were exposed to non-physiological levels of oxygen. For explanation, the fallopian tubes are physiologically hypoxic.

[0044] Gametes and / or their fertilized products containing elevated levels of mitochondrial reactive oxygen species may be characterized, for example, by a change in the epigenetic state (e.g., nuclear epigenetic state) prior to contact with a mitochondrial targeting agent according to the present invention. The altered epigenetic state may be an abnormal epigenetic state. Gametes and / or their fertilized products containing elevated levels of mitochondrial reactive oxygen species may be characterized, for example, by an altered (e.g., abnormal) DNA methylation level prior to contact with or administration of a mitochondrial targeting agent according to the present invention. The gametes and / or their fertilized products may have altered 5-methylcytosine (5mC) levels and / or altered 5-hydroxymethylcytosine (5hmC) levels. The gametes and / or their fertilized products may have increased 5-methylcytosine (5mC) levels and / or decreased 5-hydroxymethylcytosine (5hmC) levels. In particular, the zygote may have increased 5-methylcytosine (5mC) levels and / or decreased 5-hydroxymethylcytosine (5hmC) levels prior to contact with or administration of the mitochondrial targeting agent according to the present invention. The difference / change (e.g., increase or decrease) may be in comparison to a reference gamete and / or reference fertilization product (as defined herein).

[0045] The gametes and / or their fertilized products may have a 5mC level at least 10%, 20%, 30%, 40%, or 50% higher than the level of 5-methylcytosine (5mC) in the reference gametes and / or their fertilized products (as defined herein).

[0046] The gametes and / or their fertilized products may have a 5hmC level that is at least 10%, 20%, 30%, 40%, or 50% lower than the level of 5-hydroxymethylcytosine (5hmC) in the reference gametes and / or their fertilized products (as defined herein).

[0047] The gametes and / or fertilized products used in the present invention may additionally include one or more characteristics of mitochondrial dysfunction. For example, the gametes and / or fertilized products may have a reduced mitochondrial membrane potential, for example, before (and optionally during) contact with a mitochondrial targeting agent according to the present invention. Gametes and / or fertilized products containing a reduced mitochondrial membrane potential may have a mitochondrial membrane potential at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the mitochondrial membrane potential of a reference gamete and / or fertilized product (as defined herein).

[0048] Suitable methods for measuring mitochondrial membrane potential are described in the examples herein, such as tetramethylrhodamine methyl ester perchlorate (TMRM) staining.

[0049] The telomere-related disorders treated according to the present invention may be any telomere-related disorders. Telomere-related disorders may be disorders associated with short telomeres (e.g., abnormally short telomeres). Telomere-related disorders may include aging (e.g., premature aging), bone marrow failure, congenital dyskeratosis, acquired aplastic anemia, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), liver disease (e.g., associated with or resulting from congenital dyskeratosis and / or pulmonary fibrosis), cardiovascular disease, heart disease, cancer, neurodegeneration, inflammatory bowel disease, Barrett's esophagus and / or miscarriage (e.g., recurrent miscarriage).

[0050] Preferably, telomere-related disorders are aging. Therefore, in preferred embodiments, the mitochondrial targeting agent of the present invention treats aging, such as premature aging. In other words, the mitochondrial targeting agent can function as an anti-aging agent.

[0051] As used herein, the terms “to treat” or “to treat” include prophylactic treatment (e.g., to prevent the onset of telomere-related disorders) and corrective treatment (e.g., treatment of subjects already suffering from telomere-related disorders). Preferably, as used herein, “to treat” or “to treat” means prophylactic treatment. As used herein, the terms “to treat” or “to treat” may apply to telomere-related disorders and / or their symptoms.

[0052] Treatment of telomere-related disorders may encompass treatment of at least one of their symptoms. Therefore, a telomere-related disorder is preferably considered treated when at least one of its symptoms is treated. A symptom may be considered treated when its severity is reduced. Such reduction may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, preferably 100%. The severity of a symptom after treatment may be assessed by comparing it to the severity of the same symptom before treatment.

[0053] The mitochondrial targeting agent of the present invention may be administered in a therapeutically effective or prophylactically effective dose. Preferably, the mitochondrial targeting agent of the present invention is administered in a therapeutically effective dose.

[0054] The “therapeutic dose” can be any amount of a mitochondrial targeting agent sufficient to achieve such treatment when administered alone or in combination with another agent (preferably alone). For example, the “therapeutic dose” can be any amount of a mitochondrial targeting agent sufficient to increase telomere length and / or inhibit telomere shortening (e.g., during embryonic development, such as in the subject and / or subsequent embryonic development) when administered alone or in combination with another agent (preferably alone). For example, the “therapeutic dose” can be any amount of a mitochondrial targeting agent sufficient to achieve such treatment of telomere-related disorders (or their symptoms) when administered alone or in combination with another agent (preferably alone) to treat such telomere-related disorders (or their symptoms).

[0055] The “prophylactic effective dose” may be any amount of a mitochondrial targeting agent that, when administered alone or in combination with another agent (preferably alone), inhibits or delays the onset or recurrence of the disorder (or its symptoms) and / or telomere shortening. In some embodiments, the prophylactic effective dose completely prevents the onset or recurrence of the disorder and / or telomere shortening. “Inhibiting” onset may mean reducing the likelihood of the onset of the disorder (or its symptoms) and / or telomere shortening, preventing the large peak effect of the disorder (or its symptoms) and / or telomere shortening, and / or completely preventing onset.

[0056] The “prophylactic effective dose” may be any amount of a mitochondrial targeting agent that, when administered alone or in combination with another agent (preferably alone), inhibits or delays the onset or recurrence of telomere-related disorders (or their symptoms). In some embodiments, the prophylactic effective dose completely prevents the onset or recurrence of telomere-related disorders. “Inhibiting” onset may mean reducing the likelihood of developing telomere-related disorders (or their symptoms), preventing a large peak effect of telomere-related disorders (or their symptoms), and / or completely preventing onset.

[0057] Mitochondrial targeting agents can be administered to subjects. Mitochondrial targeting agents can be administered to subjects after birth. In other words, mitochondrial targeting agents can be administered to subjects at any point after birth. For example, mitochondrial targeting agents can be administered during childhood and / or adulthood. Therefore, subjects of treatment according to the present invention may be children (e.g., people under 18 years of age) or adults (e.g., people at least 18 years of age). Preferably, the subjects of treatment are adults.

[0058] As used herein, “subject” refers to a mammal. A subject may be a non-mouse mammal. A subject may be a domesticated mammal such as a livestock mammal, a pet mammal, or a wild mammal species. A subject may be a human, horse, monkey, cattle (including bulls), pig, dog, cat, sheep, goat, elephant, panda, mouse, rabbit, rat, or other mammal. Preferably, “subject” means a human subject, horse, or cattle. Most preferably, a subject is a human subject. A “subject” is preferably an adult subject, for example, a human subject at least 18 years of age. The terms “subject” and “patient” are used synonymously herein. A subject may be a subject having a telomere-related disorder as defined herein.

[0059] The gametes and / or their fertilization products may be contacted with a mitochondrial targeting agent, preferably the gametes and / or their fertilization products (preferably the gametes) containing elevated levels of mitochondrial reactive oxygen species prior to contact with the mitochondrial targeting agent. The contact may be in vivo or in vitro. In relation to the medical uses and treatment methods described herein, the contact is preferably in vivo.

[0060] Gametes and / or their fertilization products may come into contact with mitochondrial targeters upon administration of the mitochondrial targeter to a mammal. A mammal may be one that is producing, will produce, and / or has produced gametes and / or contains (e.g., at the time of administration) their fertilization products.

[0061] When administered to a mammal containing the fertilized product of gametes, the mammal is a female mammal. Contacting gametes (preferably oocytes) and / or their fertilized product with the mitochondrial targeting agent preferably increases telomere length during embryonic development (e.g., of the subject described herein). By increasing telomere length during embryonic development (e.g., of the subject), the mitochondrial targeting agent can treat telomere-related disorders (e.g., of the subject).

[0062] Gametes may be found in the testes, ovaries, fallopian tubes, or uterus of mammals. Mammals containing the fertilized product of gametes include pregnant female mammals. The fertilized product may be found in the fallopian tubes or uterus of female mammals, preferably in the uterus.

[0063] Gametes and / or their fertilization products may be contacted with mitochondrial targeting agents before, during, and / or after fertilization. For example, mitochondrial targeting agents may be administered to subjects or mammals before, during, and / or after fertilization. Preferably, this occurs before, during, and after fertilization.

[0064] The term "subject" refers to a mammal, although the terms "subject" and "mammal" may be used interchangeably herein. This is because, in some examples (e.g., increasing telomere length and / or inhibiting telomere shortening and / or treating telomere-related disorders), the subject may be the individual being treated (a subject developed from an embryo as described herein), while mitochondrial targeting agents may be administered to different mammals. "Different mammals" may be mammals that produce, will produce, and / or have produced, and / or contain (e.g., at the time of administration) gametes, as described herein.

[0065] Mitochondrial targeting agents can be administered to achieve blood or plasma concentrations in the range of 1 nM to 500 μM, 10 nM to 500 μM, 100 nM to 500 μM, 500 nM to 500 μM, 1 μM to 500 μM, 1 μM to 100 μM, 5 μM to 100 μM, 10 μM to 100 μM, 50 μM to 100 μM, 1 μM to 50 μM, 5 μM to 50 μM, 10 μM to 50 μM, 1 μM to 10 μM, 5 μM to 10 μM, or 1 μM to 5 μM. Mitochondrial targeting agents can be administered to achieve blood or plasma concentrations in the following ranges: 0.1 μM to 30 μM, 0.5 μM to 30 μM, 1 μM to 30 μM, 5 μM to 30 μM, 10 μM to 30 μM, 20 μM to 30 μM, 0.1 μM to 20 μM, 0.5 μM to 20 μM, 1 μM to 20 μM, 5 μM to 20 μM, 10 μM to 20 μM, 0.1 μM to 10 μM, 0.5 μM to 10 μM, 1 μM to 10 μM, 5 μM to 10 μM, 0.1 μM to 5 μM, 0.5 μM to 5 μM, 1 μM to 5 μM, or 0.1 μM to 0.5 μM.

[0066] Mitochondrial targeting agents may be administered to achieve blood or plasma concentrations in the range of 1–25 μM, for example, 5–15 μM, for example, 10 μM.

[0067] Preferably, the mitochondrial targeting agent may be administered to achieve a blood or plasma concentration in the range of 5 to 100 μM, for example, 20 to 80 μM. The amount may be particularly appropriate for humans. The amount may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0068] Mitochondrial targeting agents may be administered to achieve blood or plasma concentrations in the range of 175–19,000 ng / ml, 184.6–18,460 ng / ml, 250–10,000 ng / ml, 300–6,000 ng / ml, 1,000–3,000 ng / ml, or 1,200–2,500 ng / ml. For example, a mitochondrial targeting agent may be administered to achieve blood or plasma concentrations in the range of 1,400–2,200 ng / ml, for example, 1,800–1,900 ng / ml. The amounts may be particularly relevant when the mitochondrial targeting agent is metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates.

[0069] Mitochondrial targeting agents can be administered in doses spanning one of the following select ranges: 0.1 μg / kg body weight to 1000 mg / kg body weight, 0.5 μg / kg body weight to 1000 mg / kg body weight, 1 μg / kg body weight to 1000 mg / kg body weight, 1 μg / kg body weight to 100 mg / kg body weight, 1 μg / kg body weight to 10 mg / kg body weight, 1 μg / kg body weight to 1 mg / kg body weight, 1 μg / kg body weight to 100 μg / kg body weight, 10 μg / kg body weight to 1000 mg / kg body weight, 10 μg / kg body weight to 100 mg / kg body weight, 10 μg / kg body weight to 10 mg / kg body weight Body weight in kg, 10 μg / kg to 1 mg / kg, 10 μg / kg to 100 μg / kg, 10 μg / kg to 1000 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 10 mg / kg, 100 μg / kg to 1 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 1000 mg / kg, 10 mg / kg to 100 mg / kg, and 100 mg / kg to 1000 mg / kg. Mitochondrial targeting agents may be administered in doses spanning one of the following select ranges: 0.1 mg / kg to 10 mg / kg, 0.5 mg to 10 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.5 mg / kg to 5 mg / kg, 1 mg / kg to 5 mg / kg, 0.1 mg / kg to 1 mg / kg, or 0.5 mg / kg to 1 mg / kg.

[0070] Mitochondrial targeting agents may be administered in amounts ranging from 50 mg / kg to 150 mg / kg, for example, at 100 mg / kg. The aforementioned amounts may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0071] Mitochondrial targeting agents may be administered in amounts of 0.1 to 100 mg / kg or 1 to 100 mg / kg, for example, 2 to 50 mg / kg, for example, 5 to 25 mg / kg. The amounts may be particularly relevant when the mitochondrial targeting agent is MitoQ and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0072] Mitochondrial targeting agents may be administered in amounts of 1 to 600 mg / kg or 50 to 550 mg / kg, for example, 100 to 500 mg / kg, for example, 150 to 450 mg / kg. The amounts may be particularly relevant when the mitochondrial targeting agent is metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0073] Mitochondrial targeting agents may be administered once daily, twice daily, multiple times daily, or consecutively.

[0074] An appropriate duration of administration may be selected. Mitochondrial targeting agents may be administered once or over a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks. Mitochondrial targeting agents may be administered over a continuous period.

[0075] Mitochondrial targeting agents may be administered to female mammals over a period beginning before ovulation. For example, mitochondrial targeting agents may be administered to female mammals before, during, and after ovulation. Mitochondrial targeting agents may be administered to female mammals over a period beginning at least 1, 2, 3, 4, 5, 6 days, 1, 2, 3, or 4 weeks (preferably at least 5 days, e.g., 4 days) before ovulation.

[0076] Mitochondrial targeting agents are administered in doses within one of the following select ranges: 0.1 μg / kg / body weight / day to 1000 mg / kg / body weight / day, 0.5 μg / kg / body weight / day to 1000 mg / kg / body weight / day, 1 μg / kg / body weight / day to 1000 mg / kg / body weight / day, 1 μg / kg / body weight / day to 100 mg / kg / body weight / day, 1 μg / kg / body weight / day to 10 mg / kg / body weight / day, 1 μg / kg / body weight / day to 1 mg / kg / body weight / day, 1 μg / kg / body weight / day to 100 μg / kg / body weight / day, 10 μg / kg / body weight / day to 1000 mg / kg / body weight / day, 10 μg / kg / body weight / day to 100 mg / kg / body weight / day, 10 μg / kg / body weight / day to 10 mg / kg / body weight / day / day, 10μg / kg / body weight / day to 1mg / kg / body weight / day, 10μg / kg / body weight / day to 100μg / kg / body weight / day, 10μg / kg / body weight / day to 1000mg / kg / body weight / day, 100μg / kg / body weight / day to 100mg / kg / body weight / day, 100μg / kg / body weight / day to 10mg / kg / body weight / day, 100μg / kg / body weight / day to 1mg / kg / body weight / day, 1mg / kg / body weight / day to 1000mg / kg / body weight / day, 1mg / kg / body weight / day to 10mg / kg / body weight / day, 10mg / kg / body weight / day to 1000mg / kg / body weight / day, and 100mg / kg / body weight / day to 1000mg / kg / body weight / day. Mitochondrial targeting agents may be administered in doses spanning one of the following select ranges: 0.1 mg / kg / day to 10 mg / kg / day, 0.5 mg / kg / day to 10 mg / kg / day, 1 mg / kg / day to 10 mg / kg / day, 5 mg / kg / day to 10 mg / kg / day, 0.1 mg / kg / day to 5 mg / kg / day, 0.5 mg / kg / day to 5 mg / kg / day, 1 mg / kg / day to 5 mg / kg / day, 0.1 mg / kg / day to 1 mg / kg / day, or 0.5 mg / kg / day to 1 mg / kg / day.

[0077] Mitochondrial targeting agents may be administered in amounts ranging from 50 mg / kg / day to 150 mg / kg / day, for example, 100 mg / kg / day. The aforementioned amounts may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0078] Mitochondrial targeting agents may be administered in amounts of 0.1 to 100 mg / kg / day or 1 to 100 mg / kg / day, for example, 2 to 50 mg / kg / day, for example, 5 to 25 mg / kg / day. The amounts may be particularly relevant when the mitochondrial targeting agent is MitoQ and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0079] Mitochondrial targeting agents may be administered in amounts of 1 to 600 mg / kg / day or 50 to 550 mg / kg / day, for example, 100 to 500 mg / kg / day, for example, 150 to 450 mg / kg / day. The amounts may be particularly relevant when the mitochondrial targeting agent is metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0080] Preferably, the mitochondrial targeting agent may be administered in amounts of 10-1000 ng, 50-500 ng, or 100-300 ng. More preferably, the mitochondrial targeting agent may be administered in amounts of 150-250 ng (e.g., 200 ng). The amounts are preferably administered on a daily basis and most preferably orally. The amounts may be particularly suitable for humans. The amounts may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates.

