Methods for improving sperm function and fertility for assisted reproductive technology

Abstract

The present disclosure provides methods for improving sperm function and fertility for assisted reproductive technologies. In some embodiments, the methods provided by the present disclosure involve sequential changes in temperature, bicarbonate concentration, albumin concentration, and calcium ionophore.

Description

[Technical Field]

[0001] Male factors account for approximately 50% of couples' difficulty in conception. An important aspect of assisted reproduction is obtaining optimal function of male gametes (sperm) to assist in the fertilization process and maximize the availability of embryos for implantation. Therefore, there is a need for media, compositions, and methods for enhancing sperm function, and several approaches have been attempted, as detailed below.

[0002] A. Procedures for Isolating the Best Sperm Subpopulation from a Sample. This procedure is intended to enrich a sample for competent sperm. Most of these widely used methods select sperm based on sperm motility. Examples of these methods include discontinuous gradient centrifugation, swim-up, and the use of microfluidic devices. These methods are widely accepted in assisted reproductive technology (ART) clinics. Recently, Serafin Perez-Cerezales et al. (2018) reported that a high-quality sperm subpopulation was selected in vitro by thermotaxis and showed that this subpopulation had significantly higher DNA integrity and less chromatin compaction compared to raw sperm samples. In mice, these sperm produced more and better embryos through intracytoplasmic sperm injection (ICSI), doubling the number of successful pregnancies. ICSI procedures face the challenge of selecting sperm from highly heterogeneous samples. Therefore, these procedures, which enrich for populations containing competent sperm, often yield better results. However, these procedures do not improve sperm function and only isolate sperm with high fertilizing capacity.

[0003] B. Procedures intended to predict fertilization outcomes.

[0004] 1. A study by Alvarez et al. (1996) reported a method in which human sperm were subjected to heat stress to determine whether their motility increased. The results of this procedure were later correlated with in vitro fertilization (IVF) results performed using a second semen sample from the same patient. This procedure led to the introduction of a predictive tool into the clinic, which showed that patients whose sperm sample showed improved motility upon heat stress had a higher chance of fertilization.

[0005] 2. A study by Mann et al. (2002) aimed to correlate DNA integrity with sperm fertility index after heat stress. The authors showed that heat damage correlated with poor sperm characteristics.

[0006] C. A procedure for inducing an asymmetric type of sperm motility, termed hyperactivation (HA), by high-temperature incubation.

[0007] 1. Si et al. (1997). In this study, the authors investigated the response of hamster sperm to incubation at temperatures ranging from 22°C to 40°C for 3.5 hours to observe temperature-dependent HA, with higher HA being obtained at higher temperatures. This type of prolonged stress heat typically promotes DNA damage derived from reactive oxygen species.

[0008] 2. Keepler et al. (1999). In this study, the authors showed that when two halves of a frozen sperm straw were simultaneously incubated, one at 40°C and the other at 37°C, the warmer half accumulated more sperm and had higher HA levels. These results are consistent with the high HA induced at 40°C and the thermotactic effect of increasing the temperature from 37°C to 40°C.

[0009] 3. Chan et al. (1998). The authors reported that incubating human sperm at 40°C for up to 4 hours as a test to assess sperm fertilization potential improved sperm HA and increased their ability to penetrate zona-less eggs.

[0010] 4. Kucuk et al (2008) In this report, the authors showed that incubating human sperm at 40°C for 2 hours resulted in a higher percentage of motile sperm and higher pregnancy rates after intrauterine insemination (IUI) compared to sperm incubated at 37°C.

[0011] 5. Chan et al. (1998). In this study, the authors reported that high HA at 40°C correlated with IUI success (7 pregnancies out of 44 patients) when samples were processed using standard procedures at standard temperatures. This procedure has been proposed as a test to predict IUI success.

[0012] D. Calcium (Ca) to enhance sperm motility and function 2+ ) Procedures using ionophores.

[0013] 1. Tateno et al. (2012). In this paper, the authors reported that high concentrations (10-20 μM) of Ca 2+ demonstrated that application of the ionophore A23187 for 10 min immobilizes mouse sperm. After removal of the ionophore, mouse sperm acquire HA. Notably, human sperm do not regain motility after ionophore treatment, a clear difference between species. Furthermore, Sanchez Cardenas et al. (2018) demonstrated that 5–10 μM A23187 increases intracellular Ca. 2+ Substantially increasing concentrations of HA immobilized sperm within minutes, whereas lower concentrations (0.5 and 1 μM) induced sperm HA without immobilization. This is due to the increased intracellular Ca 2+ This is consistent with previous findings showing that a concentration threshold switches motility on and off.

[0014] 2. Navarrete et al. (2016). In this paper, the authors treated mouse sperm with high concentrations (10-20 μM) of Ca. 2+ CatSperT was isolated by treatment with ionophore A23187 for 10 minutes followed by subsequent removal. - / - , Slo3- / - and Adcy10 - / - We demonstrated that the infertility observed in sperm from knockout (KO) strains was overcome.

[0015] E. Other methods to improve sperm function.

[0016] 1. Navarrete et al. (2019) showed that mouse sperm ceased to move when energy was restricted in the incubation medium. When energy substrates were reintroduced to the medium, sperm motility was restored. After recovery, a significantly higher percentage of starved sperm acquired hyperactive motility, demonstrating improved in vitro fertilization capacity, when compared with sperm continuously incubated in standard capacitation medium. This technique (WO 2017173391(A1)) has been shown to have no effect on the induction of events associated with capacitation in human sperm, as further shown by Marin Briggiler et al. (2021).

[0017] The method described herein involves temperature incubation and intracellular sperm Ca 2+ The method is based on the synergistic effect of HA and blastocyst formation rate, achieved by appropriately manipulating both factors. HA is an important parameter when analyzing IVF outcomes (Wiser et al., 2014). It has been shown that patients with increased HA had a 93.3% higher fertilization rate compared to 64% in the group without increased HA. More importantly, this method improves the rate of embryo development to the blastocyst stage, resulting in a higher blastocyst score, which directly correlates with a better pregnancy rate. Summary of the Invention

[0018] i) In some embodiments, provided herein are methods for inducing improved sperm function, comprising: (a) controlling temperature, bicarbonate ions (HCO3 - ) concentration, albumin concentration and Ca 2+(b) the mammalian sperm provided by (a) is suitable for use in ART, comprising incubating mammalian sperm under specific conditions of an ionophore, thereby inducing improved sperm function compared to suitable control sperm.

[0019] ii) In some embodiments, the method is performed in vitro. In some embodiments of the method, step (b) is performed in vivo in the reproductive tract of a female subject by IUI of mammalian sperm according to step (a).

[0020] iii) In some embodiments, improved sperm function comprises improved motility as measured by computer-assisted semen analysis (CASA). In some embodiments, improved motility comprises an increase in the curvilinear velocity (VCL) of mammalian sperm, as well as an increase in the percentage of hyperactivated sperm. In some embodiments, improved sperm function comprises an increase in mammalian sperm capacitation as measured by an improved ability to fertilize an egg. In some embodiments, improved sperm function comprises the production of embryos with increased survival and / or improved implantation compared to embryos produced using suitable control sperm, or an improved ability of an embryo to develop to at least the two-cell stage, blastocyst stage, or fetus compared to embryos produced using suitable control sperm.

[0021] iv) In some embodiments, the mammalian sperm are human, non-human primate, porcine, bovine, equine, ovine, canine, feline, or murine sperm. In some embodiments, the mammalian sperm are human sperm from normozoospermic, subfertile, oligozoospermic, teratozoospermic, or asthenozoospermic men.

[0022] v) In some embodiments, the one or more sperm functions are selected from VCL, head swing amplitude (ALH), sperm capacitation, percentage of superactivated sperm. In some embodiments, the albumin is from human, bovine, or other species, or a synthetic serum replacement.

[0023] vi) In some embodiments, the temperature used after incubation of the ionophore is between 37.5°C and 45°C, preferentially between 39°C and 44°C, more precisely between 40°C and 43°C, which can be adjusted depending on the species.

