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 technology. In some embodiments, the methods provided by the present disclosure entail sequential changes in temperature, bicarbonate concentration, albumin concentration, and calcium ion carrier.

Description

Background Art

[0001] Male factor is a contributing factor in approximately 50% of couples with difficulty conceiving. An important aspect of assisted reproduction is to obtain optimal function of male gametes (sperm) to help maximize the fertilization process and availability of embryos for transfer. Therefore, there is a need for culture media, compositions and methods for increasing sperm function, although several approaches have been tried as described below:

[0002] A. Procedures to isolate the best sperm subpopulation of a sample. This procedure aims to enrich the sample for competent cells. Most of these widely used methods select sperm based on their motility. Examples of these methods are discontinuous gradient centrifugation, swim-up methods or the use of microfluidic devices. They are widely accepted in assisted reproductive technology (ART) clinics. Recently, a report by Serafín Pérez-Cerezales et al. (2018) showed that a high-quality sperm subpopulation was selected in vitro by thermotropism and that this subpopulation was the one that showed higher DNA integrity and lower chromatin compaction compared to the original sperm sample. In mice, these sperm produced more and better embryos by intracytoplasmic sperm injection (ICSI), doubling the number of successful pregnancies. ICSI procedures face the problem of selecting sperm in highly heterogeneous samples. Therefore, these procedures that enrich the population of sperm with fertilization capacity usually produce better results. However, these procedures do not enhance sperm function and only isolate those sperm with higher fertilization capacity.

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

[0004] 1. The work of Alvarez et al (1996) reported a procedure in which human spermatozoa were subjected to heat stress to see if motility was increased. The results of this procedure were later correlated with the results of in vitro fertilization (IVF) performed using a second semen sample obtained from the same patient. This procedure added a predictive tool to the clinic in that they showed that those patients whose sperm samples increased their motility under heat stress had a better chance of fertilization.

[0005] 2. The work of Mann et al. (2002) aimed to correlate DNA integrity after heat-induced stress with sperm capacitation indices. The authors showed that heat-induced damage was associated with poor sperm characteristics.

[0006] C. Procedure for inducing a type of sperm asymmetric motility called hyperactivation (HA) by incubation at elevated temperatures.

[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 over a 3.5-hour period to observe temperature-dependent HA, with higher temperatures producing higher HA. This type of prolonged heat stress generally promotes DNA damage caused by reactive oxygen species.

[0008] 2.Keepler et al. (1999). In this report, the authors showed that when cryopreserved sperm straws were split into two halves and incubated simultaneously, one half at 40°C and the other at 37°C, the warmer half accumulated more sperm and displayed higher HA values. These results are consistent with 40°C inducing higher HA and a thermotactic effect of increasing temperature from 37°C to 40°C.

[0009] 3. Chan et al. (1998). These authors reported that incubation of human sperm at 40°C for up to 4 hours improved sperm HA and resulted in higher zonular-free oocyte penetration as a test for assessing sperm fertilization capacity.

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

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

[0012] D. Use calcium (Ca 2+ ) Procedures in which ionophores enhance sperm motility and function.

[0013] 1. Tateno et al. (2012). In this article, the authors demonstrated that high concentrations (10-20 μM) of Ca 2+ The ionophore A23187 application immobilized mouse spermatozoa for 10 minutes. After the ionophore was removed, the mouse spermatozoa acquired HA. Of note, human spermatozoa did not regain motility after ionophore treatment, with clear differences between species. In addition, Sanchez-Cardenas et al. (2018) showed that although 5-10 μM A23187 significantly increased intracellular Ca within a few minutes, 2+ concentrations and immobilized sperm, but smaller concentrations (0.5 and 1 μM) elicited sperm HA without immobilization. This is consistent with the finding that intracellular Ca 2+ This is consistent with prior knowledge that concentration thresholds turn motility on and off.

[0014] 2. Navarrete et al. (2016). In this article, the authors showed that the use of high concentrations (10-20 μM) of Ca 2+ Treatment of mouse sperm with the ionophore A23187 for 10 min followed by removal overcomes the expression of CatSper1 - / - 、Slo3 - / - and Adcy10 - / - Infertility observed in sperm of knockout (KO) lines.

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

[0016] 1. Navarrete et al. (2019) showed that mouse sperm stopped moving when energy was limited in the incubation medium. Sperm motility was rescued when energy substrates were added back into the medium. After rescue, a significantly higher percentage of starved sperm acquired superactivated motility and exhibited increased in vitro fertilization capacity compared to sperm that were continuously incubated in standard capacitation medium. This technology (WO2017173391A1) has proven ineffective for inducing capacitation-related events in human sperm, as further demonstrated by Marin Briggiler et al. (2021).

[0017] The method described here is based on the regulation of temperature incubation and intracellular sperm Ca 2+ Proper manipulation of both to produce a synergistic effect HA and blastocyst formation rate. HA is a key parameter when analyzing IVF results (Wiser et al., 2014). It has been shown that among patients with increased HA, 93.3% had a high fertilization rate, compared to 64% found in the group without increased HA. More importantly, this approach improves the development rate of fertilized eggs until the blastocyst state and provides a higher blastocyst score, which is directly related to better pregnancy rates. Summary of the invention

[0018] i) In some embodiments, the present invention provides a method for inducing increased sperm function, comprising: (a) at a specific temperature, bicarbonate (HCO3 - ) concentration, albumin concentration, and Ca 2+ Incubating mammalian sperm under ionophore conditions thereby induces increased sperm function compared to suitable control sperm. (b) (a) The mammalian sperm provided is suitable for use in ART.

[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 with the mammalian sperm from step (a).

[0020] Iii) in some embodiments, the increased sperm function includes an increase in motility measured by computer-assisted semen analysis (CASA). In some embodiments, the motility increase includes an increase in the curve velocity (VCL) of mammalian sperm and a super-activated sperm percentage. In some embodiments, the increased sperm function includes an increase in the capacitation of mammalian sperm measured by an increase in the ability to fertilize an egg. In some embodiments, the increased sperm function includes producing an embryo with increased viability, and / or an improved implantation relative to an embryo produced with a suitable control sperm, or an increase in the ability of embryonic development to at least a 2-cell development stage, a blastocyst development stage, or offspring relative to an embryo produced with a suitable control sperm.

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

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

[0023] vi) In some embodiments, the temperature used after the ionophore incubation can be adjusted from 37.5°C to 45°C, preferably from 39°C to 44°C, more precisely from 40°C to 43°C, depending on the species.

