Method for preserving functional gametes

Abstract

The invention relates to the use of carbon monoxide for preserving gametes. Means and methods for preserving gametes are provided. The method of the invention comprises the steps of a) providing a gamete or a sample comprising said gamete in a container, and b) contacting said gamete in said container with carbon monoxide. The invention also provides the use of carbon monoxide in the treatment and / or prevention of diseases, such as diseases caused by or associated with elevated DNA cleavage, redox potential of gametes, and / or diseases of gametes caused by or associated with elevated ROS levels. Also provided are uses of carbon monoxide for the prevention of congenital abnormalities and / or aneuploidy, as well as for the treatment of diseases caused by and / or associated with elevated DNA cleavage and / or redox potential of gametes. The invention also relates to a method of treating congenital abnormalities or aneuploidy comprising contacting carbon monoxide gas with gametes of a patient in need thereof. Thus, the means, methods and uses of the present invention ensure that gametes are preserved in a functional / intact state, thereby ensuring maintained and / or improved gamete quality. Thus, such carbon monoxide treated / carbon monoxide exposed gametes are particularly useful in reproductive techniques, such as in vitro reproductive techniques, including in human assisted reproductive techniques and / or in animal (artificial) insemination techniques.

Description

[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 sample containing said gametes in a container, and b) contacting said gametes in said 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 increased DNA fragmentation, the redox potential of gametes, and / or diseases of gametes caused by or associated with increased ROS levels. Also provided are carbon monoxide for preventing congenital abnormalities and / or aneuploidy, and the use of carbon monoxide for treating diseases caused by and / or associated with increased DNA fragmentation and / or redox potential of gametes. The present invention also relates to a method for treating congenital abnormalities or aneuploidy, the method comprising contacting carbon monoxide gas with gametes of a patient in need thereof. Thus, the means, methods and uses of the present invention ensure that gametes are preserved in a functional / intact state, thereby guaranteeing maintained and / or improved gamete quality. Thus, such carbon monoxide-treated / carbon monoxide-exposed gametes are particularly useful in reproductive technologies, such as in vitro reproductive technologies, including assisted reproductive technologies in humans and / or in (artificial) insemination technologies in animals. The present invention relates to ex corporeal / in vitro uses, but also to in vivo uses in a medical context. In particular, desired (artificial) reproductive technologies are also described and provided herein.

[0002] Infertility is one of the most common individual health problems in the world. According to the WHO assessment, about 15% of couples of childbearing age are affected by infertility (Rutstein, "Infertility and Childlessness in Developing Countries". DHS Comparative Report No. 9. Calverton, Maryland, USA. Orc Macro and World Health Organization, 2004). These cases include, in particular, female or male infertility, as well as infertility cases including both male and female infertility. About 50% of all cases are related to male infertility. Idiopathic infertility is related to about 40% of the total number of male infertility cases (McLachlan, Med J Aust 174 (2001): 116-117). 30-85% of these cases are related to oxidative stress caused by the formation of reactive oxygen species (ROS), which leads to functionally impaired / fewer male gametes / sperms (e.g., by DNA breakage, lipid peroxidation and apoptosis) (Mannucci, Front Mol Biosci 8 (2021): 799294). Notably, male infertility rates are increasing, and sperm quality in healthy men is declining, as reflected by decreasing 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). Data from the International Committee for Monitoring Assisted Reproductive Technologies (ICMAT) indicate that an average of 3.2 million ART cycles are performed annually worldwide, including 1.6 million IVF cycles. However, despite this high level of ART, the birth rate is only 22.5%, highlighting the need to improve access to high-quality fertility treatment for those in need. This also places a significant burden on couples and, due to the high cost of ART procedures, on healthcare systems worldwide. Cost-sensitive and / or accessible approaches and methods are also needed in reproductive technologies, particularly ART.

[0003] The success rate of assisted reproductive technology (ART) is negatively affected by ROS levels / damage / non-functional gametes and leads to the failure of many ART cycles (Zorn, Int J Androl 26 (2003): 279-285). ROS levels in individuals, such as those in susceptible people who are gamete donors used in ART, may increase due to (physiological and / or psychological) stress, genetic predisposition, environmental factors (including exposure to electromagnetic waves) and / or behavioral risk factors (such as diet, smoking, drinking and / or drug abuse; see especially Agarwal, The Lancet, 2020; Agarwal, World J Mens Health, 2020; Esteves, Andrology, 2019). Further undesirable increases in ROS levels in individuals are known to those skilled in the art.

[0004] Another important health uncertainty may be the potential negative genetic changes in newborns caused by genetic changes related to ROS in gametes, especially sperm. For example, in natural fertilization, sperm with DNA damage may not fertilize the egg. In ART, the selection process is bypassed, which may lead to the unintended use of sperm with DNA damage (Zini, Canadian Medical Association Journal, 175 (5): 495, 2006). In this case, ROS and the resulting oxidative stress of gametes (especially sperm (sperm / spermatozoa)) pose a great burden on the success of reproductive technologies such as ART. In other words, it is necessary to avoid physiological stress on the gametes used in such reproductive technologies. Similarly, one of the corresponding major physiological obstacles is the exposure of gametes to oxidative stress inside and outside the body.

[0005] Antioxidants administered orally to subjects have previously been proposed to reduce ROS and improve sperm quality. A 2011 Cochrane review described studies related to these drugs and concluded that administration of these antioxidants may lead to an increase in birth rates after ART (Showell, Cochrane Database Syst Rev 1 (2011): CD007411).

[0006] However, there are also disadvantages associated with the systemic administration of antioxidants such as vitamin C, vitamin A, vitamin B complex, coenzyme Q10, etc. (Sabeti, Int J Reprod Biomed 14 (2016): 231-240). First, these antioxidants need to be administered regularly for a period of time (up to several months). In addition, these drugs can also cause side effects and are also cost-intensive for patients and payers.

[0007] Therefore, the technical problem underlying the present invention is to provide functional gametes having desirable characteristics for use in particular in reproductive technologies, such as assisted reproductive technologies.

[0008] The technical problem is solved by the embodiments and items presented herein and specifically provided in the appended claims.

[0009] Therefore, the present invention relates to means and methods for preserving gametes, comprising the following steps

[0010] a) providing / obtaining gametes or a sample comprising said gametes in a container, and

[0011] b) contacting the gametes in the container or the sample comprising the gametes with carbon monoxide.

[0012] The gametes may be brought into contact with / exposed to / treated with carbon monoxide (particularly carbon monoxide gas) for a sufficient period of time to preserve the gametes. The preservation of the gametes particularly relates to preserving the gametes in a functional / intact state. According to the invention, the gametes are brought into contact with / exposed to and / or treated with carbon monoxide (particularly carbon monoxide gas), thereby ensuring and / or supporting, maintaining and / or improving the quality of the gametes. This is also explained below and in particular illustrated in the accompanying non-limiting examples and figures.

[0013] It will be appreciated that carbon monoxide (particularly carbon monoxide gas) may be applied directly to the gametes, for example contained in, for example, a buffer solution and / or a buffer system. However, and as further described herein, carbon monoxide (particularly carbon monoxide gas) may also be in contact with the gametes contained in a sample containing the gametes. The sample may be a biological sample, such as, but not limited to, sperm cells / sperm fluid or ejaculated seminal fluid. It will be appreciated that the gametes contacted with / exposed to / treated with carbon monoxide / carbon monoxide gas may also be contained in a buffer solution and / or a buffer system. The sample containing the gametes may also be a diluted sample. For example, it is envisaged that for certain applications, the seminal fluid or ejaculated seminal fluid is further diluted in or with a buffer solution or a buffer system before and during exposure to carbon monoxide / carbon monoxide gas. Therefore, it will be appreciated that the term "gametes" also includes samples containing the gametes, such as biological samples. The terms "contacting with", "exposing to" and / or "treating with" are used interchangeably in the context of the present invention.

[0014] Thus, the present invention relates in one embodiment to means and methods for preserving gametes, wherein in one embodiment said method may comprise the following steps

[0015] a) providing gametes in a container, and

[0016] b) contacting the gametes in the container with carbon monoxide gas for a period of time sufficient to ensure preservation of the gametes.

[0017] The gametes contacted with / exposed to / treated with carbon monoxide / carbon monoxide gas include spermatids / spermatozoa or egg cells, preferably spermatids / spermatozoa. The gametes are animal gametes, preferably, but not limited to, gametes from mammals (including humans). When contacted with / exposed to / treated with carbon monoxide / carbon monoxide gas, the spermatozoa to be contacted with / exposed to / treated with carbon monoxide / carbon monoxide gas may be contained in semen, ejaculated semen, and / or a buffer / buffer system.

[0018] Carbon monoxide (CO) is an endogenous messenger molecule produced continuously in the human body. When the human physiological system is exposed to stress factors, the production of CO is increased to trigger various defense mechanisms. As an exogenous supplement of drugs, CO has been shown to have therapeutic potential (Motterlini, Nat Rev Drug Discov 9 (2010): 728-743), which has been confirmed in many preclinical studies. However, due to the lack of a safe and effective system to apply CO, it is still very lacking in conventional human use (Hopper, Curr Pharm Des 24 (2018): 2264-2282).

[0019] US 9,980,981 B2 and WO 2012 / 096912 A1 disclose compositions containing CO that can be used to treat inflammatory diseases and neurodegenerative diseases, but not for the preservation of gametes and their subsequent use in fertility medicine / ART. In addition, WO 2022 / 055991 A1 discloses CO-releasing formulations ("gas-entrapped compositions") that can be used to treat inflammatory diseases.

[0020] In the context of the present invention, the inventors surprisingly found that carbon monoxide (CO), in particular carbon monoxide (CO) in the form of carbon monoxide (CO) gas, can be successfully used for the preservation of gametes. It was found, described and explained herein that contacting gametes with carbon monoxide / carbon monoxide gas (also including exposing gametes to carbon monoxide / carbon monoxide gas or treating gametes with carbon monoxide / carbon monoxide gas) ensures the preservation of said gametes, whereby said preservation means "maintaining" in a (desired) functional / intact state. Thus, it was found and described herein that carbon monoxide (CO), in particular carbon monoxide (CO) in the form of carbon monoxide (CO) gas, can ensure / maintain and / or even improve gamete quality. For example, in particular, the inventors found that, for example, contacting sperm with carbon monoxide (gas) leads to (i) reduced ROS levels / reduced oxidative stress in sperm (see, e.g., the accompanying Figure 2 and 5to 9), and thus (ii) reduced DNA fragmentation in gametes, especially sperm (see e.g., Appendix Figure 3 、 5 8 and 10). In addition, the accompanying examples and figures also state that (iii) an improvement in the forward movement of male gametes / spermatozoa was observed compared to the control sample (not exposed to carbon monoxide) (see, for example, the accompanying Figure 4B and 5). None of these surprising technical effects have been disclosed or predicted in the art. Therefore, the inventors have found that contacting gametes such as sperm with carbon monoxide results in the preservation of gametes with ideal properties. The ideal characteristics of the present invention can be measurable characteristics, such as, in particular, reduced ROS levels / oxidative stress / redox potential, reduced DNA fragmentation levels, improved forward movement and / or maintenance of desired movement (in particular in the form of mean path velocity (VAP) of sperm). The corresponding improvements are described in the accompanying examples and illustrated therein, in particular in comparison with a control sample of gametes that were not incubated / contacted with CO. The preserved gametes exhibiting one or more of the above-mentioned ideal characteristics can then be used, for example, in assisted reproductive technology applications. Therefore, the means, methods and uses of the present invention also avoid the need for continuous systemic administration of (potentially harmful) antioxidants to subjects / patients participating in such applications. In addition, due to their gamete preservation effect, the means, methods and uses of the present invention can also be used to preserve / store gametes during extended (gamete) operation times in ART applications. The means, methods and uses provided herein are not only relevant to excorporeal or in vitro uses, but also to in vivo methods and in vivo uses of carbon monoxide. For example, as described herein, carbon monoxide can also be used to improve the quality of gametes in vivo. In particular, it is envisaged that male individuals may be treated with CO to avoid, for example, DNA breakage in gametes due to physiologically damaging or potentially damaging oxidative stress. This can be achieved by administering carbon monoxide to individuals in need of such treatment via a patch that releases CO. This embodiment of the invention is further described below and relates in particular to the inventive use of carbon monoxide in the treatment and / or prevention of diseases that are the cause of undesirable (genetic) disorders of developing embryos and / or fetuses. For example, the present invention also provides the medical use of carbon monoxide for preventing congenital anomalies and / or aneuploidy. The present invention also provides, for example, the medical use of carbon monoxide in treating and / or reducing male infertility. For example, the present invention also provides the medical use of carbon monoxide in treating and / or reducing male infertility. For example, the present invention also provides the medical use of carbon monoxide in treating and / or preventing diseases caused by / related to DNA breakage and / or redox potential of gametes that are increased. The present invention also provides, for example, the medical use of carbon monoxide in the treatment and / or prevention of gametic diseases caused by / associated with elevated ROS levels. Furthermore, this embodiment is discussed further herein below. The main point of the present invention is to avoid ROS-mediated physiological damage.

[0021] The present invention, in its broadest embodiment, relates to the use of carbon monoxide to preserve gametes. Gametes can be contacted with, exposed to, or treated with the carbon monoxide in vitro, ex vivo, and / or ex vivo. However, as described below, in vivo uses, such as for medical interventions, are also described and disclosed herein and are part of the present invention.

[0022] In the means, methods and uses of the present invention, the term "preservation" refers to a method, activity or process of keeping gametes functional, alive, intact and / or protected from damage and / or decay using carbon monoxide / carbon monoxide gas. This refers in particular to the time after the gametes have been collected from a subject, whereby the subject may be a healthy or diseased subject. The subject is preferably a mammal, most preferably a human. However, the means and methods and uses of the present invention are also readily applicable to gametes, in particular sperm, of other animals such as birds, fish and reptiles. The means and methods of the present invention, i.e. contacting the gametes with carbon monoxide, can ensure that the gametes are preserved in a functional / intact state. An object of the present invention is also illustrated in the accompanying examples, in which it is shown that exposure of gametes, in particular sperm, to carbon monoxide supports maintained and / or improved gamete quality. The preserved gametes can be successfully used, for example, in reproductive medicine, in particular assisted reproductive technology (ART) applications, but also in techniques like, for example, artificial insemination of farm animals. Therefore, the terms "gamete preservation" and "preservation of gamete quality" should be understood to mean keeping the gametes functional, alive, intact and / or protected from damage and / or decay for a given time compared to a reference time point. The reference time point can be defined by the time point at which gametes are collected from a (healthy or sick) subject (e.g., a male or female subject). However, the term "preservation" can also be understood in the context of temporary ex vivo storage of gametes during an extended (gamete) manipulation time. This is particularly true in the case of ART applications. The term "ex vivo" is known to those skilled in the art and, in the context of the present invention, specifically refers to exposing the gametes retained in the context of the present invention to carbon monoxide (CO) in an environment outside the body. In other words, gametes, in particular sperm, are exposed to carbon monoxide outside the individual's body, for example after ejaculation into a corresponding (collection) container. Therefore, the individual is preferably an individual capable of producing corresponding gametes / spermatozoa (i.e., sperm). For example, ejaculation into a corresponding container is associated with (artificial) reproductive technologies such as in vitro fertilization. It should be understood that direct ejaculation into a container is not a prerequisite for the means, methods and purposes of the present invention. It is also envisioned that the present invention is used for any seminal fluid, semen, ejaculated semen, sperm samples, separated and further purified sperm or spermatozoa, and is also used for corresponding dilution and / or further processing, processing, operation, liquefaction, pre-freezing, thawing or thawed biological samples comprising gametes, preferably sperm to be processed. The present invention is also useful and easy to apply in the process of operating any biological sample comprising gametes (preferably sperm to be preserved in the context of the present invention). Therefore, it is also envisioned that during any manipulation or operation of the biological sample, any biological sample comprising gametes is exposed to carbon monoxide / carbon monoxide gas.In the context of the present invention, the term "ex vivo" can be used interchangeably with "ex corpore". The corresponding ex vivo and ex corpore techniques and methods can be considered as "invitro" techniques / methods. Thus, in one embodiment, ex vivo treatment can refer to treating gametes provided in a container with carbon monoxide, or treating gametes that are already in a container and then exposed to carbon monoxide. However, in another embodiment of the invention described herein, the inventive concept of exposing gametes to carbon monoxide in order to advantageously preserve the gametes can also be used in the human or animal body. Therefore, the present invention also relates to the medical use of carbon monoxide in methods of treatment or prevention of disease, wherein these methods include exposing gametes in the human or animal body. Therefore, these medical uses also include the in vivo use of carbon monoxide. The corresponding exposure of the gametes to carbon monoxide can be obtained, for example but not limited to, by means of a (medical) patch, as also described below; see also WO 2021 / 180908A1 and Ruopp et al. (2023, Journal of Controlled Release) for the provision of corresponding medical patches that release carbon monoxide. In a specific aspect, in vivo treatment / exposure also refers to the exposure of gametes (in particular sperm) to carbon monoxide, whereby the gametes are still present in the gonads of an organism, preferably a eukaryotic organism, more preferably a (male) mammal, most preferably a human (man).