[0024] vii) In some embodiments, the Ca 2+ The ionophore may vary depending on the species and may be 4-bromo-A23187 (4-Br-A23187), A23187, ionomycin, or Ca 2+ Intracellular Ca, including but not limited to ionophore IV 2+ This includes any means of temporarily increasing the concentration pharmacologically. [Brief explanation of the drawings]

[0025] [Figure 1] Figures 1A-1D show how changes in incubation temperature and intracellular Ca2+ concentration in human sperm synergistically affect sperm motility parameters. In the control treatment, sperm were incubated in the presence of 0.2% DMSO for 2 min and then incubated at 37°C for an additional 30 min. In the temperature treatment, sperm were incubated in the presence of 0.2% DMSO for 2 min, followed by incubation at 40°C for 30 min. In the ionophore treatment, sperm were incubated in the presence of 5 μM 4-Br-A23187 (0.2% DMSO) for 2 min, followed by incubation at 37°C for 30 min. In the combined treatment, sperm were incubated in the presence of 5 μM 4-Br-A23187 for 2 min, followed by incubation at 40°C for 30 min. Total motility (Figure 1A), head movement amplitude (ALH) (Figure 1B), curvilinear velocity (VCL) (Figure 1C), and hyperactivation (HA) (Figure 1D) were recorded. Data are expressed as mean ± standard error of the mean (SEM), n = 7. Statistical significance was analyzed by analysis of variance (ANOVA) and Tukey's post-hoc test. Different letters indicate statistically significant differences, p < 0.05.

[0026] [Figure 2]Figures 2A and 2B show the effects of different concentrations and / or different Ca2+ ionophores on sperm hyperactivation (HA). In Figure 2A, sperm were incubated with 0.2% DMSO for 2 min, 5 μM 4-Br-A23187 for 2 min, 1 μM 4-Br-A23187 for 5 min, or 0.5 μM A23187 for 2 min, followed by exposure to 40°C for 30 min. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was analyzed by ANOVA and Tukey's post-hoc test. *p<0.05. Figure 2B analyzes the effect of different incubation temperatures on sperm hyperactivation (HA) after ionophore treatment. Sperm were incubated with 5 μM 4-Br-A23187 for 2 min, followed by incubation at either 37, 38, 39, 40, 41, 42, or 43°C for 60 min. Data are expressed as the mean ± SEM of at least three independent experiments.

[0027] [Figure 3] 3A-3C show the effect of different incubation periods on sperm hyperactivation (HA) from three donors when incubated at either 37° C. or 40° C. for 4 hours.

[0028] [Figure 4] Figures 4A and 4B show how the combined effect of temperature and Ca2+ ionophore treatment on sperm results in a higher number of human blastocysts compared to conventional IVF. Sperm from the control and combined treatments were used in clinical assisted reproductive IVF procedures. The number of fertilized eggs (two pronuclei on day 2) and the number of high-quality blastocysts on days 5 and 6 were recorded. The percentage of blastocysts per mature egg (Figure 4A) and the percentage of blastocysts per fertilized egg (Figure 4B) were determined. Statistical significance was analyzed using Fisher's exact test. *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition Sperm function i) In some embodiments, provided herein are methods for improving sperm function, the methods comprising sequentially incubating mammalian sperm under different conditions prior to insemination or injection into eggs in assisted reproduction.

[0030] The sequential incubation procedure consists of five steps: (a) Mammalian spermatozoa are incubated in HCO3 - (b) washing the mammalian spermatozoa prepared in step (a) for 5 minutes in a medium containing neither ATP nor albumin and resuspending them in the same washing medium; (c) resuspending the mammalian spermatozoa in a medium containing 0.01 to 20 μM, preferentially 0.1 to 6 μM, of Ca; 2+ (c) incubating the mammalian sperm prepared in step (b) with a medium containing up to 1% albumin for 1-10 minutes, and then resuspending them in the same washing medium and incubating them at 36-38°C for 10-60 minutes (preferentially 20-40 minutes); (d) incubating the mammalian sperm prepared in step (c) with 3-9 mM, and preferentially 6 mM HCO3 - (e) Incubating the mammalian sperm of the appropriate concentration prepared in step (d) in a medium containing 10-30 mM HCO3 - , preferentially 25 mM HCO3 - and transfer to a medium containing 0.1-1% albumin, preferentially 0.5% albumin, at 37°C. This sequential procedure improves sperm function compared to suitable control sperm, making them suitable for use in ART.

[0031] In some embodiments, fertilization of the eggs is performed in vitro (IVF or ICSI) or in vivo, for example, by intracervical insemination or IUI of pre-incubated sperm in a sequential procedure.

[0032] Improved sperm function can include one or more of the following aspects: improved motility, such as the percentage of sperm in a population exhibiting hyperactive motility, as assessed by CASA; increased capacitation; and increased fertilization rates, e.g., development to at least two cells, blastocyst development, or live births. Thus, in some embodiments, sperm function can be sperm motility, VCL, ALH, sperm capacitation, the percentage of hyperactive sperm, the ability to fertilize an egg, or embryo production. In some embodiments, embryos produced by functionally improved sperm comprise one or more characteristics selected from increased viability, increased implantation, and improved ability to develop to at least two-cell development, blastocyst development, or fetus.

[0033] ii) The improvement in one or more sperm functions contemplated herein constitutes an improvement in one or more sperm functions compared to suitable control sperm. In some embodiments, one or more sperm functions may be improved by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 100%, 200%, 300% or more. In some embodiments, one or more sperm functions may be improved by 10%-200%, 25%-150%, 50%-100%, or 70%-90%.

[0034] iii) In the context of the present disclosure, sperm "activity" and / or "function" encompass physiological processes such as, for example, sperm motility, thermotaxis, rheotaxis, and / or chemotaxis, and the ability to fertilize an egg. The terms "activity" and / or "function" may further include processes occurring before, during, and / or before or during interaction with the surrounding layers of the zygote, such as capacitation and the acrosome reaction (also known as acrosome exocytosis) or fusion with the plasma membrane, and / or processes following fertilization of the egg, such as embryo formation. In some embodiments, the embryo exhibits increased viability (longer survival), improved implantation, and / or the ability to develop to the two-cell stage, blastocyst, or fetus leading to birth.

[0035] sperm motility i) The term "motility" strictly relates to the overall movement of cells, but can also apply to other aspects of motility, such as increasing or decreasing the speed of sperm cell movement and / or the proportion of motile sperm cells in any given population. Thus, the methods disclosed herein can be used not only to improve sperm motility, but also to increase the speed of sperm cell movement and / or the proportion (percentage) of motile cells in any given sperm population.

[0036] ii) Sperm motility is expressed as the total percentage of motile sperm or the velocity of motile sperm. These measurements can be made by a variety of assays, but are conveniently measured in one of two ways: by placing sperm on a microscope slide and subjectively visually assessing them using a phase-contrast microscope, or by using CASA. Phase-contrast microscopy measures the number of motile sperm and the total sperm count, and speed is rated as fast, medium, or slow. The second method of assessing sperm motility is to use CASA to objectively determine the motility characteristics of individual sperm cells in a sample.

[0037] iii) Thus, the term "motility" encompasses the percentage of motile sperm, which can be the percentage of the total number of sperm assessed that fall into all World Health Organization (WHO) categories of motility, excluding the category designated "no motility," regardless of speed or directionality. Manual counting uses qualitative subjective selection criteria to classify sperm cells into four categories: non-motile, locally motile, non-linear, and linearly motile.

[0038] iv) The term "motility" encompasses the percentage of motile sperm, i.e., the percentage of the total number of sperm in a population that exhibit forward motility, hyperactive motility based on CASA.