[0024] vii) In some embodiments, the Ca used 2+ Ionophores can vary depending on the species and include, but are not limited to, 4-bromo-A23187 (4-Br-A23187), A23187, ionomycin, Ca 2+ Ionophore IV, including pharmacologically transient increases in intracellular Ca 2+ Concentration in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 AD describes the incubation temperature and intracellular Ca of human sperm 2+How changes in concentration produce synergistic effects in sperm motility parameters. In the control treatment, sperm were incubated in the presence of 0.2% DMSO for 2 minutes and incubated at 37°C for an additional 30 minutes. In the temperature treatment, sperm were incubated in the presence of 0.2% DMSO for 2 minutes and then incubated at 40°C for 30 minutes. In the ionophore treatment, sperm were incubated in the presence of 5 μM 4-Br-A23187 (0.2% DMSO) for 2 minutes and then incubated at 37°C for 30 minutes. In the combination treatment, sperm were incubated in the presence of 5 μM 4-Br-A23187 for 2 minutes and then incubated at 40°C for 30 minutes. Total motility ( Figure 1 A) Lateral head movement (ALH) Figure 1 B) Curve speed (VCL) Figure 1 C) and superactivated (HA) ( Figure 1 D). Data are expressed as mean ± standard error of the mean (SEM), n = 7. Statistical significance was analyzed by analysis of variance (ANOVA) and Tukey post hoc test. Different letters indicate statistically significant differences, p < 0.05.

[0026] Figure 2 AB shows different concentrations and / or different Ca 2+ Effects of ionophores on sperm hyperactivation (HA). Figure 2 In A, spermatozoa 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, and then exposed to 40 °C for 30 min. Data are presented as mean ± SEM of at least 3 independent experiments. Statistical significance was analyzed by ANOVA and Tukey post hoc test. *p < 0.05. Figure 2 In B, the effect of different incubation temperatures on sperm hyperactivation (HA) after ionophore treatment was analyzed. Spermatozoa were incubated with 5 μM 4-Br-A23187 for 2 min and then incubated for 60 min at 37, 38, 39, 40, 41, 42, or 43°C. Data are presented as mean ± SEM of at least 3 independent experiments.

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

[0028] Figure 4 AB illustrates the changes in temperature and Ca in sperm compared with conventional IVF. 2+How the combined effect of ionophore treatments produces more human blastocysts. Sperm from control and combined treatments were used in clinical assisted reproduction 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 number of blastocysts / mature eggs ( Figure 4 A) and embryonic sac / fertilized egg ( Figure 4 Fisher's exact test was used to analyze statistical significance.

[0029] *p<0.05. DETAILED DESCRIPTION

[0030] definition

[0031] Sperm function

[0032] i) In some embodiments, a method for increasing sperm function is provided herein. The method comprises incubating mammalian sperm in a sequential manner under different conditions and then fertilizing or injecting eggs in assisted reproduction.

[0033] The sequential incubation procedure consists of five steps: (a) incubating with - (b) washing the mammalian sperm with a medium also free of albumin for 5 minutes and resuspending in the same washing medium; (b) the mammalian sperm provided in step (a) is heated at 36° C. to 38° C. with 0.01 to 20 μM, preferably 0.1 to 6 μM Ca 2+ ion carrier, preferably 4-Br-A23187, for 1 to 20 minutes, preferably 3 to 6 minutes; (c) washing the mammalian sperm provided in step (b) with a medium containing up to 1% albumin for 1-10 minutes, resuspending in the same washing medium, and incubating at 36-38°C for 10-60 minutes (preferably 20-40 minutes); (d) incubating the mammalian sperm provided in step (c) at 38-43°C, preferably at 40°C, in a medium containing 3-9 mM HCO3 - (e) then transferring the appropriate concentration of the mammalian sperm provided in step (d) to a medium containing 10-30 mM HCO3 at 37°C. - (Preferably 25 mM HCO3 - ) and 0.1-1% albumin (preferably 0.5% albumin) in a culture medium. This sequential procedure increases sperm function compared to appropriate control sperm and is suitable for use in ART.

[0034] In some embodiments, fertilization of the egg is performed in vitro (IVF or ICSI) or in vivo (eg, through the cervix or IUI with sperm that has been previously incubated in a sequential procedure).

[0035] The sperm function of increase comprises one or more following aspects: the motility of increase, for example, the percentage of sperm showing super-activated motility in the colony assessed by CASA, the capacitation of increase, and the fertilization rate of increase, for example, develop to at least 2 cells, embryonic sac development or live birth.Therefore, in some embodiments, sperm function can be the percentage of sperm motility, VCL, ALH, sperm capacitation, super-activated sperm, the ability of ovum fertilization, embryonic generation.In some embodiments, the embryo produced by the sperm with increased function comprises one or more features selected from the ability of viability of increase, implantation of increase, development to at least 2 cell development stage, embryonic sac development stage or offspring of increase.

[0036] ii) As contemplated herein, an increase in one or more sperm functions is considered an increase in one or more sperm functions relative to a suitable control sperm. In some embodiments, the one or more sperm functions may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 100%, 200%, 300% or more. In some embodiments, the one or more sperm functions may be increased by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%.

[0037] iii) For purposes of this disclosure, sperm "activity" and / or "function" include physiological processes, such as sperm motility, temperature, rheology and / or chemotaxis, and the ability to fertilize an egg. The terms "activity" and / or "function" may further include processes occurring before, during, and during fertilization and / or interaction with the surrounding layers of the egg, such as capacitation and acrosome reaction (also known as acrosomal exocytosis) or fusion with the plasma membrane, and / or processes after fertilization of the egg, such as the formation of an embryo. In some embodiments, the embryo exhibits increased (longer) viability, improved implantation, and / or the ability to develop to the 2-cell stage, blastocyst, or offspring resulting in live births.

[0038] Sperm motility

[0039] i) The term "motility" is strictly related to the general movement of cells, but it can also be applied to other aspects of motility, such as the speed of movement of sperm cells and / or any increase or decrease in the proportion of sperm cells in any given population that are moving. Thus, the methods disclosed herein can be used not only to increase sperm motility, but also to increase the speed of movement of sperm cells and / or the proportion (percentage) of cells in any given sperm population that are moving.

[0040] Ii) The motility of sperm 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 determined in one of two ways. Either by subjective visual determination using a phase contrast microscope when the sperm is placed in a microscope slide, or by using CASA. Under phase contrast microscopy, a motile sperm count and a total sperm count are performed, and the velocity is assessed as fast, medium, or slow. A second method for assessing sperm motility is to use CASA, which objectively determines the motility characteristics of individual sperm cells in a sample.

[0041] iii) Therefore, the term "motility" includes the percentage of motile spermatozoa, which can be the percentage of the total number of assessed spermatozoa belonging to all World Health Organization (WHO) motility categories, regardless of speed or direction, except for the category considered "non-motile". Manual counts classify sperm cells into 4 categories (immotile, partially motile, non-linear and linear motile) using qualitative subjective selection criteria.

[0042] iv) The term "motility" includes the percentage of motile sperm, i.e. the percentage of the total sperm in a population that exhibit progressive motility, hyperactivated motility based on CASA assessment.

[0043] v) The methods disclosed herein can increase the percentage of progressive motility sperm, for example, the percentage of sperm that exhibit linear movement from one point to another, wherein the sperm has a head rotation of less than 90 degrees compared to otherwise non-progressive sperm, i.e., sperm that moves but does not move forward. In some embodiments, the increased motility includes an increase in the percentage of superactivated sperm. Superactivated sperm motility is characterized by sperm having a high amplitude, asymmetric flagellar beating pattern. Superactivated motility is characterized by violent movement, with many seemingly random changes, no clear path forward, and a rotation of the sperm head greater than 90 degrees. Superactivated sperm motility is more intense and short-term than progressive motility. Biologically speaking, superactivated sperm motility is important for enabling sperm to pass through the external blockade of the egg before fertilizing the mature egg. In some embodiments, the methods disclosed herein can increase the percentage of superactivated sperm in a given sperm population.