[0023] In the context of the present invention, the term "functionality, survival, integrity and / or protection from damage and / or decay" relates to one or more characteristics of gametes that may be negatively affected, for example, by reactive oxygen species (ROS). These properties may be preserved by the means, methods and uses of the present invention and may include, in particular, the "desirable / ideal properties" as described above. For example, as shown in the accompanying Examples 2 and 3 and Figure 2As shown in Examples 2 to 7, non-limiting (desirable) characteristics of gametes may include (reduced) redox potential / ROS levels, (reduced) DNA fragmentation, (increased) motility (in the case of sperm / spermatozoa), in particular (increased) forward motility, (reduced) lipid peroxidation, (reduced) apoptosis and (reduced) sperm degeneration. Those skilled in the art are aware of the means and methods for determining these (ideal) characteristics from gametes. However, exemplary methods and commercial kits for determining specific (static) redox potential / ROS levels, DNA fragmentation, and total and forward motility of sperm are also described in detail in Examples 2 and 3. Example 3 further describes the measurement of the mean path velocity (VAP) of sperm. VAP is defined as the speed of sperm movement, measured in microns per second (μm / s), and together with total and forward motion, is an important parameter that describes the overall movement of sperm. According to the present invention, other desired characteristics of gametes may be (maintained / increased) VAP.

[0024] For example, the Male Infertility Oxidation System (MiOXSYS, Englewood, CO) can be used to measure the (static) redox potential / ROS levels of sperm, 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, particularly total and progressive motility and VAP.

[0025] Thus, (CO-) preserved (functional, viable or intact) gametes according to the present invention may be gametes that exhibit desirable characteristics. For example, compared to non-preserved gametes (i.e., gametes that have not been exposed to CO), gametes that have been exposed to CO may, inter alia, exhibit (i) a lower redox potential, (ii) less DNA fragmentation / DNA damage, and (iii) higher or unchanged / maintained forward motility. For example, in the presence of Figures 2 to 10 A graphical representation of this (ideal) characteristic is also shown in Figure 2. Gametes preserved in CO-can be gametes comprising a haploid chromosome set, which have significantly fewer or no chromosomal abnormalities compared to unpreserved gametes. Chromosomal abnormalities can be particularly selected from quantitative abnormalities (e.g., aneuploidy), structural abnormalities (e.g., deletions, duplications, inversions, insertions, translocations, rings, isochromosomes), and / or acquired chromosomal abnormalities. According to the present invention, gametes can be healthy (no chromosomal abnormalities) or diseased gametes (chromosomal abnormalities).

[0026] In the context of the present invention, the above-mentioned term "successful use" refers to the use of the preserved gametes in reproductive medicine, in particular in ART applications or (artificial) insemination processes. The use of these preserved gametes obtained by the means, methods and uses of the present invention in these applications is specifically associated with a higher probability of a positive (i.e., desired) outcome of the application. For example, CO-preserved functional / viable / intact haploid gametes (as opposed to unpreserved gametes) can lead to a healthy diploid zygote with an increased probability (corresponding to the desired outcome) of forming during in vitro fertilization (IVF), which can lead to normal blastocyst formation and the establishment of a normal pregnancy (i.e., a pregnancy without complications that leads to the birth of a healthy newborn with a significantly reduced risk of suffering from congenital abnormalities or aneuploidy).

[0027] A "zygote" in the context of the present invention is a eukaryotic cell formed by the fertilization event between two gametes. The genome of the zygote is the combination of the DNA in each gamete and contains all the genetic information for the new individual organism. In multicellular organisms, the zygote is the earliest stage of development.

[0028] During human fertilization, the egg (haploid secondary oocyte / egg cell with duplicate chromosome copy; Female gamete) and haploid sperm cell (male gamete) of release combine to form a single diploid cell called zygote. Once a single sperm fuses with the egg cell, the latter completes the segmentation of the second meiotic division, forming a haploid daughter generation with only 23 chromosomes, almost all cytoplasm and male pronucleus. Another product of meiosis is a second polar body that only has chromosomes but can not replicate or survive. In the fertilized daughter cell, DNA is replicated in two independent pronuclei from sperm and egg, making the chromosome number of the zygote temporarily 4n diploid. About 30 hours after fertilization, pronuclear fusion and immediate mitosis produce two diploid daughter cells called blastomeres, which produce the blastocyst that eventually forms an embryo.

[0029] The term "healthy" refers to a diploid zygote and is to be construed in contrast to a diseased / abnormal zygote.

[0030] Sick / abnormal zygote may, for example, contain damaged / fractured DNA, or may not be diploid, and this is due to the result of sperm and egg cell fusion, and at least one may be (non-) euploid.Sick / abnormal zygote may not produce blastomere / blastocyst formation and embryo formation, or it may produce blastomere / blastocyst formation and embryo establishment, but compared with healthy zygote, it may cause miscarriage or suffer from the possibility of the newborn born of congenital anomaly or aneuploidy (or euploidy) higher.The non-limiting examples of congenital anomaly or aneuploidy are further described hereinafter.

[0031] Therefore, a "healthy" zygote is diploid (i.e., includes a complete set of paternal and maternal chromosomes) and can result in normal blastomere / blastocyst formation / embryo formation. Therefore, a healthy zygote has a higher probability of resulting in a newborn without congenital abnormalities or aneuploidy (or euploidy) than a diseased / abnormal zygote.

[0032] For example, according to the means, methods and purposes of the present invention, gamete can be healthy (not comprising one or more chromosomal abnormalities) or sick gamete (comprising one or more chromosomal abnormalities). Gamete defines a haploid cell, and the haploid cell is fused with another haploid cell during fertilization in the organism of sexual reproduction. Gamete is the reproductive cell of an organism, also commonly referred to as sex cell. In the species that produces two morphologically different types of gametes (for example, in the case of mammals such as people), and wherein each individual / sex only produces one type of gamete, female is any individual that produces larger type gametes (referred to as oocytes / ovum), and male produces smaller type gametes (referred to as spermatids / sperm). Therefore, in the context of the means, methods and purposes of the present invention, gamete comprises spermatids / spermatids and / or ovum / ovum, preferably spermatids / spermatids and / or ovum / ovum collected in vitro from mammalian subjects such as male or female subjects. The mammalian subject, such as male or female human subjects, can be healthy or can suffer from / be susceptible to disease, such as infertility or chromosomal abnormality. In the context of the present invention, preferably spermatozoa may be contained in semen, ejaculated semen and / or a buffer / buffer system that allows sperm cells to survive. Thus, gametes contacted with carbon monoxide may also be contained in a biological sample or a buffer / buffer system.

[0033] Those skilled in the art will appreciate exemplary buffers / buffer systems, particularly for, but not limited to, dilution, storage, washing, manipulation, processing, buffering, and purification of gametes, particularly sperm. Non-limiting examples of these buffers / buffer systems include, for example, sperm dilution buffer, sperm maintenance buffer, and sperm washing buffer. Such buffers / buffer systems may include, in particular, water / aqueous solutions, salts / ions, buffering agents / buffer solutions, amino acids (in particular, glycine and / or taurine), energy substrates, and antibiotics (in particular, gentamicin sulfate). Non-limiting examples of buffering agents / buffer solutions may include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium bicarbonate, MOPS (3-(N-morpholinyl)propanesulfonic acid), and phosphate buffered saline (PBS). Non-limiting examples of salts / ions may include potassium phosphate, sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate. Non-limiting examples of energy substrates may include sodium lactate, sodium pyruvate, and glucose. Those skilled in the art of artificial reproductive technology or related work know how to design / generate suitable buffers / buffer systems, which may include, in particular, the exemplary components / ingredients described herein. Alternatively, comparable / functionally equivalent buffers / buffer systems, e.g., Gamete and Embryo Processing Fluid (with Albumin) (FUJIFILM) and Wash (Nidacon) is readily available to those skilled in the art.

[0034] Sperm / spermatids and egg cells / oocytes are the male and female germ cells ("gametes"), respectively, in the heterogametic form of sexual reproduction in mammals.

[0035] Spermatids are formed in a process called spermatogenesis, which occurs in the seminiferous tubules of the testicles in mammals. This process involves the production of several successive spermatid precursors, starting from spermatogonia, which differentiate into spermatocytes. Spermatocytes then undergo meiosis, which reduces their chromosome number by half, producing spermatids. Spermatids then mature and, in mammals, develop tails or flagella, resulting in mature, motile spermatids.

[0036] In some embodiments, the present invention relates to spermatids and spermatozoa.Forward motion is the movement of ...

[0037] Sperm cells cannot divide and have a finite lifespan, but after fusing with an egg cell during fertilization, a new organism / embryo begins to develop, initially as a totipotent zygote. Human sperm cells are haploid (n), allowing their 23 chromosomes to join with the 23 chromosomes of the female egg cell to form a diploid (2n) cell with 46 paired chromosomes. In mammals, sperm cells / spermatozoa are stored in the epididymis and released from the penis during ejaculation in a fluid called seminal fluid / semen / ejaculate.

[0038] The number and quality of sperm cells are important measures of male fertility. However, the genetic quality of sperm cells, as well as their motility, generally decreases with age. For example, DNA damage present in sperm cells during the period after meiosis but before fertilization (i.e., the period of primary concern of the present invention) may have serious deleterious effects on fertility and the developing embryo. Mammalian sperm cells, and in particular human sperm cells, are particularly susceptible to free radical attack (e.g., reactive oxygen species (ROS)) and the resulting oxidative damage. The root of this vulnerability is the gradual loss of DNA damage repair mechanisms during spermatogenesis. Therefore, the means, methods and uses of the present invention, i.e., contact of gametes such as sperm / sperm cells with carbon monoxide gas, prevent this DNA damage (particularly during the period after meiosis but before fertilization). This is also evident, for example, from the attached Figure 3 and 5 As can be clearly seen in Figures 10 to 10, the degree of DNA damage / DNA fragmentation in sperm samples that were not exposed to carbon monoxide was higher than in sperm samples obtained from the same subjects that were exposed to carbon monoxide.

[0039] Oocyte / oocyte is produced in the ovary during female gametogenesis (also referred to as oogenesis). During oogenesis, secondary oocytes are produced, which are haploid and in which meiosis II can be arrested at metaphase II stage until fertilization. Therefore, according to the present invention, the ovum / oocyte can be a secondary oocyte.

[0040] Both spermatozoa / spermatids and egg cells / oocytes can be collected from mammalian subjects, such as healthy or diseased human subjects, prior to exposure to carbon monoxide gas. Spermatozoa / spermatids / semen can be obtained from the epididymis, for example, by ejaculation into a (sterile) container during condom collection or epididymal extraction. On the other hand, egg cells / oocytes can be obtained, for example, by transvaginal oocyte extraction.

[0041] Thus, in 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. However, in different aspects, the gametes may also be collected / obtained from other mammals, in particular also from cattle, horses, pigs, sheep, goats, camels, alpacas, dogs, cats, etc. In another aspect, the gametes may also be collected / obtained from non-mammals, in particular also from birds, fish, reptiles, etc., including but not limited to falcons and / or carp.

[0042] After collection, the collected gametes are provided in a container according to the means, methods and uses of the present invention. Such a container may be an open container or preferably a lockable / sealable (airtight) container. In a specific aspect, such a container may have a valve for connecting the CO delivery tube to the container. The container used according to the means, methods and uses of the present invention may be a container composed of polymers, elastomers, metals, glass or ceramics, but polymers are generally preferred. In one aspect, the container may be composed of polypropylene (PP), polyethylene (PE) or polystyrene (PS). In one aspect, the container to be used may be sterile / sterilized, i.e., the container may be clean and free of bacteria. The exemplary container that has also been used in the accompanying embodiments may be a sample bottle or an airtight Falcon tube. Another container used may be a (sterile) sample cup. The person skilled in the art is fully aware of the containers used in the context of the present invention.

[0043] In one embodiment, seminal fluid / semen / ejaculate can be liquefied. Liquefaction can be carried out before and / or simultaneously with the spermatozoa that gametes, particularly seminal fluid / semen / ejaculate are contained in contact with carbon monoxide gas to preserve. Liquefaction herein describes the process that destroys the gel that is formed by the protein of seminal vesicle and prostate so that seminal fluid / semen / ejaculate becomes more fluid. Those skilled in the art know the means and methods of carrying out liquefaction. For example, routinely, liquefaction occurs naturally during 30 to 60 minutes of incubation at 25 ℃ of seminal fluid / semen / ejaculate, as is also apparent from attached Example 2.

[0044] According to the means, methods and uses of the present invention, the gametes provided in the container are contacted with carbon monoxide gas for a time sufficient to ensure preservation of the gametes.

[0045] In a preferred embodiment, the gametes are contacted with carbon monoxide, in particular carbon monoxide in the form of CO gas. In this case, the gametes are first provided in a container and in a second step (such as step b above) CO gas or a CO release system (activated CORS) that releases CO gas is added to the container containing the gametes. The present invention also relates to means, methods and uses in which the gametes are allowed to contact with carbon monoxide less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute after collecting the gametes in the container. However, it is also conceivable that CO gas is directly contacted with the gametes and / or a (biological) sample containing the gametes. In all embodiments of the present invention, the sample can comprise semen, ejaculated semen and / or a buffer or buffer system containing gametes, in particular sperm. Carbon monoxide, in particular CO gas, can first be provided in a container and in a second step the gametes can be added to a container containing carbon monoxide / CO gas. Thus, the gametes may be contacted with carbon monoxide immediately after collecting the gametes in the container (or after collecting and / or providing a sample comprising the gametes, such as semen, ejaculated semen and / or a buffer or buffer system comprising the gametes). As will be apparent from the embodiments, providing carbon monoxide in the container may include activating a carbon monoxide release system (CORS) prior to collecting the gametes in the container, resulting in the formation of carbon monoxide in the container. Obviously, such a container may be sealed / enclosed between CORS activation and the collection of the gametes. In the context of the present invention, an exemplary carbon monoxide release system may be activated about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or about 1 minute before collecting the gametes. In other words, carbon monoxide / CO gas may be provided in or to a container in a first step, and the gametes or a sample comprising the gametes may be added in a second step. Vice versa, it is also within the scope of the present invention that the gametes (or a sample comprising the gametes) are first provided in a container, and in a second step, carbon monoxide / CO gas is added to the container. The addition of carbon monoxide may also include a carbon monoxide release system (ie, CORS).

