Methods for preserving functional gametes

Abstract

The present invention relates to the use of carbon monoxide for the preservation of gametes. The present invention provides means and methods for the preservation of gametes. The method of the present invention comprises the steps of: a) providing gametes or a gamete-containing sample in a container; and b) contacting the gametes in the container with carbon monoxide. The present invention also provides the use of carbon monoxide for the treatment and / or prevention of diseases, such as diseases caused by or associated with DNA fragmentation or elevated redox potential of gametes, and / or diseases of gametes caused by or associated with elevated ROS levels. Further provided are the use of carbon monoxide for preventing congenital abnormalities and / or aneuploidy, and for treating diseases caused by and / or associated with DNA fragmentation and / or elevated redox potential of gametes. The present invention also provides a method for treating congenital abnormalities or aneuploidy, comprising contacting gametes of a patient in need thereof with carbon monoxide gas. Thus, the means, methods and uses according to the present invention allow for the preservation of gametes in a functional / intact state, thereby maintaining and / or improving the quality of the gametes, and thus making such carbon monoxide-treated / carbon monoxide-exposed gametes particularly useful in reproductive technologies, for example in vitro reproductive technologies, such as assisted human reproduction technologies, and / or (artificial) insemination technologies in animals.

Description

[Technical Field]

[0001] The present invention relates to the use of carbon monoxide for preserving gametes. The present invention provides means and methods for preserving gametes. The method of the present invention comprises the steps of: a) providing gametes or a gamete-containing sample in a container; and b) contacting the gametes in the container with carbon monoxide. The present invention also provides the use of carbon monoxide in the treatment and / or prevention of diseases, such as diseases caused by or associated with DNA fragmentation or elevated redox potential in gametes, and / or diseases of gametes caused by or associated with elevated ROS levels. Further provided are carbon monoxide for use in preventing congenital abnormalities and / or aneuploidy, and carbon monoxide for use in treating diseases caused by and / or associated with DNA fragmentation and / or elevated redox potential in gametes.

[0002] The present invention further relates to a method for treating congenital abnormalities or aneuploidies, comprising contacting gametes of a patient in need thereof with carbon monoxide gas. Specifically, the means, methods, and uses of the present invention allow gametes to be preserved in a functional / intact state, thereby maintaining and / or improving gamete quality. Therefore, such carbon monoxide-treated / carbon monoxide-exposed gametes are particularly useful in reproductive technologies, such as in vitro reproductive technologies, for example, in human assisted reproduction technologies and / or animal (artificial) insemination technologies. The present invention relates to in vivo applications in addition to ex corpore / in vitro applications in the medical field. In particular, desired (artificial) reproductive technologies are also described and presented in this disclosure. [Background technology]

[0003] Infertility is one of the most common health problems worldwide. The WHO estimates that approximately 15% of couples of childbearing age suffer from infertility (Rutstein, Infecundity, Reproduction, and Childlessness in Developing Countries. DHS Comparative Reports No. 9. Calverton, Maryland, USA. orc Macro and the World Health Organization, 2004). These cases specifically include female or male infertility, as well as cases where both male and female infertility are present. Idiopathic infertility accounts for approximately 40% of all male infertility cases (McLachlan, Med J Aust 174 (2001): 116-117). Between 30 and 85% of these cases are due to oxidative stress caused by the formation of reactive oxygen species (ROS), resulting in damage / impaired function of male gametes / sperm (due to DNA fragmentation, lipid peroxidation, apoptosis, etc.) (Mannucci, Front Mol Biosci 8 (2021): 799294).

[0004] Notably, infertility rates among healthy men are increasing, and sperm quality is declining, as evidenced by declining trends in sperm concentration (1.5% per year), sperm count (1.6% per year), total motility (0.4% per year), and rapid motility (5.5% per year) (Mahrukh Hameed, Infertility and Assisted Reproduction 8, 2021). According to data from the International Committee for Monitoring Assisted Reproductive Technology (ICMART), the global average number of assisted reproductive technology (ART) cycles is 3.2 million per year, of which IVF accounts for 1.6 million. However, despite this high number of ARTs, the live birth rate remains low at 22.5%, highlighting the need to improve access to high-quality infertility treatments for those in need (Technologies ICfMAR, ICMART Preliminary World Report: ART, 2018). This places a significant burden on couples and, due to the high cost of ART treatments, also strains healthcare systems worldwide. There is a need for cost-effective and accessible tools and methods in reproductive technology, particularly assisted reproductive technology.

[0005] The higher the reactive oxygen levels / damage / dysfunction of gametes, the lower the success rate of assisted reproductive technology (ART) and the more frequently ART cycles fail (Zorn, Int J Androl 26 (2003): 279-285). ROS levels in individuals, including potential gamete donors used in assisted reproductive technology, can be increased by, among other things, (physiological and / or psychological) stress, genetic predisposition, environmental factors (e.g., exposure to electromagnetic fields), and / or behavioral risk factors (e.g., diet, smoking, alcohol, drug abuse, etc.) (Agarwal, The Lancet, 2020; Agarwal, World J Mens Health, 2020; Esteves, Andrology, 2019). Other undesirable increases in ROS levels in individuals are well known to those skilled in the art.

[0006] Another important health concern is the potential for reactive oxygen species-related genetic changes in gametes, particularly sperm, to result in negative genetic changes in the newborn. For example, in natural conception, sperm with damaged DNA are unlikely to fertilize an egg. However, in ART, the selection process is omitted, so sperm with damaged DNA may be used unintentionally (Zini, Canadian Medical Association Journal, 175(5):495, 2006). Furthermore, ROS and the resulting oxidative stress on gametes, particularly sperm / sperm cells, pose a significant obstacle to the success of assisted reproductive technologies such as ART. In other words, physiological stress must be avoided for gametes used in such reproductive technologies. Again, one physiological challenge that must be addressed is the exposure of gametes both in the body (in corpora) and outside the body (ex corpore) to oxidative stress.

[0007] It has been proposed that oral administration of antioxidants to subjects can reduce reactive oxygen species and optimize sperm quality. A 2011 Cochrane review described studies of various drug therapies and concluded that administration of such antioxidants may lead to increased live birth rates after ART (Showell, Cochrane Database Syst Rev 1 (2011): CD007411).

[0008] However, systemic administration of antioxidants such as vitamin C, vitamin A, B-complex vitamins, and coenzyme Q10 has drawbacks (Sabeti, Int J Reprod Biomed 14 (2016): 231-240). First, these antioxidants must be administered regularly and over a long period (up to several months). Furthermore, these medications can cause side effects and are costly for both patients and insurers. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the technical problem that the present invention aims to solve is to provide functional gametes with desired properties for reproductive medical techniques, particularly assisted reproduction techniques. [Means for solving the problem]

[0010] Such technical problems are solved by the aspects and items described in this specification and the appended claims.

[0011] That is, the present invention provides a means and method for preserving gametes, comprising: a) providing / obtaining gametes or a gamete-containing sample in a container; and b) contacting the gametes or the gamete-containing sample in the container with carbon monoxide The present invention relates to a means and method including:

[0012] The time for which the gametes are contacted / exposed / treated with carbon monoxide (particularly carbon monoxide gas) can be, for example, long enough to preserve the gametes. Preservation of gametes particularly relates to preserving the functionality / intactness of the gametes. By contacting / exposing / treating gametes with carbon monoxide (particularly carbon monoxide gas) in accordance with the present invention, the gametes can be maintained and / or improved, thus resulting in gametes of maintained and / or improved quality. This is also explained in the disclosure below and particularly illustrated in the accompanying non-limiting examples and figures.

[0013] As will be understood, carbon monoxide (particularly carbon monoxide gas) may be administered directly to gametes, for example, by adding it to a buffer / buffer system. However, as will also be described in the present disclosure, carbon monoxide (particularly carbon monoxide gas) may also be contacted with a sample containing said gametes. The sample may be a biological sample. Examples include, but are not limited to, sperm cells / sperm, semen, glandular fluid, etc. As will be understood, the gametes contacted / exposed / treated with carbon monoxide / carbon monoxide gas may be contained in a buffer and / or buffer system. The gamete-containing sample may also be a diluted sample. That is, for example, in certain applications, it is contemplated that the semen or glandular fluid may be diluted by being placed in or mixed with a buffer or buffer system before or during exposure to carbon monoxide / carbon monoxide gas. Therefore, it will be understood that the term "gametes" also encompasses samples containing said gametes, such as biological samples. The terms "contacted with," "exposed to," and / or "treated with" are used interchangeably herein.

[0014] Thus, according to one aspect, the present invention relates to a means and method for the preservation of gametes, said method comprising, according to one aspect: a) providing gametes in a container; and b) contacting the gametes in the container with carbon monoxide gas for a period of time sufficient to ensure preservation of the gametes. Includes.

[0015] The gametes to be contacted / exposed / treated with carbon monoxide / carbon monoxide gas include sperm cells / sperm or egg cells, preferably sperm cells / sperm. Such gametes are animal gametes, preferably from mammals, including, but not limited to, humans. The sperm to be contacted / exposed / treated with carbon monoxide / carbon monoxide gas may be contained in semen, glandular fluid, and / or a buffer / buffer system when contacted / exposed / treated with carbon monoxide / carbon monoxide gas.

[0016] Carbon monoxide (CO) is an endogenous messenger molecule constantly produced in the human body. When the human physiological system is exposed to stressors, CO production is increased, triggering various defense mechanisms. Exogenous CO supplementation as a drug has been shown to have therapeutic potential (Motterlini, Nat Rev Drug Discov 9 (2010): 728-743). This effect has been demonstrated in numerous preclinical studies. However, the lack of a safe and effective system for CO application has prevented its practical application in humans (Hopper, Curr Pharm Des 24 (2018): 2264-2282).

[0017] U.S. Patent No. 9,980,981 B2 and WO 2012 / 096912 A1 disclose CO2-containing compositions that may be used to treat inflammatory and neurodegenerative diseases, but do not disclose their use in gamete preservation or subsequent reproductive technologies / ART. WO 2022 / 055991 A1 also discloses CO2-releasing formulations ("gas entrapping compositions") that may be used to treat inflammatory diseases.

[0018] In connection with the present invention, the inventors have surprisingly found that carbon monoxide (CO), particularly carbon monoxide (CO) gas, can be successfully used to preserve gametes. It has been found and reported herein that preservation of gametes can be reliably achieved by contacting gametes with carbon monoxide / carbon monoxide gas (including exposing gametes to or treating gametes with carbon monoxide / carbon monoxide gas). That is, preservation means "maintaining" gametes in a (desired) functional / intact state. Therefore, it has been found and reported herein that carbon monoxide (CO), particularly carbon monoxide (CO) gas, can protect / maintain and even improve the quality of gametes. As a specific example, the present inventors have discovered that contacting sperm with carbon monoxide (gas) results in (i) a decrease in reactive oxygen species (ROS) levels / reduced oxidative stress in sperm (see, for example, the accompanying Figures 2 and 5-9), and as a result, (ii) a decrease in DNA fragmentation in gametes, particularly sperm (see, for example, the accompanying Figures 3, 5-8, and 10).

[0019] Furthermore, as shown in the accompanying examples and figures, (iii) an improvement in the forward motility rate of male gametes / sperm was also observed compared to control samples (not contacted with carbon monoxide) (see, for example, the accompanying Figures 4B and 5). None of these surprising technical effects have been disclosed or suggested in the art. Accordingly, the present inventors have discovered that contacting gametes, such as sperm, with carbon monoxide allows for the preservation of gametes with desired properties. Desired properties in the context of this invention include a reduction in reactive oxygen species (ROS) levels / oxidative stress / redox potential, a reduction in DNA fragmentation levels, an improvement in forward motility rate, and / or a desired maintenance of motility, particularly a property measurable in the form of sperm mean path velocity (VAP).

[0020] The corresponding improvements are described and demonstrated in the accompanying examples, particularly in comparison with a control sample of gametes that were not subjected to incubation / contact with CO. The preserved gametes exhibiting one or more of the above-mentioned desirable properties can then be used, for example, in assisted reproductive technology applications. Therefore, the means, methods, and uses of the present invention make it possible to avoid the need for continuous systemic administration of (potentially harmful) antioxidants to subjects / patients undergoing such treatment. Furthermore, due to their germ cell preservation effects, the means, methods, and uses of the present invention can also be used for the preservation / storage of germ cells in assisted reproductive technology applications, where handling of the germ cells over long periods of time is required.

[0021] The means, methods, and uses provided herein relate not only to ex corpore or in vitro applications, but also to in vivo methods and in vivo applications of carbon monoxide. As exemplified herein, carbon monoxide can also be used to improve gamete quality in vivo. In particular, it is envisioned to treat male individuals with CO to avoid gamete DNA fragmentation due to physiologically harmful (or potentially harmful) oxidative stress. This can be achieved, inter alia, by administering carbon monoxide to individuals in need of such treatment via a CO-releasing patch. Aspects of this invention, further described below, relate in particular to the inventive use of carbon monoxide in the treatment and / or prevention of diseases that cause unwanted (genetic) damage to the developing embryo and / or fetus.

[0022] For example, the present invention provides the medical use of carbon monoxide for the prevention of congenital anomalies and / or aneuploidy. Furthermore, the present invention provides the medical use of carbon monoxide, for example, in the treatment and / or alleviation of male infertility. Furthermore, the present invention provides the medical use of carbon monoxide, for example, in the treatment and / or prevention of diseases caused by / associated with DNA fragmentation and / or elevated redox potential in gametes. Furthermore, the present invention provides the medical use of carbon monoxide, for example, in the treatment and / or prevention of diseases of gametes caused by / associated with elevated ROS levels. This aspect is discussed further below. The key to the present invention is the avoidance of physiological damage mediated by reactive oxygen species.

[0023] In its broadest embodiment, the present invention relates to the preservation of gametes using carbon monoxide. Gametes can be contacted / exposed / treated with carbon monoxide in vitro, ex vivo, and / or ex corporeally. However, as explained below, in vivo uses, such as for medical intervention, are also described and disclosed herein and form part of the present invention.

[0024] The term "preservation" in the context of the means, methods, and uses of the present invention refers to a method, activity, or process that uses carbon monoxide / carbon monoxide gas to keep gametes functional, viable, intact, and / or free from damage and / or decay. This particularly refers to gametes after they have been harvested from a subject. The subject may be healthy or diseased, but is preferably a mammal, and most preferably a human. However, the means, methods, and uses of the present invention can also be readily applied to gametes, particularly sperm, of other animals, such as birds, fish, and reptiles.

[0025] That is, the means and method of the present invention, which involve contacting gametes with carbon monoxide, can reliably maintain gametes in a functional / intact state. One of the key features of the present invention is also illustrated in the accompanying examples. These examples demonstrate that contacting gametes with carbon monoxide, particularly with sperm, can maintain and / or improve gamete quality. Stored gametes may be successfully utilized, for example, in reproductive medicine, particularly assisted reproductive technology (ART), and in techniques such as artificial insemination of livestock. Thus, the terms "gamete storage" and "preservation of gamete quality" are understood to refer to maintaining gametes functional, viable, intact, and / or free from damage or decay for a certain period of time compared to a reference time point. The reference time point can be defined as the time at which gametes are collected from a subject (healthy or diseased), such as a male or female human subject. However, the term "preservation" may also be understood in the context of temporary in vitro storage of gametes for extended gamete processing periods. This may be particularly true in the context of assisted reproductive technology applications.

[0026] The term "ex vivo" is well known to those skilled in the art and, in the context of the present invention, particularly refers to the exposure of gametes to be preserved in accordance with the present invention to carbon monoxide (CO) in an ex corporeal environment. In other words, sperm, in particular, are exposed to carbon monoxide outside the individual's body, e.g., after ejaculation and release into a corresponding (collection) container. Preferably, such an individual is therefore capable of producing corresponding gametes / sperm, i.e., sperm. Ejaculation into a corresponding container is relevant for (artificial) reproductive techniques, e.g., in vitro fertilization techniques.

[0027] As will be understood, direct injection into a container is not a requirement for the means, methods, and uses of the present invention. It is also contemplated that the present invention can be used with any semen, sperm, glandular fluid, sperm sample, isolated and further purified sperm or spermatozoa, and / or corresponding biological samples that have been diluted and / or further treated, processed, treated, liquefied, or frozen, during, or after thawing, containing gametes, preferably sperm, to be treated. The present invention is also useful and readily applicable when handling biological samples containing gametes, preferably sperm, to be preserved in the context of the present invention. Therefore, it is also contemplated that any biological sample containing gametes may be exposed to carbon monoxide / carbon monoxide gas during its manipulation or handling.

[0028] In the context of the present invention, the term "ex vivo" may be used synonymously with "ex corpore." Corresponding ex vivo and ex corpore techniques and methods may be considered "in vitro" techniques / methods. Thus, according to one embodiment, ex vivo treatment refers both to treatment by providing gametes in a container with carbon monoxide, and to treatment by exposing gametes already in a container to carbon monoxide.