[0081] Mitochondrial targeting agents may be administered in appropriate forms. In this regard, the term “administering” (and similar terms) includes administering an agent that forms an effective amount of the desired mitochondrial targeting agent in vivo, or administering a prodrug or derivative. This term includes systemic (e.g., by injection, such as intravenous injection, orally in tablets, pills, capsules, or other dosage forms useful for systemic administration) and topical (e.g., creams, solutions, pastes, ointments (including solutions such as mouthwash for topical oral administration)) routes of administration.

[0082] Mitochondrial targeting agents are preferably administered orally. Mitochondrial targeting agents may be administered intravenously. Mitochondrial targeting agents may be administered by injection, such as intravenous injection. Mitochondrial targeting agents may be administered by direct introduction into the lungs. Mitochondrial targeting agents may be administered by spray, aerosolization, or infusion into the lungs. Mitochondrial targeting agents may be administered parenterally. Mitochondrial targeting agents may be administered by implant. Mitochondrial targeting agents may be administered subcutaneously (e.g., by subcutaneous injection), intratracheally, parenterally, intra-articularly, rectally, intranasally, intraocularly, vaginally, or transdermally. The methods of administration are known in the art.

[0083] "Intravenous administration" refers to the direct administration of a substance into a vein.

[0084] "Oral administration" refers to a route of administration in which a substance is taken in through the mouth, and includes buccal, sublabial, and sublingual administration, as well as enteral administration. Typical forms for oral administration of therapeutic agents include the use of tablets or capsules.

[0085] Mitochondrial targeting agents may be administered alone or in mixtures with other therapeutic agents and / or drugs that enhance, stabilize, or maintain the activity of the agent, for example. A delivery vehicle containing both the mitochondrial targeting agent and the additional agent may be used (e.g., pills, tablets, implants, injectable solutions, etc.). Mitochondrial targeting agents and additional agents may be administered together.

[0086] The present invention may also encompass combination therapies. In other words, in some embodiments, the present invention may include the simultaneous administration of a mitochondrial targeting agent and a further agent. In this regard, another drug or treatment modality may be administered in combination with the mitochondrial targeting agent. This combination therapy may be a sequential therapy in which the treatment is performed by a first treatment modality followed by the other treatment modality, or two or more treatment modalities may be administered simultaneously.

[0087] "Simultaneous administration" refers to administering two or more therapeutic agents together at one time. Two or more therapeutic agents may be co-formulated into a single dosage form or a "combination dosing unit," or they may be formulated separately and then combined into a combination dosing unit, typically for intravenous or oral administration.

[0088] When administered, the therapeutically effective dose of a drug may vary depending on the specific drug used, mode of administration, condition and its severity, and various physical factors related to the person being treated. The dose is expected to vary along with the route of administration and the properties of the drug administered and any other drugs administered. In certain embodiments, mitochondrial targeting agents may be administered in escalating and / or repeated doses.

[0089] Mitochondrial targeting agents can be administered as continuous-release formulations. Mitochondrial targeting agents can be administered as immediate-release formulations. The term "immediate-release formulation" refers to a formulation designed to rapidly release a therapeutic agent into the body over a short period of time. Immediate-release formulations are well known in the art. Mitochondrial targeting agents can be administered as sustained-release formulations. The term "sustained-release formulation" refers to a formulation designed to slowly release a therapeutic agent over a long period of time. Sustained-release formulations are well known in the art.

[0090] Mitochondrial targeting agents can be administered as pharmaceutically acceptable salts. In this context, the term "pharmaceutically acceptable salt" refers to acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of metal complexes include zinc and iron. Acids suitable for use in the preparation of pharmaceutically acceptable salts include acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, and oxo-glutamic acid. Examples include uric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, sugar acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, suberic acid, and valeric acid.

[0091] Suitable bases for use in the preparation of pharmaceutically acceptable salts include inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide, and organic bases, such as primary, secondary, tertiary, and quaternary aliphatic and aromatic amines, such as L-arginine, benetamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, iso Examples include propylamine, N-methyl-glucamine, hydravamin, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

[0092] Most preferably, the mitochondrial targeting agent may be administered as part of a pharmaceutical composition. The pharmaceutical composition may be particularly suitable for in vivo use. The pharmaceutical composition may contain an effective amount (e.g., a prophylactic or therapeutic effective dose) of the mitochondrial targeting agent. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier and / or suitable excipients. The mitochondrial targeting agent may be present in an amount that provides a suitable dose (e.g., blood or plasma concentration) as described herein. The pharmaceutical composition may be suitable for administration by one or more of the routes of administration described herein. Preferably, the pharmaceutical composition is an oral pharmaceutical composition.

[0093] The carrier may be selected based on various considerations, including the route of administration, the drug to be delivered, and the time course of drug delivery. The term “pharmaceutically acceptable carrier” refers to any type of filler, diluent, excipient, encapsulating material, or formulation aid that is substantially inert, solid, semi-solid, or liquid. An example of a pharmaceutically acceptable carrier is saline. Other physiologically acceptable carriers and their formulations are known in the art. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; surfactants, such as TWEEN 80; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer solution; and other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; as well as colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances; preservatives and antioxidants.

[0094] Suitable excipients for use in tablet form include the following tablet cores and film coats: Tablet core - corn starch, pregelatinized starch, sodium starch glycolate, povidone, glycerol dibehenate, magnesium stearate; Film coat - hypromellose, glycerol triacetic acid, talc, titanium dioxide (E171), yellow iron oxide (E172), red iron oxide (E172), and ethylcellulose. Tablets containing mitochondrial targeting agents may include a tablet core containing a certain amount of the drug as a hydrate, as well as corn starch, pregelatinized starch, sodium starch glycolate, povidone, glycerol dibehenate, and magnesium stearate, and a film coat containing hypromellose, glycerol triacetic acid, talc, titanium dioxide (E171), yellow iron oxide (E172), red iron oxide (E172), and ethylcellulose.

[0095] Mitochondrial targeting agents administered or present in pharmaceutical compositions may be pharmaceutically acceptable hydrates. Hydrates are solid adducts containing both the parent compound (e.g., the anhydrous form of the drug or excipient) and water.

[0096] The pharmaceutical compositions described herein may include other therapeutic agents and / or drugs that enhance, stabilize, or maintain the activity of mitochondrial targeting agents.

[0097] The oral formulations described herein may include any conventionally used oral form, such as tablets, capsules, buccal forms, lozenges, and oral liquids, suspensions, or solutions. Capsules may contain a mixture of the active compound with pharmaceutically acceptable starches (e.g., corn starch, potato starch, or tapioca starch), sugars, artificial sweeteners, powdered cellulose such as crystalline and microcrystalline cellulose, powders, gelatin, gum, and / or diluents. Useful tablet formulations can be prepared by conventional compression, wet granulation, or dry granulation methods, utilizing pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), suspending agents, or stabilizers, such as magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, calcium carboxymethylcellulose, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicate, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dried starch, and powdered sugar. Nonionic and anionic surface modifiers are also available. Typical examples of surface modifiers include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphate, sodium dodecyl sulfate, aluminum magnesium silicate, and triethanolamine. Oral formulations may utilize standard delayed or time-release formulations to alter peptide absorption. Oral formulations may also include mitochondrial targeting agents in water or fruit juice containing appropriate solubilizers or emulsifiers, as needed. Oral formulations are known in the art and can be formulated by those skilled in the art.

[0098] Formulations for administering aerosols are known in the art and can be formulated by those skilled in the art.

[0099] The composition may be administered parenterally (e.g., directly into the joint cavity) or intraperitoneally. For example, a solution or suspension of the drug in a non-ionized form or as a pharmacokinetically acceptable salt may be prepared by appropriately mixing it in water with a surfactant such as hydroxypropyl cellulose. Dispersions in oil may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof. Under normal storage and use conditions, these preparations typically contain preservatives to prevent microbial growth. Parenteral formulations are known in the art and can be formulated by those skilled in the art.

[0100] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, isotonic saline, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Injectable formulations are known in the art and can be formulated by those skilled in the art.

[0101] Compositions suitable for intravenous administration are known in the art and can be formulated by those skilled in the art. For example, isotonic saline can be used in intravenous compositions containing an antagonist.

[0102] Transdermal administration is understood to include all administrations that extend beyond the surface of the body and the inner layers of body passages, including epithelial and mucous membrane tissues. Such administrations may be carried out using mitochondrial targeting agents or pharmaceutically acceptable salts thereof as described herein in the form of lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal). Transdermal administration may also be achieved by the use of transdermal patches containing the mitochondrial targeting agent and a carrier that is inert to the active compound, non-toxic to the skin, and enables delivery of the drug for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms, such as creams and ointments, pastes, gels, and occlusion devices. Creams and ointments may be viscous liquids or semi-solid emulsions of either oil-in-water or water-in-oil type. Pastes consisting of absorbable powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. Various occlusion devices can be used to release the active ingredient into the bloodstream, such as a semipermeable membrane covering a reservoir containing the active ingredient with or without the carrier, or a matrix containing the active ingredient. Transdermal formulations are well known in the art and can be formulated by those skilled in the art.

[0103] Pharmaceutical compositions may also be administered by suppositories. Suppository formulations may be prepared from conventional materials containing cocoa butter, with or without the addition of waxes and glycerin to alter the melting point of the suppository. Water-soluble suppository bases such as polyethylene glycol of various molecular weights may also be used. Suppository formulations are known in the art and can be formulated by those skilled in the art.

[0104] Numerous additional excipients, dosage forms, and dispersants may be suitable for use in connection with the administration and / or formulation of mitochondrial targeting agents into pharmaceuticals or pharmaceutical compositions. Formulations are publicly known, for example, Remington's Pharmaceutical Sciences, 17 th This is described in ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference in its entirety.

[0105] Gametes can be sperm. Sperm can be obtained from male mammals. As used herein, the term "sperm" refers to male germ cells or spermatids, and includes one or more spermatids.

[0106] If the gametes are sperm, the sperm may come into contact with the mitochondrial targeting agent upon administration of the agent to a male mammal. The male mammal is producing, will produce, and / or may have produced sperm at the time of administration.

[0107] The gametes are preferably oocytes. Oocytes can be obtained from female mammals. Oocytes may be part of a cumulus-oocyte complex. In certain embodiments, the oocytes are naked oocytes. Methods for isolating cumulus cells from a cumulus-oocyte complex are known. As used herein, the term “oocyte” includes oocytes alone or oocytes associated with one or more other cells, such as oocytes as part of a cumulus-oocyte complex.

[0108] If the gametes are oocytes, the oocytes can come into contact with the mitochondrial targeting agent by administering the agent to female mammals.

[0109] Mammals can be donors. Therefore, the gametes used in this invention can be obtained from mammals. The donor can be a sperm donor or an oocyte donor.

[0110] Mitochondrial targeting agents can be administered to mammals, and after fertilization with gametes obtained from the mammals (e.g., from different mammals), telomere length may increase during embryonic development. For example, a male mammal may be administered a mitochondrial targeting agent, the male may mate with a female mammal, and after fertilization of the female's oocytes by the male's sperm, telomere length may increase during embryonic development. In another example, a male mammal may be administered a mitochondrial targeting agent, the male's sperm may be obtained and used in an assisted reproductive method, and telomere length may increase during embryonic development in the method. For example, a female mammal may be administered a mitochondrial targeting agent, the female may mate with a male mammal, and after fertilization of the female's oocytes by the male's sperm, telomere length may increase during embryonic development. In yet another example, a female mammal may be administered a mitochondrial targeting agent, the female's oocytes may be obtained and used in an assisted reproductive method, and telomere length may increase during embryonic development in the method.

[0111] The mammal may be a non-mouse mammal. The mammal may be a domesticated mammal such as a livestock mammal or a pet mammal, or a wild mammal species. Preferably, the mammal is a female mammal. The mammal may be a human, horse, monkey, cattle (including bulls), pig, dog, cat, sheep, goat, elephant, panda, mouse, rabbit, rat, or other mammal. Preferably, the mammal is a human, horse, or cattle. Most preferably, the mammal is a human. The mammal is preferably an adult mammal, for example, a human at least 18 years old.

[0112] The gametes and / or their fertilized products may be gametes and / or their fertilized products of non-mouse mammals. The gametes and / or their fertilized products may be gametes and / or their fertilized products of domesticated mammals, domesticated mammals (e.g., pets), or wild mammal species. The gametes and / or their fertilized products may be gametes and / or their fertilized products of humans, horses, monkeys, cattle (including bulls), pigs, dogs, cats, sheep, goats, elephants, pandas, mice, rabbits, or rats. Preferably, the gametes and / or their fertilized products are human, horse, or cattle gametes and / or their fertilized products. Most preferably, the gametes and / or their fertilized products are human gametes and / or their fertilized products.

[0113] Mammals are preferably of reduced fertility. The gametes used in the present invention can preferably be obtained from mammals of reduced fertility. In other words, the mammals may be suffering from or susceptible to conditions associated with reduced fertility and / or reduced gamete quality. The mammals may be overweight or obese, have abnormal levels and / or rates of gamete production, have polycystic ovary syndrome, have metabolic syndrome, have reduced fertility, have low fertility, have infertility, have ovarian dysfunction, have anovulation, have a reduced ovulation rate, have prediabetes, have diabetes, have hyperandrogenicity, have insulin resistance, have impaired glucose tolerance, have abnormal blood glucose levels (e.g., elevated blood glucose), have hyperinsulinemia, have dyslipidemia, have low ovarian reserve, have premature ovarian failure, have ovarian aging, have poor sperm quality (e.g., poor sperm motility, viability and / or fertilization ability, and insufficient sperm morphology and / or DNA integrity), have a low sperm count, have been exposed to a high-fat diet, be aged, have never smoked and / or do not smoke, and / or have a history of recurrent miscarriage.

[0114] Mammals may be aged. The gametes used in this invention can be obtained from aged mammals. Aged human mammals may be at least 30, 35, 40, or 45 years old, for example, 35–48 years old, for example, 38–45 years old. Aged human mammals may be over 30, 35, 40, or 45 years old. Mammals may be suffering from or susceptible to aging conditions associated with reduced reproductive capacity.

[0115] The mammals may be mammals that smoke and / or have smoked. The gametes used in the present invention can be obtained from mammals that smoke and / or have smoked. The mammals may be suffering from or susceptible to conditions associated with smoking-related fertility reduction and related pathologies.

[0116] Mammals can be overweight. The gametes used in this invention can be obtained from overweight mammals. Overweight human mammals weigh 25 kg / m². 2 Extremely, up to 30 kg / m 2 They may have a body mass index (BMI).

[0117] Mammals can be obese. The gametes used in this invention can be obtained from obese mammals. Obese human mammals weigh 30 kg / m². 2 They may have a body mass index (BMI) of over 10.

[0118] Depending on the circumstances, gametes and / or their fertilization products may be in contact with a mitochondrial targeting agent at one or more developmental stages. For example, gametes and their later-formed fertilization products may be in contact with a mitochondrial targeting agent. Such contact may be continuous or semi-continuous, and preferably continuous.

[0119] The fertilization product of gametes may be a zygote or an embryo. Preferably, the fertilization product is a zygote. The term “zygote” is used herein to refer to a fertilized cell containing both sperm DNA and oocyte DNA before the two-cell break stage. Thus, the term “zygote” is intended to encompass fertilized cells before, during, or after gamete fusion. “Gamete fusion” is defined as the fusion of sperm pronuclei and oocyte pronuclei. The pronuclear development stage, including PN5, occurs before gamete fusion.

[0120] The gametes and / or zygotes may be in contact with a mitochondrial targeting agent. Such contact is preferably before gamete fusion. More preferably, the contact is before PN5, and more preferably only before PN5. Thus, the contact may include contact of the mitochondrial targeting agent with the gametes and the zygotes up to the later-formed PN5 (e.g., continuous contact). Most preferably, the contact occurs between fertilization and PN5 (e.g., continuously between fertilization and PN5).