[0046] It is also envisaged that the gametes and / or the sample comprising the gametes are contacted with carbon monoxide in a repeated or iterative manner. In other words, the gametes and / or the sample comprising the gametes can be contacted with carbon monoxide multiple times. For example (but not limiting), it is also envisaged that the gametes are contacted with carbon monoxide / CO gas after at least one freeze-thaw cycle. Therefore, in the context of the present invention, the gametes or the sample comprising the gametes are contacted with carbon monoxide / CO gas after they have been preserved (for example by freezing). The exposure of the gametes or the sample comprising the gametes can occur in particular during and / or after the release of the preservation, for example by thawing the frozen gametes / frozen sample comprising the gametes. Corresponding freezing and / or thawing protocols are well known in the art (for example, but not limited to, reproductive methods such as artificial reproductive technology, in vitro fertilization, artificial insemination, etc.). These protocols can be combined with the teachings of the present invention, that is, the gametes or the sample comprising the gametes can be exposed to carbon monoxide during and after these protocol steps, so as to preserve the gametes as described and illustrated herein. Therefore, the present invention also relates to means, methods and uses for contacting frozen and / or thawed gametes, in particular spermatozoa, or frozen and / or thawed biological samples, such as semen, ejaculated semen or a buffer / buffer system comprising said gametes with carbon monoxide. In other words, the present invention is not only applicable to "fresh" gametes or (biological) samples or buffers / buffer systems comprising "fresh" gametes, but also to gametes that have been preserved (such as by freezing) and biological samples / buffers / buffer systems comprising said gametes. Preferably, carbon monoxide is used for a sufficient time / a sufficiently long time to enable and / or ensure the preservation of the gametes. The sufficient time / a sufficiently long time can be or can include the time or period from the time gametes or a sample comprising gametes (preferably sperm) are collected or obtained and preserved until they are further used for, for example, artificial reproductive technology, in vitro fertilization, for example (artificial) insemination of farm animals. Therefore, the "sufficient time" relates to the time period during which the gametes or the sample comprising said gametes are exposed to carbon monoxide / carbon monoxide gas. The (ensured) preservation of the gametes also relates to maintaining a positive and / or healthy physiological state, such as, but not limited to, a low oxidative burden (low ROS state). The (ensured) preservation also relates to further avoiding additional and / or undesirable effects on the gametes, such as exposure to further intrinsic or external adverse events, such as oxidative stress (e.g., ROS). Such undesirable effects and adverse events may have a negative impact on the gametes, in particular on sperm, and may negatively impact the "quality" of these gametes. Therefore, as shown in the accompanying examples, the "preservation of gametes" as described herein also relates to the "preservation" of the desired quality of the gametes. This quality is particularly reflected in the desired movement of the gametes (i.e., sperm), low or even non-existent DNA-fragmentation / DNA-fragmentation rate, a medically, clinically, or biologically insignificant spermatogenesis diagram, or a low oxidative stress level.Therefore, the "preservation" and / or "preservation state" of these gametes can be assessed, in particular but not limited to, by determining the movement and / or motility of the gametes (in particular spermatids / spermatozoa), the DNA fragmentation state of the gametes, by spermatogenesis diagrams and / or by determining the redox potential of the gametes. Corresponding methods are well known in the art and are easily accessible to skilled technicians, such as those working in reproductive medicine / ART, and those who use (artificial) insemination / insemination protocols, such as those who inseminate farm animals. Corresponding evaluation methods for determining the "quality" of the gametes are also described in the accompanying examples, such as keeping the gametes in a desired state. In these examples, it is demonstrated that the present invention ensures the preservation of gametes, in particular spermatozoa. The preservation may include maintaining the (desired) health state of the gametes, but as also described herein, the preservation by carbon monoxide may also provide an improvement in the quality of the gametes. In particular, it has surprisingly been shown that exposure of gametes, in particular spermatids / spermatozoa, to carbon monoxide can even increase / improve the quality of said gametes, in particular spermatids / spermatozoa; see for example the appended Examples 2 and 3, in which the "sperm / spermatozoa quality" in individual subjects was comparatively evaluated in samples treated / exposed to carbon monoxide with samples of the same subjects / individuals / donors not exposed / treated with carbon monoxide. The corresponding illustrative results are also shown in the appended. Figure 3 In the examples of the present invention (see in particular "subjects" 3 and 4), an improvement in quality, i.e. less DNA fragmentation, was recorded between samples from the same individual treated with carbon monoxide and samples not treated / exposed to carbon monoxide. A sample of gametes from the same donor was divided into two equal aliquots, the only difference being exposure to carbon monoxide or not. Figure 7D also shows a surprising improvement in the quality of the gametes (here the quality of the sperm cells / sperm). In Figure 7D it is illustrated that the negative effects of intrinsic (as well as extrinsic) oxidative stress can be avoided by the means and methods of the present invention, i.e. by exposing the gametes / samples comprising said gametes to carbon monoxide. It could surprisingly be shown that a reduced redox potential was observed in the carbon monoxide exposed / treated gamete samples / aliquots compared to untreated samples / aliquots comprising sperm cells / sperm of the same subject / individual / donor; see, for example, in particular subject 42_1 or 38_7. However, similar effects could also be observed in the other subjects.

[0047] Obviously, the maintenance and / or even certain but related improvements of gamete quality described herein are achieved by contacting / exposing the gametes (especially sperm) to / treating the gametes with carbon monoxide. Therefore, the present invention generally relates to preserving gametes in a desired state, for example preserving them in a quality acceptable for the application of gametes in artificial reproductive technology and the like. Therefore, the preservation of the gametes may include maintaining a given gamete quality, but may also include improving the quality of gametes. The preservation is particularly important in the in vitro / extracorporeal artificial reproductive technology methods described herein, for example in the preparation and ex vivo manipulation of gametes for such artificial reproductive technology methods and / or in (artificial) insemination protocols. Therefore, the "preservation" / "maintenance" of gametes refers in particular to, but is not limited to, the preservation / maintenance of gamete quality during ex vivo manipulation and / or in an in vitro environment. It is specifically but not restrictively described herein that preservation in the sense of the present invention can be successfully applied to in vitro / extracorporeal applications of gametes that require manipulation. One example is providing sperm / spermatozoa, (biological) samples or buffers / buffer systems containing them for use in artificial reproductive technologies, such as in vitro fertilization. In the accompanying examples, it can be demonstrated that the sperm tested and treated with carbon monoxide maintain (or even improve) their quality over a relatively long period of time outside the body. The results show that even within 1.5 or even 2 hours after sample collection and exposure to carbon monoxide, gamete quality remains satisfactory or even improves (compared to untreated samples). Therefore, in a non-limiting embodiment, it is envisaged that gametes (or samples / buffers / buffer systems containing gametes) are contacted with the 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 to / treatment of gametes (or samples / buffers / buffer systems containing gametes) with carbon monoxide can occur at room temperature, i.e., at about 25°C to about 35°C. Maintaining a given gamete quality, or to a certain extent, even improving the quality of gametes under certain conditions outside the body / in vitro, is one of the (non-limiting) advantages of the present invention. It is documented that preservation of the gametes is ensured, i.e., their quality is maintained, even during certain, even relatively long (in vitro) operating times (even up to 2 hours) at room temperature. Therefore, provided herein are means and methods for preserving gametes, wherein the gametes (or a sample / buffer / buffer system comprising the gametes) are contacted with carbon monoxide / carbon monoxide gas to ensure preservation of the gametes / to maintain the quality of the gametes. The means and methods may comprise contacting / treating / exposing the gametes (or a sample / buffer / buffer system comprising the gametes) to carbon monoxide / carbon monoxide gas for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, more preferably about 30 to about 60 minutes. The contacting / treatment with / exposure to carbon monoxide / carbon monoxide gas may be carried out at about 25 to about 35°C.

[0048] The present invention relates to means, methods and uses that allow gametes to be brought into contact with CO, in particular in a professional environment, such as a medical or ART laboratory, or in a non-professional environment, such as a private environment. Thus, the present invention also allows individuals, in particular people with or without any medical experience / knowledge, to perform the means, methods and uses of the present invention. Thus, these means and methods are suitable for use at home by individuals who are not familiar with or have not received training in artificial reproductive techniques and / or any related fields. Figure 1B As will be apparent from the foregoing, home use of the apparatus and method of the present invention involves collecting gametes in a container and activating a carbon monoxide release system (CORS). The activated CORS can be placed directly into the container, which can be sealed. As described in detail above, the CORS can also be activated prior to gamete collection. In any case, the gametes can be exposed to CO gas, ensuring that they are protected during liquefaction and subsequent manipulation of said gametes in assisted reproductive techniques, including cryopreservation. Figure 1B It will be apparent that persons who are not familiar with artificial reproductive technology and / or any related fields may be able to perform the means, methods and uses of the present invention.

[0049] In the context of the present invention, providing / obtaining gametes in a container and contacting the gametes with carbon monoxide can refer to providing / obtaining the patient's gametes in a container, providing / obtaining a carbon monoxide releasing system comprising a carbon monoxide releasing molecule, activating the carbon monoxide releasing system and / or molecule and thereby releasing carbon monoxide in the container, and contacting the gametes with carbon monoxide. In one aspect, the carbon monoxide releasing molecule can be obtained / provided in its activated form, so that activation is not required. In another aspect, the container can already contain carbon monoxide, carbon monoxide releasing molecules and / or carbon monoxide releasing system (such as exemplarily provided herein in the form of a carbon monoxide releasing device / capsule).

[0050] In the context of the gametes / CO gas of the present invention, the term "contacting" or "contacted" refers to the physical contact of the gametes and the carbon monoxide gas. In one aspect, the carbon monoxide gas may form a gas layer on the gametes. In some aspects, it may be necessary to actively mix the gametes when the carbon monoxide gas is spread over the gametes to ensure uniform contact of the gametes with the carbon monoxide gas. Carbon monoxide can be provided to the gametes as a solute, dissolved and / or solubilized in a liquid, which is water, an aqueous solution / suspension, including but not limited to body fluids, including but not limited to semen. It will be apparent to those skilled in the art that providing CO gas to the gametes or other tissues may result in CO dissolving and / or solubilizing in, for example, semen. Therefore, it will be apparent that the term "CO gas" may also refer interchangeably to solubilized and / or dissolved CO. The CO is preferably provided in gaseous form, whereby the provision may be direct (direct exposure to CO gas) or indirect (e.g., by exposure to CO gas released from a corresponding carbon monoxide-releasing molecule and / or carbon monoxide release system).

[0051] Carbon monoxide (CO) gas is a colorless, odorless, flammable gas with a density slightly lower than that of air, which necessitates that the container be lockable / sealable. CO consists of one carbon atom and one oxygen atom connected by a triple bond, which therefore makes it the simplest molecule of the carbon oxide family. In complexes (e.g. metal carbonyls, which are capable of releasing CO upon addition of a second compound such as FeCl3), the carbon monoxide ligand is referred to as a carbonyl. CO has important biological roles in the phylogenetic world. It is produced by many organisms, including humans. In mammalian physiology, carbon monoxide is a classic example of a stimulatory effect, where low concentrations of carbon monoxide can act as an endogenous neurotransmitter (gasotransmitter), while high concentrations can be toxic, leading to carbon monoxide poisoning. According to the present invention, this also necessitates that gametes only be exposed to the inventive amount of CO as described in detail below to prevent damage to the gametes.

[0052] As discussed herein, gametes, in particular sperm, may be negatively affected by direct and / or indirect exposure to ROS / oxidants. Without being bound by theory, whenever the amount of oxidant exceeds the maximum capacity of the cellular redox buffer, oxidative stress may occur, in particular with a negative impact on sperm motility. Conversely, whenever the amount of oxidant drops sharply below physiological levels, reductive stress may occur, in particular with a negative impact on sperm motility. It is shown herein that exposure of sperm to carbon monoxide does not negatively impact their motility. Successful exposure of gametes, in particular sperm of the present invention (exposure to CO, which has the beneficial effects described herein on the gametes) may include inhibiting or reducing undesirable DNA breakage and / or inhibiting or lowering redox potential (such as ROS). Maintaining or even increasing the motility of gametes, in particular the motility of sperm may also be one of the effects of the present invention. In the accompanying examples, corresponding technical details are provided, in particular how to measure and evaluate DNA breakage, ROS inhibition and / or gamete motility / sperm motility.

[0053] The skilled person knows that reactive oxygen species (ROS) belong to the group of reactive molecular species (RMS), which further includes, in particular, reactive nitrogen species (RNS) and reactive sulfur species (RSS). Since most RMS are highly reactive, they can react with each other, which can lead to their neutralization or the appearance of new RMS (in biological cells, in particular in gametes, such as sperm). In other words, ROS can react with other ROS, with RNS and / or with RSS, which can lead to the neutralization of the ROS or to the new appearance of ROS. In other words, the presence / appearance of ROS may 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 the level of ROS / oxidative stress / redox potential in biological cells, in particular in gametes, such as sperm, in order to prevent / avoid / reduce the appearance / presence of (more) ROS and / or RMS. As illustratively shown in the accompanying examples and figures, the present invention provides means and methods to effectively lower the redox potential of biological cells / gametes / sperms, thereby preventing / avoiding / reducing the occurrence / presence of (more) reactive oxygen species.

[0054] Thus, in the means, methods and uses provided herein, gametes are preserved, which are preferably used or intended for use in reproductive technologies, such as artificial reproductive technologies, in vitro fertilization, preferably mammals or artificial insemination technologies, preferably (farm) animals. Obviously, the "quality of the gametes" used in such reproductive technologies of humans and animals is extremely important. As demonstrated herein and as illustrated in the accompanying examples, the inventors surprisingly found that exposing gametes, in particular exposing sperm to carbon monoxide, can maintain or even improve said "quality of the gametes". In particular, it is described herein that exposing gametes, in particular sperm to carbon monoxide can mitigate and reduce the adverse effects of undesirable oxidative processes, such as the effects of reactive oxygen species (ROS). In particular, it is shown herein that exposure of gametes (here sperm) or (biological) samples containing gametes (such as semen, ejaculated semen and / or buffer / buffer system) to carbon monoxide leads to a surprisingly (and desirably) reduction in the redox potential of the gametes. It has also been shown that this contact with carbon monoxide / exposure to carbon monoxide / treatment with carbon monoxide maintains / maintains the already favorable (low) redox potential of the gametes. Therefore, the present invention provides means, methods and uses for obtaining gametes with a desired quality, for example for use in artificial reproductive technologies or for (artificial) insemination technologies, in which the quality of the gametes used is extremely important. Therefore, the present invention also relates to providing gametes useful in such technologies. Gametes (or a sample comprising the gametes) can be contacted with / exposed to carbon monoxide in a container. Contacting the gametes (or a sample comprising the gametes) in the container with carbon monoxide can result in the quality of the gametes being maintained or improved, i.e., resulting in the preservation of the gametes. The provided gametes are in contact with carbon monoxide gas in the container for a sufficiently long time to ensure the preservation of the gametes. In the context of the present invention, the term "sufficiently long time to ensure the preservation of the gametes" refers to the duration of contact of the gametes with CO gas, which is necessary for keeping the gametes functional, alive, intact or free from damage or decay after collecting the gametes from the subject. Thus, the term "sufficiently long" refers to in vitro exposure of seminal fluid / semen / ejaculated fluid and / or manipulated corresponding samples, such as liquefied seminal fluid / semen / ejaculated 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, at least 30 minutes (which may be considered the "exposure duration"). A relevant or desired exposure duration may be determined, for example, by measuring one or more characteristics of CO2-preserved gametes, such as redox potential / ROS levels, DNA fragmentation, and / or motility (in the case of sperm / spermatozoa), particularly forward motility, by one or more of the methods described herein below in Examples 2 and 3, and by comparing the results with those obtained using unpreserved gametes.In the accompanying Examples 2 and 3, the inventors found that exposing gametes to CO gas for 60 or 90 minutes resulted in CO-preserved gametes exhibiting (i) lower redox potential, (ii) less DNA breakage / DNA damage, and (iii) higher or maintained (forward) motility compared to unpreserved gametes (i.e., gametes not exposed to CO), such as from attached. Figure 2 This can also be seen in Figures 1 to 8. In the context of the present invention, contacting gametes with carbon monoxide or exposure to carbon monoxide may refer to treating the gametes with carbon monoxide and / or exposing them to carbon monoxide for 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. A non-limiting teaching of the present invention is to provide gametes, preferably spermatozoa, contacted with / exposed to / treated with carbon monoxide to remove / reduce the negative effects of ROS (on the gametes). As will be apparent from the accompanying examples, the inventors have surprisingly found that contacting gametes, especially spermatozoa, with carbon monoxide provides the beneficial effects described in detail herein in the preservation of the gametes, namely that carbon monoxide positively affects and / or maintains the quality of the gametes. In particular, as described herein, these beneficial effects of carbon monoxide on gamete quality can be shown even after 90 minutes of application of carbon monoxide in vitro, i.e. outside the body.