[0029] However, according to another aspect of the invention described herein, the inventive concept of exposing gametes to carbon monoxide for beneficial preservation can also be employed in the human or animal body. Accordingly, the present invention also relates to medical uses of carbon monoxide in methods of treatment or disease prevention, including exposing gametes in the human or animal body to carbon monoxide. These medical uses therefore also include in vivo uses of carbon monoxide. Exposure of the corresponding gametes to carbon monoxide can be achieved, for example, by a (medical) patch, as described below. See also the provisions regarding corresponding medical patches that release carbon monoxide in WO 2021 / 180908 A1 and Ruopp et al. (2023, Journal of Controlled Release).

[0030] In a particular aspect, the in vivo treatment / exposure also relates to the exposure of a pair of germ cells (especially sperm) to carbon monoxide, where germ cells refer to cells present in the gonads of an organism, preferably a eukaryote, more preferably a (male) mammal, most preferably a human (male).

[0031] The term "functional, alive, intact, and / or free from damage and / or decay" in the context of the present invention refers to one or more characteristics of gametes that may be adversely affected, for example, by reactive oxygen species (ROS). These characteristics may be maintained by the means, methods, and uses of the present invention and may therefore be included in the aforementioned "desired / desirable characteristics." For example, as shown in the accompanying Examples 2 and 3 and Figures 2 to 7, non-limiting examples of (desired) characteristics of gametes include (a reduction in) redox potential / ROS levels, (a reduction in) DNA fragmentation, (an increase in) motility (in the case of spermatozoa), particularly (an increase in) progressive motility, (a reduction in) lipid peroxidation, (a reduction in) apoptosis, and (a reduction in) sperm degeneration. Methods and means for determining such (desirable) characteristics of gametes are known to those skilled in the art. Examples 2 and 3 also provide detailed descriptions of excellent methods and commercially available kits for measuring, in particular, (static) redox potential / reactive oxygen species levels, DNA fragmentation, and sperm total and progressive motility. Example 3 also provides detailed descriptions of the measurement of sperm average path velocity (VAP). VAP is defined as the speed of sperm movement and is measured in micrometers per second (μm / s). Together with total and progressive motility, it is an important parameter that indicates overall sperm motility. According to the present invention, a further desirable characteristic of gametes is (maintaining / increasing) VAP.

[0032] For example, the Male Infertility Oxidation System (MiOXSYS, Englewood, CO) can be used to measure sperm redox potential (resting) / reactive oxygen species (ROS) levels, the Halo Sperm G2 Kit (Halotech, Madrid, Spain) can be used to measure sperm DNA fragmentation, and the CEROS II Computer Assisted Sperm Analysis (CASA) System (Hamilton Thorne, Beverly, MA) can be used to measure sperm motility, specifically total and progressive motility rates, and VAP.

[0033] That is, the (functional, viable, or intact) gametes preserved (under CO) according to the present invention may be gametes that exhibit desired characteristics. For example, gametes exposed to CO may have, in particular, (i) a lower redox potential, (ii) less DNA fragmentation / DNA damage, and (iii) a higher or unchanged / maintained rate of processive motility compared to unexposed gametes (i.e., gametes not exposed to CO). Such (desired) characteristics are illustrated, for example, in the accompanying Figures 2 to 10. Gametes preserved under CO may also be gametes that contain a haploid set of chromosomes with significantly fewer or no chromosomal abnormalities compared to unpreserved gametes. Chromosomal abnormalities may be selected from the group consisting of numerical abnormalities (e.g., aneuploidy), structural abnormalities (e.g., deletions, duplications, inversions, insertions, translocations, rings, and homozygotes), and / or acquired chromosomal abnormalities. According to the present invention, the gametes may be healthy gametes (no chromosomal abnormalities) or diseased gametes (chromosomal abnormalities).

[0034] In the present invention, the above-mentioned phrase "successfully used" refers to the use of the stored gametes in reproductive medicine, particularly in assisted reproductive technology (ART) or (artificial) insemination processes. In these applications, the use of stored gametes obtained by the means, methods, and uses of the present invention particularly increases the likelihood of a positive (i.e., desired) outcome in the application. For example, functional / viable / intact haploid gametes stored under CO2 increase the likelihood of forming healthy diploid zygotes (compared to unstored gametes), which in turn increases the likelihood of (the desired outcome) during in vitro fertilization (IVF). This allows for normal blastocyst formation and the establishment of a normal pregnancy (i.e., a complication-free pregnancy resulting in a healthy newborn and a significantly reduced risk of congenital abnormalities and aneuploidy).

[0035] As used herein, a "zygote" is a eukaryotic cell formed by fertilization between two gametes. The zygote genome is the combination of the DNA contained in each gamete and contains all of the genetic information for a new individual organism. In multicellular organisms, the zygote is the earliest developmental stage.

[0036] During human fertilization, a released egg (a haploid secondary oocyte / egg cell with duplicated chromosome copies; the female gamete) and a haploid sperm cell (the male gamete) combine to form a single diploid cell called a zygote. When a single sperm fuses with an egg, the egg undergoes a second meiotic division to produce a haploid daughter cell with 23 pairs of chromosomes, nearly all of the cytoplasm, and a male pronucleus. The other product of meiosis is the second polar body, which contains only chromosomes and is not capable of replicating or viable growth. In the fertilized daughter cell, DNA is replicated in two separate pronuclei derived from the sperm and egg, temporarily increasing the zygote's chromosome count to 4n (diploid). Approximately 30 hours after fertilization, the fusion of the pronuclei and subsequent mitotic divisions produces two diploid daughter cells called blastomeres, which form a blastocyst and ultimately an embryo.

[0037] "Healthy" here refers to a diploid zygote, which should be understood as opposed to a diseased / abnormal zygote.

[0038] Diseased / abnormal zygotes include cells that contain damaged / fragmented DNA or cells that are not diploid, such as when at least one of the fused sperm and egg cells is aneuploid or euploid. Diseased / abnormal zygotes may not result in blastomere / blastocyst formation or embryo formation, or may result in blastomere / blastocyst formation or embryo formation but may be more likely to result in miscarriage or a newborn with a congenital abnormality or aneuploidy (or euploidy) compared to a healthy fertilized egg. Non-limiting examples of congenital abnormalities or aneuploidy are provided later in this disclosure.

[0039] Thus, a "healthy" zygote is one that is diploid (i.e., contains a complete set of paternal and maternal chromosomes) and has the potential to give rise to an embryo that forms a normal blastomere / blastocyst. Healthy zygotes are more likely to result in the birth of a newborn baby without congenital abnormalities or aneuploidy (or euploidy) than diseased / abnormal zygotes.

[0040] For example, according to the means, methods, and uses of the present invention, gametes may be healthy (not containing one or more chromosomal abnormalities) or diseased (containing one or more chromosomal abnormalities). A gamete defines a haploid cell that fuses with another gamete during fertilization in sexually reproducing organisms. A gamete is an organism's reproductive cell, also commonly referred to as a sex cell. In species that produce two morphologically distinct types of gametes (e.g., mammals such as humans), each individual produces only one type of gamete, with females producing larger gametes (called oocytes / eggs) and males producing smaller gametes (called spermatids / sperm).

[0041] That is, in the context of the means, methods, and uses of the present invention, gametes include sperm / sperm cells and / or egg cells / oocytes, and preferably include sperm / sperm cells and / or egg / oocytes collected in vitro from a mammalian subject, such as a male or female subject. Such mammalian subjects, like male or female human subjects, may be healthy or may be suffering from or predisposed to a disorder such as infertility or a chromosomal abnormality. In the context of the present invention, it may be preferable for sperm to be contained in semen, glandular fluid, and / or a buffer / buffer system that allows the survival of sperm cells. Thus, the gametes to be contacted with carbon monoxide may also be contained in a biological sample or a buffer / buffer system.

[0042] Examples of buffers / buffer systems suitable for diluting, storing, washing, handling, processing, buffering, purifying, etc., gametes, such as sperm, are known to those skilled in the art. Non-limiting examples of such buffers / buffer systems include sperm dilution buffers, sperm maintenance buffers, and sperm washing buffers. Such buffers / buffer systems may contain, among others, water / aqueous solutions, salts / ions, buffers / buffering agents, amino acids (especially glycine and / or taurine), energy substrates, antibiotics (especially gentamicin sulfate), etc. Examples of buffering reagents / buffering agents include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium bicarbonate, MOPS (3-(N-morpholino)propanesulfonic acid), phosphate-buffered saline (PBS), etc.

[0043] Non-limiting examples of salts / ions include potassium phosphate, sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate. Non-limiting examples of energy substrates include sodium lactate, sodium pyruvate, and glucose. Methods for designing and producing suitable buffers / buffer systems, particularly those containing the exemplary components / ingredients described hereinabove, are known to those skilled in the art of artificial reproductive technology or related fields. Alternatively, those skilled in the art can easily design and produce suitable buffers / buffer systems, including, for example, multipurpose handling media Complete (FUJIFILM) and PureSperm (登録商標)Commercially available buffers / buffer systems of the same level / functionally equivalent quality, such as Wash (Nidacon), are readily available.

[0044] Spermatozoa / sperm cells and egg cells / oocytes are the male and female germ cells ("gametes"), respectively, in mammalian heterozygous reproduction.

[0045] Spermatocytes are formed through a process called spermatogenesis. In mammals, spermatogenesis occurs in the seminiferous tubules of the testes, starting with spermatogonia, which undergo the generation of several successive spermatid precursors before differentiating into spermatocytes. Spermatocytes then undergo meiosis, reducing their chromosome number by half and giving rise to spermatids. Spermatocytes then mature, forming tails called flagella in mammals, and becoming motile spermatids.

[0046] The motility may be described by the term "progressive motility." This refers to sperm cells / sperm that move / swim in a generally straight line or in a large circle. On the other hand, the term "non-progressive motility" refers to sperm cells / sperm that do not move / swim in a straight line or that swim in a very small circle. The combined progressive and non-progressive motility rates represent the total motility of sperm cells. These motility rates (progressive motility, non-progressive motility, total motility) can be measured using standard techniques well known in the art. For example, in the accompanying Examples 2 and 3, a computer-assisted sperm analysis (CASA) on CEROS II (Hamilton Thorne, Beverly, Massachusetts) was used to measure the progressive and total motility of sperm cells / sperm.

[0047] Sperm cells cannot divide and have a limited lifespan, but when they fuse with an egg cell during fertilization, a new organism / embryo is formed and begins development as a totipotent zygote. Human sperm cells are haploid (n), meaning their 23 chromosomes combine with the 23 chromosomes of the female egg cell to form a diploid (2n) cell with 46 pairs of chromosomes. In mammals, sperm cells / sperm are stored in the epididymis and are released from the penis during ejaculation along with a fluid called semen / sperm / glandular fluid.

[0048] The quantity and quality of sperm cells are important indicators of male fertility. However, their genetic quality, as well as their motility, generally declines with age. DNA damage present in sperm cells during the post-meiotic, pre-fertilization period (i.e., the period primarily focused on in the present invention) can have serious adverse effects on fertility and the developing embryo. Mammalian sperm cells, particularly human sperm cells, are particularly vulnerable to free radical attack (e.g., by reactive oxygen species (ROS)) and the resulting oxidative damage. This vulnerability is due to the gradual loss of DNA damage repair mechanisms during spermatogenesis. Therefore, the means, methods, and uses of the present invention, i.e., contacting gametes such as sperm / spermatocytes with carbon monoxide gas, can prevent such DNA damage (especially during the post-meiotic, pre-fertilization period). This is evident, for example, from the accompanying Figures 3 and 5-10. These figures clearly show that sperm samples taken from the same subject that were not exposed to carbon monoxide had a higher degree of DNA damage / DNA fragmentation than those that were exposed to carbon monoxide.

[0049] Egg cells / oocytes are produced in the ovaries by female gametogenesis (also known as oogenesis). During oogenesis, secondary oocytes are produced. Secondary oocytes may be haploid and may be arrested in the second division of meiosis at metaphase II until fertilization. Thus, according to the present invention, the egg cell / oocyte may be a secondary oocyte.

[0050] Both sperm / sperm cells and eggs / oocytes can be collected from a mammalian subject, such as a healthy or diseased human subject, prior to exposure to carbon monoxide gas. Sperm / spermatocytes / semen can be collected from the epididymis, for example, by masturbatory ejaculation into a (sterile) container, collection with a condom, epididymal extraction, etc. On the other hand, eggs / oocytes can be collected, for example, by transvaginal egg collection.

[0051] Thus, according to a preferred embodiment of the present invention, the gametes may be healthy or diseased human gametes collected / obtained from healthy or diseased male or female subjects. In another aspect, the gametes may be collected / obtained from other mammals, such as cattle, horses, pigs, sheep, goats, camels, alpacas, dogs, cats, etc. In another aspect, the gametes may be collected / obtained from non-mammalian animals, such as birds, fish, reptiles, and the like, including, but not limited to, peregrine falcons and / or carp.

[0052] After collection, the collected gametes are placed in a container according to the means, methods, and uses of the present invention. Such a container may be an open container, or a lockable / sealable (airtight) container. According to a particular embodiment, such a container may be equipped with a valve for connecting a CO2 transport tube. Containers used according to the means, methods, and uses of the present invention include containers made of polymer(s), elastomer(s), metal(s), glass(es), or ceramic(s), but generally, containers made of polymer(s) are preferred. According to one embodiment, the container is made of polypropylene (PP), polyethylene (PE), or polystyrene (PS). The container used may be aseptic / sterile, i.e., clean and bacteria-free. Examples of containers used in the accompanying examples include sample vials or gas-impermeable Falcon tubes. Another example of a container that may be used is a (sterile) specimen cup. Containers for use in the context of the present invention are known to those skilled in the art.

[0053] According to one embodiment, semen / sperm / glandular fluid may be liquefied. Liquefaction may be performed before and / or during contact of gametes (especially sperm) contained in the semen / sperm / glandular fluid with carbon monoxide gas for preservation. In this context, liquefaction refers to a process of breaking down the gel formed by proteins secreted from the seminal vesicles and prostate, making the semen / sperm / glandular fluid more liquid. Means and methods for liquefaction are known to those skilled in the art. For example, as is clear from the attached Example 2, liquefaction generally occurs naturally by incubating the semen / sperm / glandular fluid at 25°C for 30 to 60 minutes.

[0054] According to the means, method and use of the present invention, the gametes contained in the container are contacted with carbon monoxide gas for a sufficiently long time to ensure the preservation of the gametes.

[0055] In a preferred embodiment, the gametes are contacted with carbon monoxide, particularly in the form of CO gas. In this case, the gametes are first placed in a container, and then, as in step b) above, CO gas or a system that releases CO gas (activated CORS) is added to the container containing the gametes. The present invention further relates to means, methods, and uses that allow the gametes to be contacted with carbon monoxide within 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute after the gametes are collected in the container. However, it is also envisioned that CO gas may be directly contacted with the gametes and / or a (biological) sample containing them. In all embodiments of the present invention, the sample may be semen, glandular fluid, and / or a buffer or buffer system containing gametes, particularly sperm.

[0056] Carbon monoxide, particularly CO gas, may be supplied to the container first, and then gametes may be added to the container containing carbon monoxide / CO gas in a second step. In this case, the gametes may be contacted with carbon monoxide immediately after collection in the container (or after collection and / or supply of a sample, such as semen, glandular fluid, and / or a buffer or buffer system, containing gametes). As will be apparent from the examples, carbon monoxide may be supplied to the container by activating a carbon monoxide releasing system (CORS) to form carbon monoxide in the container before collecting the gametes in the container.

[0057] Needless to say, such a container may be sealed / closed between activation of the CORS and collection of gametes. In the present invention, an exemplary carbon monoxide releasing system can be activated about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute before collection of gametes. In other words, carbon monoxide / CO gas may be supplied to or into the container in a first step, and gametes or a gamete-containing sample may be added in a second step. It is also within the scope of the present invention to use the reverse method, i.e., first placing gametes (or a gamete-containing sample) in the container and then adding carbon monoxide / CO gas to the container in a second step. Carbon monoxide may be added using a carbon monoxide releasing system (i.e., CORS).

[0058] It is also contemplated that gametes and / or gamete-containing samples may be repeatedly or repeatedly contacted with carbon monoxide. In other words, gametes and / or gamete-containing samples may be contacted with carbon monoxide multiple times. For example (but not limited to), it is also contemplated that gametes may be subjected to at least one freeze-thaw cycle before being contacted with carbon monoxide / CO gas. Thus, it also includes contacting gametes or gamete-containing samples with carbon monoxide / CO gas after preservation (e.g., freezing). Exposure of gametes or gamete-containing samples can occur, for example, during and / or after thawing frozen gametes / gamete-containing samples to terminate preservation. Corresponding freezing and / or thawing protocols are well known in the art (e.g., in the field of reproductive methods, such as, but not limited to, artificial reproductive technology, in vitro fertilization, artificial insemination, etc.). Such protocols may be combined with the teachings of the present invention. That is, during and after such protocol steps, gametes or gamete-containing samples may be exposed to carbon monoxide and then preserved in the manner described and illustrated in this disclosure. Accordingly, the present invention also relates to means, methods, and uses for contacting frozen and / or thawed gametes, particularly sperm, or frozen and / or thawed biological samples containing gametes, such as semen, ejaculate, or buffers / buffer systems, with carbon monoxide. In other words, the present invention is not only applicable to "fresh" gametes or frozen and / or thawed biological samples containing gametes, such as semen, ejaculate, or buffers / buffer systems, but also to gametes and biological samples / buffers / buffer systems that have been preserved by methods such as cryopreservation. Carbon monoxide is preferably used for a sufficient / long enough time to allow and / or ensure the preservation of the gametes. This sufficient time / sufficiently long time may be the time or period starting from collection or acquisition of gametes or gamete-containing samples, preferably sperm, which need to be stored until further use, for example, in artificial reproductive techniques, in vitro fertilization, (artificial) insemination, etc. In other words, "sufficient time" refers to the period during which the gametes or gamete-containing samples are exposed to carbon monoxide / carbon monoxide gas.