[0044] vi) It should be understood that other standardized metrics of sperm motility parameters can also be used. Other metrics of sperm motility include "speed" and "linearity", which can be assessed using an automated semen analyzer. In some embodiments, the methods disclosed herein can increase sperm function, including increasing the average path velocity (VAP), linear velocity (VSL), VOL, ALH and beating crossover frequency (BCF) or other movement parameters of sperm, including parameters known to those skilled in the art. VAP is the speed along the average path of sperm cells. VSL is the linear or progressive velocity of cells. VCL is a measure of the travel rate of the center of mass of the sperm head in a given time period. The linearity of forward advancement (LIN) is the ratio of VSL to VCL and is expressed as a percentage. The ALH of the sperm head is calculated based on the lateral deviation amplitude around the cell advancement axis or average path. The method of measuring sperm motility by CASA is well known in the art, for example, see WO2012061578A2. As expected herein, the increase in sperm motility is considered to be an increase in sperm motility relative to a suitable control sperm.

[0045] vii) In some embodiments, sperm with increased motility are provided, which 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 increase in sperm motility can be greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% relative to suitable control sperm. In some embodiments, the increase 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 increase in sperm motility can be at least 10-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold. In some embodiments, sperm motility can be increased by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, the increased sperm function or increased sperm motility can be an increase in superactivated sperm percentage. In some embodiments, the increased sperm function or increased sperm motility can be an increase in progressive motility sperm percentage. In some embodiments, the increased sperm function or increased sperm motility can be an increase in superactivated sperm percentage. In some embodiments, the level of superactivated sperm, progressive motile sperm is increased such that superactivated sperm, progressive motile sperm, or a combination thereof, comprises 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. An increase in sperm motility indicates increased sperm function.

[0046] Sperm Capacitation

[0047] i) In some embodiments, the increased sperm function includes an increase in sperm capacitation. "Sperm capacitation" refers to the ability of sperm to undergo acrosomal exocytosis or develop hyperactive motility and bind to and penetrate the zona pellucida of an unfertilized egg. Capacitation is achieved by the ability of sperm to bind to the zona pellucida and undergo ligand-induced acrosomal exocytosis.

[0048] ii) In some embodiments, sperm with increased capacitation are provided, which are the product of a process comprising 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% relative 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 times, at least 100 times, at least 1000 times, at least 10000 times. In some embodiments, sperm capacitation can 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 so that capacitated sperm may account for 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. An increase in sperm capacitation indicates increased sperm function.

[0049] Fertilization ability

[0050] In some embodiments, sperm function includes the ability of sperm to fertilize an egg. The fertilization ability of sperm can be determined, for example, by IVF. IVF is a fertilization process in which an egg is combined with sperm in vitro ("in glass") outside the body. The process includes monitoring and stimulating the ovulation process of a woman, removing one or more eggs from the woman's ovaries, and allowing sperm to fertilize them in a culture medium in a laboratory. In embodiments, sperm with increased fertilization ability are provided, which are the products of a process of incubating sperm in one or more steps of a sequential program that enhances sperm. In some embodiments, the increase in fertilization ability can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% relative to a suitable control sperm. In some embodiments, the increase in fertilization capacity 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 fertilization capacity can be at least 10 times, at least 100 times, at least 1000 times, at least 10000 times. In some embodiments, fertilization capacity can be increased by 10% to 200%, 25% to 150%, 50% to 100%, or 70% to 90%. In some embodiments, the level of fertilizing capacity 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. An increase in fertilizing capacity indicates increased sperm function and increased fertilization.

[0051] Producing embryos

[0052] i) In some embodiments, sperm function includes producing embryos. In some embodiments, sperm with increased function prepared by the method herein is provided to enter the egg to promote fertilization, wherein promoting fertilization can include the production of embryos. In some embodiments, sperm with increased function prepared by the method herein is provided to enter the egg in vitro, thereby producing embryos in vitro. In some embodiments, sperm with increased function prepared by the method disclosed herein is provided to enter the egg in vivo through the IUI of sperm, thereby producing embryos in vivo. In some embodiments, sperm that has been incubated under one or more steps of the sequential program of enhancing sperm is fertilized in the reproductive tract of a female subject, and is provided to enter the egg to produce embryos in vivo. In some embodiments where the embryo is produced in vitro, the embryo can be cryopreserved for later use, or can be further cultured in vitro to enable the embryo to develop. In some embodiments, the embryo develops to at least the 2-cell stage, then cryopreserved and / or implanted in a female subject. In some embodiments, the embryo develops to a developmental stage greater than the 2-cell stage in vitro, then further processed. In some embodiments, the embryo develops in vitro to the blastocyst stage and is then further processed (e.g., cryopreserved or implanted in a female subject to develop into a complete offspring). For in vitro incubation and culture of embryos during the ART procedure, a series of suitable culture media can be used, 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 also include one or more growth factors. There are a variety of suitable culture media on the market, such as Earle's medium, Ham's F10 medium and human fallopian tube fluid (HTF) medium. The present disclosure also contemplates in vitro co-culture of embryos on a layer of "feeder cells" by methods known in the art. Suitable "feeder cells" for co-culture may include, for example, bovine fallopian tube cells or human fallopian tube epithelial cells.

[0053] Ii) Those skilled in the art will recognize that the advantages provided by the sperm with increased function prepared by the methods disclosed herein are not limited to increasing fertilization. On the contrary, the methods and preparations of the present invention are equally applicable to treatments that promote fertilization, whether the embryo is produced in vitro by ART or in the reproductive tract of an animal. The methods of the present invention are applicable to improving fertilization, embryo viability, embryo implantation, and pregnancy rates in assisted or other unassisted pregnancies. The embodiments of the present disclosure also provide methods for increasing the fertilizing ability of sperm in male animals.

[0054] iii) In the context of this specification, the terms "embryos with increased viability" and "embryos with longer viability" refer to an increase or enhancement in the likelihood of embryo survival produced by mammalian sperm (e.g., mammalian sperm with one or more increased sperm functions) of the methods and preparations disclosed herein compared to the likelihood of embryo survival produced by appropriate control sperm. In some embodiments, the embryo is produced by ART, such as IVF or ICSI. In some embodiments, the embryo is produced in vivo in the reproductive tract of a female mammalian subject by artificial insemination (AI).

[0055] iv) For the purposes of the present disclosure, embryo viability can be reflected in many indicators. For example, increased embryo viability can lead to increased embryo implantation rate after fertilization, reduced pre-implantation and post-implantation embryo lethality, increased clinical pregnancy rate or increased birth rate. Therefore, the present disclosure also relates to methods for preventing embryo apoptosis or developmental retardation and methods for improving animal pregnancy rates. Embryonic viability can refer to the viability of embryos in vitro or in vivo.