[0055] In a preferred embodiment of the present invention, carbon monoxide (CO) gas can be released from a carbon monoxide releasing molecule (CORM). In one aspect, a CORM that can be located inside a closed compartment (e.g., a capsule that is permeable only to CO gas) can be added to a container (containing gametes). Thus, the CORM can be out of direct contact with the gametes. The CORM can preferably be a metal carbonyl compound, even more preferably a carbonyl molybdenum compound, most preferably trisodium tricarbonyl-[tris(isocyanoethylacetate)] molybdenum (chemical formula: Na3Mo(CO)3(CNCH2CO2H)3). Na3Mo(CO)3(CNCH2CO2H)3 is also referred to as Mo-CORM hereinafter.

[0056] Carbon monoxide-releasing molecules suitable for 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.

[0057] Preferably, the carbon monoxide releasing molecule (CORM) is a metal carbonyl. The metal carbonyl can include a complex of, for example, Rh, Ti, Os, Cr, Mn, Fe, Co, Mo, Ru, W, Re, Ir, B and C group elements, more preferably a complex of Rh, Mo, Mn, Fe, Ru, B and C group elements, even more preferably a complex of Rh, Fe, Mn, Mo, B and C group elements. The metal carbonyl can be considered as a complex because they include a CO group coordinated to a metal center. However, the metal can be bonded to other groups, for example, by ionic bonds or covalent bonds, by other means other than coordination bonds. Therefore, the group forming a part of the metal carbonyl except CO need not be strictly "ligand" in the sense of being coordinated to a metal center by a lone electron pair, but is referred to as "ligand" in this article for ease of reference.

[0058] Thus, the ligands of the metal may all be carbonyl ligands. Alternatively, the carbonyl compound may contain at least one ligand that is not CO. The ligand that is not CO may typically be a neutral or anionic ligand, such as a halide, or be derived from a Lewis base and have N, P, O or S or a conjugated carbon group as a coordinating atom. Preferred coordinating atoms may be N, O and S, examples of which include, but are not limited to, sulfoxides such as dimethylsulfoxide, 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 di-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-benzylindazole), thiols and thiolates such as EtSH and PhSH, chlorides, bromides and iodides, carboxylates such as formate, acetate and oxalate, ethers such as Et2O and tetrahydrofuran, alcohols such as EtOH, and nitriles such as MeCN. Other possible ligands are conjugated carbon groups, such as dienes, for example cyclopentadiene (C5H5) or substituted cyclopentadienes. The substituents in the substituted cyclopentadienes may be, for example, alkanols, ethers or esters, such as -(CH2) n OH, where n can be 1 to 4, especially -CH2OH, -(CH2) n OR, wherein n can be 1 to 4, R can be a hydrocarbon, preferably an alkyl group of 1 to 4 carbon atoms, and -(CH2) n OOCR, wherein n may be 1 to 4 and R may be a hydrocarbon, preferably an alkyl group of 1 to 4 carbon atoms. The preferred metal in such cyclopentadiene or substituted cyclopentadiene carbonyl complexes may be Fe.

[0059] For a detailed description of carbon monoxide releasing compounds, reference is also made explicitly to WO 2008 / 130261 A1 and US 2007 / 0219120 A1, which are incorporated herein by reference.

[0060] Formula I:

[0061]

[0062] is disclosed, which can also be used as the CORM in the present invention, wherein 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 in each case can be 1-12 or 1-6 hydroxy, alkoxy, amino, alkylamino, mercapto, alkylmercapto, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, alkoxycarbonyl, acyl, acyloxy, acylamino, alkylsulfonyl, alkylsulfinyl, F, Cl, Br, NO2 and cyano; or two or more of R1, R2 and R3 can be taken together to form a substituted or unsubstituted carbocyclic or heterocyclic structure or a derivative thereof. For any substituent, the number of C atoms can be 1-12 or 1-6.

[0063] Derivatives of compounds of formula I which are acetals, hemiacetals, aminomethanols, aminals, imines, enaminones, imidates, amidines, iminium salts, sodium bisulfite adducts, hemithioacetals, dithioacetals, 1,3-dioxepane, 1,3-dioxane, 1,3-dioxalane, 1,3-dioxetane, ane), α-hydroxy-1,3-dioxepane, α-hydroxy-1,3-dioxane, α-hydroxy-1,3-dioxolane, α-keto-1,3-dioxepane, α-keto-1,3-dioxane, α-keto-1,3-dioxolane, α-keto-1,3-dioxetanes, macrocyclic esters / imines, macrocyclic esters / hemiacetals, oxazolidines, tetrahydro-1,3-oxazines, tetrahydrooxazinones, 1,3,4-oxadiazine,

[0064] Thiazolidine, tetrahydro-1,3-thiazine, thiazolinone, tetrahydro-1,3-thiazinone, imidazolidine, hexahydro-1,3-pyrimidine, imidazolidinone, tetrahydro-1,3-pyrimidinone, oxime, hydrazone, carbazone, diaminothiourea, semicarbazone, semithiocarbazone, acyloxyalkyl ester derivatives, O-acyloxyalkyl derivatives, N-acyloxyalkyl derivatives, N-Mannich base derivatives or N-hydroxymethyl derivatives can also be used as the CORM of the present invention.

[0065] The CORM of the present invention can also be, for example, trimethylacetaldehyde, 2,2-dimethyl-4-pentenal, 4-ethyl-4-formyl-hexanenitrile, 3-hydroxy-2,2-dimethylpropanal, 2-formyl-2-methyl-propylmethanoate, 2-ethyl-2-methyl-propanal, 2,2-dimethyl-3-(p-methylphenyl)propanal or 2-methyl-2-phenylpropanal.

[0066] In one aspect, oxalates, oxalate esters, or amides can be used as CORMs in the present invention.

[0067] Preferred CORMs for use in the present invention may include carbonyl molybdenum compounds, CORM-1, CORM-2, CORM-3, CORM-401, as disclosed in WO 2015 / 188941 A1, WO 2016 / 110517 A1, and DE 102017 006 393 A1.

[0068] More preferred in the context of the present invention may be molybdenum-based CORMs, such as Mo(CO)3(CNC(CH3)2COOH)3 (also known as "CORM-ALF794") and Mo(CO)3(CNCH2CO2H)3(trisodium tricarbonyl-[tris(isocyanatoethyl acetate)]molybdenum), of which Mo(CO)3(CNCH2CO2H)3 may be particularly preferred. Preferably, the trisodium salt (Na3Mo(CO)3(CNCH2CO2)3) ("Mo-CORM") may be used.

[0069] Whenever a CORM is used in the context of the means, methods, and uses of the present invention, the CORM will come into contact with a second compound, thereby releasing carbon monoxide gas. The second compound may be FeCl3, Ce(SO4)2, or H2O2. In a preferred embodiment of the present invention, the second compound may be FeCl3.

[0070] Second compounds suitable for use in the present invention are also 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.

[0071] The CO-releasing molecule (CORM) releases carbon monoxide gas upon contact with a second compound. "Contact" herein means that a reaction can occur between the CORM and the second compound, resulting in the release of CO gas. Upon contact with the second compound, the CORM begins to release (substantially) CO gas, as described in further detail below. The system (i.e., CORS) is then "activated" (activated CORS), and the released CO gas can exert its gamete-preserving activity / function.

[0072] The second compound may be a sulfur-containing compound, a nitrogen-containing compound, an oxidizing compound, an acid or a base, or water.

[0073] When the CORM is a metal carbonyl compound, the second compound may be, for example, a carbonyl substituent, such as a sulfur-containing compound or a nitrogen-containing compound. The sulfur-containing compound may be, for example, selected from an alkali metal or alkaline earth metal salt, preferably a sodium salt of a sulfite, dithionite or metabisulfite, or a compound with at least one thiol moiety, such as cysteine or glutathione.

[0074] 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 which calcium peroxide, dibenzoyl peroxide, hydrogen peroxide, urea, sodium perborate, and sodium percarbonate may be preferred. Oxidizing metal salts that can be used as the second compound may include silver (I) nitrate, iron (III) chloride, potassium permanganate, cerium (IV) sulfate, potassium dichromate, gold (III) chloride, and silver nitrate, of which iron (III) chloride, potassium permanganate, and cerium (IV) sulfate may be preferred, especially iron (III) chloride and cerium (IV) sulfate. The oxidizing metal salts can preferably be used in the form of an aqueous solution, as will be apparent from the accompanying examples.

[0075] As an acid, for example, hydrogen chloride (HCl) can be used. In another embodiment, the second compound can be a non-enzymatic compound. Preferably, the second compound can be a compound with a molecular weight of less than 10,000 g / mol, more preferably less than 7,000 g / mol or even less than 1,000 g / mol. The second compound can also be, for example, water or a solvent. A preferred CORM that releases carbon monoxide gas upon contact with water can be ALF186.

[0076] If a metal carbonyl compound is used as the CORM with a sulfur-containing compound or other electron-withdrawing compound as the second compound, for example, it is believed that when the second compound contacts the metal carbonyl compound, ligand substitution occurs, thereby triggering CO gas release.

[0077] In another embodiment of the present invention, the second compound may be selected from the group consisting of sulfur-containing compounds, nitrogen-containing compounds, oxidizing compounds and water. This is particularly the case if the CORM is a metal carbonyl compound.

[0078] In a preferred embodiment of the present invention, a carbonyl molybdenum compound may be used as the CORM, and an oxidizing compound may be used as the second compound. In a further preferred embodiment, Ru2(CO)6Cl4 may be used as the CORM, and sodium sulfite (Na2SO3) may be used as the second compound.

[0079] Particularly preferred embodiments of the present invention may include a combination of a molybdenum carbonyl compound, preferably Mo(CO)3(CNCH2COOH)3 or its trisodium salt Na3Mo(CO)3(CNCH2CO2H)3 (Mo-CORM), with iron(III) chloride (FeCl3), cerium(IV) sulfate (Ce(SO4)2), or H2O2, wherein FeCl3 and Ce(SO4)2 are used in one embodiment in the form of an aqueous solution with a concentration of about 2 to about 3 mol / L, and H2O2 is used in the form of an aqueous solution with a concentration of about 20 to 40 wt.-%, preferably about 30 wt.-%. Molybdenum carbonyl compounds may have the advantage of producing CO in high capacity (≥95%) and high purity (>95%). In order to obtain particularly high CO yields at particularly high purity, it is particularly preferred to use a combination of Mo(CO)3(CNCH2COOH)3 or its trisodium salt Na3Mo(CO)3(CNCH2CO2H)3 (Mo-CORM) and FeCl3.

[0080] The second compound may also be added to the container as part of a closed compartment (e.g., a capsule) that also contains the CORM. Thus, in a preferred embodiment, the CORM and the second compound may be contained together in a closed compartment, such as a capsule that may be added to the interior of a container containing the gametes. Such a system comprising a CORM and a second compound within a closed compartment may also be referred to as a CO release system ("CORS"). The outer sheath of such a CORS is selectively permeable to gas molecules, in particular CO gas. This also ensures that all other components contained in the CORS remain inside the CORS / corresponding closed compartment (capsule) and therefore do not come into contact with 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 diaphragm). Only physical contact of the CORM and the second compound releases CO from the CORM / CORS ("activated CORS") by diffusion of CO gas through the outer sheath into the container containing the gametes. As will also be apparent from the following Additional Example 2, the present inventors used, in an exemplary embodiment, a CORS capsule (18-22 mm in length, 6-8 mm in diameter) containing 15 mg of Mo-CORM and 150 μl of an aqueous FeCl3 solution (583.3 mg / ml), separated by a septum / membrane, to preserve a human sperm sample after liquefaction. By applying pressure along the longitudinal axis of the CORS / enclosed compartment / capsule, the CORM and FeCl3 were brought into physical contact, generating sufficient CO gas within a gas-tight 15 mL Falcon tube within 90 minutes to ensure gamete preservation.

[0081] In the context of the present invention, particularly in the in vitro and / or ex vivo methods described herein, CO releasing molecules (CORMs) such as trisodium tricarbonyl-[tris(isocyanatoethylacetate)] molybdenum may also be used. In addition, carbon monoxide releasing systems may be used. A corresponding, but illustrative, carbon monoxide releasing system (CORS) is provided in the accompanying Figure 11 and 12 In these figures, a new and inventive CORS is provided in the form of a CORS capsule, as disclosed and described in EP22216317.2 and PCT / EP2023 / 074808. The CORS capsule is also Figure 11 and 12 The device is indicated by reference numeral 40. The terms "CORS," "carbon monoxide release device," and "carbon monoxide release system" are used interchangeably in the context of the present invention. A CORS can be configured to treat biological cells, preferably living cells, ex vivo by releasing carbon monoxide ex vivo. Figure 11Reference numeral "60" in the CORS system 40 shows a corresponding configuration. "62" represents a biological cell, such as a gamete, particularly a sperm (also contained in a corresponding seminal fluid / semen / ejaculated semen). The system 60 can be configured to treat the biological cell 62, preferably a living cell, preferably a gamete, with carbon monoxide. The system 60 can be configured to provide one or more effects on the biological cell 62 that at least partially preserve the biological cell 62. The system 60 can include at least one container 64 configured to receive the biological cell 62 and at least one carbon monoxide source. The container 64 and the source of CORS 40 can be arranged relative to each other so that the biological cell 62 can come into contact with the carbon monoxide provided by CORS 40 to treat the biological cell 62. The container 64 can include a lid 66, which is preferably configured to provide a seal for the container 64 to seal the contents of the container 64 from the environment, preferably in an airtight manner. Alternatively or additionally, the treatment system "60" can be configured for ex vivo treatment of an animal and / or human body, for example by applying carbon monoxide to an external surface of the animal and / or human body, such as the skin, which can also be considered to be treating biological cells within the meaning of the present disclosure.

[0082] Figure 12 An alternative system that can be used is shown in schematic form, namely the system shown as "80" in the figure. "80" can also be used to expose living biological cells, preferably gametes, to carbon monoxide as provided herein in vitro. The exposure can include releasing carbon monoxide in an in vitro environment. System "80" can include at least one container "82" that is configured to receive biological cells "62" and at least one carbon monoxide source, Figure 12 In the illustrated configuration, for example, but not limited to, CORS 40, a container 82 and a carbon monoxide source 40 can be arranged relative to one another so that the biological cells 62 can come into contact with the carbon monoxide provided by the device 40, thereby contacting the biological cells 62 with the carbon monoxide. The container 82 can include at least one compartment 84 configured to receive and preferably contain and / or secure the device 40, preferably in a captive manner. Alternatively, or in addition to the compartment 84, the container 82 can include one or more securing devices, such as one or more clamps, configured to secure the CORS 40, preferably in a captive and / or substantially immovable manner, within the container 82. This can allow the system 80 to be preassembled, particularly by placing the CORS 40 in the compartment 84 prior to use, preferably prior to distribution to a location of application, such as one or more laboratories or medical facilities. This can facilitate operation and / or use of the system 80.

[0083] Corresponding systems for exposing CO to gametes are described and provided in EP22216317.2 and PCT / EP2023 / 074808, which are incorporated herein by reference.

[0084] The CO gas can also be supplied directly to the container via a separate tube that releases the CO gas into the container. In one aspect, the CO gas can be supplied directly from a pressurized CO gas tank, or it can be released from a liquid, such as a saturated solution, a foam, a hydrogel, or a solid in which the CO is physically bound.

[0085] The total amount of CO gas applied to the gametes in the container can be from about 20 μmol to about 500 μmol, preferably from about 30 μmol to about 450 μmol, from about 40 μmol to about 400 μmol, preferably from about 45 μmol to about 270 μmol, and preferably from about 60 μmol to about 180 μmol. The value after the term "about" in this context refers to the value itself, but also includes the (error) margin of ±10% of the value. For illustration, if each Mo-CORM molecule is completely decarbonylated, 1 mg of Mo-CORM can release about 6 μmol of carbon monoxide gas. As also illustrated in the accompanying non-limiting Example 2, in the case of a human sperm sample of about 0.7-2.9 ml volume in a 15 ml volume standard Falcon tube, 15 mg of Mo-CORM can be used for storage. This amount corresponds to 15×6 μmol=90 μmol of carbon monoxide gas released. To further illustrate, for cattle from which sperm samples having a higher average volume (5-8 mL) can be collected, four times the amount of Mo-CORM, i.e., 60 mg of MO-CORM, 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 can also depend on the volume of the collected gametes and the volume of the container. In one exemplary embodiment, i.e., Example 2, an average of approximately 8.4 mg of CORM was used per mL of collected sample (range: approximately 5.2 mg to 20.5 mg of CORM per mL of collected sample).

