WO2024105014A1

WO2024105014A1 - Antioxidant compound 10-(6'-plastoquinonyl)decyltriphenylphosphonium (skq1) for use in the prevention and/or treatment of the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal

Info

Publication number: WO2024105014A1
Application number: PCT/EP2023/081718
Authority: WO
Inventors: Ignasi Roig Navarro; Maria LOPEZ PANADES; Nikoleta NIKOU
Original assignee: Universitat Autonoma De Barcelona
Priority date: 2022-11-15
Filing date: 2023-11-14
Publication date: 2024-05-23

Abstract

The present invention relates to an antioxidant compound which is 10-(6'- Plastoquinonyl)decyltriphenylphosphonium (SkQ1) for use in the prevention and/or treatment of the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal.

Description

ANTIOXIDANT COMPOUND 10-(6'-PLASTOQUINONYL)DECYLTRIPHENYLPHOSPHONIUM (SKQ1) FOR USE IN THE PREVENTION AND/OR TREATMENT OF THE DETRIMENTAL EFFECT CAUSED BY NATURAL OR INDUCED AGING OF THE OVARIAN RESERVE OF A FEMALE MAMMAL

FIELD OF THE INVENTION

The present invention relates to the field of fertility in female mammals. In particular the present invention relates to an antioxidant compound which is 10-(6'- Plastoquinonyl)decyltriphenylphosphonium (SkQ1) for use in the prevention and/or treatment of the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal.

BACKGROUND

In mammals, oocyte development and maturation are critical processes for female fertility (Jamnongjit M, Hammes SR. Oocyte maturation: the coming of age of a germ cell. Semin Reprod Med. 2005 Aug; 23(3):234-41. doi: 10.1055/S-2005-872451. PMID: 16059829; PMCID: PMC1482430.). Lifestyle and diet habits seem to affect these processes significantly, resulting in differences in the fertility capability among populations (Sharma et al.: Lifestyle factors and reproductive health: taking control of your fertility. Reproductive Biology and Endocrinology 2013 11 :66.).

Sexual reproduction depends on the correct formation of haploid gametes. Germ cells (2n) undergo several steps to form spermatozoa or eggs (1 n). The completed chromosome complement will be reconstituted upon egg fertilization so that the new organism will contain genetic information from both parents. Mammalian gametogenesis presents extreme sexual dimorphism (Morelli, M. A., and Cohen, P. E. (2005). Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis. Reproduction 130, 761-781). For instance, while sperm are motile and have little contribution to the zygote cytoplasm, eggs are immobile and contribute to most of the zygote and embryo cytoplasm. During embryonic development, primordial germ cells (PGCs) migrate from the epiblast to reach the genital ridge. PGCs associate with somatic cells to form the embryonic gonads. At this point, male PGCs proliferate through several rounds of mitosis and then undergo an arrest at embryonic day 12, which is maintained until birth. Spermatogenesis resumes at puberty and continues cyclically through an adult's whole life producing sperm during most of the adulthood (Larose, H., Shami, A. N., Abbott, H., Manske, G., Lei, L., and Hammoud, S. S. (2019). Gametogenesis: A journey from inception to conception. Current Topics in Developmental Biology 132, 257-310. doi: 10.1016/BS.CTDB.2018.12.006.).

In contrast, female PGCs differentiate into proliferating oogonia. Around embryonic day 13, these oogonia differentiate into oocytes and enter the meiotic prophase. After completing the first meiotic prophase, oocytes arrest and surround with somatic cells to form primordial follicles. Within them, oocytes are in a quiescent state that may last decades. Associated with this process, there is a massive oocyte death that eliminates most of the fetal oocytes (Pepling, M. E. (2012). Follicular assembly: mechanisms of action. REPRODUCTION 143, 139-149. doi: 10.1530/REP-11-0299). Folliculogenesis initiates once these dormant primordial follicles are recruited to start growing. In mammals, female gametes contain most materials to maintain embryo development. Thus, in addition to forming a haploid nucleus, oogenesis has to secure the accumulation of RNAs, proteins, and organelles required to support the first stages of embryogenesis. Thus, folliculogenesis involves a remarkable increase in the oocyte size accompanied by a substantial proliferation of follicular cells surrounding it, increasing the total volume of the follicle more than 100-fold (van den Hurk, R., and Zhao, J. (2005). Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles. Theriogenology 63, 1717-1751. doi: 10.1016/j.theriogenology.2004.08.005). Folliculogenesis ends with forming a preovulatory follicle which contains an oocyte capable of resuming meiosis (Li, R., and Albertini, D. F. (2013). The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte. Nat Rev Mol Cell Biol 14, 141-52. doi: 10.1038/nrm3531.). Since the formation of the primordial follicles, some of these will be recruited to initiate folliculogenesis. Nonetheless, most of them will not succeed. In humans, it is estimated that only one in a thousand follicles will ovulate. Thus, most follicles will degenerate, a process globally called atresia (May-Panloup, P., Boucret, L., Chao de la Barca, J.-M., Desquiret-Dumas, V., Ferre-L’Hotellier, V., Moriniere, C., et al. (2016). Ovarian ageing: the role of mitochondria in oocytes and follicles. Human Reproduction Update 22, 725-743. doi: 10.1093/humupd/dmw028).