[0056] V) in some embodiments, there is provided sperm with the ability to produce an embryo with increased viability, these sperm are the products of the process of hatching sperm in one or more steps of the sequential program that strengthens sperm. In some embodiments, providing sperm with increased function to enter the egg promotes fertilization. In some embodiments, promoting fertilization includes producing an embryo with increased viability. In some embodiments, relative to the embryo produced by suitable control sperm, the increase of embryo viability produced by the sperm prepared by the method of this paper when entering the egg can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%. In some embodiments, the increase of embryo viability 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 viability can be at least 10 times, at least 100 times, at least 1000 times, at least 10000 times. In some embodiments, embryo viability 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 with increased motility 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 increased sperm function and / or increased fertilization.

[0057] vi) Typically, the cleavage stage of the embryo occurs during the first three days of culture. In vitro generated embryos can be cryopreserved and transplanted at a subsequent stage or immediately transplanted to a female subject by embryo transfer. "Embryo transfer" is a procedure in which one or more embryos and / or blastocysts are placed in the uterus or fallopian tubes. In traditional IVF processes, when each embryo is at the four (4) cell stage, the embryo is transplanted to the uterine cavity two days after fertilization, or when the embryo is at the eight (8) cell stage, the embryo is transplanted to the uterine cavity three days after fertilization. It has been recognized that it may be necessary to use embryos at the blastocyst stage reached on the 5th to 7th day of culture. The present disclosure allows embryo transfer at any time within the scope of embryo / blastocyst development. By visual observation, such as using microscopy, when the blastocyst cavity is clearly evident and accounts for more than 50% of the embryo volume, the blastocyst or embryo is considered ready to be transplanted to the uterus. In an in vivo environment, this stage is usually reached 4 to 5 days after fertilization, i.e., soon after the embryo passes through the fallopian tube and arrives at the uterus. Embryonic developmental stage can be determined by visual observation of the embryo using microscopy, and the embryo will simultaneously display certain defined physical or morphological characteristics prior to implantation in the uterus. According to the classification of Gardner et al. (1998), the embryonic sac maturity state will be determined as ranging from II AB to VI AA.

[0058] vii) Method disclosed herein causes the generation of embryo, and it develops to 2-cell development stage, embryo sac development stage or develops into the ratio of offspring and live birth and increases. In some embodiments, it is provided that sperm with the ability of embryo developed by normal development stage (for example, 2-cell stage, embryo sac stage, develops into offspring and live birth) is produced, and these sperm are the product of the process of hatching sperm in one or more steps of the sequential program of enhancing sperm. In some embodiments, sperm with increased function is provided to enter egg and promote fertilization. In some embodiments, promoting fertilization includes producing the embryo with the ability of developing by normal development stage (for example, 2-cell stage, embryo sac stage, develops into offspring and live birth) that increases. In some embodiments, relative to the embryo produced by appropriate control sperm, the increase of the ratio of embryo development by the normal development stage produced by the sperm prepared by the method can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%. In some embodiments, the increase of embryonic development through the ratio of normal development stage 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 of embryonic development through the ratio of normal development stage can be at least 10 times, at least 100 times, at least 1000 times, at least 10000 times.In some embodiments, embryonic development through the ratio of normal development stage can increase 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 with the ability to progress through normal developmental stages 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 the ability to progress through one or more normal developmental stages indicates increased sperm function and / or increased fertilization.

[0059] viii) In vivo, the embryo attaches or implants to the uterine wall, forms the placenta, and develops into a fetal offspring during pregnancy until delivery. Two weeks after transplantation, a blood test using, for example, β-hCG (human chorionic gonadotropin) and other techniques generally known in the art are tested to determine whether one or more embryos have implanted in the endometrium, i.e., whether the procedure has led to the start of a successful pregnancy. US4315908A proposes a method for detecting β-hCG in urine by radioimmunoassay. US8163508B2 provides a method and kit for predicting the pregnancy of a subject by the β-hCG method by measuring the amount of an early pregnancy-related subtype of β-hCG in a sample. Such diagnostic methods and other methods can be used within the scope of the present disclosure.

[0060] ix) In some embodiments, sperm with the ability to produce embryos with improved implantation rate or improved pregnancy rate is provided, and these sperm are the products of the process of incubating sperm in one or more steps of the sequential program of enhancing sperm. In some embodiments, providing sperm with increased function to enter the egg promotes fertilization. In some embodiments, promoting fertilization includes producing embryos with improved implantation rate or improved pregnancy rate. In some embodiments, relative to the embryo produced by suitable control sperm, the increase of the embryo implantation rate or the post-implantation pregnancy rate of the sperm prepared by the method of this paper can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%. In some embodiments, the increase of 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 rate or pregnancy rate can be at least 10 times, at least 100 times, at least 1000 times, at least 10000 times. In some embodiments, embryo implantation or pregnancy rate 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 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. Producing embryos with improved implantation (ie, increased implantation rate) or increased post-implantation pregnancy rate indicates increased sperm function and / or increased fertilization.

[0061] Mammalian sperm

[0062] i) The methods disclosed herein include increasing one or more sperm functions to facilitate fertilization. Also provided are preparations of sperm having increased functions. As described herein, the sperm may be from a vertebrate, preferably a mammal.

[0063] ii) Mammals include, but are not limited to, humans, rodents, primates, wild or domesticated animals, including wild animals, farm animals, sports animals, and pets. Rodents include, for example, mice, rats, and hamsters. Domestic and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, cat species (e.g., domestic cats) and canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). Mammalian sperm can be from non-human mammals, including ungulates, such as even-toed ungulates (e.g., pigs, boars, hippopotamuses, camels, llamas, musk deer (mouse deer), deer, giraffes, pronghorns, antelopes, mountain antelopes (including sheep, goats, etc.), or cattle) or odd-toed ungulates (e.g., horses, tapirs, and rhinos), non-human primates (e.g., monkeys, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys), canines (e.g., dogs), or cats. Mammalian sperm can be from members of the superorder Laurasiatheria. The superorder Laurasiatheria can include a group of mammals as described by Waddell et al. (1999). Members of the order Laurasiatheria may include Eulipotyphla (hedgehogs, shrews, and moles), Perissodactyla (rhinos, horses, and tapirs), Carnivora (carnivores), Cetartiodactyla (even-toed ungulates and cetaceans), Chiroptera (bats), and Pholidota (pangolins). Members of the order Laurasiatheria may be ungulates, such as odd-toed ungulates or even-toed ungulates. Mammalian sperm may be from a member of the order Carnivora, such as a cat or dog. In some embodiments, the mammalian sperm is sperm from a human, non-human primate, pig, cattle, horse, sheep, dog, cat, or mouse. In some embodiments, the mammalian sperm is human sperm.