[0059] The (reliable) preservation of gametes also relates to the maintenance of a good and / or healthy physiological state, e.g., low oxidative stress (low reactive oxygen species state). The (reliable) preservation of gametes also relates to the avoidance of additional harmful and / or undesirable effects on gametes, such as further exposure to additional endogenous or exogenous harmful events, such as oxidative stress (e.g., ROS). Such undesirable effects or adverse events may adversely affect gametes, particularly sperm, and may negatively impact the "quality" of these gametes. Therefore, as illustrated in the accompanying examples, the "gamete preservation" described in this disclosure also relates to a desirable "preservation" quality of gametes. This quality may be reflected, inter alia, in desirable gamete (i.e., sperm) motility, reduced or eliminated DNA fragmentation / DNA fragmentation rate, sperm testing that is medically, clinically, or biologically normal, or reduced levels of oxidative stress. The "preservation" and / or "preservation state" of these gametes can therefore be assessed, in particular through determining the motility and / or motility of sperm cells / sperm, the DNA fragmentation state of the gametes by spermogram, and / or determining the redox potential of the gametes. Corresponding methods are well known and readily available to those skilled in the art, not only to specialists working in reproductive medicine / ART, but also to those employing, for example, (artificial) insemination / insemination protocols for livestock. Corresponding assessment methods for determining the "quality" of gametes, e.g., methods for maintaining gametes in a desired state, are also given in the appended examples.

[0060] As shown in these examples, the present invention can ensure the preservation of gametes, particularly sperm. While such preservation may involve maintaining the (desired) health of gametes, as described in this disclosure, preservation with carbon monoxide may also result in improved gamete quality. Particularly surprisingly, it has been shown that exposure of gametes, particularly sperm cells / sperm, to carbon monoxide can enhance / even improve the quality of such gametes, particularly sperm cells / sperm. See, e.g., accompanying Examples 2 and 3. These examples evaluate samples exposed to carbon monoxide versus unexposed samples, or samples from the same subject / individual / donor that have not been exposed and / or treated with carbon monoxide. Also shown in accompanying Figure 3 (see, in particular, "Subjects" 3 and 4) are results demonstrating improved quality, such as reduced DNA fragmentation, between carbon monoxide-treated and untreated / unexposed samples. Gamete samples from the same donor were divided into two equal aliquots, the only difference being the presence or absence of carbon monoxide exposure.

[0061] Also, the accompanying Figure 7D shows a surprising improvement in the quality of gametes (here, sperm cells / sperm). Figure 7D demonstrates that the present means and methods, i.e., exposure to carbon monoxide, can avoid the negative effects of endogenous (and exogenous) oxidative stress. Surprisingly, gamete samples / aliquots exposed to / treated with carbon monoxide showed a decreased redox potential compared to untreated samples / aliquots of sperm cells / sperm from the same subject / individual / donor. See, for example, Subject 42_1 or 38_7. However, similar effects may be observed in other subjects.

[0062] As described herein, it is clear that contacting / exposing / treating gametes, particularly sperm, with carbon monoxide results in some, but significant, improvement in the preservation and / or quality of the gametes. Accordingly, the present invention generally relates to preserving gametes in a desired state, e.g., at a quality acceptable for use of the gametes in artificial reproductive technology, etc. Thus, preserving gametes may include maintaining a predetermined quality of gametes, but may also include improving the quality of gametes. Such preservation is particularly important in the in vitro / in vitro artificial reproductive technology methods described herein, e.g., gamete preparation for such artificial reproductive technology methods and in vitro manipulation and / or (artificial) insemination protocols.

[0063] Thus, "preservation" / "maintenance" of gametes refers in particular, but not exclusively, to the preservation / maintenance of the quality of gametes during ex corpore manipulations and / or in an in vitro environment. In particular, the present disclosure particularly, but not exclusively, illustrates that preservation in the sense of the present invention can be successfully used in in vitro / ex corpore manipulations where handling of gametes is essential. Examples include the provision of sperm / spermatozoa, (biological) samples, or buffers / buffer systems containing them for artificial reproductive techniques such as in vitro fertilization.

[0064] As shown in the accompanying examples, the quality of the carbon monoxide-treated sperm tested was maintained (or even improved) despite relatively long ex-corporeal handling. Even 1.5 or 2 hours after sample collection and exposure to carbon monoxide, gamete quality was shown to be well maintained or even improved (compared to untreated samples). Thus, according to a non-limiting embodiment, it is contemplated that gametes (or a sample / buffer / buffer system containing the same) are contacted with carbon monoxide for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, and more preferably about 30 to about 60 minutes. The contact / exposure / treatment of such gametes (or a sample / buffer / buffer system containing the same) with carbon monoxide can be carried out at room temperature, i.e., about 25°C to about 35°C. It is one of the (non-limiting) advantages of the present invention that the quality of a given gamete is maintained, and even improved to some extent, in a particular ex corpore / in vitro environment.

[0065] It has been reported that even a relatively long (in vitro) treatment time (up to 2 hours) at constant room temperature ensures the preservation of gametes, i.e., maintains their quality. Accordingly, provided herein are means and methods for preserving gametes, which comprise contacting gametes (or a sample / buffer / buffer system containing gametes) with carbon monoxide / carbon monoxide gas to ensure the preservation of gametes or maintain the quality of said gametes. Such means and methods may comprise contacting / treating / exposing gametes (or a sample / buffer / buffer system containing gametes) with carbon monoxide / carbon monoxide gas for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, and more preferably about 30 to about 60 minutes. The contact / treatment / exposure with carbon monoxide / carbon monoxide gas may be carried out at about 25 to about 35°C.

[0066] The present invention relates to means, methods, and uses for contacting gametes with CO, particularly in professional settings, such as medical or assisted reproductive technology (ART) laboratories, or in non-professional settings, such as private settings. That is, the present invention enables individuals, particularly those with or without medical experience or knowledge, to practice the means, methods, and uses of the present invention. Therefore, such means and methods are suitable for home use by individuals unfamiliar with or untrained in artificial reproductive technology and / or related fields. As can be seen, particularly in FIG. 1B, home use of the means and methods of the present invention involves collecting gametes in a container and activating a carbon monoxide releasing system (CORS). The activated CORS may be placed directly into the container, which may then be sealed. As described in detail above, the CORS may be activated before gamete collection. In either case, contacting the gametes with CO gas protects them during liquefaction and subsequent handling in assisted reproductive technology (including cryopreservation). As is particularly clear from FIG. 1B, even those who are not familiar with artificial reproductive technology and / or related fields can carry out the means, methods, and uses of the present invention.

[0067] In the context of the present invention, providing / obtaining gametes in a container and contacting them with carbon monoxide can refer to providing / obtaining a patient's gametes in a container, providing / obtaining a carbon monoxide-releasing system such as a carbon monoxide-releasing molecule, activating the carbon monoxide-releasing system and / or molecule, thereby releasing carbon monoxide into the container, and contacting the gametes with carbon monoxide. According to one aspect, the carbon monoxide-releasing molecule may be obtained / provided in a pre-activated form, i.e., a form that does not require activation. According to another aspect, carbon monoxide, a carbon monoxide-releasing molecule, and / or a carbon monoxide-releasing system (exemplified in this disclosure in the form of a carbon monoxide-releasing device / capsule) may be pre-contained in the container.

[0068] The term "contacting" or "contacted" with respect to the gametes / CO gas of the present invention refers to physical contact between the gametes and the carbon monoxide gas. In one aspect, the carbon monoxide gas may form a gas layer on the gametes. In another aspect, active mixing of the gametes may be required to ensure uniform contact between the gametes and the carbon monoxide gas while the carbon monoxide gas forms a layer on the gametes. Carbon monoxide may be provided to the gametes as a solute, dissolved and / or solubilized in a liquid. Such liquids include water, aqueous solutions / suspensions containing water, and body fluids, including, but not limited to, semen. As will be apparent to those skilled in the art, providing CO gas to gametes or other tissues may result in dissolving and / or solubilizing CO in, for example, semen. That is, it is apparent that the term "CO gas" may also refer to solubilized and / or dissolved CO. Preferably, CO is provided as a gas. This can be achieved either directly (by direct exposure to CO gas) or indirectly (e.g., by exposure to the corresponding carbon monoxide-releasing molecule and / or CO gas released from a carbon monoxide-releasing system).

[0069] Carbon monoxide (CO) gas is a colorless, odorless, flammable gas slightly less dense than air and therefore requires a lockable / sealable container. Carbon monoxide is the simplest molecule in the carbon oxide family, consisting of one carbon atom and one oxygen atom triple-bonded. In coordination compounds (e.g., metal carbonyl compounds, which can release CO upon addition of a second compound, such as FeCl3), the carbon monoxide ligand is called a carbonyl. CO plays an important biological role across evolutionary taxa. Carbon monoxide is produced by many organisms, including humans. Mammalian physiology suggests that carbon monoxide is a prime example of hormesis: at low concentrations, it acts as an endogenous neurotransmitter (gasotransmitter), while at higher concentrations, it is toxic and can cause carbon monoxide poisoning. Therefore, as detailed below, exposure to only the amount of CO required in accordance with the present invention is necessary to avoid damaging gametes.

[0070] As discussed in the present disclosure, gametes, particularly sperm, can be adversely affected by direct and / or indirect exposure to reactive oxygen species / oxidants. Without being bound by theory, when the amount of oxidants exceeds the maximum capacity of the cell's redox buffering capacity, oxidative stress can occur, which can adversely affect sperm motility in particular. Conversely, when the amount of oxidants is significantly below physiological levels, reductive stress can occur, which can adversely affect sperm motility in particular. The present disclosure demonstrates that exposure of sperm to carbon monoxide does not adversely affect their motility. Successful exposure of gametes, particularly sperm, to CO, as described in the present disclosure, can suppress or reduce undesired DNA fragmentation and / or suppress or reduce redox potential (e.g., ROS). Furthermore, maintaining or enhancing gamete motility, particularly enhancing sperm motility, can also be considered an effect of the present invention. The accompanying examples provide corresponding technical details on how to measure and evaluate, inter alia, DNA fragmentation, ROS inhibition and / or gamete motility / sperm motility.

[0071] Those skilled in the art are aware that the group of reactive molecular species (RMS), to which reactive oxygen species (ROS) belong, further includes reactive nitrogen species (RNS) and reactive sulfur species (RSS). Most RMS are highly reactive and can be neutralized or new RMS can be generated by reacting with each other (in biological cells, particularly gametes such as sperm). In other words, ROS can react with, among other ROS, RNS, and / or RSS, resulting in the neutralization of the ROS or the generation of new ROS. In other words, the presence / appearance of ROS can lead to the presence / appearance of more ROS and / or other RMS. Therefore, in the context of the present invention, it may be desirable to reduce ROS levels / oxidative stress / redox potential in biological cells, particularly gametes such as sperm, in order to prevent / avoid / reduce the generation / existence of ROS and / or RMS. As illustrated in the accompanying examples and figures, the present invention provides means and methods for effectively reducing the redox potential of biological cells / gametes / sperm. This can prevent / avoid / reduce the generation / presence of (further) reactive oxygen species.

[0072] Thus, within the scope of the means, methods, and uses provided herein, gametes are preserved for use or intended for use in reproductive technologies, such as artificial reproductive technologies, in vitro fertilization, preferably in mammals, or artificial insemination technologies, preferably in (livestock) animals. Naturally, in such reproductive technologies in humans and animals, the "quality" of the gametes used is of paramount importance. As shown in this disclosure and in the accompanying examples, the inventors have surprisingly discovered that exposing gametes, particularly sperm, to carbon monoxide can preserve and even improve "gamete quality." As described herein, exposing gametes, particularly sperm, to carbon monoxide can mitigate and reduce the adverse effects of undesirable oxidative processes, such as the effects of reactive oxygen species (ROS). As specifically shown herein, exposing gametes (here, sperm) or (biological) samples containing gametes (e.g., semen, glandular fluid, and / or buffers / buffer systems, etc.) to carbon monoxide surprisingly (but desired) reduces the redox potential of the gametes.

[0073] It has also been shown that gametes that already have a good (low) redox potential can be maintained / preserved by contact / exposure / treatment with carbon monoxide. Thus, the present invention provides means, methods, and uses for obtaining gametes with desired quality, for example, for artificial reproduction or (artificial) insemination techniques, where the quality of the gametes used is paramount. Thus, the present invention also relates to the provision of gametes useful for such techniques. Gametes (or a sample containing gametes) may be contacted with or exposed to carbon monoxide in a container. By contacting the gametes (or a sample containing gametes) in a container with carbon monoxide, the quality of the gametes can be maintained or improved, i.e., gamete preservation can be achieved. Provided gametes are contacted with carbon monoxide gas in the container for a time period sufficient to ensure the preservation of the gametes. "For a sufficiently long time to ensure preservation of gametes" in the context of the present invention refers to the amount of time that the gametes are in contact with CO gas after they have been removed from a subject, necessary to keep the gametes functional, viable, intact, or to protect them from damage or decay.

[0074] Thus, the term "sufficiently long time" refers to exposing semen / sperm / ejaculate and / or manipulated counterpart samples (such as liquefied semen / sperm / glandular fluid) to carbon monoxide for at least about 15 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, or at least 30 minutes (which may be considered the "exposure time"). The relevant or desired exposure period can be determined, for example, by measuring one or more properties of gametes stored under CO, such as redox potential / reactive oxygen species levels, DNA fragmentation, and / or motility (in the case of sperm), particularly forward motility rate, by one or more of the methods described in Examples 2 and 3 of the present disclosure, and comparing the results with those obtained with unstored gametes. In the accompanying Examples 2 and 3, the present inventors found that contacting gametes with CO gas for 60 or 90 minutes resulted in CO2-stored gametes with (i) a lower redox potential, (ii) less DNA fragmentation / DNA damage, and (iii) a high or maintained motility rate (high forward motility rate) compared to unstored gametes (i.e., gametes not contacted with CO). This is evident, for example, from the accompanying Figures 2 to 8.

[0075] In the context of the present invention, contacting gametes with or with carbon monoxide may refer to treating and / or exposing gametes to carbon monoxide for, for example, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, or at least 90 minutes. One non-limiting teaching of the present invention is to provide gametes, preferably sperm, that have been contacted / exposed / treated with carbon monoxide to eliminate or reduce the negative effects of reactive oxygen species (ROS). As is evident from the accompanying examples, the inventors have surprisingly found that contacting gametes, particularly sperm, with carbon monoxide can produce beneficial effects, as detailed in the present disclosure. That is, carbon monoxide favorably affects or maintains gamete quality. In particular, the present disclosure demonstrates that the beneficial effects of carbon monoxide on these gamete qualities are also exhibited after a 90 minute in vitro, i.e. ex corporeal, application of carbon monoxide.

[0076] According to a preferred embodiment of the present invention, carbon monoxide (CO) gas may be released from a carbon monoxide-releasing molecule (CORM). According to one aspect, the CORM, which may be located inside a closed compartment (e.g., a capsule permeable only to CO gas), may be added to a container (containing gametes). Thus, the CORM may not be in direct contact with the gametes. The CORM is preferably a metal carbonyl compound, more preferably a molybdenum carbonyl compound, and most preferably trisodium tricarbonyl-[tris(isocyanethylacetato)]molybdenum (chemical formula: Na3Mo(CO)3(CNCH2CO2H)3). Na3Mo(CO)3(CNCH2CO2H)3 is also referred to hereinafter as Mo-CORM.

[0077] Carbon monoxide-releasing molecules suitable for use in the present invention are described in WO 2015 / 188941 A1, WO 2016 / 110517 A1, and DE 10 2017 006 393 A1, all of which are incorporated herein by reference.

[0078] The carbon monoxide-releasing molecule (CORM) is preferably a metal carbonyl compound. Examples of metal carbonyl compounds include complexes of elements from the group consisting of Rh, Ti, Os, Cr, Mn, Fe, Co, Mo, Ru, W, Re, Ir, B, and C. The metal carbonyl compound is more preferably a complex of elements from the group consisting of Rh, Mo, Mn, Fe, Ru, B, and C, and even more preferably a complex of elements from the group consisting of Rh, Fe, Mn, Mo, B, and C. Metal carbonyl compounds may be considered complexes because they contain a CO group coordinated to the metal center. However, the metal may be bonded to other groups by bonds other than coordinate bonds, such as ionic or covalent bonds. That is, although groups other than CO that constitute part of a metal carbonyl compound are not strictly "ligands" in the sense of being coordinated to the metal center via lone electron pairs, they are referred to as "ligands" in this disclosure for ease of reference.