[0039] v) The methods disclosed herein can increase the percentage of forward-motility sperm. For example, the percentage of sperm that exhibit linear movement from one point to another with less than a 90-degree head rotation can be increased from otherwise non-progressive sperm, i.e., sperm that move but do not move forward. In some embodiments, improved motility includes an increase in the percentage of super-activated sperm. Super-activated sperm motility is characterized by sperm with a high-amplitude, asymmetric flagellar undulation pattern. Super-activated motility is characterized by vigorous movement with many seemingly random fluctuations, without a clear forward path and sperm head rotations of more than 90 degrees. Super-activated sperm motility is more vigorous and shorter-lived than progressive motility. Biologically, super-activated sperm motility is important for sperm to penetrate egg outer investment before fertilizing a mature egg. In some embodiments, the methods disclosed herein can increase the percentage of super-activated sperm in a given sperm population.

[0040] vi) It should be understood that other standardized measures of sperm motility parameters can also be used. Other measures of sperm motility include "velocity" and "straightness," which can be assessed using an automated semen analyzer. In some embodiments, the methods disclosed herein can improve sperm function, including improving other motility parameters of sperm, including mean path velocity (VAP), linear velocity (VSL), VOL, ALH, and vibration head frequency (BCF), or parameters known to those skilled in the art. VAP is the velocity along the mean path of sperm cells. VSL is the linear or forward velocity of the cells. VCL is a measure of the velocity of movement of the center of mass of the sperm head over a given period of time. Forward straightness (LIN) is the ratio of VSL to VCL, expressed as a percentage. ALH of the sperm head is calculated from the amplitude of lateral deviation relative to the axis of cell progression or mean path. Methods for measuring sperm motility by CASA are well known in the art; see, for example, WO2012061578(A2). Enhanced sperm motility as contemplated herein constitutes an enhancement of sperm motility compared to suitable control sperm.

[0041] vii) In some embodiments, sperm with improved motility are provided that are the product of a process that includes incubating sperm in one or more steps of a sequence to enhance sperm. In some embodiments, the improvement in sperm motility can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to suitable control sperm. In some embodiments, the improvement in sperm motility can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the improvement in sperm motility can be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, sperm motility may be improved by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, improved sperm function or improved sperm motility may be an increase in the percentage of super-activated sperm. In some embodiments, improved sperm function or improved sperm motility may be an increase in the percentage of forward-moving sperm. In some embodiments, improved sperm function or improved sperm motility may be an increase in the percentage of super-activated sperm. In some embodiments, the level of super-activated sperm, progressively motile sperm, or a combination thereof is increased such that the super-activated sperm, progressively motile sperm, or a combination thereof constitutes at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. Improved sperm motility indicates improved sperm function.

[0042] Sperm capacitation i) In some embodiments, improved sperm function includes increased sperm capacitation. "Sperm capacitation" refers to sperm having the ability to undergo acrosomal exocytosis or develop hyperactive motility and bind to and penetrate the zona pellucida of an unfertilized egg. Completion of capacitation is indicated by the ability of sperm to bind to the zona pellucida and undergo ligand-induced acrosomal exocytosis.

[0043] ii) In some embodiments, sperm with increased capacitation are provided that are the product of a process that includes incubating sperm in one or more steps of a sequential procedure for enhancing sperm. In some embodiments, the increase in sperm capacitation can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to suitable control sperm. In some embodiments, the increase in sperm capacitation can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the increase in sperm capacitation can be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, sperm capacitation may be increased by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, the level of sperm capacitation is increased such that capacitated sperm may constitute at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. Increased sperm capacitation indicates improved sperm function.

[0044] fertility In some embodiments, sperm function includes the ability of sperm to fertilize an egg. Sperm capacitation can be determined, for example, by IVF. IVF is a fertilization process in which an egg is combined with sperm outside the body, in vitro ("in vitro"). This process involves monitoring and stimulating a woman's ovulation process, removing an egg(s) from the woman's ovaries, and fertilizing the sperm in a culture medium in a laboratory. In one embodiment, sperm with improved capacitation are provided that are the product of a process that includes incubating sperm in one or more steps of a sequential procedure to enhance sperm. In some embodiments, the improvement in capacitation can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to suitable control sperm. In some embodiments, the increase in fertility may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the increase in fertility may be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, the increase in fertility may be 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, the level of fertility is increased such that the number of sperm capable of fertilizing an egg is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. Enhanced fertility refers to improved sperm function and increased fertilization.

[0045] Embryo generation i) In some embodiments, sperm function includes producing an embryo. In some embodiments, functionally improved sperm prepared by the methods herein are provided with access to an egg to promote fertilization. Here, promoting fertilization can include producing an embryo. In some embodiments, functionally improved sperm prepared by the methods herein are provided with access to an egg in vitro, thereby producing an embryo in vitro. In some embodiments, functionally improved sperm prepared by the methods disclosed herein are provided with access to an egg in vivo by sperm IUI, thereby producing an embryo in vivo. In some embodiments, sperm incubated in one or more steps of the sperm enhancement sequence are injected into the reproductive tract of a female subject, providing access to an egg to produce an embryo in vivo. In some embodiments, if an embryo is produced in vitro, the embryo may be cryopreserved for later use or further cultured in vitro to allow embryo development. In some embodiments, the embryo is developed to at least the two-cell stage before cryopreservation and / or implantation into a female subject. In some embodiments, the embryos are developed in vitro to a developmental stage greater than the two-cell stage before further processing. In some embodiments, the embryos are developed in vitro to the blastocyst stage before further processing (e.g., cryopreservation or transfer into a female subject for development into a complete fetus). A range of suitable media are available for in vitro incubation and culture of embryos in ART procedures, the types and compositions of which are well known to those skilled in the art. Preferably, the culture medium contains at least water, salts, nutrients, essential amino acids, vitamins, and hormones, and may further contain one or more growth factors. Various suitable culture media are commercially available, such as Earle's medium, Ham's F10 medium, and human tubal fluid (HTF) medium. The present disclosure also contemplates co-culturing embryos in vitro on a layer of "feeder cells" by methods known in the art. Suitable "feeder cells" for co-culture may include, for example, bovine oviduct cells or human oviduct epithelial cells.

[0046] ii) Those skilled in the art will understand that the benefits provided by the functionally improved sperm prepared by the methods disclosed herein are not limited to increased fertilization. Rather, the methods and preparations of the present invention are equally applicable as treatments to promote fertilization, whether the embryos are produced in vitro via ART or in the reproductive tract of an animal. The methods of the present invention are applicable to improving fertilization rates, embryo survival rates, embryo implantation rates, and pregnancy rates in assisted and unassisted pregnancies. Embodiments of the present disclosure also provide methods for improving the fertilization capacity of sperm in male animals.

[0047] iii) In the context of this specification, the terms "embryo with increased viability" and "embryo with longer survival" refer to an increase or enhancement in the likelihood of survival of an embryo(s) produced by mammalian sperm according to the methods and preparations disclosed herein, e.g., mammalian sperm with one or more improved sperm functions, compared to the likelihood of survival of an embryo(s) produced by suitable control sperm. In some embodiments, the embryos are produced by ART, e.g., IVF or ICSI. In some embodiments, the embryos are produced in vivo in the reproductive tract of a female mammalian subject by artificial insemination (AI).

[0048] iv) For the purposes of this disclosure, embryonic survival rate can be reflected by several indicators. For example, an increase in embryonic survival rate can result in an increase in embryo implantation rate after fertilization, a decrease in embryonic mortality before and after implantation, an increase in clinical pregnancy rate, or an increase in live birth rate. Therefore, the present disclosure also relates to methods for preventing apoptosis or developmental delay in embryos, and methods for increasing pregnancy rates in animals. Embryonic survival rate can refer to embryonic survival rate in vitro or in vivo.

[0049] v) In some embodiments, sperm capable of producing embryos with increased viability are provided, the sperm being the product of a process that includes incubating the sperm in one or more steps of a sperm enhancement sequence. In some embodiments, providing access to an egg for the functionally improved sperm promotes fertilization. In some embodiments, promoting fertilization includes producing an embryo(s) with increased viability. In some embodiments, the increased viability of embryos produced by access to an egg by sperm prepared by the methods herein can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to embryos produced by suitable control sperm. In some embodiments, the increased viability of embryos can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the increase in embryo survival rate can be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, the increase in embryo survival rate can be between 10% and 200%, between 25% and 150%, between 50% and 100%, or between 70% and 90%. In some embodiments, the level of sperm capable of producing embryos with increased viability is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. The production of embryos with increased viability indicates improved sperm function and / or increased fertilization.