[0064] Iii) in some embodiments, mammalian sperm is provided by healthy male mammals. In some embodiments, the mammalian sperm is from a male with certain sperm dysfunction, such as DNA mutation or few sperm counts, reduced sperm motility and abnormal sperm morphology. In some embodiments, mammalian sperm can be from a male with low fertility or few sperms. Mammalian sperm can be from a male suffering from, for example, oligospermia, teratozoospermia, asthenozoospermia or few weak teratozoospermia. Oligospermia refers to a condition characterized by a sperm concentration of <20 million / mL. Asthenozoospermia refers to a condition characterized by reduced sperm motility. Teratozoospermia refers to a condition characterized by the presence of sperm with abnormal morphology. Few weak teratozoospermia refers to a condition including oligospermia (few sperm counts), asthenozoospermia (poor sperm movement) and teratozoospermia (abnormal sperm shape). In some embodiments, the sperm is obtained from subfertile males or oligospermic males, for example, males having a sperm count of less than about: 2000, 1900, 1800, 1800, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 2 million / mL, for example less than 15 million / mL.

[0065] iv) In some embodiments, sperm is separated (or isolated) from semen prior to using the methods described herein. Any sperm enrichment or isolation method can be used in conjunction with the present invention, including density gradient centrifugation, sludge extraction, microfluidics, or a combination thereof.

[0066] v) In some embodiments, the sperm used in the methods provided herein is fresh, or is from a stored stock solution. For example, sperm is recovered from cryogenic storage prior to processing. In other embodiments, sperm is recovered from non-cryogenic storage prior to processing.

[0067] vi) In some embodiments, different numbers of sperm can be used in the methods provided herein, including a portion of a single ejaculation or the entire ejaculation. In some embodiments, sperm are pooled from at least two ejaculations (e.g., 2, 3, 4, 5, 6 or more ejaculations).

[0068] Methods of obtaining sperm samples

[0069] i) Several methods of collecting live sperm, such as masturbation into a container, the gloved hand method, the use of an artificial vagina, and electro-ejaculation. Animal semen can be collected using an artificial vagina, an electro-ejaculation device, or by hand massaging the animal's vas deferens ampulla (ampule). It can also use different methods, such as surgically puncturing the testicle or epididymis or removing the testicle or epididymis and collecting sperm in the surrounding medium, directly collecting from any part of the male reproductive tract, including testicular sperm and sperm obtained from the epididymal capus, epididymal body, or epididymal tail.

[0070] Ii) In some embodiments, the sperm used in the method provided by the invention can be a sample freshly collected from a source animal (e.g., mammal), or can be a sample thawed in advance or cryopreserved. When collecting or subsequently, the sperm collected can be merged with any of many various buffers compatible with sperm, such as trichloroacetic acid (TCA), 4-2 (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) or phosphate buffered saline (PBS). The sperm suspension collected can also contain a variety of other additives to keep sperm alive. Exemplary additives include protein sources, antibiotics, growth factors, and compositions that regulate oxidation / reduction reactions in cells and / or outside cells. Alternatively, semen can be collected in an empty container, then contacted with buffer to form a sperm suspension within a few minutes to a few hours after collection. In some embodiments, sperm cells can be collected directly into a container containing a culture medium (e.g., M199, synthetic fallopian tube fluid, PBS, BO, Test-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.).

[0071] iii) In some embodiments, sperm collection includes processing high viscosity semen or washing sperm prior to performing the methods disclosed herein. Washing includes centrifuging semen samples or thawed sperm through dilute wash medium, which allows collection of sperm-rich pellets. After the washing step, or instead of the washing step, a procedure for separating motile sperm from the sample can be performed.

[0072] iv) In some embodiments, sperm is separated from semen before being used in the methods disclosed herein. In some embodiments, sperm with increased function can be further enriched from sperm prepared according to the methods disclosed herein. Typically, sperm is separated or enriched by allowing motile sperm to swim from dead sperm, non-motile sperm and debris (sperm swimming method), by density gradient centrifugation sperm or by passing sperm through a column that binds dead sperm and debris. Separation (or enrichment) of sperm from semen is performed by a method selected from washing and spinning, sedimentation, direct swimming, precipitation and swimming, and buoyancy density gradient methods. These methods are well known in the art. They are traditionally 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 separation procedures, such as sedimentation, direct swimming, precipitation and swimming, and buoyancy density gradient methods.

[0073] v) Direct swimming method means self-selection of motile spermatozoa and generally involves placing an aliquot of culture medium layer on top of a semen sample or sperm preparation disclosed herein and allowing it to stand at room temperature for at least 30 minutes. Motile sperm cells migrate to the top layer (culture medium) from where they can be recovered. The method may also include a centrifugation step.

[0074] vi) The method can be varied depending on the number of motile cells in the sample and combined with further separation / separation techniques. For example, the swimming procedure can be performed by layering 1 mL of culture medium containing albumin onto 1 mL of bottom semen liquid in a test tube. After incubation for 1 hour at 37°C in air or in 5% CO2, the upper phase of the culture medium to which sperm with better motility properties have migrated is collected. The technique can also include a centrifugation step, such as density gradient centrifugation, or be combined with it. The separated, isolated or enriched sperm are then used in the methods disclosed herein, or can be cryopreserved, for example, before further processing. In the case of sperm preparations prepared by the methods herein, they can be used for IVF, ICSI or AI after the enrichment step, or can be cryopreserved, for example, for later use. Therefore, for any of these separation or enrichment methods, the sample can be semen, partially purified sperm, purified sperm, or sperm with increased function 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% after separation or enrichment of sperm using a separation method (e.g., direct swim-up, sedimentation and swim-up, and buoyant density gradient) compared to an untreated semen sample or an unenriched sperm preparation.

[0075] vii) In some embodiments, after separation, enrichment and washing, the sperm pellet can be resuspended in a medium suitable for further processing, including a storage medium, a HTF medium for culture, a medium for sequential procedures to enhance sperm. As it relates to sperm with increased functionality prepared by the methods disclosed herein, the sperm preparation can be resuspended in a storage medium, a HTF medium for culture, a medium for insemination, fertilization potential assays described herein, IVF, freezing, IUI, cervical cap insemination, and the like. The sperm can be added to the medium, or the medium can be added to the sperm. The medium can be a balanced salt solution, which can contain a zwitterionic buffer such as TES, HEPES, PIPES, or other buffer such as sodium bicarbonate. In general, the culture medium used for diluting sperm or culturing sperm, oocytes, embryos or embryonic stem cells is a balanced salt solution, such as M199, synthetic tubal fluid, PBS, BO, Test-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 culture medium and emCare culture medium (PETS, Canton, Tex.). In some embodiments, TALP or HTF is used for sperm culture medium, and CZB is used for embryo culture medium. The sperm or embryo of the present disclosure can be stored in a low-temperature culture medium containing a cryoprotectant.

[0076] Suitable control sperm

[0077] i) Suitable control sperm can be sperm incubated under control conditions, such as in a control buffer (such as HTF medium or modified HTF medium). HTF includes a sodium bicarbonate buffer system and can be used for purposes where a CO2 atmosphere is required during incubation. Modified HTF includes a combined sodium bicarbonate and HEPES buffer (HTF-HEPES). Suitable examples of HTF medium or modified HTF medium include commercially available medium from Irvine Scientific, Santa Ana, California. In some embodiments, HTF medium in which sodium bicarbonate is omitted is used. Sperm can be incubated for a time sufficient to provide a measurable change in sperm motility (or other characteristics); in a specific embodiment of the method, the incubation is 1 minute to 24 hours, 15 minutes to 3 hours, 30 minutes to 1.5 hours, about 1 hour, or any sub-range or sub-value thereof. It should be understood that suitable control sperm can be at least one sperm or sperm population, such as a sperm preparation or a sperm suspension.