It is commonly accepted that mammalian females are born with a pool of follicles to be used during their reproductive lifespan (Horan, C. J., and Williams, S. A. (2017). Oocyte stem cells: fact or fantasy? Reproduction 154, R23-R35. doi: 10.1530/REP-17-0008.). Thus, fertility ends once the pool of primordial follicles falls below a certain threshold (in humans -1000 primordial follicles). Moreover, associated thereto, there is a significant reduction of estrogen production that affects other organs and tissues of the body. Notably, women with an early age of menopause have an increased risk of mortality, suggesting a link between increased fertility and longevity. Indeed, the onset of menopause has a negative impact on women's health and quality of life since it supposes an increased risk of cognitive decline, insomnia, depression, and weight gain. Moreover, after menopause, women have an increased risk of suffering osteoporosis, type 2 diabetes, or cardiovascular diseases, which may partly be responsible for this increase in mortality.

Thus, exhaustion of the oocyte pool has significant implications for women's health. However, we are only beginning to understand the genetic determinants of such an important process (Day, F. R., Ruth, K. S., Thompson, D. J., Lunetta, K. L., Pervjakova, N., Chasman, D. I., et al. (2015). Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1 -mediated DNA repair. Nature Genetics 47, 1294-1303. doi: 10.1038/ng.3412.). Most reproduction problems in western societies are linked to women's fertility declines with age (Gruhn, J. R., Zielinska, A. P., Shukla, V., Blanshard, R., Capalbo, A., Cimadomo, D., et al. (2019). Chromosome errors in human eggs shape natural fertility over reproductive life span. Science (1979) 365, 1466-1469. doi: 10.1126/SCIENCE.AAV7321/

SUPPL_FILE/AAV7321S1.MOV.). In western societies, the average age of women having their first child has increased significantly over the past decades. For instance, in the USA, it raised 5.5 years from 1970 to 2018, now close to 26 years on average. But the situation is even worse in Spain, where the average age of first motherhood is ~31 years. This change has multiple causes, including social, financial, professional, and medical ones. Thus, it is a very challenging situation to be reverted. Therefore, as a result of this delay in maternity we are encountering in western societies, there is an urgent need to find efficient treatments to protect the ovarian reserve and delay ovarian aging. Furthermore, these treatments will allow extending fertility but, more importantly, delay the onset of menopause.

Natural menopause occurs in all women, and despite life expectancy as risen significantly over the last decades, the onset of menopause has remained relatively constant around 50-52 years. We recently showed that the age at natural menopause is regulated by several genetic networks that act throughout oocyte and follicle development (Ruth, K. S., Day, F. R., Hussain, J., Martinez-Marchal, A., Aiken, C. E., Azad, A., et al. (2021). Genetic insights into biological mechanisms governing human ovarian ageing. Nature 596, 393-397. doi: 10.1038/S41586-021-03779-7).

Apart from genetic determinants that influence the extent of the ovarian reserve, an earlier menopause onset can be caused by toxins' detrimental effects on the follicles, such as cigarette smoke, chemicals, pesticides, or alcohol (Hawkins Bressler, L., Bernardi, L. A., de Chavez, P. J. D., Baird, D. D., Carnethon, M. R., and Marsh, E. E. (2016). Alcohol, cigarette smoking, and ovarian reserve in reproductive-age African- American women. Am J Obstet Gynecol 215, 758.e1-758.e9. doi:

10.1016/J.AJOG.2016.07.012.). For instance, moderate alcohol consumption has been linked to a diminished ovarian reserve in humans (Li, N., Fu, S., Zhu, F., Deng, X., and Shi, X. (2013). Alcohol intake induces diminished ovarian reserve in childbearing age women. Journal of Obstetrics and Gynaecology Research 39, 516-521. doi: 10.1111/J.1447-0756.2012.01992. X).

Accordingly, there is a need to find efficient treatments to protect the ovarian reserve and delay ovarian aging. The present invention discloses compounds capable of preventing and/or treating the detrimental effect caused by aging of the ovarian reserve of a female mammal.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an antioxidant compound which is 10- (6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) for use in the prevention and/or treatment of the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Gene networks overrepresented in our GWAs analysis. The plot represents the expected number of genes found in our GWAs analysis based on their abundance on the genome (shown as “expected”) and the percentages of genes found in our GWAS corresponding to each described network.

