JP2021529554A - Compositions and methods for enhancing sperm function - Google Patents

Abstract

The present disclosure provides, among other things, methods of improving sperm function and related fertilization methods, along with activated or enhanced sperm preparations. The method provided by the present disclosure involves, in some embodiments, energy depletion followed by a gradual reintroduction of different energy sources. The present disclosure further provides products suitable for carrying out the methods provided by the present invention. The present invention provides, in some embodiments, a kit for separating sperm for IVF or IUI and processing and preparing sperm. Also provided are nutrient-free reagents useful for preparing sperm.

Description

Mutual reference This application is a US provisional patent application No. 62 / 773,448 filed on November 30, 2018, and a US provisional patent application No. 62 / 773,453 filed on November 30, 2018. Specification, US Provisional Patent Application No. 62 / 773,462 filed on November 30, 2018, US Provisional Patent Application No. 62 / 773,471 filed on November 30, 2018. , US Provisional Patent Application No. 62 / 773,433 filed on November 30, 2018, US Provisional Patent Application No. 62 / 773,440 filed on November 30, 2018, 2019 US Provisional Patent Application No. 62 / 914,803 filed on October 14, 2019, US Patent Application Publication No. 16 / 282,204 filed on February 21, 2019, 2019 2 Claiming the interests of U.S. Patent Application Publication No. 16 / 282,217 filed on 21st May and U.S. Patent Application Publication No. 16 / 282,224 filed on February 21, 2019. And each of these applications is incorporated herein by reference in its entirety.

About 50% of couples who have difficulty getting pregnant are due to male factors. An important aspect of reproductive support is maximizing the function of male gametes (sperm) and promoting maximal fertilization. Therefore, there is a need for media, compositions and methods for improving sperm function, eg, thereby promoting reproductive support.

The present invention provides, among other things, media, compositions and methods for improving sperm function, eg, thereby promoting reproductive support.

Here, in a method of enhancing fertilization, (a) incubating mammalian sperm under energy depletion conditions for a time suitable for enriching mammalian sperm, (b) from step (a). Under conditions that sequentially provide an effective amount of a first energy source and a second energy source to the enriched mammalian sperm, and (c) fertilize the mammalian sperm obtained from step (b). A method is provided in which the effective amount is sufficient to induce improved sperm function, including providing a means of reaching the egg.

Here, a method of inducing improved sperm function, (a) incubating mammalian sperm under energy-depleted conditions for a period of time suitable for producing enhanced mammalian sperm, (b). ) To provide the enhanced mammalian sperm from step (a) with an effective amount of a first energy source selected from (i) glycolytic energy sources or (ii) gluconeogenic substrates, and ( c) The mammalian sperm from step (b) is then provided with an effective amount of a second energy source selected from (i) glycolytic energy sources or (ii) gluconeogenic substrates. A method is provided in which the effective amount is sufficient to induce improved sperm function, including that the second energy source provided is not selected in step (b).

As used herein, (a) mammalian sperm are incubated under energy-depleted conditions for a time suitable for producing enhanced mammalian sperm, and (b) enhanced mammals from step (a). A sperm preparation solution containing an improved proportion of superactivated sperm prepared by the process of sequentially providing an effective amount of a first energy source and a second energy source to sperm, wherein the effective amount is 1. A sperm preparation that is sufficient to induce an increase in one or more sperm functions and contains an improved proportion of superactivated sperm is an untreated mammalian sperm, an effective amount of a first energy source or a second. The enhanced mammalian sperm of step (a), which is independently provided with the energy source of, and the enhanced amount of the first and second energy sources of the effective amount are simultaneously provided, the enhanced of step (a). A sperm preparation is provided that comprises one or more improvements in sperm function compared to suitable control spermatozoa selected from spermatozoa or mammalian spermatozoa treated with a standard fertility acquisition medium such as C-HTF.

In the present specification, a method for inducing improved sperm function is: (a) Mammalian sperm in a storage medium containing a buffer and having a pH of about 6 to 7 and a weight osmol concentration of about 300 to 400 mOsm / kg. (B) Incubating mammalian sperm for a period suitable for enriching mammalian sperm under energy-depleted conditions, and (c) Effective for enhanced mammalian sperm from step (b). A method is provided that comprises providing an amount of (i) a glycolytic energy source and / or (ii) a glycosogenic substrate, thereby inducing improved sperm function compared to a suitable control sperm.