[0078] Sperm preparation

[0079] i) In some embodiments, the invention provides sperm preparations, such as preparations of activated or enhanced sperm. These are collectively referred to as "sperm preparations provided by the invention" or "preparations provided by the invention". In some embodiments, the invention provides preparations comprising at least 5% superactivated sperm, such as 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 superactivated sperm, such as between about: 5-20, 8.5-20, 10-20 or 12.5-20% superactivated sperm preparations. As the skilled artisan will appreciate, sperm can be separated based on HA phenotype, but in some embodiments, the above percentages are based on a preparation that has not been activated and then sorted based on HA (however, in some embodiments, the sperm preparation may have been pre-treated, for example to separate or otherwise enrich sperm from other semen components, including certain irregular sperm).

[0080] ii) In some embodiments, the present invention provides a sperm preparation prepared by any one of the methods provided by the present invention.

[0081] iii) In some embodiments, the present invention provides a sperm preparation prepared by enriching sperm from the semen of a male subject, such as a normospermic male, a subfertile male or an oligospermic male, such as a subfertile (including oligospermic) male, and incubating the sperm in one or more steps of a sequential procedure for sperm enhancement.

[0082] Iv) For any preparation provided by the invention, sperm can be from any male subject, such as a mammal, and in some embodiments, can be from a human. In some embodiments, the human is a normal sperm male, or in other embodiments, the male is a subject with oligospermia or low fertility (e.g., low sperm motility).

[0083] Promote fertilization

[0084] i) The sperm preparation with increased function prepared by the method disclosed herein can be used to promote fertilization. Therefore, the present disclosure relates to a method for promoting fertilization. The method includes incubating sperm under one or more steps of a sequential procedure for enhancing cells, and providing sperm with increased function to enter the egg under conditions that promote fertilization. The sperm preparation with increased function can be applied to human IVF, ICSI, AI (e.g., IUI) and biomedical research industries of animal models of human diseases (infertility, sperm dysfunction), as well as breeding and agricultural industries. Sperm with increased function prepared by the method disclosed herein can be provided to enter unfertilized eggs of the same species as sperm to promote IVF, ICSI, or can be used for AI of female subjects of the same species as sperm, including, for example, IUI.

[0085] Internal fertilization

[0086] i) Sperm with increased function prepared by the method disclosed herein can promote in vivo fertilization by providing sperm with increased function to enter the egg in the reproductive tract of a female subject of the same species as the sperm. In vivo fertilization can be accomplished by AI of sperm, for example, by intracervical insemination or IUI. Standard AI and IUI and other methods are well known to those skilled in the art. In some embodiments, the sperm is provided to enter the egg in the reproductive tract of a female subject by IUI with increased function to promote fertilization of the egg. In other embodiments, sperm can enter the egg in vivo by IUI of mammalian sperm, and the sperm has been incubated under one or more steps of the sequential procedure for enhancing sperm. The injected sperm can be kept in a suitable liquid for use. The liquid used for this purpose can be a liquid commonly used as an AI culture medium.

[0087] In vitro fertilization (IVF)

[0088] i) Current methods and sperm preparations disclosed herein are particularly beneficial to promoting ART fertilization, such as embryo viability after ART, and specifically IVF. Other suitable ART techniques applicable to the present disclosure include, but are not limited to, gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), embryo sac transfer (BT), ICSI, gamete, embryo and cell cryopreservation, in vitro embryo preparation for embryo biopsy and other forms of embryo micromanipulation, including embryo formation by nuclear transplantation and production of transgenic lines and genetically modified lines. It may also be applicable to the production of embryonic stem cell lines.

[0089] ii) In some embodiments, sperm with increased function prepared by the methods disclosed herein can be used to fertilize eggs in vitro, such as by microinjection, including ICSI, and other methods known in the art. Typically, in IVF, after fertilization, cells are grown to the blastocyst stage and then implanted. The methods disclosed herein result in increased formation of embryos with longer viability and increased ability to develop to the 2-cell stage, blastocyst stage. Therefore, the sperm preparations disclosed herein can be used in IVF procedures, including, for example, ICSI.

[0090] iii) The method of the present disclosure includes providing sperm prepared by the method herein into an egg to promote IVF. Providing sperm into an egg in vitro can be performed in an appropriate culture medium. The culture medium used for this purpose can be a culture medium commonly used as an IVF culture medium, such as an HTF culture medium. The temperature conditions used to provide access can be a general temperature used in IVF, for example, it can be the average body temperature of a mammal or a temperature close thereto. The time for providing access can be any time generally 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 about 24 hours.

[0091] Fertilization purposes

[0092] i) The methods and sperm preparations disclosed herein are generally applicable to many species, including humans, cattle, dogs, horses, pigs, sheep, birds, rodents, etc. Although available when fertilization is needed, the current methods can be particularly used for animals and humans with fertilization dysfunction to increase the possibility of conception. These dysfunctions include low sperm count, reduced sperm motility, and abnormal sperm morphology. Therefore, the methods disclosed herein can be used to prepare sperm with increased function in infertility clinics, which are then used for IVF or IUI. The methods described herein can be used to improve AI, IVF, or ICSI of exotic and / or endangered species. Therefore, these methods can be used to promote fertilization of animals kept in zoos, as well as conservation programs aimed at improving the reproduction of endangered animals in the wild. For example, the methods and sperm preparations disclosed herein can be used to improve fertilization and pregnancy rates in animal husbandry, species with agricultural value, and species cultivated for conservation purposes.

[0093] ii) In addition, the methods and compositions of the present invention can be used in AI programs, such as in commercial breeding. The method can be performed using sperm from domestic animals (especially livestock) as well as sperm from 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 the breeding of cattle. These methods and preparations can be used for AI in the livestock production industry, where it is desired to influence the results of offspring with one or more preferred characteristics or traits by introducing specific genetically determined traits into livestock, for example, offspring of a specific sex, offspring with enhanced milk production, offspring with high-quality meat production. The use of the methods described herein will increase pregnancy rates. Mammalian sperm is often damaged by freezing and thawing, resulting in lower fertility. By improving the performance of surviving sperm, sperm prepared by the methods disclosed herein can promote higher pregnancy rates per estrus cycle when used for insemination, reducing the number of cycles required to ensure conception, thereby reducing the overall cost of AI.