[0121] Advantageously, the inventors have found that contact with mitochondrial targeting agents (particularly BGP-15) prior to gamete fusion is particularly effective in increasing telomere length and / or treating telomere-related disorders.

[0122] The term "embryo" is used herein to refer to the developmental stage beginning with a two-cell division. An embryo may be any stage, including the two-cell division stage, the four-cell division stage, the eight-cell division stage, the morula, and the blastocyst, as well as an intermediate stage.

[0123] The embryo may exhibit normal or abnormal developmental dynamics and species-specific developmental morphology. The embryo may be a blastocyst-stage embryo. The embryo may be a pre-blastocyst-stage embryo.

[0124] Preferably, the embryo is a preimplantation embryo. A preimplantation embryo may be an embryo between the two-cell cutting stage and the blastocyst stage (e.g., including the two-cell cutting and blastocyst stages). In humans, implantation typically occurs in vivo approximately 8 to 10 days after fertilization.

[0125] The gametes can be brought into contact with the mitochondrial targeting agent. Preferably, only the gametes are brought into contact with the mitochondrial targeting agent.

[0126] The fertilized product of the gametes can be contacted with a mitochondrial targeter during embryonic development from the 8-cell stage to the blastocyst stage and / or implantation (preferably before thereafter).

[0127] The term "from the 8-cell stage" is intended to include the 8-cell stage.

[0128] As used in this specification, the term "up to" includes "including up to".

[0129] The fertilized product of the gametes can be contacted with a mitochondrial targeter after the 8-cell stage of embryonic development and up to (preferably before) the blastocyst stage and / or implantation.

[0130] The fertilized product of the gametes can be contacted with a mitochondrial targeter during embryonic development, from the morula stage to the blastocyst stage and / or implantation (preferably before thereafter).

[0131] The fertilized product of the gametes can be contacted with a mitochondrial targeter during embryonic development, from the morula stage onward to the blastocyst stage and / or implantation (preferably before).

[0132] Preferably, the fertilized product of the gamete can be in contact with the mitochondrial targeting agent only during the 8-cell stage of embryonic development, the blastocyst stage, and / or before implantation.

[0133] Mitochondrial targeting agents can be administered to female mammals before implantation of the gamete fertilization product. Mitochondrial targeting agents can be administered to female mammals containing the fertilization product from the 8-cell stage to implantation (preferably before). Mitochondrial targeting agents can be administered to female mammals containing the fertilization product after the 8-cell stage and up to implantation (preferably before). Mitochondrial targeting agents can be administered to female mammals containing the fertilization product from the morula stage to implantation (preferably before). Mitochondrial targeting agents can be administered to female mammals containing the fertilization product after the morula stage and up to implantation (preferably before).

[0134] Embryos described herein preferably have increased telomere length after the invention is carried out. Telomere length may be increased compared to embryos obtained by different methodologies, for example, when the gametes and / or their fertilization products have not been in contact with the mitochondrial targeting agent of the invention. Such embryos may have telomere length that is at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% longer. Telomere length may be increased in the heart and / or liver cells of the embryo.

[0135] The embryos described herein preferably develop into fetuses having increased telomere length. Telomere length may be increased compared to fetuses obtained by different methodologies, for example, when the gametes and / or their fertilization products are not in contact with the mitochondrial targeting agent of the present invention. The fetuses may have telomere lengths that are at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% longer. Telomere length may be increased in the fetal heart and / or liver cells.

[0136] The internal cell mass (ICM) of the embryo described herein may show an increase in telomere length.

[0137] A suitable method for assaying telomere length (e.g., qPCR) is described in this example (see Figure 4).

[0138] Typically, as part of the natural aging process, telomere length decreases over time (so-called telomere loss). Mitochondrial targeting agents can inhibit this decrease (i.e., inhibit telomere shortening). Any such inhibition may be partial or complete. In other words, mitochondrial targeting agents can prevent or delay the decrease in telomere length of a subject or cell. After contact with or administration of a mitochondrial targeting agent, telomeres may become, for example, at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% longer compared to (i) the telomere length of homogeneous cells not in contact with the mitochondrial targeting agent and / or (ii) the telomere length of homogeneous subjects not administered with the mitochondrial targeting agent.

[0139] Mitochondrial targeting agents can increase the telomere length of a target or cell. This increase in telomere length can be achieved by promoting telomere elongation. Preferably, telomere length can be increased during the embryonic development of the target, for example, by promoting telomere elongation during the embryonic development of the target. The increase may be, for example, an increase of at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% of telomere length compared to (i) the telomere length of the target before administration of the mitochondrial targeting agent, (ii) the telomere length of cells before contact with the mitochondrial targeting agent, (iii) the telomere length of homogeneous cells not in contact with the mitochondrial targeting agent, and / or (iv) the telomere length of homogeneous subjects not administered with the mitochondrial targeting agent. Similarly, telomere elongation may increase by at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% compared to (i) the telomere length of the subject before administration of the mitochondrial targeting agent, (ii) the telomere length of cells before contact with the mitochondrial targeting agent, (iii) the telomere length of homogeneous cells not in contact with the mitochondrial targeting agent, and / or (iv) the telomere length of homogeneous subjects not administered with the mitochondrial targeting agent.

[0140] A “mitochondrial targeting agent” can be any agent that functionally interacts with one or more mitochondria in a cell to achieve its therapeutic effect. A mitochondrial targeting agent can physically interact with one or more mitochondria in a cell. Preferably, a mitochondrial targeting agent can reduce the level of mitochondrial reactive oxygen species in a cell, preferably a cell having an elevated level of mitochondrial reactive oxygen species. The reduction may be, for example, a reduction of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the level of mitochondrial reactive oxygen species in a cell compared to (i) the level of mitochondrial reactive oxygen species in a cell before contact with or administration of the mitochondrial targeting agent, and / or (ii) the level of mitochondrial reactive oxygen species in an equal cell that has not been contacted with or administered the mitochondrial targeting agent. Mitochondrial targeting agents can increase the mitochondrial membrane potential of cells (preferably embryos, e.g., those present in embryos at the morula stage or later, and / or preferably cells with reduced mitochondrial membrane potential) by at least 5%, 10%, 20%, 30%, 40%, or 50%, for example, by (i) the mitochondrial membrane potential of cells before contact with or administration of the mitochondrial targeting agent, and / or by (ii) the mitochondrial membrane potential of homogeneous cells that have not been contacted with or administered the mitochondrial targeting agent. Mitochondrial targeting agents can alter the epigenetic state of cells (e.g., nuclear epigenetic state), such as the DNA methylation level of gametes and / or their fertilization products, and preferably the cells are cells having an altered (e.g., abnormal) epigenetic state. For example, mitochondrial targeting agents can alter the DNA methylation level of zygotes, and preferably the zygotes are zygotes having an altered (e.g., abnormal) DNA methylation level. Mitochondrial targeting agents may reduce 5-methylcytosine (5mC) levels and / or increase 5-hydroxymethylcytosine (5hmC) levels in gametes and / or their fertilized products.For example, a mitochondrial targeting agent can reduce 5-methylcytosine (5mC) levels by at least 10%, 20%, 30%, 40%, or 50% compared to (i) the 5-methylcytosine (5mC) levels of cells before contact with or administration of the mitochondrial targeting agent, and / or (ii) the 5-methylcytosine (5mC) levels of homogeneous cells that have not been contacted with or administered the mitochondrial targeting agent. For example, a mitochondrial targeting agent can increase 5-hydroxymethylcytosine (5hmC) levels by at least 10%, 20%, 30%, 40%, or 50% compared to (i) the 5-hydroxymethylcytosine (5hmC) levels of cells before contact with or administration of the mitochondrial targeting agent, and / or (ii) the 5-hydroxymethylcytosine (5hmC) levels of homogeneous cells that have not been contacted with or administered the mitochondrial targeting agent.

[0141] Accordingly, in one embodiment, the present invention provides a mitochondrial targeting agent for use in altering the epigenetic state of a cell (e.g., nuclear epigenetic state), wherein the use preferably involves contacting a cell with the mitochondrial targeting agent. In a related embodiment, a method is provided for altering the epigenetic state of a cell (e.g., nuclear epigenetic state), comprising contacting a cell with the mitochondrial targeting agent (e.g., via administration to subjects and / or mammals as described herein). In another related embodiment, a use is provided for the use of a mitochondrial targeting agent in the manufacture of a pharmaceutical for altering the epigenetic state of a cell (e.g., nuclear epigenetic state), preferably comprising contacting a cell with the mitochondrial targeting agent. In the aforementioned embodiment, the contact is preferably in vivo. The contact can be achieved by administering the mitochondrial targeting agent to subjects and / or mammals as described herein. In another embodiment, a method is provided for altering the epigenetic state of a cell (e.g., nuclear epigenetic state) (preferably in vitro), comprising contacting a cell with a mitochondrial targeting agent in vitro. In another embodiment, a use is provided (preferably in vitro) of a mitochondrial targeter to alter the epigenetic state of a cell (e.g., nuclear epigenetic state), comprising contacting the cell with the mitochondrial targeter in vitro. The epigenetic state is preferably a DNA methylation level as described above. Such alteration is preferably a reversal (including partial or complete reversal) of an abnormal epigenetic state. The cell may be a gamete and / or its fertilization product. Preferably, the cell is a cell having an altered (e.g., abnormal) epigenetic state. More preferably, the mitochondrial targeter can alter the DNA methylation level of a zygote, and preferably the zygote is a zygote having an altered (e.g., abnormal) DNA methylation level.

[0142] Mitochondrial targeting agents may include (or consist of) mitocinone mesylate (MitoQ) and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates. For example, a mitochondrial agent may be MitoQ or its acceptable prodrugs, solvates, salts, tautomers, stereoisomers, or racemates. A mitochondrial targeting agent may be MitoQ. MitoQ (mitoquinone mesylate: [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadiene-1-yl)decyl](triphenyl)phosphonium methanesulfonate) is a ubiquinone moiety linked to a lipophilic triphenylphosphonium cation by a 10-carbon alkyl chain that preferentially accumulates in mitochondria (MP Murphy, RA Smith, Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu Rev Pharmacol Toxicol 47, 629-656 (2007)). MitoQ is commercially available from MitoQ Ltd, New Zealand. MitoQ may have CAS number 845959-50-4.

[0143] Mitochondrial targeting agents may include (or consist of) metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates. For example, a mitochondrial agent may be metformin or its acceptable prodrugs, solvates, salts, tautomers, stereoisomers, or racemates. A mitochondrial targeting agent may be metformin. Metformin (1,1-dimethyl biguanide) may exist as an HCl salt (1,1-dimethyl biguanide hydrochloride). The HCl salt of metformin has CAS number 1115-70-4. The aforementioned salt of metformin is commercially available from Sigma-Aldrich (D150959).

[0144] Preferably, the mitochondrial targeting agent is BGP-15(O-[3-piperidino-2-hydroxy-1-propyl]-nicotinamidoxime): [ka] and / or acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates thereof (or comprising thereof). For example, the mitochondrial targeting agent may be BGP-15 or an acceptable prodrug, solvate, salt, tautomer, stereoisomer or racemate thereof. Most preferably, the mitochondrial targeting agent is BGP-15.

[0145] BGP-15 is commercially available (typically as a suitable salt) or can be synthesized by methods known in the art. Examples of commercial sources of BGP-15 include Hangzhou Molcore Biopharmatech Co Ltd, Sigma-Aldrich (product number B4813 SIGMA), and Cayman Chemicals (product number 17503). BGP-15 may have a CAS number of 66611-37-8.

[0146] Derivatives of BGP-15 may include (or consist of) bimoclomol, alimochromal, NG-94, iloxanadine, and / or any pharmaceutically acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates. For example, a mitochondrial agent may be bimoclomol, alimochromal, NG-94, or iloxanadine, or an acceptable prodrug, solvate, salt, tautomer, stereoisomer, or racemate thereof.

[0147] The chemical structures of bimocromol, alimochromal, NG-94, and iloxanadine are as follows. [ka]

[0148] The above compounds can be synthesized by methods known in the art, or are commercially available.

[0149] Bimocromol ((3Z)-N-(2-hydroxy-3-piperidine-1-ylpropoxy)pyridine-3-carboximidoyl chloride) can be synthesized by methods known in the art, for example, the method described in Hungarian Patent No. 207988 (1988). Alimocromol (3-[chloro({[(2R)-2-hydroxy-3-(piperidine-1-yl)propoxy]imino})methyl]pyridine-1-ium-1-olate) can be synthesized by methods known in the art, for example, the method described in International Publication No. 0179174 or Tetrahedron: Asymmetr. 2012, 23: 1564-1570. NG-094 can be synthesized by methods known in the art. Iloxanaazine can be synthesized by methods known in the art and is commercially available from 360 Reagent.

[0150] Depending on the circumstances, multiple different types of mitochondrial targeting agents may be used in the present invention. For example, the present invention may use at least two, three, or four different types of mitochondrial targeting agents. The term “different types of mitochondrial targeting agents” is intended to include acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates of a given mitochondrial targeting agent, as well as structurally unrelated mitochondrial targeting agents. Depending on the circumstances, different types of mitochondrial targeting agents may be structurally unrelated.

[0151] Mitochondrial targeting agents, gametes, and / or their fertilized products may be used in or as part of assisted reproductive methods or techniques. Similarly, the in vitro methods or uses described herein may be assisted reproductive methods or uses. Examples of assisted reproductive techniques include artificial insemination (also known as intrauterine insemination), in vitro fertilization (IVF), intrafallopian gamete transfer (GIFT), placement of oocytes and sperm into the fallopian tube, intrafallopian zygote transfer (ZIFT), tubal embryo transfer (TET), peritoneal oocyte and sperm transfer (POST), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA). Assisted reproductive technologies are publicly known in this field and are described, for example, in Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003) Editors Gardner, DK, Weissman, A., Howies, CM., Shoham, Z. Martin Dunits Ltd, London, UK and Gordon, I. (2003) Laboratory Production of Cattle Embryos 2nd Edition CABI Publishing, Oxon, UK.

[0152] Depending on the circumstances, mitochondrial targeting agents, gametes, and / or their fertilized products may be used in or as part of in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or intrauterine insemination (IUI). Depending on the circumstances, the in vitro methods or uses described herein may include in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

[0153] IVF, which refers to the in vitro fertilization of oocytes, typically involves incubating oocytes isolated from mammals in a liquid medium containing sperm to enable fertilization. In some cases, sperm fertilization of oocytes can occur more than 24 hours after the oocyte collection step, but usually within 60 hours, ensuring that the oocytes are mature enough to maximize the success of subsequent steps in the IVF procedure.

[0154] Methods for performing assisted reproductive technologies in both humans and non-human mammals are known in the art.

[0155] By increasing telomere length according to the present invention, the developmental capacity of gametes and / or fertilized products (e.g., embryos) may be improved. For example, such improvement may be the ability of gametes to produce zygotes that complete cleavage and fertilization within the optimal time frame for the species ("on-time"), the ability of two-cell embryos to complete blastocyst development within the optimal time frame for the species ("on-time"), and / or the ability of blastocysts to initiate hatching.

[0156] In some embodiments, the gametes and / or their fertilized bodies are contacted in vitro with a mitochondrial targeting agent. In the in vitro method or use, the gametes and / or their fertilized bodies are preferably contacted in vitro with a mitochondrial targeting agent.

[0157] Gametes and / or their fertilization products may be brought into contact with a mitochondrial targeter in any suitable manner that allows the mitochondrial targeter to function. For example, contact may occur in a liquid medium in which the gametes are exposed to a precursor mitochondrial targeter that has been altered or metabolized into an activator, or in which the gametes are exposed to a mitochondrial targeter that induces further drug expression.

[0158] The gametes and / or their fertilized products may be contacted with a mitochondrial targeting agent before, during, and / or after in vitro fertilization. Preferably, this contact occurs before, during, and after in vitro fertilization.

[0159] Gametes and / or their fertilized products may be in contact with a mitochondrial targeter for at least 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 10 hours, 15 hours, 24 hours, 2 days, 4 days, 5 days, or 10 days. Gametes and / or their fertilized products may be in contact with a mitochondrial targeter for up to 10 days, 5 days, 4 days, 2 days, 24 hours, 15 hours, 10 hours, 3 hours, 2 hours, or 1 hour. Gametes and / or their fertilized products may be in contact with a mitochondrial targeter for 1 to 10 days or 5 to 8 days.