As used herein, it is a method of promoting fertilization, (a) incubating mammalian sperm under energy depleted conditions for a period suitable for sperm augmentation, and prior to incubation, mammalian sperm. (B) Effective for enhanced sperm from step (a), containing a buffer and stored in a storage medium having a weakly acidic pH and a weight osmol concentration of about 300-400 mOsm / kg. Providing a quantity of first and second energy sources in sequence, (c) independently providing an effective amount of first or second energy source to the enhanced mammalian sperm in step (a). Improving one or more of sperm function to a level that exceeds that obtained by providing or simultaneously providing an effective amount of the first and second energy sources, and (d) improved sperm. Methods are provided that include providing functional mammalian sperm with a means of reaching the egg under conditions that enhance fertilization.

In the present specification, a) a first container containing a first composition containing a buffered sperm-enhancing energy depletion composition, and b) a second composition containing at least a first energy source suitable for mammalian sperm. A kit containing a second container containing a substance, in which mammalian sperm are incubated in the first composition for a suitable period of time to give rise to enhanced mammalian sperm, at least an effective amount for the enhanced mammalian sperm. Providing a first source of energy provides a kit that enhances enhanced mammalian sperm function as compared to a suitable control.

In the present specification, a super-activated sperm preparation solution containing at least 5% of super-activated sperm, which is optionally, not preselected based on super-activation, is optionally super-activated. Sperm and / or intermediate sperm were 10, 15, 20, 25, 30, 35, 40, 45, 50% of intracellular RNA concentration (such as small non-coding RNA containing microRNA) compared to suitable controls. A super-activated sperm preparation solution is provided that has a reduction of or greater than that (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more).

In the present specification, a. Concentrating sperm from the semen of a male subject, such as a male with normal sperm, a male with low fertility or a male with oligospermia, such as a male with low fertility (including oligospermia), b. Incubating sperm under energy depletion for a time suitable for sperm augmentation, c. A first energy source that is selected from an effective amount of glycolytic energy source or an effective amount of gluconeogenic substrate for the enhanced sperm, but not both an effective amount of glycolytic energy source and a gluconeogenic substrate. The sperm preparation solution prepared by providing the above is provided.

In the present specification, a buffer is included, and the pH is weakly acidic and the pH is about 300 to 400 mOsm / kg, for example, about 300 to 380, 320 to 370, 330 to 370, 340 to 360, for example, about 320, 330, 340, 350. Provided is a sperm storage medium having a weight osmolal concentration of 360, 370 or 380, eg, about 350, which does not contain a significant amount of yolk or, in some embodiments, does not contain any yolk. Will be done.

As used herein, (a) incubating mammalian spermatozoa for a period suitable for producing enhanced mammalian spermatozoa under energy depletion, and (b) enhanced mammalian spermatozoa from step (a). A sperm preparation solution prepared by sequentially providing an effective amount of a first energy source and a second energy source, wherein the sperm of step (b) contains an epigenetic profile different from that of a suitable control sperm. , Sperm preparation solution is provided.

Here, a method of producing offspring with improved adaptability, (a) incubating a sperm sample under energy depletion for a time suitable for producing enhanced sperm. (B) providing an effective amount of a first energy source for the enriched sperm, and (c) then providing an effective amount of a second energy source for the sperm from step (b), (d). ) Improvement, including fertilizing the sperm from step (c) into an egg to generate an embryo, and (e) growing an embryo in a female subject to produce offspring with improved adaptability. The indicated indications are provided with methods that include a reduced risk of developing the condition.

The various methods, media, preparations and kits described herein can be used in combination. For example, the sperm preparation solution stored in the sperm storage medium provided by the present invention may, in some embodiments, the various methods provided by the present invention (eg, enhance sperm function, enhance fertilization, etc.). Producing offspring with improved adaptability, etc.), and in some embodiments, it can be performed using the various kits provided by the present invention, and thus in certain embodiments, It can be used in additional methods provided by the present invention, such as a method by which the sperm preparation solution provided by the present invention will be prepared and / or a fertilization method including a reproductive assisting method.