[0094] iii) Semen from animals with highly desirable traits can be used to inseminate more females, since fewer cycles are required to ensure that any one female is pregnant. For such applications, semen is obtained from males with the desired characteristics. In order to influence the gender outcome of the offspring produced, the sperm preparation can be sorted into cells carrying X and Y chromosomes, and / or enriched with sperm having one or more sperm functions disclosed herein. Sperm can be sorted into a population enriched with sperm carrying X and Y chromosomes using a flow cytometer / cell sorter by conventional methods, such as described in US5135759A. Sperm prepared by the methods disclosed herein can be sorted into a population containing a certain percentage of sperm cells carrying X chromosomes or carrying Y chromosomes. For example, one of the populations of sperm may contain at least about 65% of sperm cells carrying an X chromosome or carrying a Y chromosome, at least about 70% of sperm cells carrying an X chromosome or carrying a Y chromosome, at least about 75% of sperm cells carrying an X chromosome or carrying a Y chromosome, at least about 80% of sperm cells carrying an X chromosome or carrying a Y chromosome, at least about 85% of sperm cells carrying an X chromosome or carrying a Y chromosome, at least about 90% of sperm cells carrying an X chromosome or carrying a Y chromosome, or even at least about 95% of sperm cells carrying an X chromosome or carrying a Y chromosome. In some embodiments, sorting can be performed before preparing sperm with increased function as disclosed herein. In some embodiments, sorting can be performed before providing sperm with increased function into eggs for fertilization, such as in IVF, ICSI or AI.

[0095] iv) The methods and preparations provided by the present invention can be used for assisted fertilization, such as IVF, including through ICSI. In some embodiments, any of the methods provided by the present invention can include the step of providing sperm to the female reproductive tract. In some embodiments, the sperm preparation provided by the present invention (with increased sperm function) can be provided to enter the egg for a time sufficient to fertilize the egg, which can be in vitro (e.g., IVF, including ICSI), or in some embodiments in the female reproductive tract. In some embodiments, this method requires the subsequent implantation of the fertilized egg into a female carrier.

[0096] Example

[0097] The disclosure will be described in more detail by the following specific examples. The following examples are for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce alternative embodiments according to the present invention.

[0098] Embodiment 1: Materials and methods

[0099] Reagents and culture media: Unless otherwise specified, all reagents and chemicals were purchased from Sigma Aldrich (St. Louis, MO). The culture medium used for human sperm incubation was HTF-HEPES containing 101.5 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2, 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, 10 μg / mL gentamicin, balanced at pH 7.3-7.4. Some steps included the addition of 6 mM NaHCO3 concentration. Alternatively, the culture medium was supplemented with 0.5-1% bovine serum albumin (BSA) or 2-10% human serum substitute supplement (SSS).

[0100] For routine treatments, modified HTF from Irvine Scientific (Santa Ana, CA) supplemented with 10% SSS was used.

[0101] Gamete co-incubations were performed in Irvine Scientific HTF with 10% SSS under light oil balanced with 7.5% CO2.

[0102] Semen samples: Semen samples were obtained by masturbation into a sterile container from healthy normozoospermic men or men seeking infertility treatment. Prior to semen processing, ejaculates were liquefied at room temperature for up to 2 hours and analyzed following WHO standard procedures. Spermatozoa were separated from seminal plasma by density gradient centrifugation or direct swim-up technique to isolate high-quality viable spermatozoa.

[0103] Density gradient centrifugation: Semen samples were centrifuged at 200-500 x g for 20 min through a 40% / 80% discontinuous density gradient (PureSperm; Nidacon, Gothemburg, Sweden). The resulting sperm pellet was washed with 2-5 mL of HTF-HEPES medium (with or without HCO3 - ) resuspended, divided into several tubes according to the number of treatments, and centrifuged at 500-700 x g for 5 minutes. After removing the supernatant, each pellet was resuspended in the appropriate culture medium and the sperm concentration was adjusted to 5-20E 6 / mL.

[0104] Sperm Swimming Method: Aliquots of 0.5-1 mL of semen were overloaded 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 several tubes according to the number of treatments, and centrifuged at 400-700 x g for 5 minutes. Each pellet was then resuspended in HTF medium + 10% SSS.

[0105] Sperm motility analysis: An aliquot of 9.7 μl of sperm suspension was placed on a slide under an 18 mm x 18 mm cover slip to obtain a preparation depth of 30 μm and maintained at 37°C using a temperature-controlled stage. Sperm motility parameters were assessed using the SCA system (SCA v.6.2.0.1., Microptic SL, Barcelona, ​​Spain), acquiring 60 frames per second. At least five microscopic fields and 300 spermatozoa were analyzed. The following parameters were assessed: curvilinear velocity (VCL, μm / s), linear velocity (VSL, μm / s), average path velocity (VAP, μm / s), linearity (LIN: VSL / VAP x 100, %), straightness (STRx100, %), wobble (WOB: VAP / VCL x 100, a measure of the side-to-side movement of the sperm head, %), amplitude of lateral head displacement (ALH, μm), and beat crossing frequency (BCF, Hz). Sperm motility was measured and classified as follows: rapid forward motion (VCL ≥ 35 μm / s; STR ≥ 80%), moderate forward motion (VCL ≥ 15 μm / s; STR ≥ 80%), homeostasis (VCL < 15 μm / s; VAP ≥ 5 μm / s), and immotility (VAP < 5 μm / s). The percentages of total motility (rapid forward motion + moderate forward motion + homeostasis) and progressive motility (rapid + moderate forward motion) were recorded. The flutter was set at 25 μm / s. Sperm were considered hyperactivated when presenting VCL ≥ 150 μm / s, LIN < 50%, and ALH ≥ 3.5 μm.

[0106] Embodiment 2: Enhanced sperm motility

[0107] This example shows that precise manipulation of sperm incubation conditions, such as temperature and intracellular Ca 2+ How concentration enhances human sperm motility. Semen samples from healthy men were processed by density gradient centrifugation as described in Example 1.

[0108] In the control treatment, spermatozoa were incubated with 0.2% DMSO in HTF-HEPES for 2 min at 37°C. After centrifugation at 400 x g for 5 min, sperm cells were 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 further incubated at 37°C for 30 min in HTF-HEPES medium containing 0.5% BSA.

[0109] In the temperature treatment, spermatozoa were incubated with 0.2% DMSO in HTF-HEPES for 2 min at 37°C. After centrifugation at 400 x g for 5 min, sperm cells were 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 further incubated at 40°C for 30 minutes.

[0110] In the ionophore treatment, spermatozoa were incubated with 5 μM 4-Br-A23187 (0.2% DMSO) in HTF-HEPES for 2 min at 37°C. After centrifugation at 400 x g for 5 min, sperm cells were 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 further incubated at 37°C for 30 minutes.

[0111] In the combined treatment, spermatozoa were incubated with 5 μM 4-Br-A23187 (0.2% DMSO) in HTF-HEPES for 2 min at 37°C. After centrifugation at 400 x g for 5 min, sperm cells were 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 further incubated at 40°C for 30 minutes.

[0112] Total motility was not affected for any treatment ( Figure 1 A). The combined treatment resulted in significantly higher values ​​of HA-related kinematic parameters (e.g., VCL and ALH) compared to the other treatments ( Figure 1 BC). In particular, Figure 1 D shows that sperm treated at 40°C (temperature treatment) produced better HA values ​​(8% HA increase) compared to the control procedure. Similarly, but to a lesser extent, ionophore-treated human sperm also produced higher HA values ​​(3% HA increase). However, when sperm were first exposed to ionophores and then incubated at 40°C, a significant synergistic effect was observed (16% HA increase compared to the control procedure, 50% higher than the 11% caused by the additive increase). Values ​​are expressed as mean ± SEM of 7 independent experiments. Statistical significance of the data was analyzed using one-way ANOVA with Tukey post hoc test.