[0160] The gametes and / or their fertilized products may be in contact with the mitochondrial targeting agent for a period of 24 hours or less, 18 hours or less, 12 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. Preferably, the gametes and / or their fertilized products may be in contact with the mitochondrial targeting agent for a period of 10 minutes to 24 hours, 30 minutes to 24 hours, 1 to 20 hours, 2 to 15 hours, or 3 to 10 hours, preferably 5 to 7 hours, for example, 6 hours.

[0161] If the gamete is sperm, the sperm may be present in semen or in a diluted form thereof. Sperm may be enriched with specific characteristics such as motility, viability, vitality, fertilization ability, increased or higher DNA integrity, or reduced or lower DNA quality. For example, sperm may be sorted by density gradient centrifugation, swim-up techniques, or flow cytometry. Methods for sorting sperm are known in the art. Sperm may be present in sperm preparation media. Sperm preparation media are known in the art and are commercially available. In certain embodiments, sperm are present in sperm washing media. Sperm may be contacted with mitochondrial targeting agents before, during, and / or after fertilization ability. Sperm may be contacted with mitochondrial targeting agents before, during, and / or after maturation.

[0162] Gametes and / or their fertilized products may be present in in vitro fertilization (IVF) medium. IVF mediums are well known in the art and are commercially available.

[0163] Gametes and / or their fertilized products may be present in frozen or cryopreservation media. Freezing / cryopreservation media are known in the art and are commercially available.

[0164] Gametes and / or their fertilized products are available in the following sizes: 1 nM to 500 μM, 10 nM to 500 μM, 100 nM to 500 μM, 500 nM to 500 μM, 1 μM to 500 μM, 1 μM to 100 μM, 2 μM to 100 μM, 5 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 50 μM to 100 μM, You can come into contact with the mitochondrial targeting agent at concentrations in the following ranges: 1 μM to 50 μM, 2 μM to 100 μM, 5 μM to 50 μM, 10 μM to 50 μM, 20 μM to 50 μM, 1 μM to 20 μM, 2 μM to 20 μM, 5 μM to 20 μM, 5 μM to 10 μM, 1 μM to 5 μM, 2 μM to 5 μM, or 1 μM to 2 μM.

[0165] Preferably, the gametes and / or their fertilized products can be contacted with a mitochondrial targeting agent at a concentration in the range of 1 to 25 μM, more preferably 5 to 15 μM, for example, 10 μM. The amount may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0166] Mitochondrial targeting agents may be present in compositions containing them. Gametes and / or their fertilized products may be in the following concentrations: 1 nM to 500 μM, 10 nM to 500 μM, 100 nM to 500 μM, 500 nM to 500 μM, 1 μM to 500 μM, 1 μM to 100 μM, 2 μM to 100 μM, 5 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 50 μM to 100 μM, 1 μM to 5 Compositions containing mitochondrial targeting agents can be brought into contact with the targeting agent at concentrations in the following ranges: 0 μM, 2 μM to 100 μM, 5 μM to 50 μM, 10 μM to 50 μM, 20 μM to 50 μM, 1 μM to 20 μM, 2 μM to 20 μM, 5 μM to 20 μM, 5 μM to 10 μM, 1 μM to 5 μM, 2 μM to 5 μM, or 1 μM to 2 μM.

[0167] Preferably, the gametes and / or their fertilized products may be contacted with a composition containing a mitochondrial targeting agent at a concentration in the range of 1 to 25 μM, more preferably 5 to 15 μM, for example, 10 μM. The amount may be particularly relevant when the mitochondrial targeting agent is BGP-15 and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates.

[0168] Gametes and / or their fertilized products may be contacted with a composition containing a mitochondrial targeting agent at concentrations ranging from 175 to 19,000 ng / ml, 184.6 to 18,460 ng / ml, 250 to 10,000 ng / ml, 300 to 6,000 ng / ml, 1,000 to 3,000 ng / ml, or 1,200 to 2,500 ng / ml. For example, gametes and / or their fertilized products may be contacted with a composition containing a mitochondrial targeting agent at concentrations ranging from 1,400 to 2,200 ng / ml, for example, 1,800 to 1,900 ng / ml. The amounts may be particularly relevant when the mitochondrial targeting agent is metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers, and / or racemates.

[0169] The composition may be a culture medium. The composition may be a gamete preparation medium. Such media are known in the art and are commercially available. For example, gamete preparation media can be used for gamete collection, gamete washing and / or gamete isolation. The composition may be an in vitro fertilization medium. IVF media are known in the art and are commercially available. Such IVF media typically contain the following components: calcium chloride, gentamicin sulfate, glucose, human (or animal) albumin solution, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate, sodium pyruvate and synthetic serum substitute. The composition may be a freezing or cryopreservation medium for gametes and / or their fertilized products. Again, such media are known in the art and are commercially available. The composition may be a freezing or cryoprotection medium.

[0170] Preferably, the composition containing the mitochondrial targeting agent is a pharmaceutical composition as described herein.

[0171] Gametes and / or their fertilized products processed according to this method may be characterized by improved developmental capacity. As used herein, the term “developmental capacity” may include (i) the ability and / or likelihood of gametes to produce an embryo (e.g., immediately after oocyte fertilization or by other mechanisms such as parthenogenesis activation), (ii) one or more of the ability, likelihood and rate of oocyte development by blastocyst development immediately after embryonic formation, and (iii) one or more of the quality of the embryo achieved immediately after embryonic generation from oocyte (e.g., determined by morphological and / or biochemical evaluation). Methods for determining developmental capacity are known in the art.

[0172] For example, improved developmental capacity may include improved progression through blastocyst development, increased progression capacity through blastocyst development, increased fertilization rates, increased likelihood of progression through blastocyst development, increased rate of 2-cell embryo formation, increased rate of 4-cell embryo formation, increased rate of blastocyst formation, increased rate of progression through blastocyst development, and / or increased rate of blastocyst hatching.

[0173] References to differences / changes such as increases or decreases in this specification preferably refer to statistically significant differences / changes. Statistical significance can be determined by the methods disclosed in the examples.

[0174] Embodiments relating to various uses of the present invention (e.g., mitochondrial targeting agents for use) are intended to be equally applicable to alternative methods and / or uses, and vice versa.

[0175] sequence homology Identity percentage can be determined using any of the following sequence alignment methods, including, but not limited to, global, local, and hybrid methods, such as the segment approach. Protocols for determining identity percentage are routine procedures within the scope of those skilled in the art. The global method determines the best alignment by aligning the sequence from the beginning to the end of the molecule, adding up the scores of individual residue pairs, and imposing gap penalties. Non-restrictive methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994)) and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264 (4) J. MoI. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-restrictive methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8 (5) CABIOS 501-509 (1992)), Gibbs sampling (see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208-214 (1993)), and Align-M (see, e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20 (9) Bioinformatics: 1428-1435 (2004)).

[0176] Therefore, the sequence identity percentage can be determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992.

[0177] The "sequence identity percentage" between two or more nucleic acid sequences is a function of the number of identical positions shared by the sequences. Therefore, the identity percentage can be calculated by dividing the number of identical nucleotides by the total number of nucleotides and multiplying by 100. The calculation of sequence identity percentage may also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparison and determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms such as BLAST, which will be well known to those skilled in the art.

[0178] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general dictionary of many of the terms used herein.

[0179] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing embodiments of this disclosure. Numerical ranges include the number defining the range. Unless otherwise indicated, all nucleic acid sequences are written from left to right in a 5' to 3' orientation.

[0180] The headings provided herein are not intended to limit any particular aspect or embodiment of the disclosure.

[0181] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is subject to change. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit them, since the scope of this disclosure is defined solely by the appended claims.

[0182] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of that range is understood to be specifically disclosed to one-tenth of the lower limit. Each smaller range between any expressed value or intermediate value within the expressed range and any other expressed value or intermediate value within that range is included in this disclosure. The upper and lower limits of these smaller ranges may or may not be included in that range independently, and each range in which either or both limits are not included in the smaller range, or which limits are included in either or both, is also included in this disclosure, provided that it is subject to whichever limit is specifically excluded in the expressed range. Where the expressed range includes one or both limits, the range that excludes one or both of the included limits is also included in this disclosure.

[0183] It should be noted that the singular forms “one (a),” “one (an),” and “it” as used herein and in the appended claims include multiple references unless the context clearly indicates otherwise. Therefore, for example, a reference to “mitochondrial targeting agents” includes multiple such candidate agents, and a reference to “its mitochondrial targeting agents” includes one or more mitochondrial targeting agents and their equivalents known to those skilled in the art.

[0184] The publications discussed herein are provided only in relation to disclosures prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that such publications constitute prior art with respect to the claims attached herein.

[0185] Brief explanation of the drawing Embodiments of the present invention will be described here merely as examples with reference to the following figures and examples. Many of the figures submitted herein are better understood in color. The color versions of the drawings are part of the filed application, and the right to present color images of the drawings in later steps is reserved herein. [Brief explanation of the drawing]