The features of the methods, media, sperm preparations and kits described herein include one or more of the embodiments listed below, which can be combined and inserted. And should not be considered as a specific embodiment in the narrow sense that does not accept combinations or modifications unless specifically provided. The examples of the present disclosure provide non-limiting examples that can be easily adapted to the embodiments listed below.

1. 1. It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for enriching mammalian sperm.
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced mammalian sperm from step (a), and (c) to the mammalian sperm obtained from step (b). A method that comprises providing a means of reaching an egg under conditions that enhance fertilization, the effective amount being sufficient to induce improved sperm function.

2. Selected from curve velocity, amplifier of lateral energy, autophagy, sperm fertility acquisition, percentage of super-activated sperm, percentage of intermediate-motivated sperm and percentage of super-activated sperm and intermediate-motivated sperm One or more sperm functions, enhanced mammalian sperm provide only one of the first and second energy sources, or simultaneously provide the first and second energy sources. The method of embodiment 1, which is improved as compared to the method.

3. 3. The first energy source is a glycolytic energy source, and the second energy source is a gluconeogenic substrate, or the first energy source is a gluconeogenic substrate, and a second energy source. Is a glycolytic energy source, and further, the mammalian sperm of step (a) is a human sperm, the method of embodiment 1.

4. The method of Embodiment 3, which is carried out in vitro.

5. Step (c) is the method of embodiment 3 performed in the body in the reproductive tract of a female subject by artificial insemination of mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI).

6. Providing a second energy source in step (b) provides an effective amount of a first energy source in the body in the reproductive tract of a female subject by enhanced intrauterine insemination (IUI) of mammalian sperm. The method of Embodiment 1, which is carried out.

7. The method of embodiment 6, wherein the first energy source is a gluconeogenic substrate, which is pyruvic acid, and the second energy source is a glycolytic energy source.

8. Step (c) comprises incubating the mammalian sperm from step (b) with the egg or injecting the mammalian sperm from step (b) into the cytoplasm of the egg to enhance in vitro fertilization of the egg. The method of embodiment 4.

9. Promoting fertilization comprises the development of an embryo that exhibits improved viability and / or improved implantation as compared to an embryo generated by a suitable control sperm.

10. Promoting fertilization is the method of embodiment 1, comprising developing at least a two-cell stage, a blastocyst stage, or an embryo that develops to offspring.

11. The method of embodiment 1, wherein the mammalian sperm of step (a) is from a subject with oligospermia or a subject with low fertility.

12. The method of embodiment 1, wherein the mammalian sperm of step (a) is a human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm.

13. The method of embodiment 12, wherein the mammalian sperm of step (a) is a human sperm.

14. The method of embodiment 1, wherein the mammalian sperm of step (a) is sperm recovered from non-cold or cold storage.

15. The method of embodiment 1, wherein the mammalian sperm of step (a) is provided as a pool of two or more doses of ejaculate.

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

17. The method of embodiment 1 carried out at a weight osmolal concentration in the range of 200-280 mOsm / kg.

18. Step (b) further comprises providing one or more components upstream or downstream of glycolysis to mammalian sperm in combination with at least a first or second energy source. Method.

19. The first energy source is selected from (i) glucose or (ii) pyruvic acid, and the second energy source is selected from (i) glucose or (ii) pyruvic acid, the first and second. The energy source of is different, the method of embodiment 3.

20. The method of embodiment 1, wherein the incubation in step (a) under energy depletion conditions lasts for at least 10 minutes.

21. A method of inducing improved sperm function
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for producing enhanced mammalian sperm.
(B) To provide the enhanced mammalian sperm from step (a) with an effective amount of a first energy source selected from (i) glycolytic energy sources or (ii) gluconeogenic substrates. And (c) then by providing the mammalian sperm from step (b) with (i) an effective amount of a glycolytic energy source, or (ii) a second energy source selected from gluconeogenic substrates. The method, wherein the second energy source provided is not selected in step (b), comprises providing, and the effective amount is sufficient to induce improved sperm function.

22. Improved sperm function is improved compared to suitable control sperm, which are independently provided with untreated mammalian sperm, an effective amount of glycolytic energy source or gluconeogenic substrate. The enhanced mammalian sperm of step (a), the enhanced mammalian sperm of step (a) or the standard fertility acquisition medium (C-HTF) simultaneously provided with an effective amount of glycolytic energy source and gluconeogenic substrate. ), The method of embodiment 21.