[0079] That is, all of the ligands to the metal may be carbonyl ligands. Alternatively, the carbonyl compound may contain at least one ligand that is not CO. The non-CO ligands typically include neutral or negative ligands such as halides, or ligands derived from Lewis bases and having N, P, O, S, or conjugated carbon groups as coordination atoms. Preferred coordination atoms include N, O, and S. Examples include sulfoxides such as dimethyl sulfoxide, natural and synthetic amino acids and their salts such as glycine, cysteine, and proline, amines such as NEt3 and H2NCH2CH2NH2, aromatic bases and their analogs, such as bi-2,2'-pyridyl, indole, pyrimidine, and cytidine, pyrroles such as biliverdin and bilirubin, drug molecules such as YC-1 (2-(5'-hydroxymethyl-2'-furyl)-1-benzindole), thiols and thiolates such as EtSH and PhSH, carboxylates such as chloride, bromide, iodide, formate, acetate, and oxalate, ethers such as Et2O and tetrahydrofuran, alcohols such as EtOH, and nitriles such as MeCN. Other possible ligands include conjugated carbon groups, such as dienes, e.g., cyclopentadiene (CH5) or substituted cyclopentadienes. Substituents on substituted cyclopentadienes include, for example, alkanols, ethers, or esters, e.g., -(CH2) n OH (n is, for example, 1 to 4), particularly, —CHOH (n is, for example, 1 to 4, and R is, for example, a hydrocarbon having 1 to 4 carbon atoms, preferably alkyl), —(CH) n OR (n is, for example, 1 to 4, and R is, for example, a hydrocarbon having 1 to 4 carbon atoms, preferably alkyl), —(CH n OOCR (where n is, for example, 1 to 4, and R is, for example, a hydrocarbon having 1 to 4 carbon atoms, preferably alkyl). As the metal in such a cyclopentadiene or substituted cyclopentadiene carbonyl complex, Fe is preferred.

[0080] For detailed descriptions of carbon monoxide-releasing compounds, explicit reference is also made to WO 2008 / 130261 A1 and U.S. Patent Application Publication No. 2007 / 0219120 A1, all of which are incorporated herein by reference. In these documents, aldehydes of formula I: [ka] has been disclosed, and such compounds may also be used as CORMs of the present invention. In Formula I, R1, R2, and R3 are each independently selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, alkylheterocyclyl, substituted alkylheterocyclyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl (wherein the number of C atoms is, for example, 1 to 12 or 1 to 6, respectively), hydroxy, alkoxy, amino, alkylamino, mercapto, alkylmercapto, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, alkoxycarbonyl, acyl, acyloxy, acylamino, alkylsulfonyl, alkylsulfinyl, F, Cl, Br, NO2, and cyano. Alternatively, two or more of R1, R2, and R3 may combine to form a substituted or unsubstituted carbocyclic or heterocyclic ring structure or derivative thereof. For any substituent, the number of C atoms is for example 1-12 or 1-6.

[0081] Derivatives of compounds of Formula I include acetals, hemiacetals, aminocarbinols, amines, imines, enaminones, imidates, amidines, iminium salts, sodium bisulfite adducts, hemimercaptans, dithioacetals, 1,3-dioxepanes, 1,3-dioxanes, 1,3-dioxalanes, 1,3-dioxetanes, α-hydroxy-1,3-dioxepanes, α-hydroxy-1,3-dioxanes, α-hydroxy-1,3-dioxalananes, α-keto-1,3-dioxepanes, α-keto-1,3-dioxanes, α-keto-1,3-dioxalanes, α-keto-1,3-dioxetanes, macrocyclic esters / imines, macrocyclic esters / hemiacetals, oxazolidines, tetrahydrofurans, and the like. 1,3-oxazine, oxazolidinone, tetrahydro-oxazonone, 1,3,4-oxadiazine, thiazolidine, tetrahydro-1,3-thiazine, thiazolidinone, tetrahydro-1,3-thiazinone, imidazolidine, hexahydro-1,3-pyrimidine, imidazolidinone, tetrahydro-1,3-pyrimidinone, oxime, hydrazone, carbazone, thiocarbazone, semicarbazone, semithiocarbazone, semicarbazone, acyloxyalkyl ester derivative, O-acyloxyalkyl derivative, N-acyloxyalkyl derivative, N-Mannich base derivative, or N-hydroxymethyl derivative can also be used as a CORM in the present invention.

[0082] Examples of CORMs according to the present invention include trimethylacetaldehyde, 2,2-dimethyl-4-pentenal, 4-ethyl-4-formyl-hexanenitrile, 3-hydroxy-2,2-dimethylpropanal, 2-formyl-2-methyl-propyl methanoate, 2-ethyl-2-methyl-propionaldehyde, 2,2-dimethyl-3-(p-methylphenyl)propanal, or 2-methyl-2-phenylpropionaldehyde.

[0083] In one aspect, oxalic acid, oxalic acid esters, or amides can be used as CORMs in accordance with the present invention.

[0084] Examples of CORMs suitable for use in the present invention include molybdenum carbonyl compounds CORM-1, CORM-2, CORM-3, CORM-401, as disclosed in WO 2015 / 188941 A1, WO 2016 / 110517 A1, and DE 10 2017 006 A1.

[0085] More preferred compounds in the context of the present invention include molybdenum-based CORMs such as Mo(CO)3(CNC(CH3)2COOH)3 (also known as "CORM-ALF794") and Mo(CO)3(CNCH2CO2H)3 (tricarbonyl-[tris(isocyanatoethyl)acetato]molybdenum trisodium). Among these, Mo(CO)3(CNCH2CO2H)3 may be particularly preferred. In particular, the trisodium salt (Na3Mo(CO)3(CNCH2CO2)3) ("Mo-CORM") may be preferably used.

[0086] When a CORM is used in connection with the means, methods, and uses of the present invention, carbon monoxide gas is released by contacting the CORM with a second compound. The second compound may include FeCl, Ce(SO), or HO. According to a preferred embodiment of the present invention, the second compound includes FeCl.

[0087] Examples of second compounds suitable for use in the present invention are also described in WO 2015 / 188941 A1, WO 2016 / 110517 A1, and German Patent Application No. 10 2017 006 393 A1, all of which are incorporated herein by reference.

[0088] A CO-releasing molecule (CORM) releases carbon monoxide gas upon contact with a second compound. "Contact," in this context, means that a reaction between the CORM and the second compound occurs, resulting in the release of CO gas. Upon contact with the second compound, the CORM begins to release (substantial amounts of) CO gas, as described in more detail later in this disclosure. This "activates" the system (i.e., CORS) (activated CORS), and the released CO gas can exert gamete-preserving activity / function.

[0089] The second compound may be a sulfur-containing compound, a nitrogen-containing compound, an oxidizing compound, an acid or a base, or water.

[0090] When the CORM is a metal carbonyl compound, examples of the second compound include carbonyl-substituting agents such as sulfur- or nitrogen-containing compounds. The sulfur-containing compound can be selected from, for example, alkali metal or alkaline earth metal salts, preferably sodium salts of sulfite, dithionite, or bisulfite, or compounds having at least one thiol moiety, such as cysteine ​​or glutathione.

[0091] Examples of oxidizing compounds that can be used as the second compound in the means, methods, and uses of the present invention include peroxides, perborates, percarbonates, and nitrates. Of these, calcium peroxide, dibenzoyl peroxide, urea peroxide, sodium perborate, and sodium percarbonate are preferred. Examples of metal oxide salts that can be used as the second compound include silver(I) nitrate, iron(III) chloride, potassium permanganate, cerium(IV) sulfate, potassium dichromate, gold(III) chloride, and silver nitrate. Of these, iron(III) chloride, potassium permanganate, and cerium(IV) sulfate, especially iron(III) chloride and cerium(IV) sulfate, may be preferred. As is clear from the accompanying examples, the metal oxide salts are preferably used as an aqueous solution.

[0092] The acid may be, for example, hydrogen chloride (HCl). In another embodiment, the second compound may be a non-enzymatic compound. The second compound is preferably a compound having a molecular weight of less than 10,000 g / mol, more preferably less than 7,000 g / mol, and even more preferably less than 1,000 g / mol. The second compound may be water or a solvent. ALF186 is a preferred CORM that releases carbon monoxide gas upon contact with water.

[0093] When a sulfur-containing compound or other electron-withdrawing compound is used as a second compound in combination with a metal carbonyl compound such as a CORM, for example, it is believed that when the second compound comes into contact with the metal carbonyl compound, ligand substitution occurs, thereby inducing CO gas release.

[0094] According to another aspect of the invention, the second compound may be selected from the group consisting of sulfur-containing compounds, nitrogen-containing compounds, oxidizing compounds, and water, which may be particularly relevant when the CORM is a metal carbonyl compound.

[0095] In a preferred embodiment of the present invention, a molybdenum carbonyl compound may be used as the CORM and an oxidizing compound may be used as the second compound. In another preferred embodiment, Ru2(CO)6Cl4 may be used as the CORM and sodium sulfite (Na2SO3) may be used as the second compound.

[0096] A particularly preferred embodiment of the present invention is a combination of a molybdenum carbonyl compound, preferably Mo(CO)3(CNCH2COOH)3 or its trisodium salt (Mo-CORM), with ferric chloride (FeCl3), ceric sulfate (Ce(SO4)2), or H2O2. According to one embodiment, FeCl3 and Ce(SO4)2 are used as aqueous solutions with a concentration of about 2 to about 3 mol / L, and H2O2 is used as an aqueous solution with a concentration of about 20 to 40 wt%, preferably about 30 wt%. Molybdenum carbonyl compounds have the advantage of being able to produce CO with a high capacity (95% or higher) and high purity (95% or higher). To obtain particularly high-purity CO in a particularly high yield, it may be particularly preferable to use Mo(CO)3(CNCH2COOH)3 or its trisodium salt Na3Mo(CO)3(CNCH2CO2H)3 (Mo-CORM) in combination with FeCl3.

[0097] The second compound may be added to the container as part of a closed compartment (e.g., a capsule) containing the CORM. Thus, in a preferred embodiment, the CORM and the second compound may be encapsulated in a single closed compartment, such as a capsule, and then added to the container containing the gametes. Such a system containing a CORM and a second compound in a closed compartment is also referred to as a CO release system ("CORS"). The outer sheath of such a CORS may be selectively permeable, particularly to gas molecules such as CO gas. This allows all other components contained in the CORS to be maintained within the CORS or the corresponding closed compartment (capsule) and prevented from contacting the gametes. In one aspect, the CORM and the second compound may be physically separated within the closed compartment (e.g., by a separation membrane or barrier). Only after physical contact between the CORM and the second compound can CO gas diffuse through the outer sheath into the container containing the gametes, resulting in the release of CO from the CORM / CORS ("activated CORS"). As evidenced by the accompanying Example 2, in one illustrative embodiment, the inventors use a single CORS capsule (18-22 mm long, 6-8 mm diameter) containing 15 mg of Mo-CORM and 150 μL of 583.3 mg / mL ferric chloride solution, separated from each other by a septum / membrane, to preserve liquefied human sperm samples. By applying pressure along the longitudinal axis of the CORS / closed compartment / capsule, the CORM and FeCl3 are brought into physical contact, generating sufficient CO gas for 90 minutes in an airtight 15 mL Falcon tube, ensuring reliable gamete preservation.

[0098] In the context of the present invention, particularly in the in vitro or ex corpore methods described in this disclosure, carbon monoxide-releasing molecules (CORMs), such as trisodium tricarbonyl-[tris(isocyanethylacetate)]molybdenum, may be used. Carbon monoxide-releasing systems may also be used. Corresponding, but merely exemplary, carbon monoxide-releasing systems (CORSs) are shown in Figures 11 and 12, in which a novel and inventive CORS is provided in the form of a CORS capsule, as disclosed and described in European Patent Application No. 22216317.2 and International Patent Application No. PCT / EP2023 / 074808. The CORS capsule is designated by the reference numeral "40" in Figures 11 and 12. The terms "CORS," "carbon monoxide-releasing device," and "carbon monoxide-releasing system" may be used interchangeably in the context of the present invention. CORS can be configured to treat biological cells, preferably live cells, ex vivo by releasing carbon monoxide ex vivo. Corresponding components are shown in FIG. 11 with reference numeral "60." "62" denotes biological cells, particularly gametes such as sperm (as contained in corresponding semen / sperm / glandular fluid). System "60" may be configured to treat biological cells "62," preferably live cells, preferably gametes, with carbon monoxide. System "60" may be configured to impart one or more effects to biological cells "62." Such effects may at least partially preserve biological cells "62." System "60" may include at least one container "64" configured to receive biological cells "62" and at least one source of carbon monoxide. The container "64" and the CORS source "40" may be positioned relative to one another such that the biological cells "62" are treated by contacting them with the carbon monoxide provided by the CORS "40." The container "64" may include a lid "66." Such lid "66" is preferably configured to form a seal with the container "64" to isolate and preferably hermetically seal the contents of the container "64" from the environment.Alternatively, or in addition, the therapeutic system "60" may be configured to treat the animal and / or human body ex vivo, for example, by applying carbon monoxide to an external surface of the animal and / or human body, e.g., the skin, which may also be considered treatment of biological cells within the meaning of the present disclosure.

[0099] FIG. 12 is a schematic diagram of an alternative system. Such a system may be exemplified by "80" in the figure. "80" may also be used to expose living organisms, living cells, preferably gametes, to carbon monoxide ex vivo, as provided herein. Such exposure may include releasing carbon monoxide ex vivo. System "80" includes at least one container "82" configured to receive biological cells "62" and at least one carbon monoxide source. The configuration shown in FIG. 12 illustrates a CORS "40" as an example, but is not limited to, a carbon monoxide source. Container "82" and carbon monoxide source device "40" may be positioned relative to one another such that biological cells "62" come into contact with the carbon monoxide provided by device "40." Container "82" includes at least one compartment "84" configured to receive device "40" and to accommodate and / or secure device "40," preferably in a tethered manner. Alternatively, or in addition to compartment "84," container "82" may include one or more securing means, such as one or more clips, configured to secure CORS "40" within container "82," preferably in a tethered and / or substantially permanent manner. This allows system "80" to be pre-assembled by placing CORS "40" within compartment "84," particularly prior to use, preferably prior to distribution to the location of application, such as one or more laboratories or medical facilities. This may facilitate handling and / or use of system "80."

[0100] Corresponding systems for exposing gametes to CO are also shown and provided in European Patent Application No. 22216317.2 and International Patent Application No. PCT / EP2023 / 074808, all of which are incorporated herein by reference.

[0101] Alternatively, CO gas can be directly delivered to the container by releasing it into the container through a separate tube. In one aspect, CO gas is delivered directly from a pressurized CO gas tank or is released from a liquid, such as a saturated solution, foam, or hydrogel, in which CO is physically bound, or from a solid.

[0102] The total amount of CO gas applied to the gametes in the container is between about 20 μmol and about 500 μmol, preferably between about 30 μmol and about 450 μmol, between about 40 μmol and about 400 μmol, preferably between about 45 μmol and about 270 μmol, and preferably between about 60 μmol and about 180 μmol. In this context, a numerical value preceded by the term "about" refers to the numerical value itself but also includes a ±10% error (tolerance) of the numerical value. For example, 1 mg of Mo-CORM may release about 6 μmol of carbon monoxide gas if each Mo-CORM molecule is completely decarbonylated. As shown in the accompanying non-limiting Example 2, for a human sperm sample, 15 mg of Mo-CORM can be used when stored in a standard 15 mL Falcon tube with a volume of 0.7 mL to 2.9 mL. This amount corresponds to 15 × 6 μmol = 90 μmol of released carbon monoxide gas. By way of further example, for cattle capable of collecting larger average sperm samples (5-8 mL), four times the amount of Mo-CORM, i.e., 60 mg, can be used. This amount corresponds to a maximum release of 360 μmol of carbon monoxide. Therefore, the amount of Mo-CORM / CO used to contact the gametes may also depend on the volume of gametes collected and the volume of the container. In one exemplary embodiment, Example 2, an average of approximately 8.4 mg of CORM per mL of collected sample (range: approximately 5.2 mg to 20.5 mg of CORM per mL of collected sample) was used.

[0103] According to the means, method, and use of the present invention, gametes can be reliably preserved by contacting them with carbon monoxide gas at about 25° C. to about 35° C. for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, and more preferably about 30 to about 60 minutes. In this context, a numerical value preceded by the term "about" refers to the numerical value itself, but also includes an error (tolerance) of ±10% of the numerical value.