[0186] [Figure 1] Telomere lengths in male and female embryos produced by IVF (in vitro fertilization) or in vivo (mating). ICM telomere lengths (A) in male embryos (n=30) and female embryos (n=15) produced by IVF. Telomere lengths from IVF were compared with those from in vivo-derived 2-cell embryos (B, n=157-176) and ICM (C, n=13-22). The violin plot represents the population distribution, and the horizontal line represents the mean ± SEM. Data were logarithmically transformed for statistical analysis using an unpaired t-test. [Figure 2]Telomere elongation during preimplantation embryogenesis is impaired by oxidative stress. Mouse oocytes were in vitro fertilized (IVF), and embryos were collected at specific developmental stages at designated times (A). Telomere length (telomere / Rn18S) was measured by qPCR. Telomere length in individual oocytes / embryos (B, n=44-77). Whole blastocysts after immunosurgery (C, top) or isolated inner cell masses (ICMs) (C, bottom [all green]), showing the purity of ICM cells (green, OCT4+) without TE cells (red, CDX2+). Telomere length in ICMs and TEs removed by dissection from the same blastocysts (D, n=51). Telomere length in ICMs from blastocysts on day 5 (n=16) and day 6 (n=21) (E). MII oocytes were fertilized by IVF and cultured in vitro in either 5% or 20% oxygen (F). Telomere lengths were measured in individual 8-cell embryos (G), morulas (H), and ICMs (H, I), n=16-47 / group. Zygotes (6 hours after IVF) were labeled with the mitochondrial superoxide (mtROS) indicator MitoSox Red (plus DNA staining Hoechst-3342 (blue)), and corrected total fluorescence was determined (J, n=19 / group). Zygotes (4 hours after IVF), 8-cell embryos, morulas, and blastocysts were stained with the mitochondrial membrane potential (MMP) indicator TMRM (red) (plus Hoechst-3342 (blue)) (K, left). Corrected fluorescence was determined (right, n=4-41 / group). Zygotes (10 hours after IVF) were immunolabeled with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (L, left). Fluorescence intensity was measured and expressed as the ratio of paternal to maternal pronuclei (L, n=14 at 5% O2, n=16 at 20% O2). The violin plot shows the population distribution of telomere length, with the horizontal line representing the mean ± SEM (B, D, E, GI). For statistical analysis, qPCR data were logarithmically transformed. Statistical tests included a linear mixed-effects model (B), paired t-test (D), unpaired t-test (E, G, I, J, L), two-way ANOVA (H), or one-way ANOVA (K). Different letters indicate p<0.05, *p<0.05, ***p=0.0004, and ****p<0.0001. A representative image is shown, with a scale bar of 20 μm (C, J, K, L). [Figure 3] Pronuclear transfer between control and rotenone-exposed zygotes. In vivo fertilized zygotes were collected approximately 8 hours after fertilization and labeled with MitoSOX Red (MSR) mtROS indicator (and DNA stain Hoechst-3342, blue) (A), and red fluorescence (CTCF) was determined (A, n=25 control and n=18 rotenone). Schematic diagram of pronuclear transfer between zygotes and subsequent embryo culture (B). Zygotes (18 hours after hCG) were obtained from female mice that consumed control or rotenone-containing (150 ppm) feed for hormonal stimulation and 3 weeks prior to mating. Zygote pronuclei and cytoplasm were recombined to generate four types of embryos derived from either control or rotenone-exposed zygotes only (pronuclear transfer occurred between zygotes from the same group), or from combinations of pronuclei and cytoplasm derived from control and rotenone-exposed zygotes. Embryonic development after pronuclear transition was evaluated by the two-cell stage (C), the blastocyst stage (D), and the percentage of hatching blastocysts (E). [Figure 4]Telomere length qPCR assay. A novel qPCR assay was developed to enable robust measurement of telomere DNA per cell in individual embryos. The use of a reference gene allowed for normalization of changes in cell number during development. (A) qPCR assay design for quantifying telomeres and Rn18S sequences. 1 μL of individual oocytes or embryos were collected, and 9 μL of lysis solution was added to obtain a 10 μL sample volume for telomere and Rn18S analysis. The sensitivity of the telomere assay was validated using MII oocytes (n=15) and 2-cell stage (2C) and blastocyst stage (n=12) embryos produced by IVF. The expected decrease in Ct value with increasing cell number was demonstrated for both Rn18S (B) and telomeres (C). Specifically, doubling of cell number from 1 (MII oocyte) to 2 (2-cell embryo) was associated with a 1-cycle decrease in the Ct value for Rn18S, while blastocysts were associated with a Ct value 6 cycles lower than 2-cell embryos. Primer efficiency and linearity of sequence amplification with input DNA concentration were examined (D, E). DNA was extracted from a single mouse whole ovary and serially diluted (1:2) to produce concentrations of 6 ng / μL, 3 ng / μL, 1.5 ng / μL, 0.75 ng / μL, and 0.375 ng / μL, which were used to demonstrate the efficiency of telomere primers (D) and Rn18S reference gene primers with 36B4 as a comparator (E) under identical cycle conditions. To test linearity, DNA concentrations were log10 transformed for data plots by cycle threshold (Ct), and r² values ​​were shown. Each data point was obtained from 3 replicate PCR reactions. Each primer pair exhibited a high degree of linearity, with correlation coefficients consistently exceeding 0.995 (D, E). Comparison of the multicopy reference gene Rn18S with that of the singlecopy gene 36B4 resulted in a decrease of 1 in the detection cycle threshold (Ct), halving of the input DNA concentration for both the Rn18S and 36B4 primers, and correlation coefficients exceeding 0.995 for both primer sets (E), demonstrating the suitability of Rn18S as a reference gene.Telomere-to-reference gene ratios were calculated for 36B4 and Rn18S, and linear correlation analysis was performed (F), demonstrating the suitability of both methods for analyzing samples with limited DNA content (i.e., fetal tissue). Data are shown as mean ± SEM. [Figure 5] Hyperoxygen culture impairs fetal development after embryo transfer. A: Telomere length was evaluated using ICM derived from blastocysts that were vitrified at the "fresh" (i.e., continuous culture from fertilization) blastocyst or morula stage and then thawed to the blastocyst stage before culture, confirming that vitrification does not affect telomere elongation in embryos (A, n=25 fresh embryos and 42 vitrified embryos at 5%O2 and 27 fresh embryos and 36 vitrified embryos at 20%O2). qPCR data were logarithmically transformed for statistical analysis using one-way ANOVA, *p<0.02. Shaded areas represent population distribution, and horizontal lines are mean ± SEM (A). Blastocyst-stage embryos were transferred to pseudopregnancy recipient females (n=4 females for 5%O2 embryos and n=5 females for 20%O2 embryos) at 2.5 days of gestation, and fetuses were collected at 18.5 days (B). The number of uterine sites where embryos implanted was counted and expressed as a percentage of the transferred embryos to obtain the implantation rate (C). Whether implantation resulted in a fetus or reabsorption was recorded (D). The percentage of fetuses obtained from the total number of transferred embryos (E) and the number of fetuses per recipient (F) were calculated. Unpaired t-tests (*p<0.025) were used to evaluate embryo transfer and fetal outcomes, as well as fetal tissue length. [Figure 6]Oocyte rotenone exposure does not affect fetal outcomes after embryo transfer. A: Telomere length was assessed using ICM derived from blastocysts that were vitrified at the "fresh" (i.e., continuous culture from fertilization) blastocyst or morula stage and then thawed to the blastocyst stage before culture, confirming that vitrification does not affect embryonic telomere elongation (A, n=31 fresh control embryos and n=28 vitrified control embryos, as well as n=34 fresh rotenone-exposed embryos and n=27 vitrified rotenone-exposed embryos). ICM qPCR data were logarithmically transformed for statistical analysis using one-way ANOVA (*p<0.03). Shaded areas represent population distribution, and horizontal lines represent mean ± SEM (A). Blastocysts were transferred to pseudopregnancy recipient females (n=9 females per embryo type) on day 2.5 of gestation, and fetuses were collected for analysis on day 18.5 (B). The number of uterine sites where embryos implanted was counted and expressed as a percentage of transferred embryos to obtain the implantation rate (C). Whether implantation resulted in a fetus or reabsorption was recorded (D). The percentage of fetuses obtained from the total number of transferred embryos (E) and the number of fetuses per recipient (F) were calculated. Fetal characteristics, including body weight (G) and fetal sex (H), were analyzed (n=31 from control embryos and n=30 from rotenone-exposed embryos). Telomere length in fetal hearts (see Figure 9G) and fetal livers (I) were analyzed by qPCR, and telomere lengths between the two tissue types (J) and between male ("M") and female ("F") embryos (K) were compared. Embryo transfer and fetal outcomes (C-H), fetal liver telomere length (I), and differences in tissue telomere length between male and female embryos in the same group (K) were evaluated using unpaired t-tests. Comparison of fetal tissue types was analyzed using paired t-tests between tissues from the same fetus, with p<0.0001 (J). The violin plot represents the population distribution, and the horizontal line is the mean ± SEM (A, I~K). [Figure 7]Rotenone exposure before natural fertilization reduces telomere length in fetal hearts. A cohort of female mice, each fed either a control diet or a rotenone diet for three weeks, was paired with males (1:1) and transferred to the control diet in the presence of a vaginal mating plug to limit rotenone exposure during embryonic development. Fetuses were collected at gestational day 18.5 for analysis (A, n=33 control and n=40 rotenone fetuses). The number of fetuses per female (B), fetal weight (C), and fetal sex (D) were determined. Telomere length in fetal hearts (E) and fetal livers (F) was analyzed by qPCR, and telomere lengths between the two tissue types (G) and between male and female fetuses (H) were compared. Fetal outcomes within the same group (B-D), fetal cardiac and hepatic telomere lengths (E, F), and differences in tissue telomere lengths between male and female embryos (H) were evaluated using unpaired t-tests, *p<0.02. Comparison of fetal tissue types was analyzed using paired t-tests between tissues from the same fetus, ****p<0.0001 (G). The violin plot represents the population distribution, and the horizontal line is mean ± SEM (E-H). These data, showing a normal implantation rate but a specific reduction in fetal cardiac telomere length, are consistent with the results of experiments in which embryos derived from oocytes of rotenone-fed mice were fertilized by IVF and implanted in surrogate mothers for pregnancy (see Figures 9G and 6). [Figure 8]Hyperoxygen culture alters zygote epigenetic reprogramming. Zygotes were collected 10 hours after fertilization and immunolabeled with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies. Representative images are shown (A). Pronuclear size was measured in maternal and paternal pronuclei (n=14 5%O2 and 16 20%O2 zygotes) (B), and fluorescence signal intensity levels were quantified for 5mC (C) and 5hmC (D). Data are expressed as signal intensity for maternal (Mat.) and paternal (Pat.) pronuclei on the left axis and the ratio of fluorescence signals between paternal and maternal pronuclei of the same zygote on the right axis. Data were analyzed using paired t-tests for comparisons between maternal and paternal pronuclei of the same oocyte and unpaired t-tests for comparisons of pronuclear ratios between groups. For B, p<0.0001; for C and D, *p<0.05 and **p<0.002. [Figure 9]Mitochondrial dysfunction in oocytes reduces embryonic and fetal telomere length via nuclear modification, which is reversible with BGP-15. Mice were exposed to rotenone (150 ppm in solid feed) for three weeks prior to ovulation, IVF, and embryonic and fetal evaluation (A). Zygotes were labeled with MitoSox Red (and DNA stain Hoechst-3342) and fluorescence was measured (B, n=18 control and n=16 rotenone). Zygotes were co-stained with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (C). Fluorescence signal intensity was quantified for 5mC (left) and 5hmC (right) in maternal and paternal pronuclei (n=15 control and n=12 rotenone), and expressed as the ratio of paternal to maternal pronuclei. Zygotes and 8-cell embryos were labeled with the MMP indicator TMRM (and Hoechst-3342), and red fluorescence was measured (D, n=8-22 / group). Telomere lengths were measured in individual MII oocytes, 8-cell embryos, blastocysts (E, n=45-86 / group), and ICMs (F, n=38-42). IVF-pregnancy blastocysts from rotenone-treated (or control) mice were transplanted into surrogate females for embryonic development, and fetal tissue was collected at gestational day 18.5 for qPCR telomere length analysis of the heart (G, n=36 controls, n=35 rotenone fetuses). Pronuclei were transferred between zygotes derived from control or rotenone-exposed females in each possible combination, and reconstructed embryos were cultured to blastocysts (H). ICMs derived from blastocysts were analyzed for telomere length by qPCR(I). MitoSox Red (J, n=15-20) and telomere length (K, n=31-44) in zygotes from mice fed rotenone and treated with BGP-15 (100 mg / kg via ip injection or in a saline vehicle) for 4 days prior to gonadotropin-stimulated ovulation. Violin plots show population distribution, and horizontal lines represent the mean ± SEM (E-G, I, K). qPCR data were logarithmically transformed for statistical analysis. Statistical tests were unpaired t-tests (B, C, F, G) or one-way ANOVA (D, E, I, J, K). Different letters indicate p<0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Representative images, scale bar: 20 μm (B, C, D, J). [Figure 10] Maternal rotenone exposure alters zygote epigenetic reprogramming. Zygotes were collected 10 hours after fertilization and immunolabeled with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (A). Pronuclear size was measured in maternal and paternal pronuclei (n=12–15 zygotes per group) (B), and fluorescence signal intensity levels were quantified for 5mC (C) and 5hmC (D). Data are represented as signal intensity for maternal (Mat.) and paternal (Pat.) pronuclei on the left axis and the ratio of fluorescence signals between paternal and maternal pronuclei of the same zygote on the right axis. Data were analyzed using paired t-tests for comparisons between maternal and paternal pronuclei of the same oocyte and unpaired t-tests for comparisons of pronuclear ratios between groups. For B, p<0.02; for C, D*p<0.05, **p=0.0038, ***p=0.0003, ****p<0.0001. [Figure 11]Maternal aging impairs embryonic telomere elongation, which is restored by pre-fertilization treatment with mitochondrial-acting therapeutic agents. Female control mice of reproductive age (12 months) and young age (3-4 months) were treated with BGP-15 (100 mg / kg) or a saline vehicle before ovulation, IVF, and embryo evaluation (A). Zygotes were labeled with MitoSox Red (and Hoechst-3342 (blue)) and red fluorescence was measured (B, n=10-21). Zygotes were co-stained with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (C). Fluorescence signal intensity was quantified for 5mC (left) and 5hmC (right) in maternal and paternal pronuclei (n=12-20) and expressed as the ratio of paternal to maternal pronuclei. Morula embryos were stained with TMRM (red) and Hoechst-3342 (blue), and red fluorescence was measured (D, n=8-33). Telomere length per cell in individual MII oocytes (E) from young mice versus 12-month-old mice, and oocytes from 12-month-old nulliparous mice versus multiparous mice (F). Telomere length per cell in individual 8-cell embryos (G), whole blastocysts (H), and isolated ICMs (I). Oocytes / embryo with n≧10 per group, and ICMs with n=12-19. Reproductively aged females were administered metformin (2 mg / mL) or MitoQ (150 μM) in drinking water for 2 weeks, followed by gonadotropin stimulation and IVF of ovulated oocytes. Telomere length in individual ICMs (J, young n=47, aged ± treated n=16-25). The violin plot shows the population distribution, and the horizontal line represents the mean ± SEM. For statistical analysis, the qPCR data was logarithmically transformed. Statistical tests were one-way ANOVA (B-D, G-J) or independent t-tests (E, F), with p<0.05, p<0.01, p<0.001, and p<0.0001. Representative images and scale bars are shown at 20 μm (B, C, D). [Figure 12]Maternal aging alters zygote epigenetic reprogramming modified by BGP-15 treatment. Zygotes from young females, aged females, and aged females treated with BGP-15 (aged+B) were collected 10 hours after in vitro fertilization and immunolabeled with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (A). Pronuclear size was measured in the maternal and paternal pronuclei (n=12-20 zygotes per group) (B), and fluorescence signal intensity levels were quantified for 5mC (C) and 5hmC (D). The left axis represents the signal intensity for the maternal (Mat.) and paternal (Pat.) pronuclei, and the right axis represents the ratio of fluorescence signals between the paternal and maternal pronuclei of the same zygote. The data were analyzed using paired t-tests for comparisons between maternal and paternal prenuclei of the same zygote, and one-way ANOVA for comparisons of prenuclei and proportions between groups (*p<0.03, **p<0.005, ***p=0.0001, ****p<0.0001). [Figure 13]Maternal obesity impairs embryonic and fetal telomere elongation, which can be restored by treatment with mitochondrial-acting therapeutic agents at fertilization. Obese female mice (>36g) and lean littermates were treated with BGP-15 (100mg / kg) or saline vehicle before ovulation, IVF, and analysis of embryonic and fetal tissues (A). Zygotes were labeled with MitoSox Red and Hoechst-3342 (blue), and red fluorescence was measured (B, n=10-17). Zygotes were co-stained with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (C). Fluorescence signal intensity was quantified (n=17-33) and expressed as the ratio of paternal to maternal pronuclei (C). Oocytes, 8-cell embryos, and morulas were stained with TMRM (red) and Hoechst-3342 (blue) (D). Red fluorescence was assessed for oocytes (E, n=16-32), 8-cell embryos (F, n=14-28), and morulas (G, n=7-17). Telomere length per cell was evaluated by qPCR for individual MII oocytes (H), 8-cell embryos (I), whole blastocysts (J), and ICMs (K). Oocytes / embryo with n≧16, ICMs with n=17-31. Metformin (2 mg / mL) or MitoQ (150 μM) was administered to obese females in drinking water for 2 weeks, followed by gonadotropin stimulation and IVF of ovulated oocytes. Telomere length in individual ICMs (L, n=37-57). Blastocysts derived from oocytes of lean mice, obese mice, or BGP-15-treated (100 mg / kg) obese mice were transplanted into surrogate females for embryonic development, and fetal tissue was collected at 14.5 days of gestation for qPCR telomere length analysis of the liver and heart (M, n=6-17). Ovulated oocytes from lean or obese mice were cultured in a medium containing 10 μM BGP-15 (or vehicle) from fertilization (N-P). MitoSox Red was analyzed in zygotes (N, n=15-23), MMP in morula (O, n=17-18), and telomere length in ICM (P, n=24-43). Violin plots show population distribution, and the horizontal line represents the mean ± SEM (H-M, P). For statistical analysis, qPCR data were logarithmically transformed. The data was analyzed using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 14] Maternal obesity leads to alterations in zygote epigenetic reprogramming, which are reversed by BGP-15 treatment. Zygotes from lean females, obese (Ob) females, and obese females treated with BGP-15 (Ob+B) were collected 10 hours after in vitro fertilization and immunolabeled with anti-5-methylcytosine (5mC, red) and anti-5-hydroxymethylcytosine (5hmC, green) antibodies (A). Pronuclear size was measured in the maternal and paternal pronuclei (n=17–33 zygotes per group) (B), and fluorescence signal intensity levels were quantified for 5mC (C) and 5hmC (D). Data are expressed as signal intensity for maternal (Mat.) and paternal (Pat.) pronuclei on the left axis, and the ratio of fluorescence signal in the paternal pronucleus versus maternal pronucleus of the same zygote on the right axis. The data were analyzed using paired t-tests for comparisons between maternal and paternal prenuclei of the same zygote, and one-way ANOVA for comparisons of prenuclei and proportions between groups (*p<0.034, **p<0.008, ***p<0.0002, ****p<0.0001). [Figure 15] Exposure to BGP-15 during fertilization and embryo culture reduces superoxide production in a high-oxygen environment. Oocytes were fertilized in a medium containing 10 μM BGP-15(+) or a medium containing an equal volume of vehicle(-) at 5% O2 or 20% O2. Six hours after fertilization, zygotes were labeled with MitoSox Red (MSR) superoxide (mtROS) indicator (and DNA stain Hoechst-3342, blue) and red fluorescence (CTCF) was assessed (n=19-20). Data were analyzed using one-way ANOVA (****p<0.0001). [Figure 16]Exposure to BGP-15 during fertilization and embryo culture attenuates superoxide production in zygotes derived from rotenone-exposed mice. Oocytes from control or rotenone-exposed females were fertilized in vitro in medium containing 10 μM BGP-15(+) or equal volume of vehicle(-). Six hours after fertilization, zygotes were labeled with MitoSox Red (MSR) superoxide (mtROS) indicator (and DNA stain Hoechst-3342, blue) and red fluorescence (CTCF) was assessed (n=15-18). Data were analyzed using one-way ANOVA (****p<0.0001). [Modes for carrying out the invention]

[0187] Sequence List Sequence ID 1 (telomere forward primer) CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT Sequence ID 2 (Telomere Reverse Primer) GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT Sequence ID 3 (Rn18S forward primer) AGA AAC GGC TAC CAC ATC CAA Sequence ID 4 (Rn18S reverse primer) CCT GTA TTG TTA TTT TTC GTC ACT ACC T Sequence ID 5 (SRY forward primer) AAG CGC CCC ATG AAT GCA TT Sequence ID 6 (SRY Reverse Primer) TCC CAG CTG CTT GCT GAT CT Sequence ID 7 (36B4 forward primer) ACT GGT CTA GGA CCC GAG AAG Sequence ID 8 (36B4 reverse primer) TCA ATG GTG CCT CTG GAG ATT [Examples]

[0188] Examples Materials and methods Animals, hormone treatments, and drug administration All animal experiments were approved by the Animal Ethics Committee of the University of Adelaide and conducted in accordance with Australian standards of practice for the management and use of animals for scientific purposes. Female CBA.F1 (CBA / CaH×C57 / BL6Arc) mice aged 6-7 weeks and male mice aged 6-8 weeks were obtained from the Laboratory Animal Services of the University of Adelaide. The mice were maintained under 12-hour / 12-hour light / dark conditions and continuously fed with water and 10% fat rodent solid feed. For all drug treatments or dietary manipulations, mice were randomly assigned to experimental groups.

[0189] Obese female mice or fertile-age female mice and lean young controls were generated from the same colony (C57BL / 6JSfdAnu-Alms1bbb / Apb mouse strain maintained as a heterozygous breeding pair) and referred to as "Blobby" mice. They were fed the same standard solid diet as in (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022), LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)). Obese mice were homozygous (bbb / bbb) for the "Blobby" mutation in the Alms1 gene, which leads to overeating and severe obesity even when maintained on a standard mouse solid diet (LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)). Females were considered obese when weighing at least 36g at 4-5 months of age, and wild-type littermates were used concurrently as lean controls. Reproductively mature females were wild-type or heterozygous (+ / + or bbb / +) at 12 months of age, and young females (3-4 months of age) were used concurrently as young controls.

[0190] Rotenone (#R8875) was supplied by Sigma, and the rotenone feed was prepared and used as described in (OA Lozoya et al., Single Nucleotide Resolution Analysis Reveals Pervasive, Long-Lasting DNA Methylation Changes by Developmental Exposure to a Mitochondrial Toxicant. Cell reports 32, 108131 (2020)). Specifically, the control feed was a modified AIN-93G (TD.97184) feed, and the rotenone feed was precisely formulated with 150 ppm rotenone in TD.97184 meal feed using Teklad / Envigo as described in (OA Lozoya et al., Single Nucleotide Resolution Analysis Reveals Pervasive, Long-Lasting DNA Methylation Changes by Developmental Exposure to a Mitochondrial Toxicant. Cell reports 32, 108131 (2020)). Following previous experiments, CBA.F1 mice were randomly assigned to either a control or rotenone diet for 3 weeks (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022)).This dose of rotenone has been shown to reduce respiration driven by complex I and complex II without altering the abundance of oxidatively phosphorylated subunits or causing any apparent physiological effects (OA Lozoya et al., Single Nucleotide Resolution Analysis Reveals Pervasive, Long-Lasting DNA Methylation Changes by Developmental Exposure to a Mitochondrial Toxicant. Cell reports 32, 108131 (2020)).