Fig. 2. Effects of ethanol and SkQ1 exposition to the ovarian reserve. A) The plot shows the total number of follicles found in control, ethanol-exposed and SkQ1-Ethanol-treated mice (“SkQ1 -ethanol” herein means that ethanol is used to induce ovarian aging on mice and the antioxidant compound SkQ1 is used for the treatment of said induced ovarian aging). B) The graph shows the number of primordial, primary, secondary and antral follicles found in each group. It is noted how ethanol-exposed mice have less follicles due to a specific elimination of primordial follicles. Also, the co-administration/ treatment with SkQ1 reverts the negative effect of ethanol to the primordial follicle population.

Fig. 3. Presence of gH2AX on exposed oocytes. A) The plot shows the percentage of follicles found in control, ethanol-exposed and SkQ1-Ethanol-treated mice that contained oocytes positive for gH2AX. No significant differences are found among all studied conditions or follicle types. B) Intensity of the gH2AX signal found in control (left), ethanol-exposed (centre) and SkQ1-Ethanol-treated primordial follicles (right).

Fig. 4. Effects of ethanol and SkQ1 -Ethanol exposition to the repair of DSBs. A-D) The plots show the total number RAD51 (A; C) and 53BP1 (B; D) foci found in control (left), ethanol-exposed (centre) and SkQ1-Ethanol-treated (right) mice primordial (A-B) and growing (C-D) follicles. Note how ethanol-exposed mice tend to repair more DSBs using HR compared to control or SkQ1-Ethanol-treated mice.

Fig. 5. SkQ1 exposition can preserve the ovarian reserve. A) The plot shows the total number of follicles found in control (left), DMSO-exposed (centre) and SkQ1 -treated (right) mice. B) The graph shows the number of primordial, primary secondary and antral follicles found in each group. It is noted that the treatment with SkQ1 increases the primordial follicle population.

Fig. 6. Effect of MitoQ on the ovarian reserve. A) Plot showing the total number of follicles found in control (left), DMSO-exposed (center) and MitoQ-treated (right) mice. B) Graph showing the number of primordial, primary, secondary and antral follicles found in each group. Note how neither DMSO nor MitoQ have any effect on the follicles (P>0.05; T test). These data suggest that the treatment with MitoQ has no effect on the ovarian reserve.

Fig. 7. Effect of Trolox and Vitamin C on the ovarian reserve. A) Plot showing the total number of follicles found in control (left), DMSO-exposed (center left), Trolox-treated (center right) and Vitamin C-treated (right) mice. B) Graph showing the number of primordial, primary, secondary and antral follicles found in each group. Note how Vitamin C increases the number of primordial and primary follicles (P=0.0043; P=0.0039; T test), but exposition to Trolox only results in an increase in primary follicles (P=0.0030; T test). These data suggest that Vitamin C may also protect the ovarian reserve as well as promote the increase in growing follicles. In the long term, this promotion of folliculogenesis could be detrimental for the preservation of the ovarian reserve.

Fig. 8. Effect of different antioxidant on the ovarian reserve. Reduction of the ovarian reserve (measured as the total number of primordial follicles) in control mice and mouse treated with DMSO, SKQ1 , Vitamin C, Trolox and MitoQ. From all the abovementioned drugs, only Vitamin C and SKQ1 show a decreased reduction of the ovarian reserve over time. Having SKQ1 a higher protective effect than Vitamin C. Neither antioxidants Trolox nor MitoQ have an effect in preventing ovarian reserve decline over time.

Fig. 9. Effect of different antioxidants on the ovarian reserve. To calculate the normalized ovarian reserve, the total number of primordial follicles present in the ovaries of mice treated with the drugs, was divided by the total number of primordial follicles found in the ovaries of same age control mice. Administration of DMSO, Trolox, MitoQ or Resveratrol, had no or limited effect on the number of primordial follicles present in the ovaries. On the other hand, administration of Vitamin C or SKQ1 resulted in a significant increase of the ovarian reserve (P>0.005, T Test).

Data used to calculate the effect of Resveratrol on the ovarian reserve comes from Liu M, Yin Y, Ye X, Zeng M, Zhao Q, Keefe DL, Liu L (2013) Resveratrol protects against age-associated infertility in mice. Hum Reprod. 2013 Mar;28(3):707-17. doi: 10.1093/humrep/des437.

From thisanalysis we can conclude that SKQ1 is the antioxidant with a higher protective effect for the ovarian reserve.

Fig. 10. Expected effect of Vitamin C and SKQ1 on the ovarian function. Reduction of the ovarian reserve (measured as the total number of primordial follicles) in control mice and mouse treated with SKQ1 and Vitamin C is shown in this graph. Notice how we predict SKQ1 treatment to be more effective that the one with Vitamin C, resulting in an expected increased ovarian function over time (up to 66 weeks) in mice treated with SKQ1 compared to those treated with Vitamin C (up to 58 weeks).