[0104] Several methods can be used to determine whether gametes have been successfully preserved after contacting them with carbon monoxide gas in a container, some of which are described in Example 2 below. Gamete preservation according to the present invention can be assessed by measuring sperm / sperm cell motility, particularly forward sperm / sperm cell motility, DNA fragmentation, sperm analysis, redox potential measurement, or a combination thereof. As described in detail later in this disclosure, the Male Infertility Oxidation System (MiOXSYS, Englewood, Colorado) can be used to measure sperm (resting) redox potential / reactive oxygen species levels. As will be apparent to those skilled in the art, the terms "static redox potential," "redox potential," "reactive oxygen species level," "oxidation potential," and "redox potential" can be used interchangeably in the context of the present invention. The Halosperm G2 kit (Halotech, Madrid, Spain) can be used to measure sperm DNA fragmentation, and the CEROS II system (Hamilton Thorne, Beverly, Massachusetts) can be used to measure sperm motility, particularly total and forward motility. Meanwhile, sperm testing (also called semen analysis or semen analysis) may analyze one or more characteristics of a man's semen and the sperm cells contained therein. Some assay kits / methods assess only a few characteristics (e.g., home kits) while others assess many characteristics simultaneously (e.g., diagnostic laboratories). Non-limiting characteristics analyzed in sperm testing may include, among others, semen physical characteristics (color, odor, pH, viscosity, liquefaction), volume, sperm count, concentration, morphology, total and progressive sperm motility / progressive motility, non-progressive motility, immotile sperm, viable sperm, and normal sperm morphology. Those skilled in the art are aware of numerous commercially available kits, tools, and methods for analyzing the above characteristics.

[0105] All of the above statements relating to the means and methods of the invention also apply, where appropriate, to the uses, methods of treatment and kits detailed below.

[0106] Thus, in another aspect, the present invention also relates to the use of carbon monoxide gas to preserve gametes for the purpose of reducing the risk of congenital abnormalities and / or aneuploidy (and / or euploidy) in assisted reproductive technology applications, wherein the gametes are contacted with carbon monoxide gas in a container for a length of time sufficient to ensure preservation of the gametes.

[0107] In another aspect, the present invention relates to a method for reducing DNA fragmentation and / or redox potential of gametes in assisted reproductive technology, the method comprising contacting gametes with carbon monoxide gas. The gametes may be obtained / provided in a container and contacted with CO2 in the container. The contact of the gametes with CO2 may be performed for a time period long enough to ensure a reduction in DNA fragmentation and / or redox potential. By reducing the DNA fragmentation and / or redox potential of the gametes, motility can be maintained and / or increased. Motility includes, but is not limited to, forward motility rate, total motility rate, and / or VAP of the gametes. Furthermore, by reducing the DNA fragmentation and / or redox potential of the gametes, the risk of congenital abnormalities and / or aneuploidy in artificial reproductive technology can be reduced. In another aspect, by reducing the DNA fragmentation and / or redox potential of the gametes, the success rate of artificial reproductive technology can be improved. Therefore, by reducing the DNA fragmentation and / or redox potential of gametes, it is possible to reduce the risk of miscarriage and / or abortion in artificial reproductive techniques.

[0108] In another aspect, the present invention relates to the use of carbon monoxide in fragmenting and / or reducing the redox potential of gametes in assisted reproductive technology, comprising contacting the gametes with carbon monoxide gas. Here, the gametes may be obtained / provided in a container and contacted with CO in the container. The contact of the gametes with CO may be carried out for a time period long enough to ensure a reduction in DNA fragmentation and / or redox potential. Here, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or increase motility. Motility includes, but is not limited to, the forward motility rate, total motility rate, and / or VAP of the gametes. Furthermore, reducing the DNA fragmentation and / or redox potential of the gametes can reduce the risk of congenital abnormalities and / or aneuploidy in artificial reproductive technology. In another aspect, reducing the DNA fragmentation and / or redox potential of the gametes can improve the success rate of artificial reproductive technology. Therefore, by reducing DNA fragmentation and / or redox potential of germ cells, it is possible to reduce the risk of miscarriage and / or pregnancy loss in artificial reproductive techniques.

[0109] In another aspect, the present invention also relates to the use of carbon monoxide in the treatment / prevention of gamete-related diseases caused by / resulting from / associated with ROS / elevated ROS levels.

[0110] As detailed earlier and later in this disclosure, reactive oxygen species and / or oxidative stress (and the resulting elevated redox potential) may cause and / or be associated with congenital abnormalities and / or aneuploidy in offspring resulting from diseased gametes and / or gametes with elevated ROS levels. Consequently, exposing fertilized eggs to CO gas (in artificial reproductive technologies) may prevent or reduce DNA fragmentation and / or redox potential, thereby potentially preventing or reducing the risk of congenital abnormalities and / or aneuploidy. Thus, exposing fertilized eggs to CO gas can prevent and / or reduce the risk of congenital abnormalities and / or aneuploidy. In other words, the present invention relates to carbon monoxide for preventing abnormalities and / or aneuploidy. In another aspect, the present invention relates to carbon monoxide for use in a method for preventing abnormalities and / or aneuploidy. As is particularly evident from the examples, gametes (provided / obtained in a container) can be contacted ex vivo with carbon monoxide gas (in said container) (for a sufficiently long time). However, the present invention also relates to preventing and / or reducing the risk of birth defects and / or aneuploidy by contacting gametes in vivo with CO (for a sufficiently long time). This aspect is explained in more detail below in the present disclosure.

[0111] As detailed later in this disclosure, reactive oxygen species and / or oxidative stress (and elevated redox potential) in gametes may cause and / or be associated with male infertility in a patient. That is, it will be apparent to one skilled in the art that reducing and / or decreasing reactive oxygen species, oxidative stress, and / or redox potential in a male patient's gametes can treat / alleviate the patient's infertility. In other words, the present invention relates to carbon monoxide for use in treating / alleviating male infertility. In another aspect, the present invention relates to the use of carbon monoxide in a method for treating and / or reducing male infertility. As is apparent from the examples, in particular, gametes (provided in a container) can be contacted with CO (the gas in the container) ex vivo (for a sufficient length of time). However, the present invention also relates to contacting gametes with CO in vivo (for a sufficient length of time), thereby treating and / or reducing male infertility. This aspect will be described in more detail later in this disclosure.

[0112] In another aspect, reactive oxygen species and / or oxidative stress (and the resulting increase in redox potential) and DNA fragmentation of gametes cause and / or are associated with diseases. Such diseases may be caused by and / or associated with (elevated) stress levels (physiological and / or psychological) in patients. Such diseases may occur in patients suffering from (physiological and / or psychological) stress. By contacting gametes with CO gas (in artificial reproductive technology), DNA fragmentation and / or redox potential can be prevented and / or reduced, thereby preventing and / or treating such diseases. Therefore, by contacting gametes with CO gas, such diseases can be prevented and / or treated. In other words, CO can treat and / or prevent diseases caused by and / or associated with DNA fragmentation and / or an increase in redox potential of gametes. In other words, the present invention relates to carbon monoxide for use in treating and / or preventing diseases caused by and / or associated with (elevated) DNA fragmentation and / or redox potential of gametes. In another aspect, the present invention relates to carbon monoxide for use in a method for treating and / or preventing diseases caused by and / or associated with (elevated) DNA fragmentation and / or redox potential of gametes. As is evident, inter alia, from the examples, gametes can be provided / obtained in a container and contacted ex vivo (within said container) with CO gas (for a sufficiently long time). However, the present invention also relates to contacting gametes in vivo with CO (for a sufficiently long time), thereby preventing and / or treating such diseases. This aspect is explained in more detail below in the present disclosure.

[0113] In another aspect, elevated ROS levels may cause and / or be associated with gamete disorders. Such elevated ROS levels and resulting disorders may be caused by and / or associated with elevated (physiological and / or psychological) stress levels in patients. Such disorders may occur in patients suffering from (physiological and / or psychological) stress. Contacting gametes with CO gas (in artificial reproductive technology) may reduce (elevated) ROS levels, thereby preventing and / or treating such disorders. In other words, CO can treat and / or prevent gamete disorders caused by and / or associated with elevated ROS levels. In other words, the present invention relates to carbon monoxide for use in treating and / or preventing gamete disorders caused by and / or associated with elevated ROS levels. In another aspect, the present invention relates to carbon monoxide for use in a method for treating and / or preventing gamete disorders caused by and / or associated with elevated ROS levels. As is evident, inter alia, from the examples, gametes can be (provided / obtained in a container) and contacted (for a sufficiently long time) with CO gas ex vivo (within said container). However, the present invention also relates to contacting gametes with CO in vivo (for a sufficiently long time), thereby preventing and / or treating such diseases. This aspect is explained in more detail below in the present disclosure.

[0114] The present invention further relates to carbon monoxide for the treatment and / or prevention of miscarriage and / or abortion due to (elevated) ROS and / or (elevated) DNA fragmentation (in gametes / gametes used for fertilization).

[0115] Furthermore, it has been shown that reducing the DNA fragmentation and / or redox potential of gametes may improve the success rate and / or success rate of artificial reproductive techniques that use those gametes for fertilization. In another aspect, reducing the DNA fragmentation and / or redox potential of gametes may protect the gametes and / or prevent damage to the gametes. In yet another aspect, reducing the DNA fragmentation and / or redox potential of gametes may increase the survival rate of the gametes. In another aspect, reducing the DNA fragmentation and / or redox potential of gametes may maintain and / or improve the total motility rate of the gametes. In another aspect, reducing the DNA fragmentation and / or redox potential of gametes may maintain and / or improve the progressive motility rate of the gametes. In another aspect, reducing the DNA fragmentation and / or redox potential of gametes may maintain and / or improve the mean velocity of pathway (VAP) of the gametes.

[0116] As detailed above in this disclosure, preserving gametes in a functional, live, viable, intact, or undamaged or decayed state with carbon monoxide (gas) reduces the risk of congenital abnormalities and / or aneuploidy. As will be apparent to those skilled in the art, the terms "functional," "alive," and "viable" used herein are used interchangeably with respect to gametes. Therefore, improving and / or increasing sperm and / or sperm viability, quality, and / or survival parameters can be used interchangeably in the context of the present invention. This is particularly true when CO2-preserved gametes are used in assisted reproductive technology applications. In other words, "reducing the risk" refers to the reduction in risk associated with using functional / viable / intact gametes with CO2 preservation in assisted reproductive technology applications. When unpreserved gametes are used, the gametes are more susceptible to damage, resulting in, for example, a higher likelihood of a newborn baby being born with congenital abnormalities or aneuploidy. Therefore, contacting or exposing gametes, such as sperm, to carbon monoxide or carbon monoxide gas can maintain functional, viable, and intact gametes in a desirable manner, or even improve their quality, viability, and / or survival parameters. In the context of the present invention, the carbon monoxide-stored gametes, particularly when used in artificial reproductive techniques, may reduce the risk of miscarriage of fertilized eggs, embryos, offspring, etc. derived from the stored gametes, compared to gametes not stored / contacted / exposed to the carbon monoxide. In other words, contacting gametes, specifically sperm, that have been contacted with stored gametes with carbon monoxide (gas) may increase the success rate of artificial reproductive techniques using the stored gametes, compared to gametes that have not been contacted with carbon monoxide (gas).

[0117] Assisted reproductive technologies (ART) primarily include medical procedures aimed at treating infertility (often due to gamete dysfunction, death, or damage) and reducing the risk of congenital abnormalities or aneuploidy. Non-limiting examples of applications of assisted reproductive technologies of the present invention include, among others, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI), frozen embryo transfer (FER), preimplantation genetic diagnosis (PGT; sometimes performed in conjunction with IVF), in vitro oocyte maturation (IVM), gamete (egg and sperm) cryopreservation, frozen egg replacement (FOR), gamete intrafallopian tube transfer (GIFT), fertilized egg intrafallopian tube transfer (ZIFT), or cryopreservation. Some of these procedures are also described in Wyns, Human Reproduction Open 3 (2021): 1-17.

[0118] Congenital anomalies include a wide range of abnormalities of body structure or function that are present at birth and result from prenatal causes. For reasons of efficiency and practicality, emphasis is usually placed on major structural anomalies. These are defined as structural changes that have significant medical, social, or cosmetic consequences for the affected individual and usually require medical intervention.

[0119] That is, the one or more congenital abnormalities of the present invention can be selected from the group consisting of congenital abnormalities of the limbs, congenital abnormalities of the heart, congenital abnormalities of the nervous system, congenital abnormalities of the digestive system, and congenital abnormalities of the lungs, among others.

[0120] The congenital limb abnormalities according to the present invention may in particular be selected from the group consisting of achondroplasia, amelia, persevering amniotic membrane syndrome, brachydactyly, cleidocranial dysplasia, congenital aplasia or hypoplasia, abductor dysplasia, ectrodactyly, sepium, polydactyly, polyglottia, polydactyly and syndactyly.

[0121] The congenital cardiac abnormality according to the present invention may be selected from the group consisting of atrial septal defect, patent ductus arteriosus, tetralogy of Fallot and ventricular septal defect, among others.

[0122] The congenital abnormality of the nervous system according to the present invention may be selected from the group consisting of neural tube defects, such as agenesis of the corpus callosum, anencephaly, Arnold-Chiari malformation, Dandy-Walker malformation, encephalocele, holoprosencephaly, hydrocephalus, coloboma, megalencephaly, meningocele, meningomyelocele, micromyelopathy, polymicrogyria, and spina bifida.

[0123] The congenital anomaly of the digestive system according to the present invention may in particular be selected from the group consisting of atresia, imperforation and stenosis.

[0124] Congenital pulmonary abnormalities according to the present invention include, inter alia, congenital bronchiectasis.

[0125] In the context of the present invention, (congenital) aneuploidy refers to a cell having more or fewer chromosomes than normal, e.g., a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include cases where the complete set of chromosomes differs by one or more. Aneuploidy occurs when chromosomes do not properly separate (non-disjunction) between two cells during cell division. Most cases of autosomal aneuploidy result in miscarriage, and the most common non-autosomal chromosomes in live births are 21, 18, and 13 (Driscoll, The New England Journal of Medicine 360 ​​(2009): 2556-2562). Chromosomal abnormalities are detected in 1 in 160 live births. Non-limiting examples of (congenital) aneuploidies include, among others, trisomy 1 (1p36 deletion syndrome / 1q21.1 deletion syndrome), trisomy 2 (2q37 deletion syndrome), trisomy 3, trisomy 4 (Wolf-Hirschhorn syndrome), trisomy 5 (Cri du chat / 5q deletion syndrome), Trisomy 6, trisomy 7 (Williams syndrome), trisomy 8 (monosomy 8p / monosomy 8q), trisomy 9 (Alfie syndrome / Klefstra syndrome), trisomy 10 (monosomy 10p / monosomy 10q), trisomy 11 (Jacobsen syndrome), trisomy 12, Patau syndrome, trisomy 14, trisomy 15 (Angelman syndrome / Prader-Willi syndrome), trisomy 16, trisomy 17 (Miller-Dyker syndrome / Smith-Magenis syndrome), Edwards syndrome (distal 18q- / proximal 18q-), trisomy 19, trisomy 20, trisomy 21 (Down syndrome), cat's eye syndrome / trisomy 22 (DiGeorge syndrome / Phelan-McDermid syndrome / 22q11.2 distal deletion syndrome).

[0126] In the context of the present invention, (congenital) euploidy refers to cells that have any number of complete chromosome sets ("sets") that differ from the two chromosome sets found in normal diploid cells. Non-limiting examples of cells with a number of complete chromosome sets that differ from two include monoploid (one set), triploid (three sets), tetraploid (four sets), pentaploid (five sets), hexaploid (six sets), and heptaploid / septaploid (seven sets), among others. The general term polyploid is often used to refer to cells that have three or more chromosome sets.

[0127] In a further aspect, the present invention relates to a method for treating a congenital abnormality or aneuploidy (or euploidy), comprising contacting gametes of a patient in need thereof with carbon monoxide gas, wherein the contacting of the gametes with carbon monoxide can be performed, for example, according to the methods described above in this disclosure.

[0128] Therefore, in order to treat such congenital abnormalities or aneuploidy (or euploidy) using carbon monoxide gas, for example, before use in ART, a healthy or diseased patient, or the patient's gametes, may need to be diagnosed as needing such treatment. A patient or a patient's gametes may be in need of such treatment if the patient's existing offspring suffer from the congenital abnormality or aneuploidy (or euploidy), or if the patient's gametes are assessed to be at increased risk of developing the congenital abnormality or aneuploidy (or euploidy) compared to the gametes of a normal control patient (cohort). In the context of the present invention, the terms "patient," "disease patient," "patient in need of such treatment," "disease subject," "individual," and "individual to be treated" can be used interchangeably and may refer to a subject having gametes with DNA fragmentation and / or increased redox potential.