23. 21. The method of embodiment 21, which is performed outside the body.

24. Step (c) is the method of embodiment 21 performed in the body in the reproductive tract of a female subject by artificial insemination of mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI).

25. The method of embodiment 21, wherein the improved sperm function comprises an improvement in motility as measured by computer-aided semen analysis (CASA).

26. The method of embodiment 25, wherein the improvement in motility comprises an increase in the curvaceous rate of mammalian sperm, an increase in the proportion of superactivated sperm, an increase in the proportion of intermediate motility sperm, or a combination thereof.

27. The method of embodiment 21, wherein the improved sperm function comprises improved sperm fertility acquisition as measured by a sperm-zona pellucida binding test.

28. The method of embodiment 21, wherein the improved sperm function comprises an increase in the ability of mammalian sperm to fertilize an egg, as measured by a sperm invasion test.

29. Improved sperm function includes improved viability, improved implantation, at least 2-cell stage development, blastocyst stage development, or increased ability to develop to offspring compared to embryos developed in suitable control sperm. 21. The method of embodiment 21, comprising developing an embryo having.

30. The method of embodiment 21, wherein the mammalian sperm are human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm.

31. The method of embodiment 30, wherein the mammalian sperm is human sperm.

32. The method of embodiment 31, wherein the glycolytic energy source is glucose and the gluconeogenic substrate is pyruvic acid.

33. A sperm preparation solution containing mammalian sperm having improved sperm function prepared by the method of embodiment 21.

34. (A) Incubating mammalian sperm under energy depletion conditions for a time suitable for producing enriched mammalian sperm, and (b) an effective amount on the enriched mammalian sperm from step (a). A sperm preparation solution containing an improved proportion of superactivated sperm prepared by the process of sequentially providing a first energy source and a second energy source, the effective amount of which is one or more sperm functions. A sperm preparation solution containing an improved proportion of super-activated sperm, which is sufficient to induce an improvement in sperm,
Untreated mammalian sperm, enhanced mammalian sperm in step (a), effective amount of first energy source and second, provided independently with an effective amount of first or second energy source. One compared to a suitable control sperm selected from the enhanced mammalian sperm of step (a) or the mammalian sperm treated with standard capacitation medium (C-HTF), which is simultaneously provided with the energy source of. A sperm preparation solution containing the above-mentioned improvement in sperm function.

35. The first energy source is a glycolytic energy source, and the second energy source is a gluconeogenic substrate, or the first energy source is a gluconeogenic substrate, and a second energy source. Is the sperm preparation solution of Embodiment 34, which is a glycolytic energy source.

36. The mammalian sperm of step (a) is the sperm preparation solution of embodiment 34, which is provided as a pool of two or more ejaculatory fluids.

37. The mammalian sperm of step (a) is from a male with low fertility or an oligospermia, the sperm preparation of embodiment 34.

38. The sperm preparation solution of embodiment 34, wherein the mammalian sperm of step (a) is a human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm.

39. The sperm preparation solution of embodiment 38, wherein the mammalian sperm of step (a) is a human sperm.

40. The sperm preparation solution of embodiment 34, further comprising reduced intracellular RNA.

41. A method of inducing improved sperm function
(A) Providing mammalian sperm to a storage medium containing a buffer and having a pH of about 6-7 and a weight osmolal concentration of about 300-400 mOsm / kg.
(B) Incubating mammalian sperm under energy-depleted conditions for a time suitable for enriching mammalian sperm, and (c) an effective amount of (i) the enhanced mammalian sperm from step (b). A method comprising providing a glycolytic energy source and / or (ii) a gluconeogenic substrate, thereby inducing improved sperm function as compared to a suitable control sperm.

42. The method of embodiment 41, wherein the glycolytic energy source and the gluconeogenic substrate are provided at the same time.

43. The method of embodiment 41, wherein the glycolytic energy source and the gluconeogenic substrate are sequentially provided.

44. The method of embodiment 41, wherein the improved sperm function comprises an improvement in curve velocity, head amplitude, autophagy, sperm capacitation, superactivated sperm proportions, intermediate motile sperm proportions or a combination thereof.

45. The method of embodiment 41, wherein the improved sperm function is an increase in the proportion of superactivated sperm and intermediate motile sperm.