[0050] vi) The embryo cleavage stage typically occurs during the first three days of culture. Embryos generated in vitro may be cryopreserved and transferred to a female subject via embryo transfer at a later stage, or they may be transferred immediately. "Embryotransfer" refers to the procedure of placing one or more embryos and / or blastocysts into the uterus or fallopian tube. In traditional IVF processes, embryos are transferred to the uterine cavity two days after fertilization, when each embryo is at the four-cell stage, or three days after fertilization, when each embryo is at the eight-cell stage. It is recognized that it may be desirable to use blastocyst-stage embryos that have reached the fifth to seventh day of culture. The present disclosure allows for embryo transfer at any time along the spectrum of embryo / blastocyst development. A blastocyst or embryo is considered ready for transfer to the uterus when, by visual observation, such as with a microscope, the blastocyst is clearly visible and occupies more than 50% of the embryo's volume. In an in vivo environment, the blastocyst stage is typically achieved four to five days after fertilization, shortly after the embryo passes through the fallopian tube and reaches the uterus. The stage of embryo development can be determined by visual observation of the embryo under a microscope, which simultaneously displays certain defined physical or morphological characteristics before implantation into the uterus. The maturity state of the blastocyst is determined to range from II AB to VI AA according to the classification of Gardner et al. (1998).

[0051] vii) The methods disclosed herein result in the production of embryos with increased rates of progression to the two-cell stage, blastocyst stage, or fetal development and birth. In some embodiments, sperm capable of producing embryos capable of developing through normal developmental stages (e.g., the two-cell stage, the blastocyst stage, fetal development, and birth) are provided that are the product of a process that includes incubating sperm in one or more steps of a sperm enhancement sequence. In some embodiments, providing functionally improved sperm with access to eggs promotes fertilization. In some embodiments, promoting fertilization includes the production of embryos with increased ability to develop through normal developmental stages (e.g., the two-cell stage, the blastocyst stage, fetal development, and birth). In some embodiments, the increased rate of embryos progressing through normal developmental stages produced by sperm prepared by the methods can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to embryos produced by suitable control sperm. In some embodiments, the increase in embryos progressing through normal developmental stages can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the increase in embryos progressing through normal developmental stages can be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, the percentage of embryos progressing through normal developmental stages can be increased by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%.In some embodiments, the level of sperm capable of producing embryos capable of progressing through normal development is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. The production of embryos capable of progressing through one or more normal developmental stages indicates improved sperm function and / or increased fertilization.

[0052] viii) In vivo, the embryo attaches or implants in the uterine wall, forms a placenta, and develops into a fetus during pregnancy until term. Testing to determine whether one or more embryos have implanted in the uterine lining, i.e., whether the procedure has resulted in successful pregnancy initiation, is performed two weeks after implantation using, for example, a blood test for β-hCG (human chorionic gonadotropin) and other techniques known in the art. U.S. Patent No. 4,315,908 (A) describes a method for detecting β-hCG in urine by radioimmunoassay. U.S. Patent No. 8,163,508 (B2) provides a method and kit for predicting pregnancy in a subject by the β-hCG method by determining the amount of the early pregnancy-associated isoform of β-hCG in a sample. Such diagnostic methods and others are useful within the scope of the present disclosure.

[0053] ix) In some embodiments, sperm capable of producing embryos with improved implantation rates or improved pregnancy rates are provided, the sperm being the product of a process comprising incubating sperm in one or more steps of a sperm enhancement sequence. In some embodiments, providing functionally improved sperm with access to eggs promotes fertilization. In some embodiments, promoting fertilization includes producing embryos with improved implantation rates or improved pregnancy rates. In some embodiments, the increase in implantation rate of embryos produced by sperm prepared by the methods herein, or the increase in pregnancy rate upon embryo implantation, can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% compared to embryos produced by suitable control sperm. In some embodiments, the increase in embryo implantation rate or pregnancy rate can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the increase in embryo implantation or pregnancy rate can be at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold. In some embodiments, the increase in embryo implantation or pregnancy rate can be 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, the level of sperm capable of producing embryos with increased implantation rates or improved pregnancy rates is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the total sperm in the preparation. Improved implantation (ie, increased implantation rate) or production of embryos with increased pregnancy rates upon implantation indicates improved sperm function and / or increased fertilization.

[0054] Mammalian sperm i) The methods disclosed herein include improving one or more sperm functions to promote fertilization. Preparations of sperm with improved function are also provided. As described herein, the sperm may be from a vertebrate, preferably a mammal.

[0055] ii) Mammals include, but are not limited to, humans, rodents, primates, wild or domesticated animals (including feral animals, farm animals, sport animals, and pets). Rodents include, for example, mice, rats, and hamsters. Domestic and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, felines (e.g., house cats), and canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, salmon). The mammalian sperm can be from a non-human mammal, including an ungulate, such as an even-toed ungulate (e.g., pig, peccary, hippopotamus, camel, llama, mouse deer (mouse deer), deer, giraffe, pronghorn, antelope, caprine-antelope (including sheep, goats, and others), or cattle) or a perissodactyl ungulate (e.g., horse, tapir, and rhinoceros), a non-human primate (e.g., monkey, chimpanzee, cynomolgus monkey, spider monkey, and macaque, e.g., rhesus monkey), a canine (e.g., dog), or a cat. The mammalian sperm can be from a member of the superorder Laurasia. The superorder Laurasia includes the group of mammals described in Waddell et al. (1999). Members of the superorder Laurasiana can include Euonymus (hedgehogs, shrews, and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora (carnivores), Cetacea (even-toed ungulates and cetaceans), Chiroptera (bats), and Squamata (pangolins). Members of the superorder Laurasiana can be ungulates, e.g., ungulates of the order Artiodactyla or ungulates of the order Perissodactyla. The mammalian sperm can be from a member of the order Carnivora, such as a cat or a dog. In some embodiments, the mammalian sperm is sperm from a human, non-human primate, pig, cow, horse, sheep, dog, cat, or mouse. In some embodiments, the mammalian sperm is human sperm.

[0056] iii) In some embodiments, the mammalian sperm are provided by a healthy male mammal. In some embodiments, the mammalian sperm are from a male with a DNA mutation or some sperm dysfunction, such as low sperm count, reduced sperm motility, and abnormal sperm morphology. In some embodiments, the mammalian sperm can be from a subfertile male or a male with oligozoospermia. The mammalian sperm can be from a male suffering from, for example, oligozoospermia, teratozoospermia, asthenozoospermia, or oligoasthenoteratospermia. Oligozoospermia refers to a condition characterized by a sperm concentration of less than 20 million / mL. Asthenozoospermia refers to a condition characterized by reduced sperm motility. Teratozoospermia refers to a condition characterized by the presence of abnormally morphed sperm. Oligo-asthenozoospermia-teratozoospermia refers to conditions including oligozoospermia (low sperm count), asthenozoospermia (poor sperm motility), and teratozoospermia (abnormal sperm shape). In some embodiments, the sperm are from a subfertile or oligozoospermic male, e.g., having a sperm count of less than about 20, 19, 18, 18, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 million / mL, e.g., less than 15 million / mL.

[0057] iv) In some embodiments, sperm are separated (or isolated) from semen prior to using the methods described herein. Any method of sperm concentration or isolation, including density gradient centrifugation, swim-up, microfluidics, or a combination thereof, can be used in conjunction with the present invention.

[0058] v) In some embodiments, the sperm used in the methods provided herein are fresh or from preserved stocks. For example, the sperm are restored from cryopreservation before processing. In other embodiments, the sperm are restored from non-cryopreservation before processing.

[0059] vi) In some embodiments, different amounts of sperm, including fractions of a single ejaculate or the entire ejaculate, can be used in the methods provided by the present invention. In some embodiments, sperm are pooled from at least two ejaculates (e.g., 2, 3, 4, 5, 6, or more ejaculates).