[0129] Such gamete evaluation can be performed, inter alia, through karyotyping, DNA fragmentation analysis, and / or sperm testing (e.g., all considered standard / routine methods in the art). For example, a patient's gametes may be defined as "high risk" for congenital abnormalities or aneuploidy if they exhibit elevated levels of DNA fragmentation, elevated redox potential, elevated ROS levels, or an elevated proportion of gametes with an abnormal number of chromosomes when compared with gametes from normal control patients treated with the same method. Because haploid gametes contain 23 chromosomes, any number of chromosomes other than 23 may indicate an abnormal chromosome count in the gametes being evaluated. As previously described in detail in this disclosure, for example, the Male Infertility Oxidation System (MiOXSYS, Englewood, Colorado) can be used to measure sperm ROS levels and / or redox potential. The term "elevated" refers to the mean or median (ROS) level of a control group or a (ROS) level that is (clearly) distinguishable from a control sample. As described in more detail below in this disclosure, the accompanying Figures 7D and 8D show measurements of DNA fragmentation and redox potential of sperm samples collected from individual subjects that were not treated with carbon monoxide. Here, only a small percentage of sperm samples show high measurements of redox potential, while the majority of samples show similar (low) values ​​of redox potential. It is believed that sperm samples with low redox potential may be considered control samples.

[0130] If a patient or their gametes are identified as requiring CO treatment, the patient's gametes can be harvested and subjected to the methods described herein. As previously described in this disclosure, the patient's gametes may be contacted and / or treated in vivo with carbon monoxide. In this disclosure, carbon monoxide may be administered to the gametes systemically and / or transdermally. Systemic administration includes, but is not limited to, oral, rectal, and / or intraurethral administration, and administration of a carbon monoxide-releasing system / suppository. Transdermal and / or transcutaneous administration includes, but is not limited to, application of carbon monoxide or a carbon monoxide-releasing system to the patient's scrotum. Carbon monoxide gas may diffuse, particularly through the epithelial layer, and contact the gametes. Here, the carbon monoxide-releasing system may be attached and / or adhered to the patient's scrotum. In one aspect, the carbon monoxide-releasing system may be a patch. CO2-releasing patches are well known in the art and are described in WO 2021 / 180908 A1 and Ruopp et al. (2023, Journal of Controlled Release), which are incorporated by reference in their entireties into this disclosure.

[0131] While such patches have been used to treat wounds, inflammatory skin disorders, inflammatory disorders of subcutaneous skin tissue, joints, and tendons, their use in the treatment of gamete disorders has not previously been disclosed. To ensure sufficient in vivo treatment of gametes to achieve the desired effects of CO, the gametes can be contacted with CO (e.g., using the CO delivery system / patch described above) at least 240, 300, or 360 minutes before harvesting the gametes. In one aspect, the gametes can be treated in vivo multiple times, i.e., at least once, at least twice, at least three times, at least four times, at least five times, or at least six times, with CO treatment occurring for at least 240, 300, or 360 minutes each time before harvesting the gametes. The scrotum may be contacted with about 90 μmol to about 540 μmol of carbon monoxide. Clearly, treating gametes with CO in vivo and treating gametes with CO in vitro are not mutually exclusive. In other words, gametes may first be contacted with CO in vivo using the means and methods disclosed in this disclosure, or, after collection of the gametes, may be contacted with CO in vitro using the means and methods disclosed in this disclosure.

[0132] Administering CO to a patient's gametes in vivo may decrease the gamete's redox potential / ROS level and / or DNA fragmentation, resulting in improved gamete viability. As previously described in detail in this disclosure, various factors, including but not limited to stress, may increase and / or accumulate ROS levels in an individual's gametes. That is, an individual's ROS levels may be elevated due to, among other factors, stress (physiological and / or psychological), genetic predisposition, environmental factors (including exposure to electromagnetic fields), and / or behavioral risk factors (diet, including smoking, alcohol, and / or substance abuse). Systemic ROS levels may be related to gamete ROS levels and, therefore, may be used as an indicator of an individual's gamete ROS levels. Systemic ROS levels may be assessed by evaluating ROS levels in an individual's urine and / or blood samples, particularly serum samples. Methods and means for measuring ROS levels in, inter alia, an individual's serum sample are well known in the art and can be performed using the RedoxSys instrument from Aytu Biosciences (Aytu Biosciences, Inc, Englewood, CO 80112, USA).

[0133] ROS in gametes may affect the reproductive efficiency of artificial reproductive techniques and natural insemination, particularly during sexual intercourse. That is, gametes exposed to CO in vivo and resulting in reduced ROS levels may be particularly suitable for and / or have improved success rates in subsequent artificial reproductive techniques and / or subsequent natural insemination. As previously described in this disclosure, reducing ROS levels through carbon monoxide treatment (in vivo) may prevent congenital anomalies and / or aneuploidy, treat and / or reduce male infertility, and / or treat and / or prevent diseases caused by and / or associated with DNA fragmentation and / or elevated redox potential of gametes, or treat and / or prevent diseases of gametes caused by and / or associated with elevated ROS levels.

[0134] In another aspect, the present invention relates to a kit comprising a CORM and a second compound in a closed compartment ("CORS"), as described in detail in the present disclosure above. Preferably, such a kit may further comprise instructions describing the method and / or use as described in the present disclosure above. Such a kit may further comprise additional components, such as containers, buffers, etc., that may be used / beneficial in the above-described method. Preferably, such a kit is, for example, a home kit that can be used by anyone capable of performing the above-described method, or a kit that can be used by specially trained staff, such as in a fertility clinic.

[0135] Further embodiments are exemplified in the scientific section. The accompanying drawings illustrate the present invention. The experimental data shown in the examples and accompanying drawings should not be construed as limiting, but the technical information contained therein constitutes part of the present invention. That is, the present invention covers all additional features individually shown in the drawings, regardless of whether they are described in the description before or after this section. It should be noted that individual alternatives of the embodiments and individual alternatives of the features described in the drawings and description may be excluded from the scope of other aspects of the present invention. [Brief explanation of the drawings]

[0136] [Figure 1A] FIG. 1A: Example of the workflow of the method according to the invention for preserving sperm using carbon monoxide. [Figure 1B] Figure 1B: Example workflow of the method of the present invention for preserving sperm using carbon monoxide. The workflow shows the process of sample collection and CO treatment (using a CO2 releasing system; CORS) for options for home use and professional use such as ART laboratories. [Figure 2] Figure 2: sORP measurements of sperm samples from six subjects. Relative sORP of CO2-treated sperm (wCO2) compared to CO2-untreated sperm (woCO2, set at 1) is shown. [Figure 3]Figure 3: DNA fragmentation measurements of sperm samples from three subjects. The relative percentage of DNA fragmentation in the CO2-treated sperm group (wCO2) compared to the CO2-untreated sperm group (woCO2, set to 1) is shown. [Figure 4] Figure 4: Motility measurements of sperm samples from five subjects. A: Total motility, B: Forward motility. The relative rates of CO2-treated sperm (wCO2) compared to CO2-untreated sperm (woCO2, set at 1) are shown. [Figure 5] Figure 5: Summary of relative changes in parameters of CO2-treated sperm (wCO2) compared to CO2-untreated sperm (woCO2, set at 1). Data are expressed as mean ± SD. n = 5 (total and forward motility, see Figure 4), n = 6 (sORP, see Figure 2), n = 3 (DNA fragmentation, see Figure 3). [Figure 6] Figure 6: Relative changes in sperm viability and motility parameters in response to different carbon monoxide treatments. A: Treated / untreated ratios of total motility ("Motility"), progressive motility ("Progressive"), and VAP ("VAP") at a CO dose of 1.32 mL and different exposure times. B: Treated / untreated ratios of motility, progressive motility, and VAP at a 90-minute exposure time and different carbon monoxide doses. C: Treated / untreated ratios of oxidation-reduction potential ("sORP") and DNA fragmentation ("SDF") at a CO dose of 1.32 mL and different exposure times. D: Treated / untreated ratios of oxidation-reduction potential and DNA fragmentation at a 90-minute exposure time and different CO doses. Values ​​are shown as mean ± SD, n=5. [Figure 7A-B]Figure 7: Paired measurements from individual subjects in CO2-treated and untreated groups with different incubation periods. Glandular fluid from each subject was collected in a sample cup, aliquoted, and then treated with CORS (1.32 mL of CO2 gas) for different times (60 to 270 minutes). After each time point, total motility (A), forward motility (B), and mean velocity (VAP) (C) were analyzed using computer-assisted sperm analysis. Sperm DNA fragmentation (SDF) (E) was measured microscopically, and static oxidation-reduction potential (sORP) (D) was measured using the MiOXSYS® system. Numbers on the x-axis indicate subject ID. The data from Figure 7 are summarized in Figure 6, and portions of the data are summarized in Figures 9 and 10. [Figure 7C-D] Same as above. [Figure 7E] Same as above. [Figure 8A-B] Figure 8: Pairwise measurements of individual subjects in the CO2-treated and untreated groups. Glandular fluid from each subject was collected in a sample cup, aliquoted, and then treated with different concentrations of CORS (0.44, 1.32, or 2.64 mL of CO2 gas) for 90 minutes. After the 90-minute period, total motility rate (A), forward motility rate (B), and mean velocity profile (VAP) (C) were analyzed using computer-assisted sperm analysis. Sperm DNA fragmentation (SDF) (E) was measured microscopically, and static oxidation-reduction potential (sORP) (D) was measured using a MiOXSYS® system. Numbers on the x-axis indicate subject ID. The data from Figure 8 are summarized in Figure 6. [Figure 8C-D] Same as above. [Figure 8E] Same as above. [Figure 9] Figure 9: Linear correlation between sORP levels in untreated sperm samples and the effect of CO treatment on sORP levels in the sperm samples. Evaluation of the correlation between resting oxidation-reduction potential (sORP) values ​​in the untreated group and delta sORP values ​​(difference between untreated and treated groups) after 60 minutes (A) (according to WHO guidelines) or 90 minutes (B) (maximum time in ART lab) of incubation with CORS (15 mg CORM), and for the entire 90 minutes (C). r: correlation coefficient, p: significance level. Units on the x- and y-axes are [mV / 106 sperm]. [Figure 10]Figure 10: Linear correlation between SDF levels in treated sperm samples and the effect of CO treatment on the SDF levels of the sperm samples. Evaluation of the correlation between sperm DNA fragmentation (SDF) values ​​in the untreated group and the delta SDF values ​​(difference between the untreated and treated groups) after 60 minutes (A) (according to WHO guidelines) or 90 minutes (B) (the maximum time in the ART laboratory) of incubation with CORS (15 mg of CORM), and for the entire 90 minutes (C). r: correlation coefficient, p: significance level. The units on the x- and y-axes are [%]. [Figure 11] 11: A side view of an exemplary processing system according to one embodiment of the present invention. The highlighted (technical) features are explained in detail in this disclosure. [Figure 12] 12: A side view of an exemplary processing system according to a further embodiment of the present invention. The highlighted (technical) features are explained in detail in this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0137] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described in this disclosure can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0138] The methods and techniques of the present invention are generally carried out according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, e.g., WHO laboratory manual for the examination and processing of human semen, 6th edition; WHO 2021; Madigan et al., Brock Biology of Microorganisms, 15th ed., Pearson (2018); Berg et al., Stryer Biochemie, 7th ed., Springer Spektrum (2013).

[0139] While the invention has been illustrated and described in detail in the drawings and foregoing description, it is to be understood that such illustration and description are by way of illustration or example only and not of limitation. Those skilled in the art will recognize that changes and modifications can be made within the spirit and scope of the following claims. In particular, the invention covers all aspects in any combination of features from the different aspects described above and below.

[0140] The present invention also covers all additional features individually shown in the drawings, whether or not they are described in the description before or after this section, and it should be noted that individual alternatives of the embodiments and individual alternatives of the features thereof shown in the drawings and description may be excluded from the scope of other aspects of the present invention.

[0141] Furthermore, in the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single unit may fulfill the functions of several features recited in a claim. Furthermore, the terms "essentially," "about," "approximately," and the like, used in connection with an attribute or a value, precisely define the corresponding attribute or value. Reference signs in the claims are not to be construed as limiting the scope.

[0142] Numerous documents have been cited herein, including scientific papers, patent applications, and manufacturer's manuals. The disclosures of these documents should not be deemed relevant to the patentability of the present invention, but are incorporated by reference in their entirety into this disclosure. More specifically, all documents referenced are incorporated by reference into this disclosure to the same extent as if each document were individually incorporated by reference.

[0143] In accordance with the above, the present invention relates inter alia to the following: Further aspects of the invention relating to these are as set out above and as shown in the accompanying examples and figures.

[0144] 1. A method for preserving gametes, comprising: a) providing / obtaining gametes or a gamete-containing sample in a container; and b) contacting the gametes or the gamete-containing sample in the container with carbon monoxide A method comprising:

[0145] 2. The method of paragraph 1, wherein the gametes or the gamete-containing sample comprises sperm or egg cells, preferably wherein the sperm are contained in semen, glandular fluid, and / or a buffer / buffer system.

[0146] 3. The method according to paragraph 1 or 2, wherein carbon monoxide is released from a carbon monoxide-releasing molecule and / or carbon monoxide is supplied directly to the container through a separate pipe.

[0147] 4. The method according to any one of items 1 to 3, wherein carbon monoxide forms a gas layer above the gametes or the gamete-containing sample.

[0148] 5. The method according to item 3 or 4, wherein the carbon monoxide-releasing molecule is preferably a metal carbonyl compound, more preferably a molybdenum carbonyl compound, and most preferably Na3Mo(CO)3(CNCH2CO2H)3.

[0149] 6. The method according to any one of items 3 to 5, wherein carbon monoxide is generated by contacting the carbon monoxide-releasing molecule with FeCl3, Ce(SO4)2, or H2O2, preferably FeCl3.

[0150] 7. The method according to any one of items 1 to 6, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, and more preferably about 30 to about 60 minutes.

[0151] 8. The method according to any one of items 1 to 7, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide at about 25 to about 35°C.

[0152] 9. The method according to any one of items 1 to 8, wherein the amount of carbon monoxide applied to the gametes or the gamete-containing sample is about 20 μmol to about 500 μmol, about 40 μmol to about 400 μmol, preferably about 45 μmol to about 270 μmol, and preferably between about 60 μmol and about 180 μmol.

[0153] 10. The method according to any one of items 1 to 9, wherein the gametes are human gametes.

[0154] 11. The method according to any one of items 1 to 9, wherein the gametes are derived from a cow, horse, pig, sheep, goat, camel, alpaca, dog, cat, bird, or fish.

[0155] 12. The method according to any one of items 1 to 11, wherein the preservation state of the gametes is assessed by measuring the motility, DNA fragmentation, spermiogram, and / or redox potential of the gametes.

[0156] 13. A method for reducing DNA fragmentation and / or redox potential of gametes in artificial reproductive techniques, comprising contacting said gametes or a sample containing said gametes with carbon monoxide.

[0157] 14. Use of carbon monoxide to reduce DNA fragmentation and / or redox potential of gametes in assisted reproductive techniques, comprising contacting said gametes or a sample containing said gametes with carbon monoxide.

[0158] 15. The method of item 13 or the use of item 14, wherein the assisted reproductive technology comprises in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, frozen embryo transfer, preimplantation genetic testing, in vitro maturation of oocytes, frozen oocyte transfer, gamete intrafallopian tube transfer, zygote intrafallopian tube transfer, and / or cryopreservation.

[0159] 16. The method according to item 13 or 15, or the use according to item 14 or 15, wherein the motility of gametes is maintained / enhanced by reducing DNA fragmentation and / or redox potential of the gametes.

[0160] 17. The method of paragraph 13, 15, or 16, or the use of paragraph 14, 15, or 16, wherein the risk of congenital abnormalities and / or aneuploidy in artificial reproductive technologies is reduced by reducing DNA fragmentation and / or redox potential of gametes.

[0161] 18. Carbon monoxide for use in preventing birth defects and / or aneuploidy.

[0162] 19. Carbon monoxide for use in treating / improving male infertility.

[0163] 20. Carbon monoxide for use in treating / ameliorating diseases caused by / associated with DNA fragmentation and / or elevated redox potential in gametes.

[0164] 21. Carbon monoxide for use in treating / ameliorating gamete disorders caused by / associated with elevated ROS levels.

[0165] 22. Carbon monoxide for use according to item 20 or 21, wherein the disease is caused by / associated with elevated stress levels or the subject to be treated is suffering from stress.

[0166] 23. Carbon monoxide for use according to item 22, wherein the disease is caused by / associated with an increased level of psychological and / or physiological stress, or the subject to be treated has psychological and / or physiological stress.

[0167] 24. Carbon monoxide for use according to any one of items 18 to 23, wherein the prevention of congenital abnormalities and / or aneuploidy, the treatment / amelioration of male infertility, or the treatment / prevention of the above diseases comprises contacting gametes with carbon monoxide.

[0168] 25. Carbon monoxide for use according to paragraph 24, wherein the gametes are contacted with carbon monoxide in vitro / ex corpore.

[0169] 26. Carbon monoxide for the method according to any one of items 13 and 15 to 17, the use according to any one of items 14 to 17, or the application according to item 25, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide for less than 10 minutes, preferably less than 5 minutes, more preferably less than 3 minutes, more preferably less than 2 minutes, and more preferably less than 1 minute after the gametes are collected in the container.

[0170] 27. Carbon monoxide for the use according to item 24, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide in vivo.

[0171] 28. Carbon monoxide for the use according to item 27, wherein carbon monoxide is administered to the gametes systemically or transdermally.