46. The method of embodiment 41, wherein the mammalian sperm of step (a) is provided as a pool of two or more doses of ejaculate.

47. The method of embodiment 41, wherein the storage medium does not contain egg yolk.

48. The method of embodiment 41, wherein the storage medium further comprises an antibiotic.

49. The method of embodiment 41, wherein the storage medium further comprises serum albumin.

50. The method of embodiment 41, wherein the buffer is HEPES, MOPS or a combination thereof.

51. The method of embodiment 41, wherein the storage medium has a pH of about 6.6 to 6.9 and a weight osmolal concentration of about 330 to 370 mOsm / kg.

52. The method of embodiment 41, wherein the storage medium further comprises one or more carbon sources selected from the group consisting of glucose, fructose, mannose and sucrose.

53. The method of embodiment 41, wherein the mammalian sperm in the storage medium are stored under non-cold conditions prior to step (a).

54. It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy depletion conditions for a time suitable for sperm augmentation, prior to incubation, the mammalian sperm contains a buffer and has a weakly acidic pH of about 300. Incubating, stored in storage medium with a weight osmolal concentration of ~ 400 mOsm / kg,
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced sperm from step (a).
(C) Independently providing an effective amount of the first or second energy source to the enhanced mammalian sperm of step (a), or an effective amount of the first and second energy sources. To improve one or more of sperm function to a level higher than that obtained by simultaneously donating, and (d) reach the egg under conditions that promote fertilization of mammalian sperm with improved sperm function. Methods involving providing means.

55. The method of embodiment 54, wherein the mammalian sperm of step (a) is provided as a pool of two or more doses of ejaculate.

56. One or more sperm functions include curve velocity, head amplitude, autophagy, sperm fertility acquisition, superactivated sperm ratio, intermediate motile sperm ratio and superactivated sperm and intermediate motile sperm ratio. 54. The method of embodiment 54, selected from.

57. The method of embodiment 54, which is performed extracorporeally.

58. Step (d) is the method of embodiment 54, wherein step (d) is performed in the body in the reproductive tract of a female subject by endometrial insemination (IUI) of mammalian sperm with improved sperm function from step (c).

59. Providing a second energy source in step (b) is by insemination (IUI) of enhanced mammalian sperm from step (b) provided with an effective amount of first energy source in a female. The method of embodiment 54, which is performed in the body in the reproductive tract of the subject.

60. Step (d) comprises incubating mammalian sperm with improved sperm function with the egg or injecting mammalian sperm with improved sperm function into the cytoplasm of the egg to enhance in vitro fertilization of the egg. The method of embodiment 57.

61. a) a first container containing a first composition containing a buffered sperm-enhancing energy depleting composition, and b) a second containing a second composition containing at least a first energy source suitable for mammalian sperm. It is a kit that includes the container of
Incubation of mammalian sperm in the first composition for a suitable period of time yields enhanced mammalian sperm.
A kit that provides an effective amount of at least a first energy source for enhanced mammalian sperm to improve the function of the enhanced mammalian sperm as compared to a suitable control.

62. The kit of embodiment 61, wherein the first composition comprising the energy depletion composition for buffered sperm enhancement is a nutrient-free synthetic human oviductal fluid.

63. Enhanced mammalian sperm function includes curve velocity, head amplitude, autophagy, sperm fertility acquisition, superactivated sperm ratio, intermediate motile sperm ratio, superactivated sperm and intermediate motile sperm ratio, or A kit according to any one of embodiments 61 to 62, which is a combination thereof.

64. The energy depletion composition for buffered sperm enhancement is about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0. 05, 0.04, the kit of any one of embodiments 61-63, comprising a glucose concentration of less than or less than 0.03 mM.

65. The energy depletion composition for buffered sperm enhancement is about 0.15, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.03. 02, 0.01, 0.005, 0.003, 0.002 Any one of the kits of embodiments 61-64, comprising a pyruvic acid concentration of less than or less than 0.002 mM.

66. The kit of any one of embodiments 61-65, wherein the preferred time is at least about 10, 20, 30, 40, 45, 50, 55, 60, 90, 120, 150 or 180 minutes.

67. The kit of any one of embodiments 61-66, wherein the buffered sperm-enhancing energy depletion composition comprises a volumetric osmolality in the range of about 200-280 mOsm, eg, about 220-260, 225-255, 230-250 mOsm.