DETAILED DESCRIPTION

In another embodiment, the antioxidant compound of the present invention (SkQ1) is combined with at least one another active ingredient.

In a preferred embodiment, said at least one another active ingredient is another antioxidant compound selected from the group consisting of triphenylphosphonium (TPP)-based antioxidants, mitochondria-targeted antioxidants, Trolox, Vitamin C, Vitamin E, Propyl Gallate, Resveratrol, DMU-212, Rhapontigenin, Beta Carotene, Quercetin, and Pentoxyfylline. In the context of the present invention, the term “triphenylphosphonium (TPP)-based antioxidant” is intended to mean a compound whose chemical structure contains a triphenylphosphonium moiety and has the ability of acting as an antioxidant.

In the context of the present invention, the term “mitochondria-targeted antioxidant” is intended to mean a compound with the ability of acting as an antioxidant at mitochondrial level.

In a further preferred embodiment, said triphenylphosphonium (TPP)-based antioxidant is selected from, SkQPalm, Mito-apocynin (C11) and MitoQ. The compound of the present invention (SkQ1) belongs to this family and contains a lipophilic cation linked to an antioxidant, plastoquinone, via a saturated hydrocarbon chain. Due to its lipophilic properties, the triphenylphosphonium (TPP)-based antioxidant family can effectively penetrate cell membranes. The positive charge directs the whole molecule's transport, including antioxidant moiety, into the negatively charged mitochondrial matrix.

In another further preferred embodiment, said mitochondria-targeted antioxidant is selected from Tiron, elamipretide (SS-31) and XJB-5-131.

It is noted that triphenylphosphonium (TPP)-based antioxidants could also be included in the group of mitochondria-targeted antioxidants (for example, SkQ1), but not all the mitochondria-targeted antioxidant encompassed by the present invention are considered as triphenylphosphonium (TPP)-based antioxidants.

In this disclosure and in the claims, terms such as "comprises," "comprising," "containing" and "having" are open-ended terms and can mean "includes," "including," and the like; while terms like "consisting of" or "consists of" refer to the mentioned elements after these terms and others which are not mentioned are excluded.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise.

It should be noted that the term "approximately" or “about” applied to the values used earlier and later in this document includes a margin of error of ± 5 %, such as, for example, ± 4 %, ± 3 %, ± 2 %, ± 1 %.

As used herein, the term "treat" and its cognates refer to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, "treat" refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient, in another embodiment, "treat" refers to inhibiting the progression of a disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, in another embodiment, "treat" refers to slowing the progression or reversing the progression of a disease or disorder. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease or disorder. In the context of the present invention said “disease or disorder” would be the detrimental events caused by aging of the ovarian reserve of a female mammal.

As used herein, the term “preventing and/or treating” includes “preventing and treating” and “preventing or treating”.

The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a disease or disorder. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

In another preferred embodiment, said induced aging of the ovarian reserve of a female mammal is due to the intake of ethanol. In a further preferred embodiment said intake of ethanol is a chronic intake of ethanol. In a further preferred embodiment, said intake of ethanol is an acute intake of ethanol. By “induced” is understood any external agent or conditions.

In another preferred embodiment, the antioxidant compound of the present invention is included in a pharmaceutical composition which may also include at least one another active ingredient as mentioned above. Said composition may further comprise a pharmaceutically acceptable vehicle, adjuvant, diluent or excipient, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

In another preferred embodiment, the antioxidant compound of the present invention is included, alone or combined with at least one another active ingredient, in a functional food or a dietary supplement. “Alone” is meant to be as the unique active ingredient but including any acceptable vehicle, adjuvant, diluent or excipient suitable for such functional food or dietary supplement.

In another preferred embodiment, said prevention and/or treatment comprises orally, transdermally, topically or parenterally administering the antioxidant compound of the present invention or the pharmaceutical composition comprising thereof.

In another preferred embodiment, the antioxidant compound of the present invention or the pharmaceutical composition comprising thereof is administered in the form of an aqueous solution or DMSO solution.

In another preferred embodiment, said prevention and/or treatment comprises parenterally, preferably intravenously, administering the antioxidant compound of the present invention or the pharmaceutical composition comprising thereof.

In another preferred embodiment, the antioxidant compound for use according to the present invention is administered to a female mammal, wherein said mammal is a human being, i.e. a woman.

The present disclosure also relates to a method of preventing and/or treating the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal according to the embodiments included herein comprising the step of administering a therapeutically effective amount of an antioxidant compound which is 10- (6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1), according to the embodiments included herein, to a female mammal in need thereof, wherein preferably said mammal is a human being, i.e. a woman.