[0086] According to the means, methods and uses of the present invention, gametes can be contacted with carbon monoxide gas at about 25 to about 35° C. for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, more preferably about 30 to about 60 minutes to ensure preservation of the gametes. The value followed by the term “about” in this context also refers to the value itself, but also includes a margin of error of ±10% of the value.

[0087] After the gametes have been exposed to carbon monoxide gas in the container, there are several methods that can be used to determine whether the gametes have been successfully preserved, some of which are also described in the accompanying Example 2. The preservation of gametes according to the present invention can be assessed by measuring (sperm / spermatid) motility, in particular forward (sperm / spermatid) motility, DNA-fragmentation, spermatogenesis diagrams, redox potential measurements or a combination thereof. As described above and below in detail, the Male Infertility Oxidation System (MiOXSYS, Englewood, CO) can be used, for example, to measure the (static) redox potential / ROS level of sperm. It will be apparent to those skilled in the art that the terms static redox potential, redox potential, ROS level, oxidation potential and redox potential can be used interchangeably in the context of the present invention. The Halo Sperm G2 Kit (Halotech, Madrid, Spain) can be used to measure DNA-fragmentation of sperm, and the CEROS II system (Hamilton Thorne, Beverly, MA) can be used to measure sperm motility, in particular total motility and forward motility. On the other hand, spermatogenesis diagram (also referred to as seminogram or semen analysis) can analyze one or more characteristics of male semen and the spermatid therein comprised.According to measurement kit / method, can only assess a few characteristics (for example, using home test kit) or can assess many characteristics (for example, by diagnostic laboratory) simultaneously.For spermatogenesis diagram, non-limiting characteristics to be analyzed especially can comprise the physical characteristics (color, smell, pH, viscosity and liquefaction) of semen, volume, sperm count, concentration, form, sperm total movement and advance / forward motion, non-forward motion, the percentage ratio of immobile sperm, the percentage ratio of live sperm and the percentage ratio of normal form of sperm.Those skilled in the art know many commercially available test kits and the means and methods of analyzing above-mentioned characteristics.

[0088] All the above explanations described in the context of the means and methods of the present invention also apply to the following uses, methods of treatment and kits, which are described in detail below.

[0089] Therefore, in another embodiment, the present invention also relates to the use of carbon monoxide gas to preserve gametes in assisted reproductive technology (applications) to reduce the risk of congenital abnormalities and / or aneuploidy (and / or euploidy). To this end, the gametes are exposed to carbon monoxide gas in a container for a sufficient period of time to ensure the preservation of the gametes.

[0090] In another embodiment, the present invention relates to a method for reducing DNA fragmentation and / or redox potential of gametes in assisted reproductive technology, wherein the gametes are contacted with carbon monoxide gas. The gametes can be obtained / provided in a container and contacted with CO in the container. The gametes can be contacted with CO for a sufficiently long time (to ensure that the DNA fragmentation and / or redox potential of the gametes are reduced). Here, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or increase the movement of the gametes, including but not limited to forward movement, total movement and / or VAP. In addition, 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. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can improve the success / success rate of artificial reproductive technology. Therefore, reducing the DNA fragmentation and / or redox potential of the gametes can reduce the risk of miscarriage and / or pregnancy failure in artificial reproductive technology.

[0091] In another embodiment, the present invention relates to the use of carbon monoxide in reducing the fragmentation and / or redox potential of gametes in assisted reproductive technology, wherein the gametes are contacted with carbon monoxide gas. The gametes can be obtained / provided in a container and contacted with CO in the container. The gametes can be in contact with CO for a sufficiently long time to ensure that the DNA fragmentation and / or redox potential of the gametes are reduced. Here, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or increase the movement of the gametes, including but not limited to forward movement, total movement and / or VAP. In addition, 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. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can increase the success / success rate of artificial reproductive technology. Therefore, reducing the DNA fragmentation and / or redox potential of the gametes can reduce the risk of miscarriage and / or pregnancy failure in artificial reproductive technology.

[0092] In another embodiment, the present invention also relates to the use of carbon monoxide for the treatment / prevention of gamete-related diseases caused by / associated with ROS / elevated ROS levels.

[0093] As described in detail above and below, reactive oxygen species and / or oxidative stress (and the resulting increased redox potential) can lead to / are associated with congenital abnormalities and / or aneuploidy in offspring caused by diseased gametes and / or gametes with high ROS levels. Therefore, contacting gametes with CO gas can prevent / reduce DNA breakage and / or redox potential, and can therefore prevent and / or reduce the risk of congenital abnormalities and / or aneuploidy (in artificial reproductive technology). Therefore, contacting gametes with 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 in a method for preventing abnormalities and / or aneuploidy. It will be apparent from the examples that gametes can be (provided / obtained in a container) in vitro (in the container) in contact with CO gas (for a sufficiently long time). However, the present invention also relates to contacting gametes with CO in vivo (for a sufficient period of time) to prevent and / or reduce the risk of congenital abnormalities and / or aneuploidy, as described in more detail below.

[0094] As already described in detail above and below, reactive oxygen species and / or oxidative stress (and thereby also elevated redox potential) of the gametes can cause / be associated with male infertility in the patient. It will therefore be apparent to one skilled in the art that depleting and / or reducing reactive oxygen species, oxidative stress and / or redox potential in the gametes of a male patient can treat / reduce the infertility of said patient. In other words, the present invention relates to carbon monoxide for use in treating / reducing male infertility. On the other hand, the present invention relates to carbon monoxide for use in a method for treating and / or reducing male infertility. As will be apparent from the examples, the gametes can be contacted (provided in a container) ex vivo (in said container) with CO gas (for a sufficiently long time). However, the present invention also relates to contacting the gametes with CO in vivo (for a sufficiently long time) to thereby treat and / or reduce male infertility, as described in more detail below.

[0095] In another aspect, reactive oxygen species and / or oxidative stress (and thereby also increased redox potential) and DNA breakage of gametes can cause diseases and / or be associated with diseases. These diseases can be caused by and / or associated with the patient's (elevated) (physiological and / or psychological) stress level. These diseases can occur in patients who are subject to (physiological and / or psychological) stress. Exposure of gametes to CO gas can prevent and / or reduce DNA breakage and / or redox potential and can therefore prevent / treat these diseases (in artificial reproductive technologies). Therefore, exposure of gametes to CO gas can prevent and / or treat these diseases. In other words, CO can treat and / or prevent diseases caused by and / or associated with (elevated) DNA breakage and / or redox potential of gametes. In other words, the present invention relates to carbon monoxide for the treatment and / or prevention of diseases caused by and / or associated with (elevated) DNA breakage 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 will be apparent from the examples, gametes can be contacted (provided / obtained in a container) with CO gas ex vivo (in said container) for a sufficient period of time. However, the present invention also relates to contacting gametes with CO in vivo (for a sufficient period of time) to prevent and / or treat such diseases, as described in more detail below.

[0096] On the other hand, elevated ROS levels can cause gamete diseases and / or be associated with gamete diseases. Such elevated ROS levels and such diseases may be caused by or associated with elevated stress levels (physiological and / or psychological) in the patient. These diseases may occur in patients suffering from (physiological and / or psychological) stress. Gametes can be contacted with CO gas to reduce (elevated) ROS levels and can therefore prevent and / or treat such diseases (in artificial reproductive technology). In other words, CO can treat and / or prevent gamete diseases caused by elevated ROS levels and / or associated with elevated ROS levels. In other words, the present invention relates to carbon monoxide, which is used to treat and / or prevent gamete diseases caused by elevated ROS levels and / or associated with elevated ROS levels. On the other hand, the present invention relates to carbon monoxide, which is used to treat and / or prevent gamete diseases caused by elevated ROS levels and / or associated with elevated ROS levels. As will be apparent from the examples, gametes can be (provided / obtained in a container) in vitro (in the container) in contact with CO gas (for a sufficiently long time). However, the present invention also relates to contacting gametes with CO in vivo (for a sufficient period of time) to prevent and / or treat such diseases, as described in more detail below.

[0097] The present invention also relates to carbon monoxide for use in the treatment and / or prevention of pregnancy failure and / or miscarriage caused by (elevated) ROS and / or (elevated) DNA fragmentation (in gametes / gametes used for fertilization).

[0098] It is further apparent that reducing the DNA fragmentation and / or redox potential of the gametes can increase the success and / or success rate of artificial reproductive technologies using the gametes for fertilization. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can protect the gametes and / or prevent damage to the gametes. In a further aspect, reducing the DNA fragmentation and / or redox potential of the gametes can increase the vitality of the gametes. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or improve the (total) movement of the gametes. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or improve the forward movement of the gametes. On the other hand, reducing the DNA fragmentation and / or redox potential of the gametes can maintain and / or improve the average path velocity (VAP) of the gametes.

[0099] As already described in detail above, the use of carbon monoxide (gas) to maintain gametes in a functional, viable, energetic, intact state or a state free of damage or decay reduces the risk of congenital abnormalities and / or aneuploidy. It will be apparent to those skilled in the art that the terms "functional", "viable" and "vigorous" as used herein are interchangeable with respect to gametes. Thus, in the context of the present invention, improving and / or increasing sperm and / or sperm motility, quality and / or vitality parameters may be used interchangeably. This is particularly the case when CO-preserved gametes are used in assisted reproductive technology applications. Thus, the term "reduced risk" refers to the reduced risk associated with the use of CO-preserved functional / viable / intact gametes in assisted reproductive technology (applications) compared to the use of unpreserved gametes, which may be more severely damaged, resulting in a higher likelihood of newborns suffering from congenital abnormalities or aneuploidy. Thus, contacting / exposing gametes, in particular sperm, to carbon monoxide / carbon monoxide gas can preserve functional / viable / intact gametes while maintaining or even improving (desirable) quality, vitality and / or viability parameters. In the context of the present invention, the carbon monoxide-preserved gametes can also reduce the risk of miscarriage of fertilized eggs, embryos, offspring, etc. derived from one of the preserved gametes when used in (especially but not limited to) artificial reproductive techniques, compared to gametes that have not been preserved / contacted / exposed to the carbon monoxide. In other words, contacting gametes, in particular sperm, with carbon monoxide (gas) can increase the success / success rate of artificial reproductive techniques using the preserved gametes, compared to gametes that have not been treated with carbon monoxide (gas).

[0100] Assisted reproductive technology (ART) includes medical procedures primarily used to address infertility (which is typically the result of non-functional / dead / damaged gametes) and to reduce the risk of congenital abnormalities or aneuploidy. Non-limiting examples of ART applications according to the present invention may include in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI), frozen embryo replacement (FER), preimplantation genetic testing (PGT; which can be performed in conjunction with IVF), in vitro maturation of oocytes (IVM), cryopreservation of gametes, frozen oocyte replacement (FOR), gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFT) or cryopreservation, some of which are also described in Wyns, Human Reproduction Open 3 (2021): 1-17.

[0101] Congenital anomalies encompass a wide range of abnormalities of body structure or function that are present at birth and that originate prenatally. For the sake of efficiency and practicality, the focus is generally on major structural anomalies. These are defined as structural changes that have significant medical, social, or cosmetic consequences for the affected individual and generally require medical intervention.

[0102] Thus, the congenital anomaly according to the invention may in particular be selected from congenital anomalies of the limbs, congenital anomalies of the heart, congenital anomalies of the nervous system, congenital anomalies of the gastrointestinal system and congenital anomalies of the lung.

[0103] Congenital anomalies of the limbs according to the invention may be selected in particular from the group consisting of achondroplasia, ectromelia, amniotic syndrome, brachydactyly,

[0104] Cranio-clavicular dysplasia, congenital hypoplasia or agenesis, ectrodactyly, oligodactyly, phocomelia, polydactyly, polymelia, polysyndactyly, syndactyly.

[0105] The congenital anomaly of the heart according to the invention may in particular be selected from atrial septal defect, patent ductus arteriosus, tetralogy of Fallot and ventricular septal defect.

[0106] Congenital anomalies of the nervous system according to the invention may in particular be selected from neural tube defects such as agenesis of the corpus callosum, anencephaly, Arnold-Chiari malformation, Dandy-Walker malformation, encephalocele, holoprosencephaly, hydrocephalus, anencephalocele, megalencephalocele, meningocele, myelomeningocele, microcephaly, polymicrogyria and spina bifida.

[0107] The congenital anomaly of the gastrointestinal system according to the invention may in particular be selected from atresia, imperforate or stenosis.

[0108] The congenital anomaly of the lung according to the invention may in particular be congenital bronchiectasis.

[0109] (Congenital) aneuploidy in the context of the present invention relates to the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46 chromosomes. It does not include differences in one or more complete sets of chromosomes. Aneuploidy originates during cell division when chromosomes do not separate properly between two cells (non-disjunction). Most cases of aneuploidy in autosomes lead to miscarriage, and the most common extra autosomes in live births are 21, 18 and 13 (Driscoll, The New England Journal of Medicine 360 (2009): 2556-2562). One in every 160 live births has a chromosomal abnormality detected. Non-limiting examples of (congenital) aneuploidy can be selected in particular from 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 (Alfi's syndrome / Kleefstra 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-Dieker syndrome / Smith-Magenis syndrome), Edwards syndrome (distal 18q- / proximal 18q-, trisomy 19, trisomy 20, trisomy 21 (Down syndrome), cat eye syndrome / trisomy 22 (DiGeorge syndrome / Phelan-McDermid syndrome / distal 22q11.2 deletion syndrome).

[0110] (congenital) euploidy in the context of the present invention relates to cells with a complete chromosome set (" set ") of any number, which is different from the 2 chromosome sets contained in normal diploid cells. The non-limiting examples of euploid cells with a complete chromosome set of different numbers can especially include haploid (1 set), triploid (3 sets), tetraploid (4 sets), pentaploid (5 sets), hexaploid (6 sets), heptaploid (7 sets) cells, and the complete chromosome set is different from two chromosome sets. The general term polyploid can be used to describe cells with three or more chromosome sets.

[0111] In another embodiment, the present invention relates to a method for treating congenital anomalies or aneuploidy (or euploidy), the method comprising contacting carbon monoxide gas with gametes of a patient in need thereof. Contacting the gametes with carbon monoxide can be performed, for example, according to the method described above.

[0112] Therefore, in order to treat the above-mentioned congenital abnormalities or aneuploidy (or euploidy) with carbon monoxide gas, it may be necessary to diagnose whether a healthy or sick patient or the patient's gametes need such treatment before being used for ART. If the patient's existing offspring suffer from congenital abnormalities or aneuploidy (or euploidy), or the patient's gametes are assessed to be at a higher risk of causing congenital abnormalities or aneuploidy (or euploidy) than the gametes of a normal control patient (group), the patient or the patient's gametes may need such treatment. In the context of the present invention, the terms "patient", "sick patient", "patient in need of such treatment", "sick subject", "individual" and "individual to be treated" can be used interchangeably and can refer to a subject with gametes having increased DNA breakage and / or redox potential.

[0113] This gamete assessment can be performed, in particular, by, for example, karyotyping, DNA fragmentation, and / or spermatogenesis diagram analysis, all of which are considered standard / conventional methods in the art. A "higher risk" of causing congenital anomalies or aneuploidy can be defined as, for example, a higher proportion of gametes having elevated levels of DNA fragmentation, elevated redox potential, elevated ROS levels, or an unusual number of chromosomes when the patient's gametes are compared to gametes of a normal control patient treated in the same manner as the patient's gametes. Since haploid gametes contain 23 chromosomes, any number of chromosomes different from 23 can represent an unusual number of chromosomes in the gametes being assessed. As described in detail above, for example, the male infertility oxidation system (MiOXSYS, Englewood, CO) can be used to measure (elevated) ROS levels and / or (elevated) redox potential of sperm. The term "elevated" refers to a (ROS) level that is (significantly) different from the average or median (ROS) level of a control group or control sample. As described in detail below, Figures 7D and 8D show DNA fragmentation and oxidation-reduction potential measurements for sperm samples from individual subjects that were not treated with carbon monoxide. Here, only a few sperm samples showed high values for oxidation-reduction potential, while the majority of samples showed comparable (low) oxidation-reduction potential values. It is conceivable that sperm samples with low oxidation-reduction potentials could be considered control samples.