[0172] 29. Carbon monoxide for the method according to item 17, the use according to item 17, or the application according to any one of items 17 and 24 to 28, wherein the congenital abnormality is selected from congenital abnormalities of the limbs, congenital abnormalities of the heart, congenital abnormalities of the nervous system, congenital abnormalities of the digestive system, and congenital abnormalities of the lungs.

[0173] 30. The method according to Item 29, the use according to Item 29, or carbon monoxide for the use according to Item 29, wherein the congenital abnormality is a congenital limb abnormality selected from the group consisting of anchondrosis, amelia, amniotic band syndrome, brachydactyly, cleidocranial dysplasia, congenital aplasia or hypoplasia, abduction, ectrodactyly, sepia, polydactyly, polyglottia, polydactyly, and syndactyly.

[0174] 31. The method according to item 29, the use according to item 29, or carbon monoxide for the application according to item 29, wherein the congenital abnormality is a congenital cardiac abnormality selected from the group consisting of atrial septal defect, patent ductus arteriosus, tetralogy of Fallot, and ventricular septal defect.

[0175] 32. The method according to Item 29, the use according to Item 29, or carbon monoxide for the use according to Item 29, wherein the congenital abnormality is a congenital abnormality of the nervous system selected from the group consisting of neural tube defects, such as agenesis of the corpus callosum, anencephaly, Arnold-Chiari malformation, Dandy-Walker malformation, encephalocele, holoprosencephaly, hydrocephalus, lissencephaly, megalencephaly, meningocele, meningomyelocele, micromyelopathy, polymicrogyria, and spina bifida.

[0176] 33. The method according to item 29, the use according to item 29, or carbon monoxide for the application according to item 29, wherein the congenital abnormality is a congenital abnormality of the digestive system selected from the group consisting of atresia, imperforation, and stenosis.

[0177] 34. Carbon monoxide for the method according to item 29, the use according to item 29, or the application according to item 29, wherein the congenital abnormality is a congenital pulmonary abnormality, preferably congenital bronchiectasis. [Example]

[0178] Example 1 This example outlines an exemplary workflow of a method according to the present invention for preserving sperm using carbon monoxide, which is also shown in accompanying Figure 1.

[0179] In the first step, a carbon monoxide (CO) releasing system (CORS) containing (i) a carbon monoxide-releasing molecule (CORM, e.g., Na3Mo(CO)3(CNCH2CO2H)3; Mo-CORM) and (ii) a second compound, e.g., FeCl3, Ce(SO4)2, or HO2, preferably FeCl3, is provided in a CO gas permeable sealed compartment (e.g., capsule). The (Mo-)CORM and the second compound are not in contact with each other within the sealed compartment at this point. In the next step, sperm are collected from a healthy or diseased subject and transferred to a container. Simultaneously, force is applied to both ends of the capsule's long axis, bringing the (Mo-)CORM into contact with the second compound (e.g., an aqueous solution of FeCl3, Ce(SO4)2, or HO2) within the closed compartment / capsule ("activated CORS"). This releases carbon monoxide gas from the (closed) compartment / capsule. Next, the sealed compartment releasing carbon monoxide gas is added to a container containing a sperm sample. The container is sealed, and the sample is incubated for a given time (preferably 30-60 minutes at 25-35°C) before being transported to an assisted reproductive technology (ART) laboratory. During incubation, liquefaction occurs naturally under given conditions. Liquefaction in this context refers to the process of breaking down the gel formed by proteins secreted by the seminal vesicles and prostate, making the semen / glandular fluid more liquid. Finally, the CORS (= a closed compartment containing Mo-CORM and a CORM such as FeCl3, Ce(SO4)2, or H2O2, preferably FeCl3) is removed from the container, and the sample is either further processed for ART or cryopreserved.

[0180] The dosage (number of CORS added / amount of (Mo-)CORM) depends on the loading of Mo-CORM added to the system and the maximum amount of CO released. 1 mg of Mo-CORM can release up to 6 μmol of CO gas. The Mo-CORM loading of CORS depends on the size of the system. Detailed specifications regarding the size and loading of Mo-CORM and FeCl3 are described in International Patent Application No. PCT / EP2020 / 078794, published as WO 2021 / 074159 A1. Further details are described in European Patent Application No. 22216317.2, filed December 23, 2022, and International Patent Application No. PCT / EP2023 / 074808, filed September 8, 2023.

[0181] Example 2 Example 2 shows the improvement of sperm characteristics (oxidation-reduction potential, DNA fragmentation, and / or sperm motility, particularly forward motility) when exposed to carbon monoxide gas, compared with "untreated" control sperm from the same patient that were not exposed to carbon monoxide gas. The results of this example are shown in the accompanying Figures 2 to 5.

[0182] method In this example, a CORS capsule measuring 18–22 mm in length and 6–8 mm in diameter was filled with 15 mg of Mo-CORM and 150 μL of a 583.3 mg / mL FeCl3 solution, and used to store samples of 0.7 mL to approximately 2.9 mL in a standard 15 mL Falcon tube. CORS, Mo-CORM, and FeCl3 were prepared as follows:

[0183] Preparation of CORS The CORS was fabricated using additive manufacturing. Two compartments, separated by a partition, were 3D printed within one compartment. The compartment containing the ferric chloride solution was printed using a drop-on-demand (PolyJet modeling process) method using an Objet Eden 350 printer (96 nozzles, 40 μM droplet size, resolution: X = 600 dpi, Y = 600 dpi; Stratasys, Deden Prairie, MN, USA). The photopolymerizable resin VeroBlackPlus (Stratasys, Deden Prairie, MN, USA) was used, and the structure was cured by UV polymerization at 365 nm using a UV lamp. A water-soluble support structure (SUP705; Stratasys, Deden Prairie, MN, USA) was used and removed by washing (potable water; isopropanol) after the printing process.

[0184] The compartment housing the Mo-CORM was fabricated by laser sintering (LS) using polyamide PA2200 (EOS GmbH, Krailing, Germany). Processing was performed using a Formiga P110 3D printer (EOS GmbH, Krailing, Germany) with a CO2 laser (wavelength 10.6 μm, layer thickness 0.1 mm). The silicone membrane (SIK8649) surrounding the two compartments was obtained from RAUMEDIC AG (Helmbreckt, Germany). CAD software (VISI 2019 & 2020, Vero UK, Cheltenham, UK) was used for the production-ready design.

[0185] Preparation of Mo-CORM The synthesis of trisodium tricarbonyl-[tris(isocyanethylacetato)]molybdenum CORM (NaMo(CO)(CNCHCO)), Mo-CORM, was carried out with some modifications according to a previous protocol (Achatz, D et al. Zeitschrift für anorganische und allgemeine Chemie 2005, 631 (12), 2339-2346). Briefly, under dry and inert conditions, 2.64 g (10.0 mmol) of molybdenum hexacarbonyl was dissolved in 35 mL of anhydrous acetonitrile (99.8%; 90 °C; 22 h) to form the intermediate acetonitrile complex. Subsequently, 4.7 mL of ethyl isocyanate (3.5 equiv, 35 mmol, 4.86 g) was added at 55 °C to displace the acetonitrile and form the complex. The ester was then hydrolyzed with 20 mL of NaOH (16 equiv.) in tetrahydrofuran at room temperature. The free acid was formed by adding aqueous hydrochloric acid. Finally, the trisodium salt was obtained by adding NaOH in ethanol (5 mmol NaOH / 1 mmol Mo-CORM). All reagents were purchased from Sigma Aldrich (Schnelldorf, Germany) and used without further purification.

[0186] Preparation of FeCl3 aqueous solution FeCl3·6H2O was purchased from Sigma Aldrich (Schnelldorf, Germany) and prepared in deionized water to a concentration of 583.3 mg / mL to prepare an aqueous solution.

[0187] sperm analysis Semen / sperm volumes of 1.46 mL to 5.7 mL were collected in 100 mL sample vials from healthy men aged 25 to 50 years. The samples were then divided into two equal fractions, each incubated in the presence (wCO) and absence (woCO). To estimate the sperm concentration in the samples and establish an initial baseline for each sample, a routine sperm analysis was performed before transferring the samples to gas-tight 15 mL Falcon tubes. In a sealed container, the samples were incubated at 25–35°C for 90 minutes with and without an activated CORS system. Liquefaction occurred naturally during this incubation period. The samples were then analyzed for total motility, forward motility, resting redox potential, and DNA fragmentation.

[0188] Sample total motility, forward motility, and concentration were assessed using the CEROS II (Hamilton Thorne, Beverly, MA) computer-assisted sperm analysis (CASA) system. Six microliters of sperm sample diluted 1:1 with Multipurpose Handling Medium-Complete (MHM-C, Fujifilm / Irvine Scientific, Santa Ana, CA) were measured to assess concentration and motility (total motility and forward motility).

[0189] Static oxidation-reduction potential (sORP) levels were measured using the Male Infertility Oxidative System (MiOXSYS, Englewood, CO). A 30 μL sperm sample was transferred to the sample application port of the MiOXSYS sensor and analyzed for 2 minutes.

[0190] A count-based microscopy assay was performed to measure DNA fragmentation. The sperm samples were diluted to a maximum of 20xE6 sperm per mL using MHM-C (Multipurpose Handling Medium-Complete, Fujifilm, Irvine Scientific) and analyzed using the Halo Sperm G2 kit (Halotech, Madrid, Spain). Prepared agarose was mixed with the sample according to the manufacturer's instructions. 8 μL of the resulting mixture was placed on a microscope slide, removed from the heat source, and allowed to cool for 5 minutes. The prepared samples were treated with denaturant for 7 minutes, lysis solution for 20 minutes, distilled water for 5 minutes, 70% ethanol for 2 minutes, 100% ethanol for 2 minutes, eosin stain for 7–10 minutes, and thiazine stain for 7–10 minutes according to the manufacturer's instructions. At least 300 sperm were then analyzed using bright-field microscopy.

[0191] result 1. (Static) Oxidation-Reduction Potential (sORP) Level The "relative redox potential" data points shown in Figure 2 are derived for the following ratio per subject: sORP wCO (Subject n) / sORP woCO (Subject n), where sORP woCO (Subject n) is set to 1. The sORP data, which is an indicator of oxidative stress (e.g., due to ROS) in sperm (spermatozoa), tend to be lower in samples exposed to CO (wCO) than in samples not exposed to CO (woCO). This is evident from the location of the data points; four of these values ​​are located below the dashed reference line for woCO = 1. Therefore, without being bound by theory, these results indicate that the levels of reactive oxygen species (ROS) in sperm are reduced compared to sperm exposed to CO and sperm not exposed to CO (Figure 2).

[0192] 2. DNA Fragmentation The "relative DNA fragmentation" data points shown in Figure 3 are the ratios per subject: DNA fragmentation wCOSubject n / DNA fragmentation woCOSubject n, where DNA fragmentation woCOSubject n is set to 1. As is clear from Figure 3, samples exposed to CO have reduced DNA fragmentation compared to samples not exposed to CO. This is evident from the location of the data points; all of these data points are located below the dashed reference line for woCO=1.

[0193] 3. Sperm motility (total motility and forward motility) The "relative total motility rate" or "relative forward motility rate" data points shown in Figure 4A and B, respectively, are the following percentages per subject: a) Relative value of total motility rate: total motility wCOSubject n / total motility woCOSubject n b) Relative value of progressive motility rate: progressive motility wCOSubject n / progressive motility woCOSubject n These are values ​​derived by setting Subject n to 1. No difference in total motility rate was observed between samples exposed to CO (wCO) and those not exposed to CO (woCO) (most data points are located close to the dashed woCO = 1 reference line; see Figure 4A), but progressive motility rate was increased in wCO compared to woCO (as evidenced by all data points, except for Subject 6, located on or above the dashed woCO = 1 reference line; see Figure 4B).

[0194] In this context, total motility refers to the percentage of sperm that exhibit some movement, including non-progressive movement. Progressive motility refers to sperm that move in a roughly straight line or a large circle. Therefore, the progressive motility rate can be an important parameter for predicting the success of an ART cycle.

[0195] Therefore, compared with sperm that had not been incubated in the presence of CO, the rate of forward motility was increased in sperm that had been incubated in the presence of CO.

[0196] In summary, these results demonstrate that contacting sperm with CO2 results in gametes with desirable / improved characteristics: reduced oxidative stress / oxidation-reduction potential / reactive oxygen levels (Figure 2), DNA fragmentation (Figure 3), and improved forward motility (Figure 4B). All measured parameters are again comprehensively depicted in Figure 5. Figure 5 shows the average values ​​of the data points shown in Figures 2–4 for each measured parameter / characteristic. The reference level for samples not exposed to CO2 (woCO2) was again set at 1 (dashed line). As can be seen from Figure 5, contacting sperm with CO2 resulted in (i) a decrease in oxidative stress / reactive oxygen ("ORP"; the average values ​​below the reference dashed line indicate that the average value of wCO2 is less than the average value of woCO2), (ii) a decrease in DNA fragmentation (the average values ​​below the reference dashed line indicate that the average value of wCO2 is less than the average value of woCO2), and (iii) an improvement in forward motility (the average values ​​above the dashed line indicate that the average value of wCO2 is greater than the average value of woCO2).

[0197] Thus, the present inventors have surprisingly found that contacting gametes, such as sperm, with carbon monoxide (CO) preserves the sperm. Incubating gametes with CO has shown that these sperm exhibit desirable properties (compared to sperm that have not been exposed to CO), making them particularly useful for ART.

[0198] Example 3 Example 3 demonstrates the improvement of sperm viability parameters (e.g., reductions in DNA fragmentation and redox potential) in response to different CO / Mo-CORM regimens (carbon monoxide incubation time and CO / Mo-CORM challenge). Furthermore, this example demonstrates the maintenance of sperm motility (e.g., total motility, forward motility, and mean path velocity (VAP)) in response to different CO / Mo-CORM regimens (CO incubation time and CO / Mo-CORM challenge). Sperm from the same subjects were treated with CO gas or left untreated as a "non-treated" control. CO treatment was performed for various durations (60, 90, 180, or 270 minutes) and CO / Mo-CORM challenges (0.44 mL, 1.32 mL, or 2.64 mL of CO, i.e., 5 mg, 15 mg, or 30 mg of Mo-CORM). The results of this example are shown in Figures 6-10.

[0199] Figure 6 shows the ratios of the measured values ​​of CO-treated and untreated sperm samples obtained from the same subject. Here, a value of 1 indicates a lack of effect of CO treatment, as indicated by the horizontal dotted line. Values ​​above the dotted line represent an increase in each measured value due to CO treatment compared to the untreated control, while values ​​below the dotted line represent a decrease in each measured value due to CO treatment compared to the untreated control. Figures 7 and 8 show the individual measurements of the data summarized in Figure 6. In Figures 7 and 8, each bar corresponds to the measurement value of an individual, and the adjacent white and black bars correspond to pairs of CO-treated and untreated sperm samples, respectively, obtained from the same subject. That is, each point in Figure 6 corresponds to the ratio of the pair of CO-treated and untreated samples shown in Figures 7 and 8. That is, Figures 7 and 8 clearly support the observations based on Figure 6. Panels D and E of Figures 7 and 8 show the redox potential and DNA fragmentation measurements of sperm samples from individual subjects, respectively. These results clearly show that high variability in both redox potential and DNA fragmentation was observed among untreated sperm samples from different subjects, and that in some subjects, leaving the sperm untreated exposed the sperm to extremely high oxidative stress. Figures 7 and 8 further clearly show that CO treatment reduces redox potential and DNA fragmentation in these subjects. This is particularly true for sperm samples treated for 60 or 90 minutes. Figures 9 and 10 show correlation analyses between the effects of CO treatment (differences between treated and untreated samples) and each measurement of untreated sperm samples. Figures 9 and 10 demonstrate that CO treatment is effective, especially in subjects with high sperm redox potential and / or DNA fragmentation.

[0200] method In this example, sperm samples were treated with one, three, or six CORS capsules, each loaded with 5 mg of Mo-CORM (equivalent to 0.44 mL of CO), for 60 to 270 minutes, or left untreated for the same periods.

[0201] CORS and Mo-CORM were prepared using the materials described below: Mo-CORM was activated using FeCl3 (prepared as described in Example 2).