The compound of the present invention is capable of being administered in unit dose forms, wherein the term "unit dose" means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active compound itself, or as a pharmaceutically acceptable composition. The compound as provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable vehicle, adjuvant, diluent or excipient. Such compositions may be prepared for use in oral administration, particularly in the form of tablets or capsules; or parenteral administration, particularly in the form of liquid solutions, suspensions or emulsions.

In the case of oral administration, the tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatine blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colourings, and flavourings. In addition, the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bimodal.

Liquid preparations for administration include sterile aqueous or non-aqueous (for example, DMSO) solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavouring and colouring agents, and the like.

A number of examples will be provided below that are intended to illustrate the invention and in no way limit the scope of the invention, which is established by the attached claims.

EXAMPLES

Materials and Methods

Animals and treatment We used 109 four weeks old female mice from strain C57BL/6JOIaHsd for the treatment. The animals used were kept in the UAB animal house, and the Ethics Committee for Animal Experimentation of the Autonomous University of Barcelona (UAB) and the Catalan Government approved all procedures.

The 109 mice were randomly assigned to eleven different groups and two different rounds of treatments. We used 10 animals to analyze the time zero (4 weeks of age) of the experimental process, so they did not receive and treatment. Then, 69 animals were used in a 14 weeks treatment, divided in the following groups: Control, Ethanol, SkQ1+EtOH, DMSO, SkQ1+DMSO (SkQ1), Vitamin C+DMSO (Vitamin C), Trolox +DMSO (Trolox). All mice were housed in groups of five. Control mice were housed in a controlled environment for 14 weeks with food and water ad libitum. Ethanol mice were administered a solution of water containing 0.1% of ethanol ad libitum for 14 weeks. DMSO mice were administered a solution of water containing 0.1% of DMSO ad libitum for 14 weeks. We dissolved SkQ1 in ethanol or DMSO and incorporated it into the mice's drinking water bottles at a final ethanol concentration of 0.1% and a SkQ1 concentration of 7.5 pM. The SkQ1-Ethanol and SkQ1-DMSO mice had undisturbed access to the drinking bottle for 14 weeks. The Vitamin C-DMSO mice were administered via drinking water Vitamin C diluted in DMSO, at final concentration of 500mg/ml. The Trolox-DMSO mice were administered with through their drinking water at a final concentration of 48mg/mL. The final age of the animals is 18 weeks old.

We used 30 animals for a treatment 7 weeks long. The animals were randomly assigned to three different groups: Control, DMSO, MitoQ-DMSO (MitoQ). All mice were housed in groups of five, in a controlled environment for 14 weeks with food and water ab libitum. The Control mice were administered with water and food. The DMSO mice were administered a solution of water containing 0.1 % of DMSO ad libitum for 7 weeks. We dissolved the MitoQ in DMSO and incorporated it into the mice’s drinking water bottles at a final concentration of DMSO 0.1 % and a MitoQ concentration of 100pmol/L.

Histology and oocyte quantification

Harvested ovaries were immediately fixed overnight in 4% paraformaldehyde in PBS or in Bouin solution (70% saturated picric acid solution, 25% formaldehyde, 5% glacial acetic acid). Samples were then dehydrated, cleared, and embedded in paraffin using standard procedures. The whole ovary was sectioned at 7 pm thickness, and a fifth of the ovary (every fifth section) was processed for immunostaining as follows: the sections were deparaffinized and stained with PAS-H as previously performed (Ruth et al., 2021 see above).

Follicles were manually counted and classified under the microscope (Olympus CH-2) with the 100x objective with oil. The analyzed follicles were classified as previously reported Pacheco S, Maldonado-Linares A, Garcia-Caldes M, Roig I (2019) ATR function is indispensable to allow proper mammalian follicle development. Chromosoma . 2019 Sep;128(3):489-500. doi: 10.1007/s00412-019-00723-7.

To predict the effect of the treatment on the extension of the ovarian reserve, first we multiplied the number of observed primordial follicles in control and treated mice at 5 and 19 weeks by 5 to extrapolate the total number of primordial follicles found in the ovaries. Then, we performed a lineal regression with the data obtained per each condition to predict the expected exhaustion of the ovarian reserve.

Immunolabeling

Immunofluorescence is a procedure used to detect a target molecule with a primary antibody conjugated with a fluorophore directly or/ and a secondary antibody that recognizes an epitope in the primary which is chemically conjugated with a fluorochrome, detectable for an epifluorescence microscope. Thus, this staining can detect specific molecules in a fixed tissue or cell. In that project, immunofluorescence staining was used in order to detect and analyze the DNA damage with the use of the DNA Double Strand Breaks marker, anti-gH2AX, and the DNA repair mechanisms with the use of the RAD51 , for detection of the Homologous Recombination pathway activation, and the 53BP1 , for Non-Homologous End Joining activation.