[0191] BGP-15 ([(O-[3-piperidino-2-hydroxy-1-propyl]-nicotinamidoxime)], CAS 66611-37-8, Hangzhou Molcore Biopharmatech Co Ltd) was administered intraperitoneally (ip) at a dose of 100 mg / kg body weight in physiological saline for 4 days, starting the day before PMSG injection (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022), LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)). Controls were injected with an equal volume of 0.9% physiological saline vehicle relative to body weight.

[0192] Metformin (1,1-dimethyl biguanide hydrochloride, D150959, Sigma-Aldrich) was freely administered to mice in drinking water at a concentration of 2 mg / mL for two weeks prior to PMSG and hCG treatment (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022)).

[0193] MitoQ (mitoquinone mesylate: [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadiene-1-yl)decyl](triphenyl)phosphonium methanesulfonate) is a ubiquinone moiety linked to a lipophilic triphenylphosphonium cation by a 10-carbon alkyl chain that preferentially accumulates in mitochondria (MP Murphy, RA Smith, Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu Rev Pharmacol Toxicol 47, 629-656 (2007)). MitoQ (generously donated by MitoQ Ltd, New Zealand) was freely delivered in 150 μM drinking water for two weeks prior to PMSG and hCG treatment (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022)).

[0194] Female mice were administered 5 IU / 12g body weight of pregnant mare serum gonadotropin (PMSG, #493-10, Lee BioSolutions) followed 47.5 hours later by an equal dose of human chorionic gonadotropin (hCG, Pregnyl), both via intraperitoneal (IP) injection.

[0195] Origin of oocytes, early embryos, and blastocysts To collect meiotic II (MII) oocytes, mice were euthanized by cervical dislocation 15 hours after hCG administration, and the ovaries and fallopian tubes were collected and placed in pre-warmed (37°C) αMEM-HEPES handling medium supplemented with 1% FCS. Ovulated cumulus-oocyte complexes (COCs) were isolated by opening the fallopian tubes using a 30G needle. Cumulus cells were completely removed from the MII oocytes by aspiration using a glass pipette pulled to the appropriate diameter, following treatment with hyaluronidase (Seikagaku, #100741) at 37°C for 5-10 minutes.

[0196] To generate embryos via in vitro fertilization (IVF), ovulated COC clusters were gently washed twice in pre-warmed (37°C) fertilization medium (Vitro Fertilization, Cook Australia, Brisbane, Australia), then placed in 100 μL droplets of fertilizable sperm from a fertile male (referred to as "fertilization time"), and then returned to an incubator (37°C, 5% O2, 6% CO2) for 4 hours. Subsequently, the fertilized oocytes were cleaned of all excess sperm and cumulus cells by gentle aspiration, transferred to a culture dish containing cleavage medium (Vitro Cleave, Cook Australia, Brisbane, Australia, 10 embryos per 20 μL droplet of cleavage medium), and returned to the incubator for 24 hours post-fertilization. At this point, the number of embryos that successfully reached the 2-cell stage was scored. For in vitro BGP-15 treatment of oocytes and embryos, a 0.5 M BGP-15 stock in sterile H2O was diluted to 10 μM in the respective culture media (fertilization medium or cleavage medium). For the control, an equal volume of sterile H2O was added to the culture medium. For the high-oxygen (20% O2) experiment, fertilization incubation and subsequent culture in cleavage medium were performed at 37°C, 20% O2, and 6% CO2.

[0197] To ensure accurate embryo collection at the correct stage, the number of ovulated oocytes per mouse and on-time embryonic development were monitored in each experiment. Four-cell embryos were collected at 39 hours post-fertilization, eight-cell embryos at 55 hours, morulas at 77 hours, and blastocysts at 96 hours post-fertilization. All morulas collected for analysis had similar cell counts, and all blastocysts were at similar developmental stages (late expansion or early hatching) and were morphologically normal.

[0198] For in vivo embryos, female mice were housed with male mice whose reproductive capacity had been proven after hCG administration (1:1 ratio), and separated after 16 hours. To collect two-cell embryos, the females were euthanized by cervical dislocation 24 hours later (40 hours after hCG administration), and the two-cell embryos were dissected and extracted from the fallopian tubes. To collect blastocysts, the females were euthanized 82 hours after hCG administration, and the embryos were flushed from the uterus using a 1cc syringe containing pre-warmed αMEM-HEPES + 1% FCS handling medium. Telomere lengths in embryos induced via IVF were found to be no different from those of embryos obtained through pregnancy by in vivo fertilization (i.e., mating) (Figure 1B, Figure 1C).

[0199] Isolation of blastocyst cell populations The paired collection of inner cell mass (ICM) and trophectoderm (TE) cell populations (Figure 2D) was performed by manual dissection. The zona pellucida was removed using an acid tyrofoam solution of Embryomax (Millipore, #MR-004D), and the blastocysts were transferred to individual 5 μL pre-warmed (37°C) droplets of αMEM handling medium containing 1% FCS in 50 × 9 mm Petri dishes (Falcon, #351006). The blastocysts were manually separated into two cell populations using an Eppendorf TransferMan 4r system and standard holding pipettes with 15 μM inner and 120 μM outer diameters (The Pipette Company, #LHC-ID15), as well as a biopsy pipette with a 30 μM tip (OD) and a 90° bevel (The Pipette Company, #LBC-OD30-BA90). The cell populations were separated and frozen individually for each blastocyst, but paired for analysis.

[0200] The purified ICM population was isolated using immunosurgery (see Figure 2C). The zona pellucida of the blastocysts was removed via treatment with an acidic tyrofoam solution of Embryomax, washed twice in cleavage medium, and transferred to cleavage medium containing 20% ​​heat-inactivated rabbit anti-mouse serum (Sigma, #M5774). After 1 hour of incubation (37°C, 5% O2, 6% CO2), the blastocysts were washed three times in cleavage medium and transferred to cleavage medium containing 20% ​​guinea pig serum (Sigma, #G9774) and incubated for 15 minutes. The blastocysts were washed three times in cleavage medium and incubated in this medium for a further 30 minutes. Lysified TE cells were extracted by gently aspirating the ICM into a finely stretched glass pipette.

[0201] Zygote-pronucleus transfer Female mice that had consumed a control or rotenone-containing diet for 3 weeks were given PMSG and hCG (as described above) and housed with 8-week-old CBA.F1 males for mating (1:1). 18 hours after hCG administration (approximately 6 hours after fertilization), the females were humanely killed to collect the zygotes, completely exposing the cumulus cells, and transferred to cleavage medium, where they were incubated in an incubator for 1.5–2 hours (approximately 8 hours after fertilization). The zygotes were then incubated for 20 minutes in cleavage medium containing 1 μg / mL cytochalasin D (Sigma, #C2618) and 0.3 μg / mL nocodazole (Sigma, #SML1665). Subsequently, the zygotes were transferred to αMEM-HEPES handling medium containing 60 μg / mL BSA, 1 mg / mL PVP, and cytochalasin D and nocodazole at the aforementioned concentrations. Both pronuclei were removed from the zygote using a micropipette (Eppendorf, #5195000079), and a small amount of inactivated Sendai Virus (Cosmo Bio, USA, approximately 3000 hemagglutination units / ml) was pipetteed into the pipette. The virus and pronuclei were injected into the periuterine space of a second enucleated single-cell embryo (see Figure 3B). The reconstructed embryos were washed in cleavage medium to remove the inhibitor and cultured in the same cleavage medium until the blastocyst stage. The blastocyst development rate was similar to that of the unreconstructed embryos, and there was no difference between the groups (Figures 3C-3E).

[0202] Telomere and Rn18S qPCR using DNA derived from individual oocytes and embryos Individual oocytes or embryos for qPCR analysis were washed three times in 1× phosphate-buffered saline (PBS) containing 1 mg / mL polyvinylpyrrolidone (PVP, Sigma). 1 μL of each sample was transferred to a 0.5 mL PCR tube (Axygen, #PCR-05-C), rapidly frozen in liquid nitrogen (LN2), and stored at -80°C until use. 9 μL of lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 μg / mL proteinase K, 0.5% Tween-20) was added to each sample (final volume 10 μL), and DNA was extracted by heating at 55°C for 2 hours, followed by heating at 95°C for 10 minutes. The samples were cooled to 4°C and briefly centrifuged. The ICM samples were further diluted with 10 μL of sterile H2O to obtain a total volume of 20 μL.

[0203] To generate "calibrator" DNA for use as a standard in all assays, whole ovaries were collected from 6-week-old PMSG- and hCG-stimulated CBA female mice, and total DNA was extracted using the QIAmp DNA Micro Kit (Qiagen, #56304) "Isolation of Genomic DNA from Small Volumes of Blood" protocol according to the manufacturer's instructions. DNA concentration and purity were quantified by NanoDrop One Microvolume UV-Vis spectrophotometer and diluted to 2.5 ng / μL.

[0204] qPCR was performed using the Applied Biosystems 7900HT Fast Real-Time PCR system or the Quantstudio 12K Flex (ThermoFisher) with Power SYBR Green PCR Master Mix (Applied Biosystems, #4367659), standard MicroAmp Optical 96-well Reaction Plates (Life Technologies, #4306737), and custom-made primers (Sigma). All primers were cycled for 40 cycles of 10 minutes at 95°C, followed by 15 seconds of annealing at 95°C, 30 seconds at 60°C, and 30 seconds of extension at 72°C, before standard melting curve cycling was performed. Two replicates were performed per sample and per primer pair using 2 μL of sample per reaction (see Figure 4D). The final reaction mixture for each well consisted of 10 μL of Power SYBR Green PCR Master Mix, 0.5 μL each of forward and reverse primers (10 μM stock), 2 μL of sample, and 20 μL of sterile H2O up to the final volume. The telomere primer sequences were telomere forward 5'-CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT-3' (SEQ ID NO: 1) and telomere reverse 5'-GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT-3' (SEQ ID NO: 2) (RJ Callicott, JE Womack, Real-time PCR assay for measurement of mouse telomeres. Comparative medicine 56, 17-22 (2006)).The reference gene primer sequences were Rn18S forward 5'-AGA AAC GGC TAC CAC ATC CAA-3' (SEQ ID NO: 3) and Rn18S reverse 5'-CCT GTA TTG TTA TTT TTC GTC ACT ACC T-3' (SEQ ID NO: 4) (C. de Frutos et al., Spermatozoa telomeres determine telomere length in early embryos and offspring. Reproduction (Cambridge, England) 151, 1-7 (2016)). To minimize plate-to-plate variability, Rn18S and telomere reactions for each sample were performed on the same plate, and calibration DNA was included on all plates. Telomeres relative to the Rn18S copy were 2. -ΔΔCt The method was used to calculate and obtain ΔΔCt by subtracting the ΔCt of the sample (telomere Ct - Rn18S Ct) of the calibration material (telomere Ct - Rn18S Ct).

[0205] Sex-determining qPCR of embryos was performed using the same reagents and system as before, and two reactions were performed for all samples. The final reaction mixture for each sample consisted of 10 μL of Power SYBR Green PCR Master Mix, 0.5 μL each of forward primer and reverse primer (SRY primer, 10 μM stock, forward sequence 5'-AAG CGC CCC ATG AAT GCA TT-3' (SEQ ID NO: 5), reverse sequence 5'-TCC CAG CTG CTT GCT GAT CT-3' (SEQ ID NO: 6)), 4 μL of sample, and sterile H2O up to the final volume of 20 μL. To confirm the size of the qPCR product, the reaction mixture was flowed onto a 4% agarose (Promega, #V3125t) gel containing Gel Red (Biotium, #41003). 2.5 μL of 6× loading dye (New England Bio Labs, #B70245) was added to each sample, and 10 μL was run through each lane, with a 100 bp ladder (ThermoFisher Scientific, #SM0243) used for reference. The gel was run at 100 V for 1 hour and imaged via a Biorad Gel Doc EZ Imager with Image Lab software. Male embryos were distinguished by a 105 bp PCR product. Telomere lengths did not differ between ICMs from male and female embryos (see Figure 1A), so unless otherwise specified, male and female embryos were grouped for analysis.

[0206] MitoSOX Red (MSR) staining Zygotes were collected and incubated with Hoescht-3342 in handling medium containing 60 μg / mL BSA, 1 mg / mL PVP, and 5 μM MitoSOX Red (Invitrogen, #M36008) at 37°C for 20 minutes. After a brief wash in handling medium, the samples were mounted in pre-warmed handling medium (containing BSA and PVP as described above) and imaged using an Olympus FV3000 Confocal Microscope. Images were acquired using the same confocal microscope settings, with the operator blinded to the treatment group. Total cell fluorescence (CTCF) was calculated using Image J and adjusted for background fluorescence to obtain corrected total cell fluorescence (CTCF).

[0207] Mitochondrial membrane potential assay Completely exposed MII oocytes, fertilized zygotes, or indicated developmental embryos were washed twice in pre-warmed (37°C) handling medium containing 60 μg / mL BSA and 1 mg / mL PVP. These were then incubated for 30 minutes at 37°C in handling medium containing BSA and PVP as described above, along with 25 μM TMRM (tetramethylrhodamine methyl ester perchlorate, Sigma, #T5428), with the addition of 1:250 Hoescht-3342 for the last 15 minutes. After a brief rinse in handling medium, the samples were mounted in pre-warmed handling medium (as described above) and imaged using a Cell Voyager CV1000 spinning disk confocal microscope (Yokogawa) with a 40x objective lens. Images were acquired using the same confocal microscope setup, with the operator blinded to the treatment group. Total cell fluorescence (CTCF) was calculated and adjusted for background fluorescence using Image J to obtain corrected total cell fluorescence (CTCF).

[0208] Pronuclear cytosine methylation immunofluorescence Fertilized zygotes (stages PN3 to PN4) were collected 10 hours after fertilization. The zona pellucida was dissolved using an acid tyrofoam solution of Embryomax (Millipore, #MR-004D), and then briefly washed in phosphate-buffered saline (PBS) containing 1 mg / ml polyvinylpyrrolidone (PVP, Sigma) (PBS-PVP). The zygotes were fixed in 3.7% PFA-PBS at room temperature (RT) for 20 minutes, then washed twice for 5 minutes in PBS-PVP, followed by permeability in 0.2% Triton X-100 (in PBS) for 10 minutes at room temperature, and then washed three times for 5 minutes in PBS-PVP. The zygotes were incubated in 4N HCl solution at RT for 10 minutes and thoroughly washed in 0.05% Tween-20 in PBS (PBS-T). The zygotes were blocked overnight at 4°C in a blocking solution (1% BSA in PBS, 0.02% Triton X-100), and then incubated overnight at 4°C with mouse anti-5mC (1:200, Biorad, #ab10805) and rabbit anti-5hmC (1:600, Active Motif, #39769) (diluted in the blocking solution). After washing three times for 10 minutes in PBS-T, the samples were incubated for 1 hour at RT with Alexa Fluor 594 conjugate goat anti-mouse (1:500, Invitrogen) and Alexa Fluor 488 conjugate goat anti-rabbit secondary antibody (1:1000, Invitrogen), protected from light. Samples were mounted on slides with Prolong® Diamond Antifade Mountant (Invitrogen, #P36965) and examined using a Cell Voyager CV1000 spinning disk confocal microscope (Yokogawa) with a 40x objective lens. Images were acquired using the same confocal microscope setup, with the operator blinded to the treatment group. Pronuclear size and staining intensity were calculated using ImageJ software. The mean signal intensity was calculated by first subtracting the signal from the cytoplasmic area (representing background staining) and then dividing by the pronuclear area.To quantify the standard differences between 5mC and 5hmC in male and female pronuclei (L. Han et al., Embryonic defects induced by maternal obesity in mice derive from Stella insufficiency in oocytes. Nature genetics 50, 432-442 (2018)), the signal intensity of each marker was also expressed as the ratio of values ​​within the paternal to maternal pronuclei of the same zygote.

[0209] Transfer of blastocyst embryos into pseudopregnant female mice Embryos derived from hyper-oxygen culture or in vivorotenone experiments were vitrified at the morula stage, then thawed and cultured to the blastocyst stage before being transplanted into pseudo-pregnant females. To determine whether embryonic vitrification affects ICM telomere length, vitrified morulae were thawed, cultured overnight, and then ICM was collected and analyzed. There was no difference in ICM telomere length between those derived from fresh morulae and vitrified morulae, regardless of oxygen culture conditions (Figure 5) or pre-fertilization rotenone exposure (Figure 6A).