[0060] How to Obtain a Sperm Sample i) Several methods of collecting viable sperm, such as masturbation into a container, by gloved hands, the use of an artificial vagina, or an electric ejaculator. Animal semen can be collected by using an artificial vagina, an electric ejaculator, or by manually massaging an animal's ampoule. Sperm can also be collected directly from any part of the male reproductive tract, such as testicular sperm or sperm obtained from the caput, epididymis, or cauda epididymis using different methods, such as testicular or epididymal puncture, removal of the testicles or epididymis, and collection of sperm in the surrounding medium.

[0061] ii) In some embodiments, the sperm used in the methods provided herein may be a sample freshly collected from a source animal (e.g., a mammal) or may be a sample that has been previously thawed or cryopreserved. At the time of collection or thereafter, the collected sperm may be combined with any of several different buffers compatible with sperm, such as trichloroacetic acid (TCA), 4-2(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or phosphate-buffered saline (PBS). The collected sperm suspension may also contain various other additives to keep the sperm viable. Exemplary additives include protein sources, antibiotics, growth factors, and compositions that modulate intracellular and / or extracellular oxidation / reduction reactions. Alternatively, semen can be collected into an empty container and then contacted with a buffer solution within minutes to hours of collection to form a sperm suspension. In some embodiments, sperm cells can be collected directly into a container containing medium (e.g., M199, synthetic oviductal fluid, PBS, BO, test egg yolk, Tyrode's, HBSS, Ham's F10, HTF, Menezo's B2, Menezo's B3, Ham's F12, DMEM, TALP, Earle's Buffered Salts, CZB, KSOM, BWW medium, and emCare medium, in some embodiments, TALP, CZB, etc.).

[0062] iii) In some embodiments, sperm collection involves processing of viscous semen or washing of sperm prior to performing the methods disclosed herein. Washing involves centrifuging a sample of semen or thawed sperm through a dilute washing medium, allowing for collection of a sperm-enriched pellet. After or instead of the washing step, a procedure can be performed to isolate motile sperm from the sample.

[0063] iv) In some embodiments, sperm are isolated from semen before use in the methods disclosed herein. In some embodiments, functionally improved sperm can be further concentrated from sperm prepared according to the methods disclosed herein. Generally, sperm are isolated or concentrated by swimming motile sperm away from dead sperm, non-motile sperm, and debris (sperm swim-up), by centrifuging sperm through a density gradient, or by passing sperm through a column that binds dead sperm and debris. Isolation (or concentration) of sperm from semen is carried out by a method selected from wash and spin, sedimentation, direct swim-up, pellet and swim-up, and buoyancy density gradient methods. These methods are well known in the art. These methods have long been used in ART and are described in detail in Brinsden (1999). In some embodiments, sperm prepared by the methods disclosed herein can be further enriched for motile sperm by isolation procedures such as sedimentation, direct swim-up, pellet and swim-up, and buoyant density gradient methods.

[0064] v) The direct swim-up method refers to the self-selection of motile sperm and essentially involves layering an aliquot of medium on top of a semen sample or a sperm preparation as disclosed herein and allowing it to stand at room temperature for at least 30 minutes. Motile sperm cells migrate to the upper layer (medium) from which they can be recovered. This method can also include a centrifugation step.

[0065] vi) Depending on the number of motile cells in the sample, the method can be modified and combined with additional isolation / separation techniques. For example, a swim-up procedure can be performed by overlaying 1 mL of albumin-containing medium on 1 mL of semen in a test tube. After incubation in air or 5% CO2 at 37°C for 1 hour, the upper layer of medium, into which sperm with better motility characteristics migrate, is collected. This technique may also include or be combined with a centrifugation step, such as centrifugation on a density gradient. The separated, isolated, or concentrated sperm can then be used in the methods disclosed herein or, for example, cryopreserved before further processing. In the case of sperm preparations prepared by the methods herein, the sperm may be used for IVF, ICSI, or AI after the concentration step, or may be cryopreserved for later use, for example. Thus, for any of these isolation or concentration methods, the sample can be semen, partially purified sperm, purified sperm, or functionally enhanced sperm prepared by the methods herein. In some embodiments, the percentage of motile cells is increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, or about 100% compared to an unprocessed semen sample or unconcentrated sperm preparation after isolating or concentrating sperm using isolation methods such as direct swim-up, pellet and swim-up, and buoyant density gradient methods.

[0066] vii) In some embodiments, after isolation, concentration, and washing, the sperm pellet can be resuspended in a medium suitable for further processing, including storage medium, HTF medium for culture, and medium for sequential procedures to enhance sperm. In connection with the functionally enhanced sperm prepared by the methods disclosed herein, the sperm preparation can be resuspended in storage medium, HTF medium for culture, artificial insemination medium, fertility assays described herein, IVF, freezing, IUI, cervical cap artificial insemination medium, and the like. The sperm may be added to the medium, or the medium may be added to the sperm. The medium may be a zwitterionic buffer such as TES, HEPES, PIPES, or HCO3. -The medium may be a balanced salt solution that may contain other buffers such as sodium. Generally, media for diluting sperm or culturing sperm, oocytes, embryos, or embryonic stem cells are balanced salt solutions, such as M199, synthetic oviductal fluid, PBS, BO, test egg yolk, Tyrode's, HBSS, Ham's F10, HTF, Menezo's B2, Menezo's B3, Ham's F12, DMEM, TALP, Earle's Balanced Salts, CZB, KSOM, BWW medium, and emCare medium (PETS, Canton, Texas). In some embodiments, TALP or HTF is used for sperm culture medium, and CZB is used for embryo culture medium. The sperm or embryos of the present disclosure can be stored in a cryo-medium containing a cryoprotectant.

[0067] Suitable control sperm i) Suitable control sperm may be sperm incubated under control conditions, for example, in a control buffer, such as HTF medium or modified HTF medium. - It contains a sodium buffer system and can be used in applications that require a CO2 atmosphere during incubation. - In some embodiments, HTF medium or modified HTF medium includes those commercially available from Irvine Scientific (Santa Ana, CA). - Sodium-free HTF medium was used. The sperm can be incubated for a period of time sufficient to result in a measurable change in sperm motility (or other characteristics). In certain embodiments of the method, the incubation is for 1 minute to 24 hours, 15 minutes to 3 hours, 30 minutes to 1.5 hours, about 1 hour, or any subrange or subvalue thereof. It is understood that a suitable control sperm can be at least one sperm or sperm population, such as a sperm preparation or sperm suspension.

[0068] sperm preparation i) In some embodiments, the present invention provides sperm preparations, such as activated sperm or enhanced sperm preparations, collectively "sperm preparations provided by the present invention" or "preparations provided by the present invention." In some embodiments, the present invention provides a super-activated sperm preparation comprising at least 5% super-activated sperm, e.g., at least about 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0% or more, e.g., about 5-20, 8.5-20, 10-20, or 12.5-20% super-activated sperm. As will be appreciated by those skilled in the art, sperm may be separated based on HA phenotype, although in some embodiments the foregoing percentages are based on preparations that have not been activated and then sorted based on HA (although in some embodiments the sperm preparation may have been pre-treated, e.g., to separate or otherwise concentrate sperm from other semen components, including certain irregular sperm).

[0069] ii) In some embodiments, the present invention provides a preparation of sperm prepared by any one of the methods provided by the present invention.

[0070] iii) In some embodiments, the present invention provides a sperm preparation prepared by concentrating sperm from the semen of a male subject, e.g., a normospermic, subfertile, or oligozoospermic male, e.g., a subfertile (including oligozoospermic) male, and incubating the sperm in one or more steps of a sequential procedure to enhance the sperm.

[0071] iv) For any of the preparations provided by the present invention, the sperm can be from any male subject (e.g., a mammal, in some embodiments, a human). In some embodiments, the human is a normozoospermic male, or in other embodiments, the male is a subject with oligozoospermia or low fertility (e.g., low sperm motility).