68. Providing an effective amount of at least the first energy source to the enriched mammalian sperm is at least at a volumetric osmolal concentration of about 270, 275, 280, 285, 290 or 295 mOsm, any of embodiments 61-67. Or one kit.

69. The first composition is, for example, 7.5 to 12.5 mM, for example, about 5 to 20 mM HEPES, 90 to 100 mM, for example, about 80 to 120 mM sodium chloride, 4 to 7 mM mM, for example, about 3 to 8 mM. Potassium chloride, 1.5-2.5 mM, for example about 1-5 mM calcium chloride, 0.3-0.4 mM, etc., for example about 0.1-0.5 mM potassium phosphate, 0.16-0. The kit of any one of embodiments 61-68, comprising one or more of, for example, about 10-50 mM sodium chloride, such as 35 mM, eg 0.1 to 0.5 mM magnesium sulfate, 15-30 mM.

70. The first composition is, for example, about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, The kit of any one of embodiments 61-69, further comprising 0.0009% or 0.001% phenol red.

71. The first composition and / or the second composition has a concentration of, for example, about 1-20 μg / ml, 2-18 μg / ml, 4-16 μg / ml, 6-14 μg / ml or 8-12 μg / ml. The kit of any one of embodiments 61-70, further comprising an antibiotic such as gentamicin.

72. The first energy source is the kit of any one of embodiments 61-71, which is a glycolytic energy source or a gluconeogenic substrate.

73. The kit of any one of embodiments 61-72, wherein the second container containing the second composition comprising at least the first energy source further comprises an energy depletion composition for buffered sperm enhancement.

74. Embodiment 61, wherein the first composition, the second composition, or both the first composition and the second composition are, for example, aqueous solutions such as sterile aqueous solutions that have been pre-sterilized by sterile filtration. One of the kits from ~ 73.

75. The kit of any one of embodiments 61-73, wherein the first composition and / or the second composition is a lyophilized composition.

76. Further comprising a third container containing a third composition containing at least a second energy source suitable for mammalian sperm to provide an effective amount of a second energy source for enhanced mammalian sperm can provide sperm function. The kit of any one of embodiments 61-75, wherein the improved and effective amount of the second energy source is provided simultaneously or sequentially with the effective amount of the first energy source.

77. The third composition is, for example, the kit of embodiment 76, which is an aqueous solution such as a sterile aqueous solution that has been pre-sterilized by sterile filtration.

78. The third composition is the kit of embodiment 76, which is a lyophilized composition.

79. A third container containing a third composition comprising at least a second energy source suitable for mammalian sperm is a kit of any one of embodiments 76-78, further comprising an energy depletion composition for buffered sperm enhancement. ..

80. The third composition further comprises an antibiotic such as gentamicin at a concentration of, for example, about 1-20 μg / ml, 2-18 μg / ml, 4-16 μg / ml, 6-14 μg / ml or 8-12 μg / ml. , Any one of embodiments 76-79.

81. One kit of embodiments 76-80, wherein the second energy source is a glycolytic energy source or a gluconeogenic substrate, and the second energy source is not selected as the first energy source.

82. Glycolytic energy sources are, for example, about 100 mM to 1 M, 200 to 900 mM, 300 to 800 mM, 400 to 600 mM or 500 mM, for example at least about 100, 200, 300, 400, 500, 600, 700, 800, 900 mM or 1 M. The kit of any one of embodiments 72-81, wherein the concentration of glucose is.

83. The gluconeogenic substrate is, for example, pyruvic acid at a concentration of about 10-50 mM, 15-45 mM, 20-40 mM or 25-35 mM, for example at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50 mM. A kit of any one of embodiments 72-82.

84. The first energy source or optionally the second energy source is an effective amount of glucose, the effective amount of glucose being about 0.6 mM to 10.0 mM, for example about 1.0 to when introduced into the diluent. The kit of any one of embodiments 71-83, which is 7.0 mM, 2.5-7.0 mM, 3.5-6.5 mM or about 5 mM, eg, at least about 1, 2, 3 or 4 mM.

85. The first energy source or optionally the second energy source is an effective amount of pyruvic acid, the effective amount of pyruvic acid being about 0.15-0.66 mM at the time of introduction into the diluent, eg about 0. The kit of any one of embodiments 71-83, which is 20-0.50 mM, 0.25-0.40 mM or about 0.30 mM.