[0202] material All chemicals required for the synthesis and activation of Mo-CORM (molybdenum hexacarbonyl, ethyl isocyanoacetate, acetonitrile, anhydrous tetrahydrofuran, sodium hydroxide (pa), hydrochloric acid (pa), absolute ethanol, ferric chloride 6H2O, and nitric acid (65%, pa)) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Multipurpose Handling Medium (MHM) for sperm dilution was purchased from Irvine Scientific (Santa Ana, CA). 270 ppm CO2 calibration gas was purchased from Linde AG (Munich, Germany). Polyamide (PA2200) was obtained from EOS GmbH (Kreiling, Germany), and Duroplast photopolymerizable resin MED610 + VeroBlackPlus (RGD875) was obtained from Stratasys Ltd. (Rehovot, Israel). Silicone R 6.65 × 0.4 mm was purchased from RAUMEDIC AG (Helmbrechts, Germany). SF33 2K silicone (mixing ratio 1:1, viscosity before mixing: 7000–8000 cP, hardness after mixing: 33 ShA, density 1.11 g / cc, break point: 4.7 N / mm) was used. 2 ) and SF45 2K silicone (mixing ratio 1:1, viscosity before mixing: 8500 cP (23°C), hardness after mixing: 45 ShA, density: 1.12 g / cc, breaking point: 3.5 ± 0.5 N / mm 2 ) were purchased from Siliconfabrik (Ahrensburg, Germany). All molds were made from polytetrafluoroethylene (Vink, Germany) using a CNC machine type neo (Datron AG, Germany). Loctite SI 5248, Loctite 4902, Loctite HY 4011, and Loctite SF7701 were purchased from Henkel (Düsseldorf, Germany). Sterican(登録番号) Needles 0.9 x 40 mm and Omnican (registration number) U100 insulin needles 0.3 x 8 mm were purchased from B. Braun (Melsung, Germany). All other reagents were purchased from Sigma Aldrich Chemie GmbH and were of at least pharmaceutical grade unless otherwise stated.

[0203] CORS manufacturing All parts were molded using custom-designed Teflon molds. After pouring the material into the mold, a vacuum was applied for five minutes to remove any air bubbles. The material was then poured again to the rim and vacuumed for another five minutes. After this time, a corresponding mold was pressed into the silicone and secured with a clamp. After drying at 40°C for 24 hours, the mold was separated from the mold with compressed air. The container and lid were molded separately. The container was filled with Mo-CORM. For some versions, an adhesive primer was then applied to the lid and top edge of the container. The adhesive was then applied, and both parts were crimped together. A circular motion was used to distribute the adhesive evenly.

[0204] Synthesis of Mo-CORM The CORM trisodium tricarbonyl-[tris(isocyanethylacetate)]molybdenum (Na3Mo(CO)3(CNCH2CO2)3, Mo-CORM) was synthesized as previously described (Reilaender, ACS Biomaterials Science & Engineering, 2022). Briefly, molybdenum hexacarbonyl was stirred in anhydrous acetonitrile, and the acetonitrile was exchanged with three ligands. These ligands were then exchanged with the EICA ligand. Purification and cation exchange afforded Mo-CORM as a white solid.

[0205] Sperm preparation Human sperm were collected from healthy volunteers in 100 mL containers. The glandular fluid was then aliquoted and placed in 15 mL gas-impermeable Falcon tubes. One sample was exposed to one or more activated CORSs. After a set time, both samples were analyzed for motility parameters using Computer-Aided Sperm Analysis (CASA), resting oxidation-reduction potential (sORP) using MiOXSYS, and sperm DNA fragmentation (SDF) using a dispersion test using the Halosperm G2 kit.

[0206] Computer-assisted sperm analysis (CASA) Sample total motility, forward motility, VAP, and concentration were measured using a CEROS II (Hamilton Thorne, Beverly, MA) computer-assisted sperm analysis (CASA) system. Six microliters of sperm sample was diluted 1:1 with multipurpose treatment medium (MHM-C, Fujifilm / Irvine Scientific, Santa Ana, CA) and analyzed with the CASA system to assess motility.

[0207] Static Oxidation-Reduction Potential (sORP) Analysis Static oxidation-reduction potential (sORP) levels were measured using an Oxidative Male Infertility Measurement System (MiOXSYS, Englewood, Colorado) according to the manufacturer's instructions. A 30 μL sample was transferred to the sample application port of the MiOXSYS sensor and analyzed for 2 minutes. Results were expressed in millivolts (mV) and expressed as semen sperm concentration (mV / 10 6 Normalized to sperm / mL.

[0208] DNA fragmentation (SDF) Sperm DNA fragmentation (SDF) was assessed by a dispersion test using the Halosperm G2 kit (Halotech, Madrid, Spain) according to the manufacturer's specifications. Briefly, samples were diluted to a maximum of 20 million cells / mL. Then, the samples were mixed with prepared agarose. 8 μL of the mixture was transferred onto a slide and allowed to cool for 5 minutes. Different reagents were added and discarded after the designated time: denaturant for 7 minutes, lysis solution for 20 minutes, distilled water for 5 minutes, 70% ethanol for 2 minutes, 100% ethanol for 2 minutes, eosin stain for 7–10 minutes, and thiazine stain for 7–10 minutes. A minimum of 400 sperm were analyzed using an Olympus IX73 inverted LED fluorescence microscope.

[0209] result In this example, the effects of carbon monoxide released from a carbon monoxide releasing system (CORS) on sperm viability and motility parameters were investigated using an experimental design approach based on different amounts of CO gas (0.44–2.64 mL) and effective incubation times (60–270 min). Based on the fact that sperm cells require a constant balance of reactive oxygen species (ROS) to maintain motility, a key finding was that exposure to CO gas concentrations ranging from 0.44 to 2.64 mL for extended periods of up to 90 min did not impair sperm motility, forward motility rate, or velocity compared to untreated sperm. Oxidation-reduction potential (sORP) and DNA fragmentation (SDF) were also assessed during the same process. According to WHO guidelines, liquefaction and processing should ideally be performed within the first 60 min. However, this process may take longer than 60 min. Therefore, longer CO treatment times were also considered.

[0210] 1. Sperm motility (total motility, forward motility, average path speed) Sperm samples were exposed to three CORS treatments or a control (15 mg Mo-CORM; 1.32 mL CO) for different exposure times: 60, 90, 180, and 270 minutes (Figures 6, 7, 9, and 10). Motility rates changed 1.13 ± 0.41-fold at 60 minutes, 0.99 ± 0.03-fold at 90 minutes, 1.16 ± 0.37-fold at 180 minutes, and 0.94 ± 0.20-fold at 270 minutes. Progressive motility rates changed 0.85 ± 0.13-fold at 60 minutes, 0.92 ± 0.17-fold at 90 minutes, 0.72 ± 0.19-fold at 180 minutes, and 1.04 ± 0.48-fold at 270 minutes. The sperm cell velocity was also measured using CASA, and the change was 0.94 ± 0.09 fold at 60 min, 0.96 ± 0.04 fold at 90 min, 0.79 ± 0.13 fold at 180 min, and 0.97 ± 0.08 fold at 270 min (Figure 6A). To examine the effect of varying dose, an additional experiment was performed with a 90-min irradiation time. Here, sperm samples were irradiated with 1, 3, 6, or no CORS as a control (5 mg, 15 mg, 30 mg, or 0 mg Mo-CORM; equivalent to 0.44 mL, 1.32 mL, 2.64 mL, or 0 mL CO, respectively; Figures 6 and 8). The maximum CORS that could be maintained in the setup at one time was 6. The change in motility was 0.98 ± 0.12-fold at 0.44 mL and 0.90 ± 0.12-fold at 2.64 mL. The change in forward motility rate was 0.97 ± 0.12-fold at 0.44 mL and 0.88 ± 0.10-fold at 2.64 mL. The change in VAP was 1.01 ± 0.08-fold at 0.44 mL and 1.03 ± 0.03-fold at 2.64 mL (Figure 6B). Figure 7 clearly supports these observations based on Figure 6 .

[0211] 2. Sperm viability parameters (oxidation-reduction potential and DNA fragmentation) The redox potential and DNA fragmentation were evaluated using the same process. The effect of CO on redox potential was 0.57 ± 0.35-fold at 60 minutes, 0.91 ± 0.12-fold at 90 minutes, 1.02 ± 0.32-fold at 180 minutes, and 1.13 ± 0.28-fold at 270 minutes. The changes in DNA fragmentation were 0.77 ± 0.07-fold, 0.67 ± 0.24-fold, 0.90 ± 0.11-fold, and 0.90 ± 0.19-fold at 60, 90, 180, and 270 minutes, respectively (Figure 6C). In the dose-varying sample, the redox potential changed 0.89 ± 0.17-fold at 0.44 mL and 0.59 ± 0.24-fold at 2.64 mL. DNA fragmentation changed 0.83 ± 0.09-fold at 0.44 mL and 1.31 ± 0.55-fold at 2.64 mL (Figure 6D). In summary, in line with the WHO scheme, after 60 min, the redox potential was observed to change approximately 0.57 ± 0.35-fold (1.32 mL). Furthermore, DNA fragmentation changed approximately 0.77 ± 0.07-fold (1.32 mL). At a longer time point of 90 min, the change in redox potential was 0.91 ± 0.12-fold (1.32 mL). DNA fragmentation changed 0.67 ± 0.24-fold (1.32 mL).

[0212] In summary, the CO2 / Mo-CORM regimens tested in this study (CO2 incubation time and CO2 / Mo-CORM challenge) were demonstrated to improve sperm viability parameters. Specifically, redox potential and sperm DNA fragmentation (SDF) decreased when the CO2 incubation time was 60 and 90 minutes and the CO2 dose was 0.44 mL and 1.32 mL. This confirms the results shown in Figures 2, 3, and 5. Therefore, the regimens of incubation with 0.44 mL or 1.32 mL of CO2 for 60 or 90 minutes appear to be favorable for sperm viability parameters.

[0213] Overall, incubation with 0.44 mL or 1.32 mL of CO (equivalent to 5 mg or 15 mg of Mo-CORM and 1 or 3 CORS, respectively) for 60 or 90 minutes does not adversely affect sperm motility, but is considered to be a suitable method for reducing sperm DNA fragmentation and redox potential. The results shown in Figures 6 and 7 clearly demonstrate that incubation with 0.44 mL or 1.32 mL of CO (equivalent to 5 mg or 15 mg of Mo-CORM and 1 or 3 CORS, respectively) for 60 or 90 minutes (the preferred regimen) can improve sperm viability parameters without adversely affecting sperm motility.

[0214] Furthermore, the 60-minute incubation time perfectly fits into the routine laboratory work of IVF laboratories in accordance with WHO guidelines (WHO laboratory manual for the examination and processing of human semen, World Health Organization, 2021). This treatment is likely to improve the success rate of artificial reproductive technology (ART) applications (e.g., in vitro fertilization: IVF), which are limited by sperm DNA fragmentation and high redox potential.

[0215] Thus, the present inventors have surprisingly discovered that contacting gametes such as sperm with carbon monoxide (CO) contributes to the preservation of sperm, particularly when the sperm are contacted with 0.44 mL or 1.32 mL of CO for 60 or 90 minutes. As a result, sperm contacted with 0.44 mL or 1.32 mL of CO for 60 or 90 minutes, among others, have been shown to exhibit desirable properties (compared to sperm not contacted with CO), making them particularly useful for ART.

[0216] 3. Effect of CO treatment on sperm viability in subjects with reduced sperm viability Panels D and E of Figures 7 and 8 show the redox potential and DNA fragmentation measurements of untreated and CO-treated sperm samples collected from individual subjects. High variability in both redox potential and DNA fragmentation was observed between untreated sperm samples from different individuals, clearly indicating that sperm from some test subjects were exposed to extremely high oxidative stress when untreated. Here, untreated sperm samples 42_1 and 38_7 in particular exhibited significantly elevated redox potential and DNA fragmentation levels compared to the majority of samples. These samples are believed to have been collected from subjects suffering from infertility or below-average fertility. CO treatment significantly reduced the redox potential and DNA fragmentation values ​​of samples 42_1 and 38_7. This clearly indicates that CO treatment may be effective for subjects with sperm with significantly reduced viability parameters. Therefore, CO treatment may potentially be used to treat / prevent (gamete) diseases characterized by elevated redox potential and / or DNA fragmentation in gametes. This includes, among other things, the treatment or reduction of male infertility, and potentially the prevention of congenital anomalies and / or aneuploidy in offspring derived from CO-treated gametes.

[0217] Figures 9 and 10 further demonstrate the effectiveness of CO treatment on both redox potential and DNA fragmentation. These figures show correlation analyses between the effect of CO treatment (difference between treated and untreated samples; y-axis) and each measurement of untreated sperm samples (x-axis). Both figures focus on 60- and 90-minute incubations, regimens that demonstrated significant effectiveness of CO treatment (Figure 7). Figures 9 and 10 demonstrate that CO treatment is particularly effective in subjects with high sperm redox potential and / or DNA fragmentation.

Claims

1. 1. A method for preserving gametes, comprising: a) providing / obtaining gametes or a gamete-containing sample in a container; and b) contacting the gametes or the gamete-containing sample in the container with carbon monoxide A method comprising:

2. 2. The method of claim 1, wherein the gametes or the gamete-containing sample comprises sperm or egg cells, preferably the sperm being present in semen, glandular fluid, and / or a buffer / buffer system.

3. 3. The method of claim 1 or 2, wherein carbon monoxide is released from a carbon monoxide-releasing molecule and / or carbon monoxide is supplied directly to the vessel through a separate pipe.

4. 4. The method according to claim 1, wherein carbon monoxide forms a gas layer above the gametes or the gamete-containing sample.

5. The carbon monoxide-releasing molecule is preferably a metal carbonyl compound, more preferably a molybdenum carbonyl compound, and most preferably a Na ３ Mo(CO) ３ (CNCH ２ CO ２ H) ３ The method according to claim 3 or 4, wherein

6. The carbon monoxide releasing molecule is FeCl ３ , Ce(SO ４ ) ２ , or H ２ O ２ and preferably FeCl ３ The method according to any one of claims 3 to 5, wherein carbon monoxide is released by contacting the catalyst with the catalyst.

7. 7. The method according to any one of claims 1 to 6, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide for a period of about 15 to about 120 minutes, preferably about 30 to about 90 minutes, more preferably about 30 to about 60 minutes.

8. 8. The method of claim 1, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide at about 25 to about 35°C.

9. 9. The method according to any one of claims 1 to 8, wherein the amount of carbon monoxide applied to the gametes or the gamete-containing sample is in the range of from about 20 μmol to about 500 μmol, from about 40 μmol to about 400 μmol, preferably from about 45 μmol to about 270 μmol, preferably from about 60 μmol to about 180 μmol.

10. The method according to any one of claims 1 to 9, wherein the gametes are human gametes.

11. 10. The method according to any one of claims 1 to 9, wherein the gametes are from a cow, a horse, a pig, a sheep, a goat, a camel, an alpaca, a dog, a cat, a bird, or a fish.

12. 12. The method according to any one of claims 1 to 11, wherein the preservation state of the gametes is assessed by measuring the motility, DNA fragmentation, spermiogram, and / or redox potential of the gametes.

13. 1. A method for reducing DNA fragmentation and / or redox potential of gametes in artificial reproductive techniques, the method comprising contacting said gametes or a sample containing said gametes with carbon monoxide.

14. 1. Use of carbon monoxide to reduce DNA fragmentation and / or redox potential of gametes in assisted reproductive techniques, comprising contacting said gametes or a sample containing said gametes with carbon monoxide.

15. 15. The method of claim 13 or the use of claim 14, wherein the assisted reproductive techniques comprise in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, frozen embryo transfer, preimplantation genetic testing, in vitro maturation of oocytes, frozen oocyte transfer, gamete intrafallopian tube transfer, zygote intrafallopian tube transfer, and / or cryopreservation.

16. 16. The method of claim 13 or 15 or the use of claim 14 or 15, wherein the motility of gametes is maintained / enhanced by reducing DNA fragmentation and / or redox potential of said gametes.

17. 17. The method of claim 13, 15 or 16, or the use of claim 14, 15 or 16, wherein the risk of congenital abnormalities and / or aneuploidy in artificial reproductive technologies is reduced by reducing DNA fragmentation and / or redox potential of gametes.

18. Carbon monoxide for use in preventing birth defects and / or aneuploidy.

19. Carbon monoxide for use in treating / improving male infertility.

20. Carbon monoxide for use in treating / ameliorating diseases caused by / associated with DNA fragmentation and / or elevated redox potential of gametes.

21. Carbon monoxide for use in treating / ameliorating gamete disorders caused by / associated with elevated ROS levels.

22. Carbon monoxide for use according to claim 20 or 21, wherein the disease is caused by / associated with elevated stress levels or the subject to be treated has stress.

23. Carbon monoxide for use according to claim 22, wherein the disease is caused by / associated with elevated levels of psychological and / or physiological stress or the subject to be treated has psychological and / or physiological stress.

24. Carbon monoxide for the use according to any one of claims 18 to 23, wherein the prevention of congenital abnormalities and / or aneuploidy, the treatment / amelioration of male infertility, or the treatment / prevention of said diseases is carried out by contacting gametes with carbon monoxide.

25. Carbon monoxide for use according to claim 24, wherein the gametes are contacted with carbon monoxide in vitro / ex corpore.

26. 28. Carbon monoxide for the method according to any one of claims 13 and 15 to 17, the use according to any one of claims 14 to 17 or the application according to claim 25, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide in less than 10 minutes, preferably less than 5 minutes, more preferably less than 3 minutes, more preferably less than 2 minutes, more preferably less than 1 minute after the gametes have been collected in the container.

27. 25. Carbon monoxide for the use according to claim 24, wherein the gametes or the gamete-containing sample are contacted with carbon monoxide in vivo.

28. 28. Carbon monoxide for use according to claim 27, wherein the carbon monoxide is administered to the gametes systemically or transdermally.

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