[0113] like Figure 2 As shown in A, pharmacological methods were used to temporarily increase intracellular Ca 2+ The described synergistic effects were observed with different concentrations. For example, incubation with 5 μM 4-Br-A23187 for 2 minutes, 1 μM 4-Br-A23187 for 5 minutes or 0.5 μM A23187 for 2 minutes followed by incubation at 40°C for 30 minutes produced higher levels of HA compared to the control.

[0114] In addition, different temperatures of 38°C or higher can be used after the ionophore pulse. Incubation of sperm cells at temperatures between 38°C and 43°C for 60 minutes produced an increase in the percentage of HA compared to the control ( Figure 2B).

[0115] Due to the heterogeneity of human semen, the effect of temperature on HA increase is variable between donors ( Figure 3 AC). Incubation at 40°C produced higher levels of HA at each time point compared to 37°C, especially during the first hour.

[0116] Example 3 : Methods for treating sperm to improve fertilization rates and embryo development in human IVF

[0117] This example describes the use of sperm treated according to certain embodiments of the present invention to enhance fertility in human subjects undergoing IVF.

[0118] The subjects were adult women who underwent anonymous egg donation (i.e., 9 subjects aged between 18 and 35 years). Before egg retrieval, the subjects were treated with standard procedures (e.g., ovulation inhibition, followed by ovulation stimulation, hCG trigger injection as indicated). The 14 retrieved eggs were divided into two groups: a control group and a combined treatment group. The semen samples of patients who visited the reproductive clinic were divided into two halves and processed by density gradient centrifugation. In the half assigned to the control group (control treatment), the sperm was placed in HTF supplemented with 10% SSS for 2-4 hours at room temperature. For the treated sperm (combined treatment), the sperm was kept at room temperature for 0.5-2 hours in HTF-HEPES containing 2% SSS. Subsequently, the sperm was washed with a medium without SSS and incubated in HTF-HEPES at 37°C for 2 minutes with 5 μM 4-Br-A23187. After centrifugation at 400x g for 5 minutes, 1 mL of 10% SSS and 6 mM HCO3 were added. - Sperm cells were washed with HTF-HEPES medium and resuspended in 6 mM HCO3 - The sperm cells were further incubated at 37°C for 30 minutes and at 40°C for 60 minutes.

[0119] For the combined treatment group and the control group, sperm and eggs were incubated in vitro. The number of fertilized eggs (two pronuclei the next morning) and the number of high-quality embryo sacs at 5 and 6 days were recorded (see Table I). High-quality embryo sacs were embryo sacs with a score of 3BB or above according to the Gardner grading system (Gardner, 2016). Compared with the control group, the number of fertilized eggs ( Figure 4 A) and total mature eggs ( Figure 4 B), The combined treatment produced a greater number of high-quality embryo sacs.

[0120] Table I. Preliminary clinical study results of combination treatment.

[0121]

[0122] Embodiment 4: Time-lapse monitoring of human embryos

[0123] The fertilized egg (Vitrolife, The cells were cultured in an incubator in Sweden and monitored over time for 5-6 days until the embryo sac stage. The timing of each developmental event was determined using EmbryoViewer image analysis software (Vitrolife): tPNf, time of pronuclear fading; 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 embryo sac. The KIDScore developed by EmbryoScope and based on a very large multicenter dataset TM Day 5 (version 2) was used as a predictive model for the post-implantation potential of embryos on day 5 (Gazzo et al., 2020). According to KIDScore, the combined treatment produced more embryos with high implantation potential compared to the control group (see Table I).

[0124] Conclusion

[0125] For all numerical limits describing certain parameters in this application, such as "about", "at least", "less than", and "more than", the description also necessarily includes any ranges bounded by the stated values. Thus, for example, the description "at least 1, 2, 3, 4, or 5" also specifically describes ranges of 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, etc.

[0126] The headings used in this application are for convenience only and shall not affect the interpretation of this application.

[0127] The preferred features of each aspect provided by the present invention (e.g., culture media, compositions, preparations and methods) are applicable to all other aspects of the present invention after appropriate modifications, and are illustrated by the dependent claims without limitation, and also include the combination and arrangement of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of specific embodiments and aspects of the present invention (including working examples). For example, without departing from the present invention, the specific experimental parameters exemplified in the working examples can be adapted piecemeal for use in the claimed invention. For example, for the disclosed materials, although the specific reference to each of the various individual and collective combinations and arrangements of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Therefore, if a class of elements A, B and C, and a class of elements D, E and F are disclosed, and examples of combinations of elements AD are disclosed, then each is contemplated individually and collectively even if each is not recorded individually. Therefore, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE and CF is explicitly contemplated and should be considered to be disclosed from the disclosure of A, B and C; D, E and F; and example combination AD. Likewise, any subset or combination of these is also expressly contemplated and disclosed. Thus, for example, the subgroup of AE, BF, and CE is expressly contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and example combination AD. This concept applies to all aspects of this application, including elements of the compositions and steps in methods of making or using the compositions.

[0128] The above aspects of the invention, as recognized by one of ordinary skill in the art following the teachings of the specification, may be claimed in any combination or arrangement as long as they are novel and non-obvious over the prior art - thus, as long as an element is described in one or more references known to one of ordinary skill in the art, it may be specifically excluded from the claimed invention by a negation or disclaimer of a feature or combination of features.

[0129] literature

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Claims

1. A method for preparing mammalian sperm, wherein the method comprises: (a) In Ca 2+ incubating the mammalian sperm in the presence of an ionophore, (b) Washing the mammalian sperm from step (a) with fresh medium to remove Ca 2+ Ionophores, (c) treating the mammalian sperm of step (b) at a temperature between 38°C and 43°C in a solution containing HCO3 - incubated in medium, and (d) transferring an appropriate concentration of the mammalian sperm of step (c) into an appropriate culture medium, wherein the mammalian sperm is suitable for use in assisted reproductive technology (ART).

2. The method of claim 1, wherein one or more sperm functions are selected from the group consisting of: curvilinear velocity, amplitude of lateral head displacement, sperm capacitation, percentage of superactivated sperm, and wherein the sperm function is improved relative to suitable control sperm.

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

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

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

6. The method according to claim 1, wherein albumin of steps ac) is present or absent.

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

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

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

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

11. The method of claim 1, wherein the mammalian sperm of step (a) is recovered from cryogenic storage.

12. The method of claim 1, wherein the mammalian sperm of step (a) is recovered from non-cryogenic storage.

13. The method according to claim 1, wherein the mammalian sperm of step (a) is from a subject with normozoospermia, oligozoospermia, asthenozoospermia or teratozoospermia or a subject with subfertility.

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

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

16. The method according to claim 1, wherein the mammalian sperm of step (a) is enriched from semen by density gradient centrifugation, sludge method or microfluidics technology before step (a).

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