First, the ovary sections were deparaffinized and rehydrated as previously performed (Ruth et al., 2021 , see above). Then, the slides were subjected to antigen retrieval, to unmask epitopes by placing them in a plastic container fully submerged with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20 in MilliQ water, pH 6.0), and they were microwaved, at the highest power for 15 minutes. By the end of that time, the lunch box was removed from the microwave and let stand at Room Temperature (RT) to cool down for 15 minutes.

Next, the slides were washed with 0.1% Tween-20 in PBS 1x, and permeabilization with 0.5% Triton X-100 in 1x PBS was performed for 15 minutes. Then, they were washed one more time and incubated with a blocking solution. To analyze DNA damage, the slides were blocked for 1 hour using Bovine Serum Albumin blocking solution (0,4% BSA, 0,05% Tween-20 in PBS 1x). For the immunofluorescence protocol of the RAD51 and 53BP1 detection, a goat serum blocking buffer was used (1% goat serum, 0.01% Tween- 20, 0.3% BSA, 0.0252% Glycine, 1% PBS x10, 8% MilliQ water, 0.4% 5% TritonX-100 in PBS 1x). After blocking the slides, primary antibodies diluted in blocking buffer (Table 1) were added to each section (5 pl per section), covered with parafilm, and incubated in a humid chamber at 4°C overnight.

Then, the slides were washed two times, 3 minutes each time, with 0.1 % Tween-20 in PBS 1x. A fluorescence conjugated secondary antibody diluted in blocking buffer was added (5 pl per section), and the slides were covered with parafilm and incubated at 37 °C in a humid chamber for 1 hour. After the incubation, they were washed three times, 3 minutes each time, with 0.1% Tween-20 in PBS 1x. Finally, the slides were drained, and 8 pg/ml DAPI (Sigma-Aldrich) was added to Vectashield mounting medium (Vector Labs). Finally, a coverslip was added, and the sections were analyzed. The slides were stored at -20 °C for long-term storage or at 4°C for short periods.

The labeled sections for the imaging yH2AX were analyzed with the epifluorescence microscope (Zeiss Axiophot). The sections labeled for the RAD51 and 53BP1 were analyzed with a confocal microscope (Leica SP5).

Table 1 : Antibodies use in this study

Antibody Concentration Reference

(in blocking solution)

Cy3-conjugated anti-mouse (goat)

#115-165-003, Jackson

ImmunoResearch

Anti-53BP1 (mouse) 1:200 MAS 3802, Millipoie

Alexa Fluor 488 anti-Rabbit (goat)

Example 1. Alcohol and Oxidative stress regulate the age of natural menopause in humans.

To reveal the role of ROS and alcohol consumption in the regulation of the ovarian reserve in humans, we used our previously reported genome-wide association study (GWAS) data on the age of natural menopause (ANM) in women between 40 and 60 years (Ruth et al., 2021 , see above).

As mentioned above, among the >250 loci we detected to control ANM, 85 were involved in the DNA damage response (DDR), representing the most relevant pathway detected in our analysis. However, other pathways were also overrepresented in our GWAS. Thus, first, we studied the presence of alcohol-related genes in our GWAS hits. Interestingly, we found an overrepresentation of genes related to alcohol metabolism (6.9%, P=0.0016, Chi-square with Yates correction test, Fig. 1). More importantly, since alcohol has been known to cause ROS and oxidative stress in several cell types and tissues, we wondered if genes related to these were also overrepresented in our GWAS hits. Indeed, both genes related to ROS (6.5%, P=0.0301 , Chi-square with Yates correction test) and oxidative stress (10.7%, P=0.0105, Chi-square with Yates correction test) were overrepresented in our GWAS positive hits (Fig. 1). Finally, since ROS is a by-product of the cell metabolism originated in the mitochondria, we wondered if the genes-related to mitochondria were overrepresented in our GWAS. Again, we found a very significant overrepresentation of mitochondria-related genes in our list of loci regulating ANM (10.7%, P<0.0001 , Chi-square with Yates correction test).

Altogether, these data suggest that alcohol, ROS, oxidative stress, and mitochondria regulate the ANM and thus are relevant to controlling the human ovarian reserve.

Example 2. Daily ethanol consumption results in a significant decrease in the ovarian reserve.

Next, we tested if daily ethanol consumption could hamper the ovarian reserve in mice. To do so, we administered 0.1% ethanol dissolved in the drinking water of young female mice ad libitum for 14 weeks. After this treatment, we obtained the mouse ovaries and compared the number of follicles found in mice exposed to ethanol versus a control group that only drank water. We found a significant reduction in the total number of follicles present in the ovaries of mice exposed to ethanol (Fig. 2; P=0.0157, T test). Interestingly, this reduction was due to a decrease in almost 40% of the number of primordial follicles (Fig. 2; P=0.001 , T test). Contrastingly, growing follicles, which include primary, secondary, and antral follicles, were not affected by the exposure to ethanol (Fig. 2; P>0.05, T test). These data show that chronic alcohol consumption reduces the ovarian reserve by harming the primordial follicles. More importantly, this reduction in the ovarian reserve most likely decreases females' reproductive lifespan. Example 3. SkQ1 treatment protects the ovarian reserve from ethanol.