[0114] Once it is determined that the patient or the patient's gametes require CO treatment, the patient's gametes can be collected and processed as described above. As described above, the patient's gametes can also be contacted with CO and / or treated with CO in vivo. Here, CO can be administered systemically and / or transdermally to the gametes. Systemic administration may include, but is not limited to, administering a CO release system / CO release suppository by oral, rectal and / or intraurethral administration. Transdermal and / or percutaneous administration of CO may include, but is not limited to, applying CO or a CO release system to the patient's scrotum. CO gas can diffuse through, in particular, the epithelial layer and come into contact with the gametes. Here, the CO release system can be attached and / or adhered to the patient's scrotum. In one aspect, the CO release system can be a patch. CO release patches are well known in the art and are described in WO 2021 / 180908 A1 and Ruopp et al. (2023, Journal of Controlled Release), the entire contents of which are incorporated herein by reference. Such patches can be used to treat wounds, inflammatory diseases of the skin, and inflammatory diseases of the subcutaneous skin tissue, joints, and tendons, however, their use in treating gamete diseases has not been previously disclosed. In order to allow the gametes to be fully treated so as to obtain the desired effect of CO in vivo, the gametes can be exposed to CO (e.g., using the above-mentioned CO delivery system / patch) for at least 240 minutes, at least 300 minutes, at least 360 minutes before the gametes are collected. In one aspect, the in vivo treatment of the gametes can be performed multiple times, i.e., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 times with CO each time for at least 240 minutes, at least 300 minutes, at least 360 minutes, before the gametes are collected. The scrotum can be exposed to about 90 μmol to about 540 μmol of carbon monoxide. Clearly, treating gametes with CO in vivo is not mutually exclusive with treating gametes with CO in vitro. In other words, the gametes can first be contacted with CO in vivo using the means and methods disclosed herein, and the gametes can be contacted with CO in vitro using the means and methods disclosed herein after the gametes are collected.

[0115] Administration of CO to a patient's gametes can reduce the redox potential / ROS levels and / or DNA breakage of the gametes, thereby increasing their vitality. As described in detail above, ROS levels can increase and / or accumulate in individual gametes due to various factors, including but not limited to stress. That is, ROS levels in an individual may increase due to, in particular, (physiological and / or psychological) stress, genetic predisposition, environmental factors (including exposure to electromagnetic waves) and / or behavioral risk factors (diet, including smoking, alcohol consumption and / or drug abuse), etc. Systemic ROS levels can be associated with gamete ROS levels and can therefore be used as an indicator of individual gamete ROS levels. Systemic ROS levels can be evaluated by evaluating ROS levels in individual urine and / or blood samples, particularly in serum samples. Means and methods for determining ROS levels in individual serum samples are well known in the art and can be performed using Aytu Biosciences' RedoxSys device (Aytu Biosciences, Inc, Englewood, CO 80112, USA).

[0116] ROS in gametes can affect, among other things, the reproductive efficiency of artificial reproductive techniques and natural fertilization during mating. It is therefore clear that gametes that have been exposed to CO in vivo and thus exhibit reduced ROS levels are particularly suitable for subsequent artificial reproductive techniques and / or subsequent natural fertilization, and can improve their success rate and / or success rate. As already described above, reducing ROS levels by (in vivo) CO treatment can prevent congenital anomalies and / or aneuploidy, treat and / or reduce male infertility, treat and / or prevent diseases caused by and / or associated with increased DNA fragmentation and / or redox potential in gametes, or treat and / or prevent gamete diseases caused by / associated with elevated ROS levels.

[0117] In another embodiment, the present invention relates to a kit comprising a CORM and a second compound in a closed compartment ("CORS") as described in detail above. Preferably, such a kit may further comprise instructions describing the method and / or use as described above. Such a kit may also contain additional components, such as containers, buffers, etc., which can be used / useful in the method as described above. Such a kit may preferably be a home kit that can be used by everyone who can perform the above method, or a kit that can be used by specially trained personnel, for example in a fertilization clinic.

[0118] Further embodiments are exemplified in the scientific section. The accompanying drawings provide an illustration of the invention. Although the experimental data in the examples and the experimental data shown in the drawings are not to be considered restrictive, the technical information contained therein forms part of the present invention. Therefore, the present invention also covers all other features shown individually in the figures, even though they may not be described in the preceding or following description. In addition, individual alternatives to the embodiments described in the drawings and the description and individual alternatives to their features may be excluded from the subject matter of another aspect of the present invention.

[0119] Figure 1A : An illustrative workflow of the method of the present invention for preserving sperm using carbon monoxide.

[0120] Figure 1B : Illustrative workflow of the method of the present invention for preserving sperm using carbon monoxide. This workflow shows the process of sample collection and CO treatment (using a CO release system; CORS) for home use and options for use in professional fields (e.g., ART laboratories).

[0121] Figure 2 : Results of sORP measurements on sperm samples from 6 subjects. Shown are the relative sORP of the sperm fraction treated with CO (wCO) compared to the sperm fraction not treated with CO (woCO, set to 1).

[0122] Figure 3 : Results of DNA fragmentation measurements of sperm samples from 3 subjects. Shown are the relative rates of DNA fragmentation in the sperm fraction treated with CO (wCO) compared to the sperm fraction not treated with CO (woCO, set to 1).

[0123] Figure 4 : Results of sperm motility measurements from sperm samples from 5 subjects. A: Total motility, B: Progressive motility. Relative values are shown for the fraction of sperm treated with CO (wCO) compared to the fraction of sperm not treated with CO (woCO, set to 1).

[0124] Figure 5 : Summary of relative changes in sperm parameters in CO-treated (wCO) compared to sperm without CO (woCO; set to 1). Data are presented as mean ± SD; n = 5 (total and progressive motility, see Figure 4 ), n=6 (sORP, see Figure 2 ), n=3 (DNA fragmentation, see Figure 3 ).

[0125] Figure 6Figure 3: Relative changes in sperm motility and motility parameters in response to different CO protocols. A. Ratios of motility ("Motility"), progressive motility ("Progressive"), and ventricular apomorphy ("VAP") treated / untreated with 1.32 mL CO and different exposure times; B. Ratios of motility, progressive motility, and VAP treated / untreated with different CO doses and 90-min exposure time; C. Ratios of redox potential ("sORP") and DNA fragmentation ("SDF") treated / untreated compared with the control group (1.32 mL CO and different exposure times); D. Ratios of redox potential and DNA fragmentation treated / untreated with different CO doses and 90-min exposure time; Values are expressed as mean ± SD, n = 5.

[0126] Figure 7: Paired measurements of individual subjects who did and did not receive CO treatment during different incubation periods. Ejaculated semen from each subject was collected in a sample cup, divided into equal parts, and treated with CORS (1.32 mL CO gas) and without CORS for different periods of time (60-270 minutes). After different time periods, motility (A), progressive motility (B), and average velocity (VAP) (C) were analyzed by computerized sperm analysis. Sperm DNA fragmentation (SDF) was determined by microscopy (E), and sperm DNA fragmentation was determined by The system determines the static oxidation-reduction potential (sORP) (D). The numbers below the x-axis represent the subject ID. The data for Figure 7 are summarized in Figure 6 Part of the summary is in Figure 9 and 10 middle.

[0127] Figure 8: Paired measurements of individual subjects who did and did not receive CO treatment at different concentrations. Ejaculated semen from each subject was collected in a sample cup, divided into equal parts, and treated with and without different concentrations of CORS (0.44 mL, 1.32 mL, or 2.64 mL CO gas) for 90 minutes. After 90 minutes of treatment, motility (A), forward motion (B), and mean velocity profile (VAP) (C) were analyzed by computerized semen analysis. Sperm DNA fragmentation (SDF) was determined by microscopy (E), and sperm DNA fragmentation was determined by The system determines the static oxidation-reduction potential (sORP) (D). The numbers below the x-axis represent the subject ID. The data for Figure 8 are summarized in Figure 6 middle.

[0128] Figure 9: Linear correlation between sORP levels in untreated sperm samples and the effectiveness of CO treatment on the sORP levels of said sperm samples. Correlation between static redox potential (sORP) values in the untreated group and ΔsORP values (difference between untreated and treated) after incubation with CORS (15 mg CORM) for 60 minutes (A) (according to WHO guidelines) or 90 minutes (B) (maximum duration in ART laboratories), as well as during the entire 90-minute period (C) was evaluated. r: correlation coefficient; p: significance level. Measurements on the x- and y-axes are expressed as [mV / 10 6 Sperm] indicates.

[0129] Figure 10 : Linear correlation between SDF levels in untreated sperm samples and the effectiveness of CO treatment on their SDF levels. Correlation was assessed between sperm DNA fragmentation (SDF) values in the untreated group and ΔSDF values (difference between untreated and treated) after incubation with CORS (15 mg CORM) for 60 minutes (A) (according to WHO guidelines) or 90 minutes (B) (maximum duration in ART laboratories), as well as over the entire 90-minute period (C). r: correlation coefficient; p: significance level. Measurements on the x- and y-axes are expressed in [%].

[0130] Figure 11 : Schematic diagram of a side view of an exemplary treatment system according to an embodiment of the present invention. The salient (technical) features are described in detail above.

[0131] Figure 12 : A schematic diagram of a side view of an exemplary treatment system according to another embodiment of the present invention. The salient (technical) features are described in detail above.

[0132] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not restrictive.

[0133] Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, WHO laboratory manual for the examination and processing of human semen, 6th edition; WHO 2021; Madigan et al., Brock Biology of Microorganisms, 15th edition, Pearson (2018); Berg et al., Stryer Biochemie, 7th edition, Springer Spektrum (2013).

[0134] Although the present invention has been shown and described in detail in the drawings and the foregoing description, such illustration and description should be considered illustrative or exemplary rather than restrictive. However, it should be understood that changes and modifications may be made by one skilled in the art within the scope and spirit of the appended claims. In particular, the present invention covers further embodiments having any combination of features from the various embodiments described above and below.

[0135] The invention also covers all other features shown individually in the drawings, even though they may not be described in the preceding or following description. In addition, individual alternatives of the embodiments and individual alternatives of their features described in the drawings and the description may be excluded from the subject matter of another aspect of the invention.

[0136] 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 the claims. Terms such as "substantially," "approximately," and "roughly" relating to an attribute or value also precisely define the attribute or exact value, respectively. Any reference signs in the claims should not be construed as limiting the scope.

[0137] Throughout this specification, numerous references are cited, including scientific publications, patent applications, and manufacturer's manuals. The disclosures of these references, while not considered relevant to the patentability of the present invention, are hereby incorporated by reference in their entirety. More specifically, all references are incorporated by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference.

[0138] In light of the above, the present invention relates in particular to the following projects, further embodiments of which have been explained above and are also illustrated in the accompanying examples and figures:

[0139] 1. A method for preserving gametes, comprising the following steps:

[0140] a) providing / obtaining gametes or a sample comprising said gametes in a container, and

[0141] b) contacting the gametes in the container or the sample comprising the gametes with carbon monoxide.

[0142] 2. The method according to item 1, wherein the gametes or the sample containing the gametes comprise sperm or egg cells, preferably wherein the sperm are contained in semen, ejaculated semen and / or a buffer / buffer system.

[0143] 3. The method according to items 1 and 2, wherein the carbon monoxide is released from a carbon monoxide-releasing molecule and / or wherein the carbon monoxide is supplied directly to the container via a separate pipe.

[0144] 4. The method according to any one of items 1 to 3, wherein the carbon monoxide forms a gas layer on the gametes or on the sample comprising the gametes.

[0145] 5. The method according to items 3 and 4, wherein the carbon monoxide-releasing molecule is preferably a metal carbonyl compound, even more preferably a molybdenum carbonyl compound, most preferably Na3Mo(CO)3(CNCH2CO2H)3.

[0146] 6. The method according to any one of items 3 to 5, wherein the carbon monoxide-releasing molecules are contacted with FeCl3, Ce(SO4)2 or H2O2, preferably with FeCl3, thereby releasing the carbon monoxide.

[0147] 7. The method according to any one of items 1 to 6, wherein the gametes or the sample comprising the gametes is contacted with the carbon monoxide for about 15 to about 120 minutes, preferably about 30 to about 90 minutes, more preferably about 30 to about 60 minutes.

[0148] 8. The method according to any one of items 1 to 7, wherein the gametes or the sample comprising the gametes is contacted with the carbon monoxide at about 25°C to about 35°C.

[0149] 9. The method according to any one of items 1 to 8, wherein the amount of carbon monoxide applied to the gametes or the sample containing the gametes is 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.

[0150] 10. The method according to any one of items 1 to 9, wherein the gametes are human gametes.

[0151] 11. The method according to any one of items 1 to 9, wherein the gametes are gametes from cattle, horses, pigs, sheep, goats, camels, alpacas, dogs, cats, birds or fish.

[0152] 12. The method according to any one of items 1 to 11, wherein the preservation of the gametes is assessed by measuring motility, DNA fragmentation, spermatogenesis diagram and / or redox potential of the gametes.

[0153] 13. A method for reducing DNA fragmentation and / or redox potential of gametes in artificial reproductive technology, wherein the gametes or a sample containing the gametes are contacted with carbon monoxide.

[0154] 14. Use of carbon monoxide for reducing DNA fragmentation and / or redox potential of gametes in assisted reproductive technology, wherein the gametes or a sample comprising the gametes are contacted with carbon monoxide.

[0155] 15. The method according to item 13 or the use according to item 14, wherein the assisted reproductive technology comprises in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, frozen embryo replacement, preimplantation genetic testing, in vitro maturation of oocytes, frozen oocyte replacement, gamete intrafallopian transfer, fertilized egg intrafallopian transfer and / or cryopreservation.

[0156] 16. The method according to item 13 or 15, or the use according to item 14 or 15, wherein reducing DNA fragmentation and / or redox potential of the gametes maintains / enhances the motility of the gametes.

[0157] 17. The method according to any one of items 13, 15 and 16, or the use according to any one of items 14 to 16, wherein reducing DNA fragmentation and / or the redox potential of gametes reduces the risk of congenital anomalies and / or aneuploidy in artificial reproductive techniques.

[0158] 18. Carbon monoxide for use in preventing congenital anomalies and / or aneuploidy.

[0159] 19. Carbon monoxide for use in treating / reducing male infertility.

[0160] 20. Carbon monoxide for use in the treatment and / or prevention of diseases caused by / associated with elevated DNA fragmentation and / or redox potential of gametes.

[0161] 21. Carbon monoxide for use in the treatment and / or prevention of gametic diseases caused by / associated with elevated ROS levels.

[0162] 22. Carbon monoxide for use according to item 20 or 21, wherein the disease is caused by / associated with elevated stress levels, or wherein the individual to be treated is subject to stress.

[0163] 23. Carbon monoxide for use according to item 22, wherein the disease is caused by / associated with elevated levels of psychological and / or physiological stress, or wherein the individual to be treated is subject to psychological and / or physiological stress.

[0164] 24. Carbon monoxide for use according to any one of items 18 to 23, wherein the prevention of congenital anomalies and / or aneuploidy, the treatment / reduction of male infertility or the treatment / prevention of said diseases is characterized in that the gametes are exposed to carbon monoxide.

[0165] 25. Carbon monoxide for use according to item 24, wherein the gametes are contacted with carbon monoxide in vitro / extracorporeally.

[0166] 26. 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 carbon monoxide for use in item 25, wherein the gametes or the sample containing the gametes are contacted with the carbon monoxide 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 collecting the gametes in the container.

[0167] 27. Carbon monoxide for use according to item 24, wherein the gametes or the sample comprising the gametes are contacted with carbon monoxide in vivo.