[0072] Promoting fertilization i) Sperm preparations with improved function prepared by the methods disclosed herein may be useful for promoting fertilization. Accordingly, the present disclosure relates to a method for promoting fertilization. The method includes incubating sperm in one or more steps of a sequential procedure to enhance the cells and providing the improved sperm with access to eggs under conditions to promote fertilization. The preparation of improved sperm can be applied in IVF, ICSI, and artificial insemination (AI) (e.g., IUI) in humans, as well as in the biomedical research industry for animal models for human diseases (infertility, sperm dysfunction), and in the reproductive and agricultural industries. The improved sperm prepared by the methods disclosed herein may be provided with access to unfertilized eggs of the same species as the sperm to promote IVF, ICSI, or may be used for AI, including, for example, IUI, in female subjects of the same species as the sperm.

[0073] In vivo fertilization i) Functionally improved sperm prepared by the methods disclosed herein may be useful for promoting in vivo fertilization by providing functionally improved sperm with access to eggs in the reproductive tract of a female subject of the same species as the sperm. In vivo fertilization can be achieved by artificial insemination of the sperm, for example, by intracervical insemination or intrauterine insemination (IUI). Standard AI and IUI, as well as other methods, are well known to those skilled in the art. In some embodiments, functionally improved sperm are provided with access to eggs in the reproductive tract of a female subject by IUI to promote fertilization of the eggs. In other embodiments, sperm can access eggs in vivo by IUI of mammalian sperm that have been incubated in one or more steps of a sperm enhancement sequence. The injected sperm can be used while retained in a suitable liquid. The liquid used for this purpose may be a liquid commonly used as a medium for AI.

[0074] In vitro fertilization (IVF) i) The methods and sperm preparations disclosed herein are particularly useful for fertilization by ART, e.g., promoting embryo survival rates after ART, particularly IVF. Other suitable ART techniques to which the present disclosure is applicable include, but are not limited to, gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), blastocyst transfer (BT), ICSI, cryopreservation of gametes, embryos, and cells, in vitro preparation of embryos for embryo biopsy, and other forms of embryo micromanipulation, including the formation of embryos by nuclear transfer and the production of transgenic and genetically modified lines. They are also applicable to the production of embryonic stem cell lines.

[0075] ii) In some embodiments, the functionally improved sperm prepared by the methods disclosed herein can be used to in vitro fertilize eggs, for example, by microinjection, including ICSI, and other methods known to those skilled in the art. Typically, in IVF, after fertilization, the cells are expanded to the blastocyst stage before implantation. The methods disclosed herein result in increased formation of embryos with longer survival and improved ability to develop to the two-cell, blastocyst stage. Thus, the sperm preparations disclosed herein can be useful in IVF procedures, including, for example, ICSI.

[0076] iii) The method of the present disclosure also includes providing sperm prepared by the method herein with access to eggs to promote IVF. Providing sperm with in vitro access to eggs can be carried out in an appropriate medium. The medium used for this purpose can be a medium commonly used for IVF, such as HTF medium. The temperature conditions for providing access can be temperatures commonly used for IVF, such as average mammalian body temperature or a temperature close to it. The time for providing access can generally be any time required for IVF, but is not particularly limited, and is preferably 6 to 24 hours. The IVF rate can be determined by incubating one or more sperm with a mature oocyte for approximately 24 hours.

[0077] Use of fertilization i) The methods and sperm preparations disclosed herein are generally applicable to many species, including humans, cattle, dogs, horses, pigs, sheep, birds, rodents, and the like. While useful whenever fertilization is desired, the methods of the present invention are particularly useful for increasing the likelihood of conception in animals and humans with fertility dysfunctions. Such dysfunctions include low sperm count, reduced sperm motility, and abnormal sperm morphology. Thus, the methods disclosed herein may be useful for preparing functionally improved sperm prior to use in IVF or IUI in fertility clinics. The methods described herein can be used to improve AI, IVF, or ICSI in exotic and / or endangered species. Therefore, the methods can also be used to promote fertility in animals kept in zoos and in conservation programs aimed at improving reproduction in endangered animals in the wild. For example, the methods and sperm preparations disclosed herein can be used to improve fertility and pregnancy rates in animal husbandry, for agriculturally valuable species and in species bred for conservation purposes.

[0078] ii) Furthermore, the methods and compositions of the present invention are useful, for example, for AI procedures in commercial breeding. The methods can be performed using sperm from livestock, particularly livestock, and wild animals (e.g., endangered species). For example, as disclosed herein, embodiments of the methods and compositions of the present disclosure can be applied to cattle breeding. The methods and preparations can be useful for AI in the livestock production industry, where it is desirable to introduce specific genetically determined traits into livestock to influence the outcome and obtain offspring with one or more desirable characteristics or traits, e.g., offspring of a specific sex, offspring with enhanced milk production, or offspring for quality meat production. Use of the methods described herein improves pregnancy rates. Mammalian sperm are often damaged by freezing and thawing, resulting in reduced conception rates. By improving the performance of viable sperm, the use of sperm prepared by the methods disclosed herein for insemination can improve pregnancy rates per estrous cycle and reduce the number of cycles required to ensure conception, thus reducing the overall cost of AI.

[0079] iii) Semen from animals with highly desirable traits can be used to inseminate more females, as fewer cycles are required to ensure conception of any one female. In such applications, semen is obtained from males with the desired characteristics. To influence the sex of the resulting offspring, sperm preparations can be sorted into X- and Y-chromosome-bearing cells and / or enriched for sperm with one or more of the improved sperm functions disclosed herein. Sperm can be sorted into X- and Y-chromosome-enriched populations by commonly used methods, for example, using a flow cytometer / cell sorter as described in U.S. Pat. No. 5,135,759(A). Sperm prepared by the methods disclosed herein can be sorted into populations containing a specific percentage of X- or Y-chromosome-bearing sperm cells. For example, a population of sperm may include at least about 65% X- or Y-chromosome sperm cells, at least about 70% X- or Y-chromosome sperm cells, at least about 75% X- or Y-chromosome sperm cells, at least about 80% X- or Y-chromosome sperm cells, at least about 85% X- or Y-chromosome sperm cells, at least about 90% X- or Y-chromosome sperm cells, or at least about 95% X- or Y-chromosome sperm cells. In some embodiments, sorting can be performed before preparing functionally improved sperm as disclosed herein. In some embodiments, sorting can be performed before providing functionally improved sperm with access to eggs for fertilization, such as in IVF, ICSI, or AI.

[0080] iv) The methods and preparations provided by the present invention can be used in assisted fertilization, such as IVF, including by ICSI. In some embodiments, any of the methods provided by the present invention can include providing sperm to the female reproductive tract. In some embodiments, the sperm preparations (with improved sperm function) provided by the present invention can be provided with access to an egg for a time sufficient to fertilize the egg, which may be ex vivo (e.g., IVF, including ICSI) or, in some embodiments, within the female reproductive tract. Such methods, in some embodiments, involve subsequent transfer of the fertilized egg into a female carrier. [Example]

[0081] The present disclosure will be described in more detail by the following specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to obtain alternative embodiments according to the present invention.

[0082] Example 1: Materials and Methods Reagents and Media: All reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. The medium used for human sperm culture was HTF-HEPES, containing 101.5 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl, 0.37 mM KH2PO4, 0.2 mM MgSO4, 0.33 mM sodium pyruvate, 2.77 mM glucose, 21.4 mM sodium lactate, 21 mM HEPES, and 10 μg / mL gentamicin, equilibrated at pH 7.3–7.4. At some steps, 6 mM NaHCO3 concentrate was added. Alternatively, the medium was supplemented with either 0.5–1% bovine serum albumin (BSA) or 2–10% human serum substitute (SSS).

[0083] For conventional treatment, modified HTF from Irvine Scientific (Santa Ana, CA) supplemented with 10% SSS was used.

[0084] Gamete co-culture was carried out in HTF containing 10% SSS (Irvine Scientific) under light oil equilibrated with 7.5% CO2.

[0085] Semen samples: Semen samples were obtained by masturbation into a sterile container from healthy normospermic men or men seeking infertility treatment. Before semen processing, the ejaculate was liquefied at room temperature for up to 2 hours and analyzed according to WHO standard procedures. Sperm separation from seminal plasma was performed by density gradient centrifugation or direct swim-up technique to isolate viable sperm of good quality.