[0210] Mice were humanely euthanized by cervical dislocation at 18.5 days of gestation. Implantation sites were counted and the outcome (i.e., fetus or reabsorption) was recorded. Fetal weight, length from crown to rump, placental weight, and fetal sex were recorded. Fetuses were humanely euthanized, and multiple tissues (heart, liver, kidney, tail, gonads, brain) were collected, individually rapid-frozen in liquid N2, and stored at -80°C until use. Embryos cultured in 20% O2 showed reduced implantation ability compared to those cultured in 5% O2 (Figure 5), so direct comparison of fetal tissues from the two groups was not possible and was not investigated further. Embryos from rotenone-fed mice showed the same implantation rate as those from control mice (Figures 6 and 7).

[0211] In experiments involving obese mouse models, embryos produced by IVF were cultured to the blastocyst stage under atmospheric oxygen, as previously described, and then immediately transplanted into surrogate females (LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)). Briefly, female mice confirmed to have mated with fertile, vasectomized males were considered to be at 0.5 days pregnant and selected as embryo recipients. At 2.5 days of pseudopregnancy, 7–10 fresh (i.e., continuously cultured) blastocysts were transplanted into the uterus (3–5 blastocysts / horn) of each pseudopregnancy mouse anesthetized by aveltin iP injection (0.5 mg / g body weight, Sigma-Aldrich, St. Louis, MO). An analgesic carprofen (5 mg / kg) (Rimadyl Pfizer) was administered subcutaneously once postoperatively. Fetal tissue was collected and extracted on day 14.5 of gestation (LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)).

[0212] In vivo pregnancy fetus To produce naturally conceived (in vivo) fetuses, females fed either rotenone or a control diet for 3 weeks were housed (1:1) with 8-week-old CBA.F1 males, and the presence of a mating plug was considered day 0.5 of gestation. Males were not exposed to the rotenone diet (i.e., were given the control diet), and all females were placed on the control diet after the presence of a mating plug to limit rotenone exposure during the pre-fertilization period. Fetuses were collected at embryonic day 18.5, and there were no differences in fetal number, weight, and sex ratio (Figure 7). Placental weight, length from crown to rump, and fetal-to-placental weight ratio were also unchanged. Fetal tissue collection was as described above.

[0213] Extraction of fetal tissue DNA and qPCR Fetal heart and liver tissues were dissolved overnight at 55°C in 250 μL of lysis buffer (20 mM EDTA pH 8.0, 50 mM Tris pH 8.0, 120 mM NaCl, 1% SDS) containing 5 μL of proteinase K (10 mg / mL) with constant shaking (100 rpm). The following morning, 250 μL of 4 M ammonium acetate was added, vortexed briefly, and incubated at RT for 15 minutes with shaking, followed by a further 10 minutes of incubation without shaking. The samples were centrifuged at 10,000 rpm for 10 minutes, and 400 μL of supernatant was collected and added to a clean tube. 800 μL of 100% ethanol was added, vortexed briefly, and then centrifuged at 10,000 rpm for 8 minutes to pelletize the DNA. The ethanol was removed, and the pellet was washed with 500 μL of 70% ethanol. The excess ethanol was removed, and the DNA was resuspended by incubation at 55°C with shaking (100 rpm) in 300 μL of sterile H2O. The DNA was quantified using a ThermoFisher Nanodrop One UV-Vis spectrophotometer and diluted to 4 ng / μL with sterile water for use.

[0214] Telomere length in fetal tissue was analyzed via qPCR using Quantstudio 12K Flex (ThermoFisher) as described above. The primer sequences for telomeres and the reference gene (Rn18S) were as described above. Fetal tissue derived from oocytes of obese (and lean control) mice was analyzed using the reference gene 36B4. The primer sequences were 36B4 forward: 5'ACT GGT CTA GGA CCC GAG AAG 3' (SEQ ID NO: 7) and 36B4 reverse: 5'TCA ATG GTG CCT CTG GAG ATT 3' (SEQ ID NO: 8) (RJ Callicott, JE Womack, Real-time PCR assay for measurement of mouse telomeres. Comparative medicine 56, 17-22 (2006)). The cycling conditions were as described above. The final reaction mixture for each well consisted of 10 μL of Power SYBR Green PCR Master Mix, 0.2 μL each of forward and reverse primers (10 μM stock), 4 μL of sample, and 20 μL of sterile H2O up to the final volume. Two replicates were performed for each sample. Telomere length (T / S ratio) relative to the reference gene copy was calculated using formula 1 / (telomere Ct / reference Ct) (RJ Callicott, JE Womack, Real-time PCR assay for measurement of mouse telomeres. Comparative medicine 56, 17-22 (2006)).

[0215] When comparing telomere lengths between cardiac and hepatic tissues, it was found that telomeres were significantly longer in the liver than in the heart, and this difference did not differ between males and females, regardless of whether the fetus was induced via IVF and embryo transfer (Figures 6J, 6K) or in vivo fertilization (Figures 7G, ​​7H).

[0216] statistical analysis Results are presented as mean ± SEM. qPCR data are displayed as mean ± SEM superimposed on a violin plot to show the distribution of data points. All data points represent independent biological replicates. Normality analysis indicated that logarithmic transformation was necessary for telomere data for statistical analysis. Statistical analysis was performed using GraphPad Prism version 8 for Windows (GraphPad Software Inc., La Jolla, CA) and SPSS Statistics 26 (IBM, Armonk, NY). Paired two-tailed t-tests, unpaired two-tailed t-tests, one-way and two-way ANOVA (comparison of means), and linear mixed-effects models were used as instructed, with statistical significance considered to be P-value < 0.05.

[0217] Example 1 8. Telomere elongation between cell formation and blastocyst formation is slowed by oxidative stress. Pre-implantation development involves complete reprogramming of hierarchical nuclear DNA between fertilization and ICM formation. However, the dynamics of telomere elongation are not well characterized. To comprehensively map the dynamics of telomere length throughout this embryogenetic stage, mouse MII stage oocytes were in vitro fertilized and collected at precise developmental milestones between 2-cell stage (24 hours after fertilization) and blastocyst formation (96 hours after fertilization) (Figure 2A). Telomere DNA was measured in individual oocytes and embryos and normalized against a reference gene using a validated quantitative PCR-based assay to account for cell number increase (Figure 4). Telomere elongation was detected up to the 2-cell stage (Figure 2B) and again at the 8-cell stage (Figure 2B). Between the 8-cell stage and the blastocyst stage, telomere elongation was rapid (occurring within approximately 40 hours) and on a larger scale (Figure 2B). This is consistent with the well-established telomerase-mediated elongation that occurs between the morula and blastocyst stages. To verify telomere elongation in proper embryos compared to the trophectoderm (TE, placental precursor) of individual blastocysts, telomere length in isolated ICMs (Figure 2C) was measured. This confirmed that telomere length was longer in the ICM (Figure 2D). At day 6 of culture, the point of normal implantation (Figure 2E), there was no further change in ICM telomere length, confirming that the ICM telomere length in blastocysts at day 5 was the maximum value achieved during pre-implantation development.

[0218] Oxidative stress is a critical determinant of telomere stability. Specifically, because telomeres are GC-rich, they are particularly sensitive to telomere shortening induced by oxidative stress. In contrast, the potential effects of oxidative stress on telomere elongation during preimplantation embryogenesis are unclear. Therefore, to better understand the regulation of telomere elongation rates, we investigated the effects of inducing oxidative stress during embryo culture on telomere length. Oxidative stress was induced by exposing mouse MII oocytes to 20% O2 (similar to ambient air) during in vitro fertilization (IVF) and embryo culture (Figure 2F). This was compared to embryos fertilized and cultured in 5% O2 (this is the gold standard for mouse and human in vitro embryo culture to better mimic the female reproductive tract). Telomere length in 8-cell embryos was unaffected by culture in 20% O2 (Figure 2G), showing no defects in the early stages of elongation (and no acceleration of telomere shortening). However, by the morula stage, embryos cultured under high oxygen conditions showed shorter telomeres (Figure 2H). The normal increase in telomere length per cell between the morula stage and the intracellular blastocyst (ICM) was stunted in embryos exposed to oxidative 20% O2 culture conditions (Figure 2H). Validation in a larger cohort of embryos clearly demonstrated a significant reduction in telomere DNA per cell in ICMs derived from embryos cultured under high oxygen conditions (Figure 2I). These results indicate a previously uncharacterized association between oxidative stress and telomere biology. Specifically, in contrast to the well-established mechanisms of ROS-induced telomere shortening, this study demonstrates that oxidative stress impairs telomere elongation in embryos, particularly during morula and blastocyst development.

[0219] Mitochondria are a major site of ROS production, and more recently, they have been identified as an intracellular source of metabolites and enzymes that influence epigenetic reprogramming and zygote genome activation, processes that directly control developmental gene expression networks. Therefore, we investigated whether mitochondrial dysfunction may be a potential mechanistic link between oxidative stress and impaired telomere elongation. Mitochondrial ROS (mtROS, reflecting mitochondrial superoxide production) and mitochondrial membrane potential (MMP, an indicator of oxidative phosphorylation) were measured to characterize the effect of 20% O2-induced oxidative stress on mitochondrial bioenergy. At 6 hours post-IVF, mtROS was significantly increased in zygotes exposed to 20% O2 (Figure 2J). In contrast, MMP, detected using TMRM potentiometric dye, was unchanged in zygotes or 8-cell embryos, but the significant increase in MMP observed at the morula and blastocyst stages was significantly blunted in embryos cultured at 20% O2 (Figure 2K).

[0220] To investigate whether the disruption of oocyte mitochondrial activity is associated with zygote nuclear changes, the overall methylation status was measured by immunohistochemistry as a general assessment of the epigenetic reprogramming state in pronuclear-stage zygotes (10 hours post-IVF). As expected (Figure 2L, Figure 8), the paternal pronucleus (the larger of the two) was preferentially stained for 5-hydroxymethylcytosine (5hmC), the oxidized form of 5-methylcytosine (5mC) produced during Tet-mediated demethylation. In zygotes exposed to 20% O2, the difference in 5hmC abundance between paternal and maternal pronuclei was reduced (Figure 2L, Figure 8). This indicates that oxidative stress alters zygote nuclear processes, potentially involving epigenetic reprogramming or transcriptional activation. Therefore, disruption of mitochondrial function and / or pronuclear reprogramming may be a cause of telomere elongation impairment during late pre-transplant development.

[0221] Example 2 In later generations, telomere length is regulated by the reprogramming of mitochondria and zygote nuclei in oocytes. To directly test whether mitochondrial function regulates telomere elongation during embryogenesis, mitochondrial bioenergy activity was specifically targeted using a complex I inhibitor (rotenone), and telomere lengths were measured in oocytes, 8-cell embryos, and blastocysts. Low doses of rotenone (150 ppm in feed (OA Lozoya et al., Single Nucleotide Resolution Analysis Reveals Pervasive, Long-Lasting DNA Methylation Changes by Developmental Exposure to a Mitochondrial Toxicant. Cell reports 32, 108131 (2020))) were provided to 6-week-old mice for 3 weeks prior to IVF under the same conditions as the control (Figure 9A). Six hours after IVF, zygotes derived from oocytes of rotenone-exposed females showed a dramatic increase in mtROS (Figure 9B). Disrupted 5mC / 5hmC epigenetic patterning was also evident in zygotes derived from oocytes of rotenone-fed mice (Figure 9C, Figure 10), indicating that disrupted oocyte mitochondrial activity directly influences pronuclear reprogramming events. Rotenone-induced disruption of MMPs persisted in 8-cell embryos (Figure 9D).

[0222] Telomere length was shorter in MII oocytes derived from rotenone-exposed females compared to controls (Figure 9E), likely due to ROS-mediated telomere shortening during follicular formation. However, this deficit reversed by the 8-cell stage (Figure 9E), again suggesting that the earliest telomere elongation stage is not affected by mitochondrial bioenergy or oxidative stress. In embryos derived from rotenone-exposed female oocytes, some telomere elongation occurred between the 8-cell and blastocyst stages, but blastocyst telomeres were significantly shorter, particularly within the ICM (Figure 9F) (Figure 9E). These results demonstrate that direct perturbation of pre-fertilization oocyte mitochondrial function impairs subsequent telomere elongation capacity, especially between the 8-cell and blastocyst stages of preimplantation, which is the same phenotype as that observed in embryos exposed to in vitro oxidative stress.

[0223] Blastocysts derived from control or rotenone-exposed females were transplanted into surrogate uteruses (to replicate the same embryonic environment), and the fetuses were examined near birth (see Figure 6) to determine whether the ICM telomere length deficit persisted in the progeny. Telomeres were shorter in the progeny's hearts (Figure 9G), reflecting the relative difference in ICM. These data demonstrate that the relative difference in telomere length in blastocyst pluripotent stem cells persists throughout fetal development. Furthermore, since the progeny from both groups conceived under identical conditions for fertilization, embryo culture, and embryonic development, differential molecular signals determining telomere length at birth were present within the ovulated egg.

[0224] To elucidate the role of mitochondrial versus nuclear events in regulating telomere elongation in the intracellular mass cell (ICM), the inventors performed reciprocal pronuclear translocation. At the one-cell stage (approximately 8 hours post-fertilization), pronuclei derived from either rotenone-fed mice or control mice were microinjected into the enucleated cytoplasm of rotenone-exposed or control zygotes to obtain four types of reconstructed embryos, which were cultured to the blastocyst stage (Figure 9H, Figure 3). The ICMs of embryos reconstructed from rotenone-exposed pronuclei plus rotenone-exposed cytoplasm had shorter telomeres than those of embryos reconstructed from control components (Figure 9I), consistent with the relative differences in unreconstructed embryos (Figure 9F). Telomere length was also reduced in embryos reconstructed from the pronuclei of rotenone-exposed zygotes, but not in those that received cytoplasm from rotenone-exposed zygotes (Figure 9I). This demonstrates that nuclear material contains molecular signals that impair telomere elongation ability. Therefore, disruption of mitochondrial function in oocytes at fertilization leads to pronuclear epigenetic changes before the first mitosis, which are responsible for impaired telomere elongation in the ICM during the blastocyst stage.

[0225] We investigated whether ICM telomere length could be restored by regulating mitochondrial activity in oocytes. We used BGP-15, a hydroxyl acid compound that improves mitochondrial bioenergy, prevents fragmentation associated with induced oxidative stress, and has shown therapeutic utility in preclinical lesions with mitochondrial dysfunction. Mice fed rotenone were treated with BGP-15 (100 mg / kg by ip injection) or a saline vehicle for 4 days prior to gonadotropin-stimulated ovulation (T. Umehara et al., Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Science advances 8, eabn4564 (2022), LL Wu et al., Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors. Development (Cambridge, England) 142, 681-691 (2015)), and IVF was performed under the same conditions (Figure 9A). This pre-fertilization BGP-15 treatment significantly reduced mtROS levels in zygotes of rotenone-fed mice (Figure 9J). BGP-15 treatment in rotenone-fed females also reduced defects in ICM telomere length (Figure 9K). Cumulatively, these data suggest that ICM telomere length is tightly linked to oocyte mtROS levels at fertilization, and that this biology can be modified to influence telomere setpoints.

[0226] Example 3 Maternal aging or obesity impairs telomere elongation in the oocyte mitochondrial cell mass (ICM), which is reversible by restoring mitochondrial function in the oocyte. Two physiological conditions affect oocyte mitochondrial function are aging and obesity. We investigated whether telomere elongation is affected in relation to these conditions and whether it is reversible using BGP-15 to restore mitochondrial function. Reproductively aged female mice (12 months old, estimated to be 38-45 years old for females) or young (3-4 months old) female mice were treated with BGP-15 (100 mg / kg / day for 4 days) or vehicle, and ovulated oocytes were subjected to IVF under identical conditions (Figure 11A). Ovulated oocytes from aged mice showed high levels of mtROS, which was reduced in females treated with BGP-15 (Figure 11B). Zygotes from aged females tended to show a change in the relative levels of 5mC and 5hmC in the paternal pronucleus compared to the maternal pronucleus, while those pre-treated with BGP-15 showed the same ratio as zygotes from young mice (Figure 11C). Mitochondrial membrane potential (MMP) was reduced in morulae derived from aged mice. This deficiency was normalized in female embryos treated with BGP-15 prefertilization (Figure 11D).