[0168] 28. Carbon monoxide for use according to item 27, wherein the carbon monoxide is administered to the gametes systemically or transdermally.

[0169] 29. The method according to item 17, the use according to item 17 or the carbon monoxide according to any one of items 17 and 24 to 28, wherein the congenital anomaly is selected from congenital anomalies of the limbs, congenital anomalies of the heart, congenital anomalies of the nervous system, congenital anomalies of the gastrointestinal system and congenital anomalies of the lungs.

[0170] 30. The method according to item 29, the use according to item 29 or the carbon monoxide used according to item 29, wherein the congenital anomaly is a limb congenital anomaly selected from the group consisting of achondroplasia, ectromelia, amniotic syndrome, brachydactyly,

[0171] Cranio-clavicular dysplasia, congenital hypoplasia or agenesis, ectrodactyly, oligodactyly, phocomelia, polydactyly, polymelia, polysyndactyly, syndactyly.

[0172] 31. The method according to item 29, the use according to item 29 or the carbon monoxide used according to item 29, wherein the congenital anomaly is a congenital anomaly of the heart selected from atrial septal defect, patent ductus arteriosus, tetralogy of Fallot and ventricular septal defect.

[0173] 32. The method according to claim 29, the use according to claim 29 or the carbon monoxide used according to claim 29, wherein the congenital anomaly is a congenital anomaly of the nervous system selected from neural tube defects such as agenesis of the corpus callosum, anencephaly, Arnold-Chiari malformation, Dandy-Walker malformation, encephalocele, holoprosencephaly, hydrocephalus, anencephalocele, megalencephalocele, meningocele, myelomeningocele, microcephaly, polymicrogyria and spina bifida.

[0174] 33. The method according to item 29, the use according to item 29 or the carbon monoxide used according to item 29, wherein the congenital anomaly is a congenital anomaly of the gastrointestinal system selected from atresia, imperforate or stenosis.

[0175] 34. The method according to item 29, the use according to item 29 or the use of carbon monoxide according to item 29, wherein the congenital anomaly is a congenital anomaly of the lung, preferably wherein the congenital anomaly of the lung is congenital bronchiectasis.

[0176] Example 1

[0177] This example outlines an illustrative workflow for the method of the present invention for preserving sperm using carbon monoxide. This exemplary workflow is also shown in FIG1 .

[0178] In a first step, a carbon monoxide (CO) releasing system (CORS) comprises (i) a carbon monoxide releasing molecule (CORM, such as Na3Mo(CO)3(CNCH2CO2H)3; Mo-CORM) and (ii) a second compound such as FeCl3, Ce(SO4)2 or H2O2, preferably FeCl3, which are provided in a closed compartment (such as a capsule) that is permeable to CO gas. The (Mo-)CORM and the second compound are now not in contact with each other within the closed compartment. In a next step, sperm are harvested from / by a healthy or diseased subject / patient and transferred to a container. Simultaneously, the (Mo-)CORM is brought into contact with the second compound (such as an aqueous solution of FeCl3, Ce(SO4)2 or H2O2) within the closed compartment / capsule by applying a force to both ends of the longitudinal axis of the capsule ("activated CORS"). This causes the carbon monoxide gas to be released from the (closed) compartment / capsule. The closed compartment that releases carbon monoxide gas is then added to the container containing the sperm sample. The container is sealed, the sample is incubated for a given time (preferably 25-35°C, 30-60 minutes) and transferred to the assisted reproductive technology (ART) laboratory. During incubation, liquefaction occurs naturally under given conditions. Liquefaction in this article describes the process of breaking down the gel formed by proteins of the seminal vesicles and prostate to make semen / ejaculate more fluid. Finally, the CORS (= closed compartment containing CORM, such as Mo-CORM and FeCl3, Ce(SO4)2 or H2O2, preferably FeCl3) is removed from the container and the sample is further processed for ART or cryopreservation.

[0179] The dosage (the amount of CORS added / the amount of Mo-CORM) depends on the Mo-CORM loading of the system and the maximum amount of CO that can be 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. A detailed description of the size and loading of Mo-CORM and FeCl3 is described in PCT / EP2020 / 078794, which is published as WO 2021 / 074159A1. Further description can be found in EP22216317.2, filed on December 23, 2022, and in PCT / EP2023 / 074808, filed on September 8, 2023.

[0180] Example 2

[0181] Example 2 demonstrates that sperm properties (such as redox potential, DNA fragmentation, and / or sperm motility, particularly forward motility) are improved when exposed to carbon monoxide gas, compared to "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 middle.

[0182] method

[0183] In this example, a CORS capsule (18-22 mm in length, 6-8 mm in diameter) filled with 15 mg of Mo-CORM and 150 μl of aqueous FeCl 3 solution (583.3 mg / mL) was used to store 0.7 mL to approximately 2.9 mL of sample in a standard 15 mL Falcon tube.

[0184] CORS, Mo-CORM and FeCl3 were prepared as follows:

[0185] Preparation of CORS

[0186] CORS was produced by additive manufacturing. Two compartments separated by a diaphragm contained in one of the compartments were 3D printed. The compartment containing the FeCl3 aqueous solution was printed in a drop-on-demand manner (multi-jet modeling process) using an Objet Eden 350 printer (96 nozzles, droplet size 40 μM, resolution X = 600 dpi, Y = 600 dpi; Stratasys, Deden Prairie, Minnesota, USA). As a resin, the photopolymer VeroBlackPlus (Stratasys, DedenPrairie, Minnesota) was used, and the structure was cured by UV polymerization at 365 nm using a UV lamp. In addition, a water-soluble support structure (SUP705, Stratasys, Deden Prairie, Minnesota) was used, which was removed by a washing step (drinking water; isopropyl alcohol) after the printing process.

[0187] The compartments containing Mo-CORM were produced by laser sintering (LS) using polyamide PA2200 (EOS GmbH, Krailing, Germany) as the material and processed by a Formiga P110 3D printer (EOS GmbH, Krailing, Germany) including 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 (Helmbrechst, Germany). CAD software (VISI 2019 & 2020, Vero UK, Cheltenham, UK) was used for production-ready design.

[0188] Preparation of Mo-CORM

[0189] The synthesis of trisodium tricarbonyl-[tris(isocyanoethyl acetate)]molybdenum (Na3Mo(CO)3(CNCH2CO2)3, Mo-CORM) was modified from a previous method (Achatz, D et al. Zeitschrift für anorganische undallgemeine Chemie 2005, 631(12), 2339-2346). Briefly, 2.64 g (10.0 mmol) of molybdenum hexacarbonyl was dissolved in 35 mL of anhydrous acetonitrile (99.8%; 90°C; 22 h) under dry and inert conditions to obtain the intermediate acetonitrile complex. Then, 4.7 mL of ethyl isocyanoacetate (3.5 eq.; 35 mmol; 4.86 g) was added at 55°C to displace the acetonitrile and obtain the complex. The ester was then hydrolyzed with NaOH (16 eq.) in 20 mL of tetrahydrofuran at room temperature. The free acid was formed by the addition of aqueous HCl. Finally, NaOH ethanol solution (5 mmol NaOH / 1 mmol Mo-CORM) was added to obtain the trisodium salt. All reagents were purchased from Sigma Aldrich (Schnelldorf, Germany) and used without further purification.

[0190] Preparation of FeCl3 aqueous solution

[0191] FeCl3*6H2O was purchased from Sigma Aldrich (Schnelldort, Germany) and prepared into an aqueous solution with a concentration of 583.3 mg / mL using deionized water.

[0192] Sperm analysis

[0193] Between 1.46 mL and 5.7 mL of human semen / sperm were collected from healthy men aged 25 to 50 years and placed in 100 mL sample bottles on site. The samples were then divided into two equal parts, each for subsequent incubation with CO2 (wCO2) and without CO2 (woCO2). To estimate the sperm concentration in the samples and establish an initial baseline for each sample, a routine spermatography analysis was performed before the samples were transferred to 15 mL gas-tight Falcon tubes. The samples were incubated at 25-35°C in a sealed container with and without an activated CORS system for 90 minutes. During this incubation period, liquefaction occurred naturally. The samples were then analyzed for total motility, forward motility, static redox potential, and DNA fragmentation.

[0194] Total motility, progressive motility, and concentration of the samples were evaluated using the CEROS II (Hamilton Thorne, Beverly, Massachusetts) computer-assisted sperm analysis (CASA) system. A 6-μL sperm sample was diluted 1:1 with gamete and embryo processing medium (containing albumin) (MHM-C, FujiFilm / Irvine Scientific, Santa Ana, California), and concentration and motility levels (total and progressive motility) were assessed.

[0195] Static redox potential (sORP) levels were determined using a male infertility oxidation system (MiOXSYS, Englewood, CO). 30 μL of sperm sample was transferred to the sample application port on the MiOXSYS sensor and analyzed for 2 minutes.

[0196] To determine DNA fragmentation, count-based microscopic analysis was performed. For this purpose, sperm samples were diluted to a maximum of 20xE6 sperm per mL with MHM-C (Gamete and Embryo Processing Medium (with albumin), FujiFilm, Irvine Scientific) and analyzed using the Halo Sperm G2 Kit (Halotech, Madrid, Spain). The samples were mixed with the prepared agarose according to the manufacturer's instructions. 8 μL of the resulting mixture was then placed on a microscope slide and cooled for 5 minutes by removing the heat source. The prepared samples were further processed according to the manufacturer's instructions: denaturant treatment for 7 minutes, lysis solution treatment for 20 minutes, distilled water treatment for 5 minutes, 70% ethanol treatment for 2 minutes, 100% ethanol treatment for 2 minutes, eosin staining solution for 7-10 minutes, and thiazide staining solution for 7-10 minutes. At least 300 sperm were then analyzed using bright field microscopy.

[0197] result

[0198] 1. (Static) Oxidation Reduction Potential (sORP) Level

[0199] Figure 2 The “Relative Redox Potential” data points in the table are derived from the following components of each test subject: sORPwCO 受试者n / sORP woCO 受试者n , where sORP woCO 受试者nis set to 1. As a measure of oxidative stress (caused by ROS, etc.) in sperm (spermatozoa), the sORP data show a trend toward lower levels in CO2-exposed samples (wCO) compared to samples not exposed to CO (woCO). This is evident from the position of the data points, four of which lie below the dashed woCO = 1 reference line. Therefore, without being bound by theory, these results suggest that CO2 exposure results in reduced levels of reactive oxygen species (ROS) in sperm compared to sperm not exposed to CO ( Figure 2 ).

[0200] 2. DNA breakage

[0201] Figure 3 The “Relative DNA Fragmentation” data points shown in the table are derived from the following fractions of DNA fragmentation wCO for each subject: 受试者n / DNA fragmentationwoCO 受试者n , while DNA breaks woCO 受试者n Set to 1. Figure 3 It is clear from the graph that DNA fragmentation is reduced in samples exposed to CO compared to samples not exposed to CO. This is evident from the position of the data points, which all lie below the dashed woCO = 1 reference line.

[0202] 3. Sperm motility (total and forward movement)

[0203] Figure 4 The "Relative Total Motion" or "Relative Forward Motion" data points shown in A and B, respectively, are derived from the following scores for each subject:

[0204] a) For relative total motion: total motion wCO 受试者n / Total Movement woCO 受试者n

[0205] b) For relative forward motion: forward motion wCO 受试者n / Forward movement woCO 受试者n ,

[0206] The total motion / forward woCO are set to 1. Although for the total motion (most of the data points are located near the dashed woCO=1 reference line, see Figure 4 A) No differences were observed between samples with CO exposure (wCO) and without CO exposure (woCO), but forward locomotion was increased in wCO compared to woCO (this is evident from the fact that, with the exception of subject 6, all data points lie on or above the dashed woCO = 1 reference line, see Figure 4 B).

[0207] In this context, total motility refers to the percentage of sperm that perform any type of movement. This movement can include non-progressive movement. Progressive motility refers to sperm swimming primarily in straight lines or large circles and can therefore be an important predictor of ART cycle success.

[0208] The forward motility of sperm incubated with CO was increased compared to sperm not incubated with CO.

[0209] In summary, the results indicate that exposure of sperm to CO leads to desirable / improved characteristics of gametes (such as reduction in oxidative stress / redox potential / ROS levels ( Figure 2 ) and reduction of DNA fragmentation ( Figure 3 ) and their improvement in forward motion ( Figure 4 B)). All measured parameters are again in Figure 5 is fully shown in . Figure 5 For each measurement the parameters / characteristics are shown, e.g. Figures 2 to 4 The mean of the data points shown in , where the respective reference level of the samples not in contact with CO (woCO) was again set to 1 (dashed line). Figure 5 As can be clearly seen in the figures, exposure of sperm to CO results in (i) reduced oxidative stress / ROS (“ORP”; mean values below the dashed reference line indicate mean wCO values less than mean woCO values), (ii) reduced DNA fragmentation (mean values below the dashed reference line indicate mean wCO values less than mean woCO values), and (iii) improved forward motility (mean values above the dashed reference line indicate mean wCO values greater than mean woCO values).

[0210] Thus, the present inventors have surprisingly discovered that carbon monoxide (CO) can lead to the preservation of sperm when in contact with gametes such as sperm. As a result of the gametes being incubated with CO, these sperm exhibit desirable characteristics (compared to sperm not exposed to CO) and are therefore particularly useful for ART.

[0211] Example 3

[0212] Example 3 demonstrates that sperm motility parameters (such as DNA fragmentation and reduction in redox potential) are improved in response to different CO / Mo-CORM protocols (CO incubation duration and CO / Mo-CORM load). In addition, this example demonstrates that sperm motility (such as total motility, forward motility, and mean path velocity (VAP)) is maintained in response to different CO / Mo-CORM protocols (CO incubation duration and CO / Mo-CORM load). Sperm from the same subject were treated with CO gas or remained untreated as "untreated" controls. The duration of CO treatment (60 min, 90 min, 180 min, or 270 min) and CO / Mo-CORM load (0.44 min, 1.32 min, or 2.64 mL of CO, corresponding to 5 mg, 15 mg, or 30 mg Mo-CORM) were varied. The results of this example are shown in the accompanying Figures. Figures 6 to 10 middle.

[0213] Figure 6 The ratio of the respective measurements of CO-treated and untreated sperm samples obtained from the same subject is depicted. Here, no effect of CO treatment corresponds to a value of 1, as indicated by the dashed horizontal line. Any value above the dashed line corresponds to an increase in the corresponding measurement by CO treatment compared to the untreated control, while any value below the dashed line corresponds to a decrease in the corresponding measurement by CO treatment compared to the untreated control. Figures 7 and 8 show Figure 6 In Figures 7 and 8, each bar corresponds to a single measurement, where the adjacent white and black bars correspond to paired CO-treated and non-CO-treated sperm samples obtained from the subject, respectively. Figure 6 Each point in corresponds to the ratio of the paired CO-treated and untreated samples shown in Figures 7 and 8. Therefore, Figures 7 and 8 clearly confirm that the Figure 6 Observations made. Figures 7 and 8, panels D and E, respectively, depict measurements of redox potential and DNA fragmentation in sperm samples from individual subjects. High variations in redox potential and DNA fragmentation can be observed between untreated sperm samples from different subjects, clearly demonstrating that, among the subjects tested, sperm from several individuals experienced particularly high levels of oxidative stress when untreated. Figures 7 and 8 further clearly demonstrate that CO treatment was able to reduce redox potential and DNA fragmentation in these subjects. This was particularly true for sperm samples treated for 60 or 90 minutes. Figure 9 and 10 Correlation analysis of the CO treatment effect (difference between treated and untreated samples) with the respective measurements of untreated sperm samples is described. Figure 9 and 10 CO treatment proved particularly effective in subjects suffering from high sperm redox potential and / or DNA fragmentation.

[0214] method

[0215] In this example, sperm samples were treated with one, three, or six CORS capsules, each containing 5 mg Mo-CORM (equivalent to 0.44 mL CO), for 60-270 minutes, or left untreated for the same period of time.

[0216] Using these materials, CORS and Mo-CORM were prepared as follows: FeCl3 (prepared as described in Example 2) was used for Mo-CORM activation.