Since ethanol exposition has been linked to increased production of ROS in cells, we wondered if an antioxidant treatment could revert the toxic effect of ethanol in the ovarian reserve. Because the oocytes are the cell type of the body with more mitochondria, we decided to use the antioxidant SkQ1. Thus, we administered 7.5 M SkQ1 in a solution of 0.1 % ethanol in drinking water ad libitum for 14 weeks to young female mice. The treatment with SkQ1 rescued the oocytes from the adverse effects of ethanol since SkQ1-Ethanol-treated mice showed control levels of follicles in their ovaries (Fig. 2; P=0.7635, T test). Furthermore, the population of primordial follicles was significantly higher than the one found in ethanol-exposed mice (Fig. 2; P=0.0049, T test). Moreover, the follicle population was restored entirely in SkQ1 -treated mice (Fig. 2; P=0.8994, T test). These data suggest that chronic ethanol exposition causes oxidative stress, which might be detrimental to primordial follicles. Furthermore, it suggests that SkQI can revert the toxic effect of chronic ethanol consumption on the ovarian reserve.

Example 4. Resting and growing follicles have marks of DNA damage.

Due to the vast implication of the DDR in regulating the ovarian reserve, we investigated the presence of DNA damage in our control, ethanol-exposed and SkQ1-Ethanol-treated mouse oocytes. We expected to find an increased presence of DNA damage on ethanol- expose follicles. However, we found a high presence of gH2AX, a standard marker of DNA damage, in all follicles studied (Fig. 3A). No significant differences were found among the different groups of mice studied (P>0.05, T test). The absence of differences among all studied groups could be justified because oocytes that accumulated DNA damage in ethanol-exposed samples could have been apoptosed already. Interestingly, when we analyzed the intensity of the gH2AX signal (Fig. 3B), we observed that control and SkQ1 -treated cells had higher levels of gH2AX, in agreement with the idea that ethanol-exposed samples had already lost most of the oocytes with higher doses of DNA damage.

Example 5. DNA damage repair mechanisms

Since the analysis of gH2AX revealed no differences between the different studied groups, we studied the presence of markers for homologous recombination (RAD51) or non-homologous end joining), which are the most important mechanisms responsible for the repair of double-stranded DNA breaks (DSBs). Firstly, we found that most oocytes (-76% of primordial and 84% of growing follicles) presented either RAD51 foci, 53BP1 foci, or both kinds of foci, with no statistically significant differences among groups. Interestingly, we observed a shift in how DSBs were repaired in the different groups. While control mice presented more often 53BP1 foci than RAD51 foci in both primordial (percentage of oocytes with 53BP1 vs. RAD51 : 61 % vs. 44%) and growing follicles (82% vs. 58%), ethanol-exposed mice showed a different trend where RAD51 was most commonly found in primordial (percentage of oocytes with 53BP1 vs. RAD51 : 36% vs. 65%) and growing follicles (38% vs. 71%). Remarkably, SkQ1 -treated mice showed a control-like trend in primordial follicles (percentage of oocytes with 53BP1 vs. RAD51 : 52% vs. 38%). However, growing follicles showed a similar preference for NHEJ and HR (59% vs. 60%). These tendencies led to increased RAD51 foci in ethanol-exposed primordial follicles as compared to control mice (P=0.0101 , T test, Fig. 4). At the same time, control and SkQ1-Ethanol-treated mice have an increased presence of 53BP1 foci (P=0.0492, T test, Fig. 4). These data reflect that exposition to ethanol changes how DNA is repaired in primordial follicles, which could explain why there is a significant loss of primordial follicles in ethanol-exposed mice. Remarkably, treatment with SkQ1 reverts this change in the DNA damage repair dynamics.

Example 6. SkQ1 can preserve the follicle pool in control mice

Since our data suggested that an SkQ1 treatment could revert the effects of oxidative stress on the primordial follicle population, and oxidative stress has been commonly accepted as one of the significant causes of aging, we wondered if SkQ1 could prevent follicle loss associated with aging. To test this, we administered 7.5 pM SkQ1 in a solution of 0.1% DMSO in drinking water ad libitum for 14 weeks to young female mice. A group of mice was administered 0.1 % DMSO in drinking water to detect any effect of DMSO on the ovarian reserve. Although treatment with DMSO did not affect the ovarian reserve (Fig. 5), SkQ1 -treated mice presented more follicles than controls or DMSO- treated mice. This increase was due to a significant rise in primordial follicles (P=0.0022, T test). These data suggest that oxidative stress may be a key factor limiting the extent of mammalian female fertility. And more importantly, treatment with SkQ1 can protect the ovarian reserve, thus most likely extending fertility and ovarian function.