[0086] Density gradient centrifugation: Semen samples were centrifuged at 200–500 × g for 20 min through a 40% / 80% discontinuous density gradient (PureSperm; Nidacon, Gothenburg, Sweden). The resulting sperm pellet was then placed in HTF-HEPES medium (HCO3 - The sperm were resuspended in 2–5 mL of PBS (with or without supplementation), divided into multiple tubes depending on the number of treatments, and centrifuged at 500–700 × g for 5 minutes. After removing the supernatant, each pellet was resuspended in the appropriate culture medium and the sperm concentration was adjusted to 5–20E6 / mL.

[0087] Sperm swim-up: 0.5-1 mL of semen aliquot was overlaid with 1-2 mL of HTF-HEPES supplemented with 10% SSS and incubated at 37°C for 1 hour. The supernatant was then carefully collected, divided into multiple tubes depending on the number of treatments, and centrifuged at 400-700 x g for 5 minutes. Each pellet was then resuspended in HTF medium + 10% SSS.

[0088] Sperm motility analysis: A 9.7 μl aliquot of sperm suspension was placed on an 18 mm × 18 mm slide under a coverslip, resulting in a preparation depth of 30 μm, and maintained at 37°C using a temperature-controlled stage. Sperm motility parameters were assessed using the Sperm Class Analyzer® system (SCA v.6.2.0.1, Microptic SL, Barcelona, ​​Spain), acquiring 60 frames per second. At least five microscopic fields and 300 sperm were analyzed. The following parameters were assessed: curvilinear velocity (VCL, μm / s), linear velocity (VSL, μm / s), mean path velocity (VAP, μm / s), straightness (LIN: VSL / VAP × 100, %), linearity (STR × 100, %), wobble (WOB: VAP / VCL × 100, a measure of sperm head movement from side to side, %), head amplitude (ALH, μm), and head frequency (BCF, Hz). Sperm motility was measured and classified as follows: fast progressive (VCL ≥ 35 μm / s; STR > 80%), medium progressive (VCL ≥ 15 μm / s; STR > 80%), in situ (VCL < 15 μm / s; VAP ≥ 5 μm / s), and immobile (VAP < 5 μm / s). The percentages of total (fast progressive + medium progressive + in situ) and progressive (fast progressive + medium progressive) motility were recorded. Drifting was set at 25 μm / s. Sperm were considered hyperactivated if they exhibited a VCL ≥ 150 μm / s, LIN < 50%, and ALH ≥ 3.5 μm.

[0089] Example 2: Enhancement of sperm motility This example describes the incubation conditions of sperm, such as temperature and intracellular Ca 2+ We demonstrate how precisely manipulating concentrations can enhance human sperm motility. Healthy male semen samples were processed by density gradient centrifugation as described in Example 1.

[0090] In the control treatment, sperm were incubated with 0.2% DMSO in HTF-HEPES for 2 min at 37°C. Sperm cells were centrifuged at 400 × g for 5 min and then washed by adding 1 mL of HTF-HEPES medium containing 1% BSA, and the pellet was resuspended in 6 mM HCO3 - The sperm cells were resuspended in HTF-HEPES medium containing 0.5% BSA and further incubated at 37°C for 30 minutes. For temperature treatment, sperm were incubated with 0.2% DMSO in HTF-HEPES for 2 min at 37°C. Sperm cells were centrifuged at 400 × g for 5 min and then washed by adding 1 mL of HTF-HEPES medium containing 1% BSA. The pellet was then resuspended in 6 mM HCO3 - The sperm cells were resuspended in medium containing 0.5% BSA and further incubated at 40°C for 30 minutes.

[0091] For ionophore treatment, sperm were incubated with 5 μM 4-Br-A23187 in HTF-HEPES (0.2% DMSO) for 2 min at 37 °C. Sperm cells were centrifuged at 400 × g for 5 min and then washed by adding 1 mL of HTF-HEPES medium containing 1% BSA. The pellet was then resuspended in 6 mM HCO3 - The sperm cells were resuspended in medium containing 0.5% BSA and further incubated at 37°C for 30 minutes.

[0092] For the combined treatment, sperm were incubated with 5 μM 4-Br-A23187 (0.2% DMSO) in HTF-HEPES for 2 min at 37 °C. Sperm cells were centrifuged at 400 × g for 5 min and then washed by adding 1 mL of HTF-HEPES medium containing 1% BSA. The pellet was then resuspended in 6 mM HCO3 - The sperm cells were resuspended in medium containing 0.5% BSA and further incubated at 40°C for 30 minutes.

[0093] Overall motility was unaffected by any of the treatments (Figure 1A). The combined treatment showed significantly higher values ​​of kinetic parameters related to HA (e.g., VCL and ALH) compared with the other treatments (Figures 1B-1C). Notably, Figure 1D shows that sperm treated at 40°C (temperature treatment) showed better HA values ​​(8% increase in HA) compared with the control procedure. Similarly, to a lesser extent, ionophore-treated human sperm also showed higher HA values ​​(3% increase in HA). However, when sperm were first exposed to the ionophore and then incubated at 40°C, a significant synergistic effect was observed (16% increase in HA compared with the control procedure, 50% higher than the 11% increase with the addition). Values ​​are expressed as the mean ± SEM of seven independent experiments. Statistical significance of the data was analyzed using one-way ANOVA and Tukey's post-hoc test.

[0094] As shown in Figure 2A, the described synergistic effect was observed using different means of pharmacologically transiently increasing intracellular Ca2+ concentrations. For example, incubation with 5 μM 4-Br-A23187 for 2 min, 1 μM 4-Br-A23187 for 5 min, or 0.5 μM A23187 for 2 min, prior to incubation at 40°C for 30 min, resulted in higher levels of HA compared to the control.

[0095] Furthermore, different temperatures above 38°C can be used after the ionophore pulse. Incubating sperm cells at temperatures between 38°C and 43°C for 60 min increased the percentage of HA compared to the control (Figure 2B).

[0096] Because human semen is heterogeneous, the effect of temperature on HA increase varied among donors (Figures 3A-C). Incubation at 40°C produced higher levels of HA compared with 37°C at all time points, especially in the first hour.

[0097] Example 3: Methods for treating sperm to improve fertilization rates and embryo development in human IVF This example describes the use of sperm treated according to certain embodiments of the present invention to improve conception rates in human subjects undergoing IVF.

[0098] The subjects were anonymous adult female egg donors (i.e., nine subjects aged 18–35 years). Prior to egg collection, the subjects underwent standard procedures (e.g., ovulation suppression followed by ovulation stimulation with hCG trigger injections as directed). The 14 collected eggs were divided into two groups: a control group and a combined group. Semen samples from patients attending a reproductive clinic were divided into two and processed by density gradient centrifugation. Half of the sperm assigned to the control group (control treatment) were incubated in HTF supplemented with 10% SSS for 2–4 hours at room temperature. For the treated sperm (combined treatment), the sperm were kept in HTF-HEPES containing 2% SSS for 0.5–2 hours at room temperature. The sperm were then washed in SSS-free medium and incubated with 5 μM 4-Br-A23187 in HTF-HEPES for 2 minutes at 37°C. The sperm cells were centrifuged at 400 × g for 5 min and then resuspended in 10% SSS and 6 mM HCO3 - Wash by adding 1 mL of HTF-HEPES medium containing 6 mM HCO3 - The sperm cells were resuspended in a medium containing 10% SSS and further incubated at 37°C for 30 minutes and at 40°C for 60 minutes.

[0099] For both the combined treatment and control groups, sperm were incubated with eggs in vitro. The number of fertilized eggs (with two pronuclei the next morning) and the number of high-quality blastocysts on days 5 and 6 were recorded (see Table 1). High-quality blastocysts were those with a score of 3BB or higher according to the Gardner grading system (Gardner, 2016). The combined treatment produced a greater number of high-quality blastocysts per fertilized egg (Figure 4A) and per fully mature egg (Figure 4B) compared to the control group.