[0217] Material

[0218] All chemicals required for the synthesis and activation of Mo-CORM, including molybdenum hexacarbonyl, ethyl isocyanoacetate, acetonitrile, anhydrous tetrahydrofuran, sodium hydroxide (pa), hydrochloric acid (pa), anhydrous ethanol, FeCl₃⁻¹H₂O, and nitric acid (65%, pa), were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Gamete and embryo handling medium (containing albumin) (MHM) for sperm dilution was purchased from Irvine Scientific (Santa Ana, CA). 270 ppm CO calibration gas was purchased from Linde AG (Munich, Germany). Polyamide (PA2200) and duroplast photopolymer MED610+VeroBlackPlus (RGD875) were provided by EOS GmbH (Krailling, Germany) and Stratasys Ltd. (Recovot, Israel), respectively. Silicone R 6.65 × 0.4 mm was purchased from RAUMEDIC AG (Helmbrechts, Germany). SF332K-Silicone (mixing ratio 1:1, viscosity before mixing: 7000-8000 cP, stiffness after mixing: 33 ShA, density 1.11 g / cc, breaking point: 4.7 N / mm 2 ) and SF45 2K-silicone (mixing ratio 1:1, viscosity before mixing: 8500 cP (at 23°C), stiffness after mixing: 45 ShA, density: 1.12 g / cc, breaking point: 3.5 ± 0.5 N / mm 2) were purchased from Silikonfabrik (Ahrensburg, Germany). All casting molds were constructed from polytetrafluoroethylene (Vink, Germany) on a neo CNC machine (Datron AG, Germany). Loctite SI 5248, Loctite 4902, Loctite HY 4011, and Loctite SF7701 were purchased from Henkel (Düsseldorf, Germany). Needle 0.9*40mm and U100 insulin needles 0.3*8 mm were purchased from B. Braun (Melsungen, Germany). Unless otherwise stated, all other reagents were purchased from Sigma Aldrich Chemie GmbH and were of at least pharmaceutical grade.

[0219] CORS Manufacturing

[0220] All parts are molded using custom-designed Teflon molds. After filling the mold, vacuum is applied for 5 minutes to extract any air bubbles. The casting is then refilled to the brim and vacuum is applied for another 5 minutes. The respective casting is then pressed into silicone and secured with a clamp. After drying at 40°C for 24 hours, the mold is separated from the casting using compressed air. The container and lid are cast separately. The container is filled with Mo-CORM. In some cases, the lid and upper rim of the container are then prepared by applying an adhesive primer. Adhesive is then applied and the two parts are pressed together. Moving the two parts relative to each other in a circular motion ensures even distribution of the adhesive.

[0221] Synthesis of Mo-CORM

[0222] The CORM trisodium tricarbonyl-[tris(isocyanoethyl acetate)]molybdenum (Na3Mo(CO)3(CNCH2CO2)3, Mo-CORM) was synthesized as previously published. Briefly, molybdenum hexacarbonyl was stirred in anhydrous acetonitrile to exchange three of its ligands for acetonitrile. These ligands were exchanged for the EICA ligand. Purification and cation exchange afforded Mo-CORM as a white solid.

[0223] Sperm preparation

[0224] Human sperm was collected from healthy volunteers in 100 mL sample containers. Ejaculated semen was then aliquoted into 15 mL gas-tight Falcon tubes, and one sample was exposed to one or more activated CORS. After a predetermined time, both samples were analyzed for motility parameters using computer-assisted sperm analysis (CASA), static redox potential (sORP) using MiOXSYS, and sperm DNA fragmentation (SDF) using a diffusion assay using the Halosperm G2 Kit.

[0225] Computer-Assisted Sperm Analysis (CASA)

[0226] The samples were tested for total motility, progressive motility, VAP, and concentration using the CEROS II (Hamilton Thorne, Beverly, MA) computer-assisted sperm analysis (CASA) system. Six μL of sperm samples diluted 1:1 with Gamete and Embryo Processing Medium (with albumin) (MHM-C, Fuji Film / Irvine Scientific, Santa Ana, CA) were analyzed using the CASA system and assessed for motility.

[0227] Static Oxidation Reduction Potential (sORP) Analysis

[0228] Static redox potential (sORP) levels were determined using an oxidative male infertility measurement system (MiOXSYS, Englewood, CO) according to the manufacturer's instructions. A 30 μL sample was transferred to the sample application port on the MiOXSYS sensor and analyzed for 2 minutes. Results were expressed in millivolts (mV) and normalized to semen sperm concentration (mV / 10 6 sperm / mL).

[0229] DNA fragmentation (SDF)

[0230] Sperm DNA fragmentation (SDF) was assessed by diffusion testing using the Halosperm G2 Kit (from Halotech, Madrid, Spain) according to the manufacturer's instructions. Briefly, the sample was diluted to a maximum of 20 mIo / mL. The sample was then mixed with the prepared agarose. 8 μL of the mixture was transferred to a glass slide and allowed to cool for 5 minutes. Different reagents were added and discarded after a set time: denaturant was added for 7 minutes, lysis solution for 20 minutes, distilled water for 5 minutes, 70% ethanol for 2 minutes, 100% ethanol for 2 minutes, eosin staining solution for 7-10 minutes, and thiazide staining solution for 7-10 minutes. A minimum of 400 spermatozoa were analyzed using an Olympus IX73 inverted LED fluorescence microscope.

[0231] result

[0232] This study investigated the effects of CO released from a CO release system (CORS) on sperm motility and motility parameters using an experimental design based on varying amounts of CO gas (0.44–2.64 mL) and effective incubation times (60–270 minutes). Based on the fact that sperm cells require a certain balance of reactive oxygen species (ROS) to maintain their motility, a key finding was that exposure to CO at concentrations of 0.44–2.64 mL CO did not impair sperm motility, progressive movement, or velocity during extended treatment times of up to 90 minutes compared to untreated sperm. In the same approach, the redox potential (sORP) and DNA fragmentation (SDF) were evaluated. According to WHO guidelines, liquefaction and processing should ideally be performed within the first 60 minutes. However, this process can take longer than 60 minutes. Therefore, longer time periods were considered when considering CO treatment.

[0233] 1. Sperm motility (total motion, forward motion, and average path speed)

[0234] Sperm samples were exposed to three CORS or no CORS as a control (15 mg Mo-CORM; 1.32 mL CO) and tested at different exposure durations including 60-min, 90-min, 180-min, and 270-min ( Figure 6 , 7, 9 and 10). At 60 minutes, the motility increased by 1.13 ± 0.41 times, at 90 minutes, the motility increased by 0.99 ± 0.03 times, at 180 minutes, the motility increased by 1.16 ± 0.37 times, and at 270 minutes, the motility increased by 0.94 ± 0.20 times. Forward motility increased by 0.85 ± 0.13 times at 60 minutes, by 0.92 ± 0.17 times at 90 minutes, by 0.72 ± 0.19 times at 180 minutes, and by 1.04 ± 0.48 times at 270 minutes. CASA was also used to measure the velocity of sperm cells, which increased by 0.94 ± 0.09 times at 60 minutes, by 0.96 ± 0.04 times at 90 minutes, by 0.79 ± 0.13 times at 180 minutes, and by 0.97 ± 0.08 times at 270 minutes ( Figure 6 A). To determine the effects of varying doses, we selected 90 minutes as the exposure time for additional experiments. Here, sperm samples were exposed to one, three, six, or no CORS as controls (5 mg, 15 mg, 30 mg, or 0 mg Mo-CORM, corresponding to 0.44 mL, 1.32 mL, 2.64 mL, or 0 mL CO, respectively; Figure 6and 8). Six is the upper limit of the CORS that the device can accommodate at one time. The change in motion is 0.98 ± 0.12 times for 0.44 mL and 0.90 ± 0.12 times for 2.64 mL, while the change in forward motion is 0.97 ± 0.12 times for 0.44 mL and 0.88 ± 0.10 times for 2.64 mL. The change in VAP is 1.01 ± 0.08 times for 0.44 mL and 1.03 ± 0.03 times for 2.64 mL ( Figure 6 B).

[0235] Figure 7 clearly confirms that Figure 6 These observations made.

[0236] 2. Sperm motility parameters (redox potential and DNA fragmentation)

[0237] During the same process, redox potential and DNA fragmentation were evaluated. The effect of CO on redox potential showed a change of 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. DNA fragmentation changes were 0.77 ± 0.07-fold, 0.67 ± 0.24-fold, 0.90 ± 0.11-fold, and 0.90 ± 0.19-fold at 60 minutes, 90 minutes, 180 minutes, and 270 minutes, respectively. Figure 6 C). For the dose-varying samples, we observed changes in redox potential of 0.89 ± 0.17-fold for 0.44 mL and 0.59 ± 0.24-fold for 2.64 mL. DNA-fragmentation changed to 0.83 ± 0.09-fold for 0.44 mL and 1.31 ± 0.55-fold for 2.64 mL ( Figure 6 D). In summary, consistent with the WHO protocol, after 60 minutes, a redox potential change of approximately 0.57 ± 0.35-fold (1.32 mL) was observed. In addition, DNA fragmentation showed a change of approximately 0.77 ± 0.07-fold (1.32 mL). At an extended time of 90 minutes, a redox potential change of 0.91 ± 0.12-fold (1.32 mL) was observed. DNA fragmentation became 0.67 ± 0.24-fold (1.32 mL).

[0238] In summary, the results demonstrate that the CO / Mo-CORM protocols tested in the present invention (CO incubation duration and CO / Mo-CORM loading) improve sperm motility parameters. Namely, redox potential and sperm DNA fragmentation (SDF) were reduced at 60 and 90 minutes CO incubation time and 0.44 mL and 1.32 mL CO. This confirms that Figure 2 、 3and the results described in 5. Therefore, with regard to sperm motility parameters, incubation durations of 60 min or 90 min with 0.44 mL or 1.32 mL CO may be preferred protocols.

[0239] In conclusion, incubation times of 60 or 90 min with 0.44 or 1.32 mL CO (corresponding to 5 or 15 mg Mo-CORM and 1 or 3 CORS, respectively) may be preferred because they have no negative effect on sperm motility, yet decrease sperm DNA fragmentation and redox potential. Figure 6 The results in Figures 4 and 5 clearly indicate that incubation times of 60 or 90 min with 0.44 or 1.32 mL of CO (corresponding to 5 or 15 mg of Mo-CORM and 1 or 3 CORS, respectively) may be preferred protocols as they improve sperm motility parameters without negatively affecting sperm movement.

[0240] Furthermore, the 60-minute incubation duration is fully compatible with routine laboratory procedures in IVF laboratories following the WHO guidelines (WHO laboratory manual for the examination and processing of human semen, World Health Organization, 2021). Therefore, this treatment has the potential to improve the success rate of artificial reproductive technology (ART) applications (e.g., in vitro fertilization; IVF) that are limited by high sperm DNA fragmentation and redox potential.

[0241] Thus, the present inventors have surprisingly found that carbon monoxide (CO) when in contact with gametes, such as sperm, results in the preservation of sperm, particularly when the sperm are contacted for 60 minutes or 90 minutes with 0.44 mL or 1.32 mL CO. Thus, sperm that have been contacted for 60 minutes or 90 minutes with, in particular, 0.44 mL or 1.32 mL CO, exhibit desirable characteristics (compared to sperm that have not been contacted with CO) and are therefore particularly suitable for ART.

[0242] 3. Effect of CO treatment on sperm motility from subjects with reduced sperm motility

[0243] Panels D and E of Figures 7 and 8 depict measurements of redox potential and DNA fragmentation for untreated / CO-treated sperm samples from individual subjects. Here, a high degree of variability in redox potential and DNA fragmentation can be observed between untreated sperm samples from different individuals, clearly demonstrating that among the test subjects, the sperm of several subjects were subjected to particularly high oxidative stress during their untreated periods. Here, in particular, untreated sperm samples 42_1 and 38_7 showed significantly elevated redox potential and DNA fragmentation levels compared to the majority of samples. It is conceivable that these samples were from subjects with infertility / sub-average fertility. CO treatment caused a substantial reduction in the redox potential and DNA fragmentation values for samples 42_1 and 38_7. This clearly demonstrates that CO treatment can help subjects with severely impaired sperm motility parameters. Therefore, it is conceivable that CO treatment could be used to treat / prevent (gamete) diseases characterized by elevated redox potential and / or DNA fragmentation in gametes. This includes, in particular, treating or reducing male infertility. Furthermore, it is conceivable that CO treatment of gametes may prevent congenital abnormalities and / or aneuploidy in any offspring derived from the CO-treated gametes.

[0244] Figure 9 and 10 The effectiveness of CO treatment on redox potential and DNA fragmentation, respectively, was further confirmed. These figures depict the effect of CO treatment (difference between treated and untreated samples; y-axis) in correlation with the corresponding measurements for untreated sperm samples (x-axis). Both figures focus on incubation durations of 60 and 90 minutes, as these protocols previously demonstrated significant effects of CO treatment (Figure 7). Figure 9 and 10 CO treatment proved particularly effective in subjects suffering from high sperm redox potential and / or DNA fragmentation.

Claims

1. A method for preserving gametes, comprising the following steps: a) providing / obtaining gametes or a sample comprising said gametes in a container, and b) contacting the gametes in the container or the sample comprising the gametes with carbon monoxide.

2. The method according to claim 1, wherein said gametes or said sample comprising said gametes comprises sperm or egg cells, preferably wherein said sperm are contained in semen, ejaculated semen and / or a buffer / buffer system.

3. The method according to claim 1 and 2, wherein the carbon monoxide is released from a carbon monoxide releasing molecule and / or wherein the carbon monoxide is supplied directly to the container via a separate pipe.

4. The method according to any one of claims 1 to 3, wherein the carbon monoxide forms a gas layer on the gametes or on the sample comprising the gametes.

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

6. The method according to any one of claims 3 to 5, wherein the carbon monoxide-releasing molecules are contacted with FeCl3, Ce(SO4)2 or H2O2, preferably with FeCl3, thereby releasing the carbon monoxide.

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

8. The method according to any one of claims 1 to 7, wherein the gametes or the sample comprising the gametes are contacted with the carbon monoxide at a temperature of about 25°C to about 35°C.

9. The method according to any one of claims 1 to 8, wherein the amount of carbon monoxide applied to the gametes or the sample containing the gametes is 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. The method according to any one of claims 1 to 9, wherein the gametes are gametes from cattle, horses, pigs, sheep, goats, camels, alpacas, dogs, cats, birds or fish.

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

13. A method for reducing DNA fragmentation and / or redox potential of gametes in artificial reproductive technology, wherein the gametes or a sample containing the gametes are contacted with carbon monoxide.

14. Use of carbon monoxide for reducing DNA fragmentation and / or redox potential of gametes in assisted reproductive technology, wherein the gametes or a sample comprising the gametes are contacted with carbon monoxide.

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

16. The method according to claim 13 or 15, or the use according to claim 14 or 15, wherein reducing DNA fragmentation and / or redox potential of the gametes maintains / increases the motility of the gametes.

17. The method according to any one of claims 13, 15 and 16, or the use according to any one of claims 14 to 16, wherein reducing DNA fragmentation and / or the redox potential of the gametes reduces the risk of congenital anomalies and / or aneuploidy in artificial reproductive techniques.

18. Carbon monoxide for use in preventing congenital anomalies and / or aneuploidy.

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

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

21. Carbon monoxide for use in the treatment and / or prevention of gametic diseases 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 wherein the individual to be treated is subject to stress.

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

24. Carbon monoxide for use according to any one of claims 18 to 23, wherein the prevention of congenital anomalies and / or aneuploidy, the treatment / reduction of male infertility or the treatment / prevention of said diseases is characterized in that the gametes are exposed to carbon monoxide.

25. Use of carbon monoxide according to claim 24, wherein the gametes are contacted with carbon monoxide in vitro / extracorporeally.

26. 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 carbon monoxide according to claim 25, wherein the gametes or the sample comprising the gametes are contacted with the carbon monoxide 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 collecting the gametes in the container.

27. Carbon monoxide for use according to claim 24, wherein said gametes or said sample comprising said gametes are contacted with carbon monoxide in vivo.

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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