Since SkQ1 is a TTP-based antioxidant that targets the mitochondria, these data suggest that other TTP-based antioxidants and mitochondria-targeting antioxidants, and even other general antioxidants, can protect the ovarian reserve from its decline associated to aging and the exposure to some toxic agents, like ethanol.

Example 7. SkQ1 is the best antioxidant to protect the ovarian reserve from the effects of aging.

Because we observed that SkQ1 could protect the ovarian reserve from induced and natural aging, we wondered if other antioxidants could have similar effects. To test this, we treated young mice with MitoQ (Fig. 6), Trolox and Vitamin C (Fig. 7) (see Materials and Methods for details). Interestingly, MitoQ and Trolox had no effect on the ovarian reserve. However, Vitamin C-treated mice showed a slight increase in the number of primordial follicles present in their ovaries. Nonetheless, this increase was smaller than the observed with SkQ1 (Fig. 8 and 9). Our data predicts that SKQ1 treatment to be more effective that the one with Vitamin C, resulting in an expected increased ovarian function over time (Fig. 10). We also compared our results with the effect of resveratrol on the ovarian reserve published elsewhere (Liu M, Yin Y, Ye X, Zeng M, Zhao Q, Keefe DL, Liu L (2013) Resveratrol protects against age-associated infertility in mice. Hum Reprod. 2013 Mar;28(3):707-17. doi: 10.1093/humrep/des437). This comparison clearly shows that SkQ1 treatment is significantly more effective to protect the ovarian reserve than resveratrol (Fig. 9).

Conclusions

1. The human ovarian reserve is regulated by oxidative stress, alcohol metabolism, and mitochondria-related genes.

2. Daily intake of ethanol causes a severe reduction of the ovarian reserve due to the selective elimination of primordial follicles, which will limit ovarian function and fertility.

3. The treatment with the antioxidant compound of the present invention (SkQ1) reverts the adverse effects of chronic ethanol intake on the ovarian reserve.

4. Exposition to ethanol leads to a change in the repair pathway choice in the oocytes favouring homologous recombination instead of non-homologous end joining, which is reversible with treatment with the antioxidant compound of the present invention (SkQ1).

5. Oxidative stress is an essential factor limiting the extent of the ovarian reserve and, thus, regulating the aging process of the ovary.

6. The treatment with the antioxidant compound of the present invention (SkQ1), protects the ovarian reserve from natural or induced aging.

Claims

1. An antioxidant compound which is 10-(6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) for use in the prevention and/or treatment of the detrimental effect caused by natural or induced aging of the ovarian reserve of a female mammal.

2. The antioxidant compound for use according to claim 1 , wherein said antioxidant compound is combined with at least one another active ingredient.

3. The antioxidant compound for use according to claim 2, wherein said at least one another active ingredient is another antioxidant compound selected from the group consisting of triphenylphosphonium (TPP)-based antioxidants, mitochondria-targeted antioxidants, Trolox, Vitamin C, Vitamin E, Propyl Gallate, Resveratrol, DMU-212, Rhapontigenin, Beta Carotene, Quercetin, and Pentoxyfylline.

4. The antioxidant compound for use according to claim 3, wherein said triphenylphosphonium (TPP)-based antioxidant is selected from SkQPalm, Mito- apocynin (C11) and MitoQ.

5. The antioxidant compound for use according to claim 3, wherein said mitochondria- targeted antioxidant is selected from Tiron, elamipretide (SS-31) and XJB-5-131.

6. The antioxidant compound for use according to any of the preceding claims, wherein said antioxidant compound according to claim 1 or the combination of the antioxidant compound with at least one another active ingredient according to any of claims 2 to 5, is included in a pharmaceutical composition.

7. The antioxidant compound for use according to any of the preceding claims, wherein said prevention or treatment comprises orally, transdermally, topically or parenterally administering said antioxidant compound or the pharmaceutical composition comprising thereof.

8. The antioxidant compound for use according to any of the preceding claims, wherein said antioxidant compound or the pharmaceutical composition comprising thereof is administered in the form of an aqueous solution or DMSO solution.

9. The antioxidant compound for use according to any of claims 1-8, wherein said antioxidant compound according to claim 1 or the combination of the antioxidant compound with at least one another active ingredient according to any of claims 2 to 5 is included in a functional food or a dietary supplement.

10. The antioxidant compound for use according to any of the preceding claims, wherein said aging is due to the intake of ethanol.

11. The antioxidant compound for use according to claim 10, wherein said intake of ethanol is a chronic intake of ethanol.

12. The antioxidant compound for use according to claim 10, wherein said intake of ethanol is an acute intake of ethanol.

13. The antioxidant compound for use according to any of the preceding claims, wherein said female mammal is a woman.