[0227] Ovulating (MII) oocytes from aged mice had shorter telomeres (Figure 11E), and interestingly, oocyte telomere length was maintained in breeding pairs and was significantly shorter in nulliparous females (who had experienced consecutive ovarian cycles) compared to littermates who were continuously pregnant (Figure 11F). Therefore, in animals of precisely the same age, limiting the number of estrous cycles appears to have a protective effect on oocyte telomere length. After IVF, there was no difference in telomere length between 8-cell embryos from aged and juvenile mice (Figure 11G), indicating normalization of defects in oocytes. However, blastocysts from aged mice had shorter telomeres than those from juvenile mice (Figure 11H), indicating a defect in the later stages of elongation. BGP-15 treatment of aged mice (for 4 days before ovulation) restored telomere length in blastocysts (Figure 11H). Telomere length in the ICM population reflects that of the entire blastocyst, maternal reproductive aging reduces telomere length in ICMs, and BGP-15 treatment restores this deficiency (Figure 11I).

[0228] Given the remarkable effect of BGP-15 on restoring ICM telomere length, we tested two other pharmaceuticals characterized by improved mitochondrial bioenergy. MitoQ is a commercially available mitochondrial-targeted CoQ10 ubiquinone, while metformin is commonly prescribed for the treatment of insulin resistance, including in women seeking pregnancy. Reproductively aged (12 months) female mice were administered either metformin (2 mg / mL) or MitoQ (150 μM) in drinking water for two weeks prior to gonadotropin-stimulated ovulation, followed by IVF under standard conditions. Treatment of aged females with metformin restored ICM telomere length, resulting in embryos indistinguishable from those derived from younger females (Figure 11J). These results further demonstrate that telomere elongation capacity during preimplantation development is regulated by maternal factors contained within ovulated oocytes, particularly mitochondrial activity, and provide proof of concept that this biology can be targeted to influence embryonic telomere length.

[0229] The effects of maternal obesity on telomere elongation dynamics in offspring were investigated by comparing obese mice (due to overeating) with lean littermates, using ovulated oocytes from all groups that underwent IVF under identical conditions in mice treated with BGP-15 for 4 days pre-ovulation (Figure 13A). High mtROS (Figure 13B) and modified 5mC / 5hmC pronuclear epigenetic patterns (Figures 13C, 14) were observed in zygotes from obese mice and were normalized by pre-fertilization treatment with BGP-15. Mitochondrial membrane potential (MMP) was reduced in 8-cell embryos and morulae, and this deficiency was normalized in female embryos treated with pre-fertilization BGP-15 (Figures 13D-13G).

[0230] In MII oocytes derived from obese mice, telomere length per cell was reduced compared to lean mice, but this was not affected by pre-fertilization treatment with BGP-15 (Figure 13H). At the 8C stage, telomere length did not differ between groups, as did normalization in embryos exposed to other mitochondrial stressors (i.e., 20% O2, rotenone, and aging) (Figure 13I). At the blastocyst stage, telomeres in embryos from obese mice were shorter than those from lean controls (Figure 13J), and this was a clear defect in the ICM (Figure 13K). BGP-15 treatment of obese mice restored blastocyst telomere length, including the ICM (Figure 13K), to the same level as lean controls (Figure 13J). In a manner precisely similar to maternal reproductive aging, obese female mice were administered either metformin (2 mg / mL) or MitoQ (150 μM) in their drinking water for two weeks prior to gonadotropin-stimulated ovulation, followed by IVF. Both treatments restored ICM telomere length to levels comparable to those of embryos derived from lean mice (Figure 13L). Cumulatively, these data demonstrate that physiological disruption of oocyte mitochondria (due to aging or obesity) is responsible for impaired telomere elongation and reduced telomere length in the progeny's ICM.

[0231] Blastocysts derived from oocytes of lean mice, obese mice, and obese mice treated with BGP-15 (in vivo for 4 days) were transplanted into lean surrogate parents of the same strain, and telomere length was analyzed in fetal tissue at day 14.5. Telomere length was shorter in both the heart and liver of fetuses derived from obese female oocytes (Figure 13M), demonstrating that the relative difference in ICM (Figure 13K) was maintained throughout fetal development. Furthermore, fetuses derived from obese female oocytes treated with pre-fertilization BGP-15 showed increased telomere length in both the heart and liver compared to progeny from untreated obese controls (Figure 13M). Therefore, maternal obesity results in a fundamental defect in oocytes that impairs telomere elongation in the ICM, i.e., a deficiency in telomere setpoints that persists throughout the embryo. This molecular change is preventable by systemic treatment with mitochondrial-targeting therapeutics on the day before ovulation.

[0232] To further investigate the developmental timeframes affecting telomere elongation capacity, we tested whether direct acute manipulation of mitochondrial bioenergy at fertilization via in vitro administration of BGP-15 could restore telomere elongation in blastocysts. When oocytes from lean control mice or obese mice were treated with BGP-15 (10 μM) in IVF and embryo culture medium, mtROS levels in zygotes derived from obese mouse oocytes were reduced within 6 hours (Figure 13N). The same response to BGP-15 was observed in zygotes prepared from oocytes exposed to 20% O2 (Figure 15) or from oocytes from rotenone-fed mice (Figure 16), demonstrating the effectiveness of BGP-15 in directly influencing mtROS production. Treatment of obese mouse oocytes with BGP-15 during IVF and embryo culture also normalized MMPs at the morula stage (Figure 13O). Most notably, this in vitro BGP-15 treatment normalized ICM telomere length defects in blastocysts (Figure 13P). These results demonstrate that telomere elongation capacity can be acutely regulated even from fertilization. This information, combined with the results of pronuclear translocation experiments (Figures 9H-9I), identifies the time between fertilization and PN5 (approximately 10 hours post-IVF) as a critical developmental window in which mitochondrial-pronuclear signaling determines telomere elongation capacity 4 days later, establishing ICM telomere setpoints.

[0233] conclusion Therefore, new discoveries have been made regarding the mechanisms controlling telomere regeneration in the early postnatal period and the resetting of telomere length in progeny. Specifically, between fertilization and the first zygote cell division, optimal mitochondrial function and epigenetic remodeling are linked to induce telomere elongation in the later stages of preimplantation development and determine the ICM telomere setpoint. Through this process, within the first few days after fertilization, relative differences in telomere length at birth, which are major determinants of lifelong health and lifespan, are established. These findings have significant clinical and public health implications. They offer a potential mechanistic explanation for the development of shorter telomeres in some infants and identify oxidative stress to blastocysts in culture as a possible basis for shorter telomeres in IVF-pregnancy infants. More broadly, they demonstrate that maternal health and environmental conditions at fertilization have long-term consequences, including, now, influence the progeny's susceptibility (or resilience) to aging and age-related diseases in later life.

[0234] Different regulatory signaling pathways have been identified between the two organelles, with appropriate mitochondrial bioenergy promoting telomere elongation capacity. While proper regulation of mitochondrial metabolism is central to the establishment of embryonic stem cell pluripotency and subsequent cell fate acquisition, telomere elongation is a prominent and essential component of nuclear reprogramming. Therefore, the identification of this new link between mitochondria and telomeres during in vivo embryogenesis may extend to other contexts of cell reprogramming and stem cell biology. While we do not wish to be constrained by theory, the molecular signaling queue between mitochondrial activity at fertilization and the telomere elongation mechanism, which is active several days later, likely involves mtROS (or other mitochondrial metabolite) signaling to nuclear effectors that actively reprogram DNA epigenetic marks and histone modifications to regulate subsequent gene expression.

[0235] These findings provide new evidence that the pre- and post-fertilization period between ovulation and gamete fusion is a critical developmental period in which maternal environmental signals are transmitted to the next generation. They reveal novel mechanisms by which oocyte mitochondria acutely respond to maternal physiological cues, initiating specific nuclear responses that shape the lifelong health trajectory of offspring. Demonstrating the plasticity of oocyte mitochondrial responses and identifying specific pharmaceutical compounds that can modulate these responses pre- and immediately after fertilization means that therapeutic opportunities exist to optimize this biology, which is a major determinant of chronic disease risk.

[0236] All publications referenced herein are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the invention, which will be apparent to those skilled in the art in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

Claims

1. A mitochondrial targeting agent used to treat telomere-related disorders.

2. A method for treating telomere-related disorders, comprising the administration of a mitochondrial targeting agent.

3. Use of mitochondrial targeting agents in the manufacture of pharmaceuticals to treat telomere-related disorders.

4. A mitochondrial targeting agent, method, or use according to any one of claims 1 to 3, comprising administering the mitochondrial targeting agent to a subject having the telomere-related disorder after the subject's birth.

5. The subject is a child or an adult, preferably an adult, mitochondrial targeting agent, method or use for use according to claim 4.

6. Mitochondrial targeting agents used to increase telomere length or inhibit telomere shortening.

7. A method for increasing telomere length or inhibiting telomere shortening, comprising the administration of a mitochondrial targeting agent.

8. The use of mitochondrial targeting agents in the manufacture of pharmaceuticals for increasing telomere length or inhibiting telomere shortening.

9. A mitochondrial targeting agent, method, or use for use according to any one of claims 6 to 8, comprising administering the mitochondrial targeting agent to a subject after the birth of the subject.

10. The subject is a child or an adult, preferably an adult, mitochondrial targeting agent, method or use for use according to claim 9.

11. A mitochondrial targeting agent, method, or use for use according to any one of claims 6 to 10, wherein increasing telomere length or inhibiting telomere shortening is used to treat telomere-related disorders.

12. A mitochondrial targeting agent, method, or use for use according to any one of claims 1 to 3, 6 to 8, or 11, comprising contacting gametes and / or their fertilized products with the mitochondrial targeting agent.

13. The telomere-related disorder is prevented and / or treated by increasing telomere length during embryonic development, as described in any one of claims 1 to 3, 11, or 12, a mitochondrial targeting agent, method, or use for use according to claim 1 to 3, 11, or 12.

14. The telomere-related disorder is aging (e.g., premature aging), bone marrow failure, congenital dyskeratosis, acquired aplastic anemia, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), liver disease (e.g., associated with or resulting from congenital dyskeratosis and / or pulmonary fibrosis), cardiovascular disease, heart disease, cancer, neurodegeneration, inflammatory bowel disease, Barrett's esophagus and / or miscarriage (e.g., recurrent miscarriage), a mitochondrial targeting agent, method or use for use according to any one of claims 1 to 5 or 11 to 13.

15. A mitochondrial targeting agent for use in increasing telomere length during embryonic development, wherein such use comprises contacting gametes and / or their fertilized products with the mitochondrial targeting agent.

16. A method for increasing telomere length during embryonic development, comprising contacting gametes and / or their fertilized products with a mitochondrial targeting agent.

17. The use of a mitochondrial targeting agent in the manufacture of a pharmaceutical product for increasing telomere length during embryonic development, comprising contacting gametes and / or their fertilized products with the mitochondrial targeting agent.

18. The mitochondrial targeting agent, method or use for use according to any one of claims 12 to 17, wherein the gametes and / or their fertilized products are brought into contact with the mitochondrial targeting agent by administration of the mitochondrial targeting agent to a female mammal containing the gametes and / or their fertilized products, and the gametes are oocytes.

19. The gametes are produced, produced, and / or produced by administration of the mitochondrial targeting agent to a male mammal, and the gametes are sperm, a mitochondrial targeting agent, method or use for use according to any one of claims 12 to 17.

20. The mitochondrial targeting agent, method, or use for use according to claim 18 or 19, wherein the embryo subsequently produced by the gametes and / or their fertilized product has an enlarged telomere length.

21. The mammal has reduced reproductive capacity, the mitochondrial targeting agent, method or use for use according to any one of claims 18 to 20.

22. A method for increasing telomere length during embryonic development, comprising contacting gametes and / or their fertilized products with a mitochondrial targeting agent in vitro.

23. Use of a mitochondrial targeting agent to increase telomere length during embryonic development, comprising contacting gametes and / or their fertilized products with the mitochondrial targeting agent in vitro.

24. A method for generating an embryo, comprising contacting gametes and / or their fertilized products with a mitochondrial targeting agent in vitro.

25. The gametes are used in an in vitro fertilization method, according to or use according to any one of claims 22 to 24.

26. A method for increasing telomere length during embryonic development, comprising using a gamete and / or its fertilized product that has been in contact with a mitochondrial targeting agent in an assisted reproductive method, preferably an in vitro fertilization method.

27. Use of a mitochondrial targeting agent to increase telomere length during embryonic development, comprising using a gamete and / or its fertilized product that has been in contact with the mitochondrial targeting agent in an assisted reproductive method, preferably an in vitro fertilization method.

28. A method for generating an embryo, comprising using a gamete and / or its fertilized product that has been in contact with a mitochondrial targeting agent, in an assisted reproductive method, preferably an in vitro fertilization method.

29. The method or use according to any one of claims 26 to 28, wherein the gametes are in contact with the mitochondrial targeting agent by administering the mitochondrial targeting agent to a mammal.

30. The method or use according to any one of claims 26 to 28, wherein the gametes are in contact with the mitochondrial targeting agent in vitro.

31. The method or use according to any one of claims 22 to 30, wherein the gametes are oocytes obtained from female mammals having reduced reproductive capacity.

32. The method or use according to any one of claims 22 to 30, wherein the gametes are sperm obtained from a male mammal having reduced reproductive capacity.

33. The aforementioned mammals, (a) Preferably an aging mammal which is a human being at least 30, 35 or 40 years old, and / or (b) Preferably 25 kg / m ２ A mitochondrial targeting agent, method, or use for use according to any one of claims 18-21 or 31 or 32, wherein the human is an overweight or obese mammal having a body mass index (BMI) greater than 1.

34. The fertilization product of the gamete is a zygote or embryo, preferably a zygote, the mitochondrial targeting agent, method or use for use according to any one of claims 12 to 33.

35. A mitochondrial targeting agent, method or use for use according to any one of claims 12 to 34, wherein at least the gametes and the fertilized product of the gametes, which are zygotes, are contacted with the mitochondrial targeting agent (preferably before gamete fusion), and preferably only the gametes and the zygotes are administered with or contacted with the mitochondrial targeting agent, most preferably before gamete fusion.

36. The fertilization product of the gamete is a zygote, and at least the zygote is contacted with the mitochondrial targeting agent prior to PN5, preferably the fertilization product of the gamete is a zygote, and only the zygote is contacted with the mitochondrial targeting agent prior to PN5, the mitochondrial targeting agent, method or use for use according to any one of claims 12 to 35.

37. The fertilized product of the gamete is contacted with the mitochondrial targeting agent from the 8-cell stage of embryonic development to the blastocyst stage and / or before implantation, preferably, the fertilized product of the gamete is contacted with the mitochondrial targeting agent only from the 8-cell stage of embryonic development to the blastocyst stage and / or before implantation, according to any one of claims 12 to 35.

38. A mitochondrial targeting agent, method, or use for use according to any one of claims 12 to 37, wherein the gametes and / or the fertilized product thereof contain elevated levels of mitochondrial reactive oxygen species prior to contact with the mitochondrial targeting agent (e.g., contact therewith).

39. The aforementioned mitochondrial targeting agent is (a) BGP-15 and / or acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates, (b) Mitoquinone mesylate (MitoQ) and / or acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates, (c) Metformin and / or its acceptable derivatives, prodrugs, solvates, salts, tautomers, stereoisomers and / or racemates The mitochondrial targeting agent, method, or use for use according to any one of claims 1 to 38.

40. The aforementioned mitochondrial targeting agent is (a) BGP-15 or its derivatives, (b) Mitoquinone mesylate (MitoQ) or its derivatives, (c) Metformin or its derivatives The mitochondrial targeting agent, method, or use for use according to any one of claims 1 to 39.

41. The embryo develops into a fetus having increased telomere length, according to any one of claims 13 to 40, mitochondrial targeting agent, method or use for use.

42. An embryo that can be obtained by the method described in any one of claims 24 or 25 or 28 to 41.

43. A mitochondrial targeting agent for use in altering the epigenetic state of a cell (e.g., nuclear epigenetic state), wherein such use preferably involves contacting the cell with the mitochondrial targeting agent.

44. A method for altering the epigenetic state of a cell (e.g., nuclear epigenetic state), comprising contacting the cell with a mitochondrial targeting agent (e.g., in vivo).

45. Use of a mitochondrial targeting agent in the manufacture of a pharmaceutical product for altering the epigenetic state of a cell (e.g., nuclear epigenetic state), preferably comprising contacting the cell with the mitochondrial targeting agent.

46. A method for altering the epigenetic state of a cell (e.g., nuclear epigenetic state), comprising contacting the cell with a mitochondrial targeting agent in vitro.

47. Use of a mitochondrial targeting agent to alter the epigenetic state of a cell (e.g., nuclear epigenetic state), comprising contacting the cell with the mitochondrial targeting agent in vitro.

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