[0100] [Table 1] Example 4: Time-lapse monitoring of human embryos The fertilized eggs were cultured in an EmbryoScope® (Vitrolife, Gothenburg, Sweden) incubator and monitored for 5–6 days until the blastocyst stage. The EmbryoViewer image analysis software (Vitrolife) was used to determine the time of each developmental event: tPNf: time of pronuclear loss; t2: time of 2 cells; t3: time of 3 cells; t4: time of 4 cells; t5: time of 5 cells; t8: time of 8 cells; tM: time of morula; tB: time of blastocyst. The KIDScore™ Day 5 (Version 2) model, developed by EmbryoScope and based on a very large multicenter dataset (Gazzo et al., 2020), was used as a predictive model for the implantation potential of embryos after day 5 transfer. Compared to the control group, the combined treatment produced a greater number of embryos with high implantation potential according to KIDScore (see Table 1).

[0101] Final remarks In this application, for all numerical boundaries describing any parameter, such as "about," "at least," "less than," and "greater than," the description necessarily encompasses any range bounded by the recited values. Thus, for example, a description of "at least 1, 2, 3, 4, or 5" also describes ranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, and 4 to 5, among others.

[0102] The headings used in this application are for convenience only and shall not affect the interpretation of this application.

[0103] Preferred features of each aspect (e.g., media, compositions, preparations, and methods) provided by the present invention are applicable mutatis mutandis to all other aspects of the invention and include, without limitation, combinations and permutations of the individual features (e.g., elements, including numerical ranges and exemplary embodiments) of specific embodiments and aspects of the invention as exemplified by the dependent claims and including the Examples. For example, specific experimental parameters illustrated in the Examples can be adapted piecemeal for use in the claimed invention without departing from the invention. For example, for the disclosed materials, specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C, and a class of elements D, E, and F, and an example combination of element A, D, and A, are disclosed, each is individually and collectively contemplated, even if each is not individually mentioned. Thus, in this example, from the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF should be considered specifically contemplated and disclosed. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, from the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD, the subgroups of, for example, AE, BF, and CE should be considered specifically contemplated and disclosed. This concept applies to all aspects of this application, including elements of compositions of matter and steps in methods of making or using the compositions.

[0104] The foregoing aspects of the invention, as recognized by one of ordinary skill in the art following the teachings herein, may be claimed in any combination or permutation, so long as they are novel and unobvious over the prior art. Thus, to the extent that an element is described in one or more documents known to those of ordinary skill in the art, aspects may be excluded from the claimed invention, particularly by a negative proviso or disclaimer of a feature or combination of features.

[0105] References Alvarez JG, Minaretzis D, Barrett CB, Mortola JF, Thompson IE. The sperm stress test: a novel test that predicts pregnancy in assisted reproductive technologies. Fertil Steril. 1996 Feb;65(2):400-5. doi:10.1016 / s0015-0282(16)58107-2。

[0106] Brinsden PR, Bourn Hall Clinic. A textbook of in vitro fertilization and assisted reproduction: the Bourn Hall guide to clinical and laboratory practice. The Parthenon Publishing Group. 1999;564。<{

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[0108] Chan PJ, Corselli JU, Patton WC, Jacobson JD, King A. Enhanced fertility after heat-induced hyperactivation. Fertil Steril. 1998 Jan;69(1):118-21. doi:10.1016 / s0015-0282(97)00440-8。

[0109] Gardner DK,Schoolcraft WB.Culture and transfer of human blastocysts.Curr Opin Obstet Gynecol.1999 Jun;11(3):307-11.doi:10.1097 / 00001703-199906000-00013。

[0110] Gazzo E,Pena F,Valdez F,Chung A,Bonomini C,Ascenzo M,Velit M,Escudero E.The KIDScoreTM D5 algorithm as an additional tool to morphological assessment and PGT-A in embryo selection:a time-lapse study.JBRA Assist Reprod.2020 Jan 30;24(1):55-60.doi:10.5935 / 1518-0557.20190054。

[0111] Keppler EL,Chan PJ,Patton WC,King A.Aggregation of human sperm at higher temperature is due to hyperactivation.Arch Androl.1999 Jan-Feb;42(1):35-9.doi:10.1080 / 014850199263020。

[0112] Kucuk T,Sozen E,Buluc B.Effect of heat-induced hypermotility on pregnancy rate in intrauterine insemination for male factor infertility associated with asthenospermia:a prospective,randomized,controlled study.J Assist Reprod Genet.2008 Jun;25(6):235-8.doi:10.1007 / s10815-008-9226-1。

[0113] Mann SL,Patton WC,King A,Chan PJ.Comparative genomic hybridization analysis of sperm DNA apoptosis after exposure to heat shock.J Assist Reprod Genet.2002 Apr;19(4):195-200.doi:10.1023 / a:1014846113170。

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[0115] Navarrete FA,Alvau A,Lee HC,Levin LR,Buck J,Leon PM,Santi CM,Krapf D,Mager J,Fissore RA,Salicioni AM,Darszon A,Visconti PE.Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models.Sci Rep.2016 Sep 15;6:33589.doi:10.1038 / srep33589。

[0116] Navarrete FA, Aguila L, Martin-Hidalgo D, Tourzani DA, Luque GM, Ardestani G, Garcia-Vazquez FA, Levin LR, Buck J, Darszon A, Buffone MG, Mager J, Fissore RA, Salicioni AM, Gervasi MG, Visconti PE. Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies. Front Cell Dev Biol. 2019 Nov 5;7:262. doi:10.3389 / fcell.2019.00262.

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Claims

1. 1. A method for preparing mammalian sperm, comprising: (a) Ca ２＋ incubating said mammalian sperm in the presence of an ionophore; (b) washing the mammalian sperm of step (a) with fresh medium to remove Ca ２＋ Removing ionophores, (c) treating the mammalian sperm of step (b) with HCO ３ － incubating in a medium containing the serovar at a temperature of 38°C to 43°C; (d) transferring the mammalian sperm of step (c) at an appropriate concentration into a suitable medium; The method, wherein the mammalian sperm is suitable for use in assisted reproductive technology (ART).

2. 10. The method of claim 1, wherein one or more sperm functions are selected from the group consisting of curvilinear velocity, head bob amplitude, sperm capacitation, percentage of superactivated sperm, and said sperm functions are improved compared to suitable control sperm.

3. 10. The method of claim 1, further comprising transferring the mammalian sperm of step d) to a fertilization medium, a storage medium, or a culture medium.

4. 4. The method of claim 3, wherein the fertilization medium is any sperm culture medium.

5. 4. The method of claim 3, further comprising the step of cryopreserving the mammalian sperm prior to use in the ART.

6. 10. The method of claim 1, wherein steps a) to c) may or may not include albumin.

7. 2. The method of claim 1, wherein the ART comprises IVF of an egg with sperm from the mammal in step (d) to produce an embryo.

8. 2. The method of claim 1, wherein the ART is selected from the group consisting of frozen embryo transfer (FET), IVF, ICSI, gamete intrafallopian tube transfer (GIFT), and zygote intrafallopian tube transfer (ZIFT).

9. 2. The method of claim 1, wherein the ART is AI of the mammalian sperm in step (d).

10. 10. The method of claim 9, wherein the AI ​​is IUI or intracervical artificial insemination.

11. 10. The method of claim 1, wherein the mammalian sperm of step (a) are restored from cryogenic storage.

12. 10. The method of claim 1, wherein the mammalian sperm in step (a) is restored from non-cryopreservation.

13. 2. The method of claim 1, wherein the mammalian sperm in step (a) are from a normal, oligozoospermic, asthenozoospermic, or teratozoospermic subject, or from a subfertile subject.

14. 2. The method of claim 1, wherein the mammalian sperm in step (a) are human sperm or sperm of any mammalian species.

15. 10. The method of claim 1, wherein the mammalian sperm in step (a) is provided as an individual sample or as a pool of two or more ejaculates.

16. 10. The method of claim 1, wherein the mammalian sperm of step (a) are concentrated from semen by density gradient centrifugation, swim-up, or microfluidics prior to step (a).

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