86. The kit of any one of embodiments 71-85, wherein the first energy source is, optionally, pyruvic acid in the form of sodium pyruvate.

87. The kit of any one of embodiments 76-86, wherein the second energy source is glucose.

88. The first composition is any one of the kits of embodiments 71-87, comprising, for example, human serum albumin at a concentration of about 1-10 mg / ml, 2-8 mg / ml or 3-7 mg / ml.

89. The kit of any one of embodiments 71-88, comprising an additional container containing human serum albumin.

90. The first and / or second composition comprises, for example, NaCl at a concentration of about 97.8 mM, KCl at a concentration of about 4.7 mM _{, for example CaCl 2} , at a concentration of about 2 mM, for example at a concentration of about 0.37 mM. KH ₂ PO ₄ , eg, silicate at a concentration of about 0.2 mM ₄ . 7H ₂ O, consists essentially for example of about 4 mg / ml concentration of HSA, for example, gentamicin at a concentration of about 10 [mu] g / ml, for example from phenol red HEPES and for example a concentration of about 0.0006% to about 10mM concentration, performed A kit of any one of the forms 71 to 89.

91. The third composition is, for example, NaCl at a concentration of about 97.8 mM, KCl at a concentration of about 4.7 mM, CaCl _{2 at a concentration of about 2 mM, for example KH 2} PO _{4 at} a concentration of about 0.37 mM. for example MgSO ₄ · _7H 2 O of about 0.2mM concentration, for example, HSA at a concentration of about 4 mg / ml, for example gentamycin at a concentration of about 10 [mu] g / ml, for example about the 10mM concentrations HEPES and for example from about 0.0006% The kit of any one of embodiments 76-90, essentially consisting of a concentration of phenol red.

92. Further comprising a means for selecting sperm selected from microfluidic devices, density gradient solutions, sperm isolation matrices (optionally suspended in nutrient-free synthetic human oviduct fluid, such as silanized silica) or combinations thereof. Any one kit of embodiments 71-91.

93. The kit of any one of embodiments 71-92, further comprising instructions for use, such as instructions for performing the methods as described herein, such as starvation-rescue / starvation-renutrition.

94. The kit of any one of embodiments 71-93, further comprising a collection container for collecting a sample of mammalian sperm from a mammalian donor.

95. The kit of any one of embodiments 71-94, wherein the first container and / or the second container is a bottle, vial, syringe or test tube.

96. The third container is a kit of any one of embodiments 76-95, which is a bottle, vial, syringe or test tube.

97. The first container and / or the second container is a kit of any one of embodiments 71-96, which is a multipurpose container.

98. The third container is a kit of any one of embodiments 76 to 97, which is a multipurpose container.

99. A way to improve sperm function
i) Incubating sperm under energy depletion for a time suitable for sperm augmentation,
ii) For enhanced sperm, an effective amount of first, selected from glycolytic energy sources or gluconeogenic substrates, but not both an effective amount of glycolytic energy sources and an effective amount of gluconeogenic substrates. Providing an energy source, and iii), including providing the sperm with an effective amount of a second energy source, such as providing both an effective amount of a gluconeogenic substrate and a glycolytic energy source. A method of improving sperm function.

100. Energy depletion is, for example, about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04. , A method of embodiment 99, comprising a low glucose concentration of, for example, less than about 0.01 mM, such as glucose less than 0.03 mM or lower, about 0.02 or less than 0.01 mM.

101. Energy depletion is, for example, about 0.15, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01. , 0.005, 0.003, 0.002 mM or less, comprising a low pyruvic acid concentration, the method of embodiment 99 or 100.

102. Energy depletion is at least about 10, 20, 30, 40, 50, 60 minutes, eg at least about 30, 40, 45, 50, 55, 60, 90, 120, 150 or 180 minutes or 1, 1.5, 2 , 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or over 10 hours The method of any one of embodiments 99 to 101.

103. The time between providing an effective amount of the first energy source and providing an effective amount of the second energy source after sperm enrichment is at least about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, eg at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, One method of any one of embodiments 99-102, which is 18, 19 or 20 minutes, eg, at least about 5 to 15 minutes.

104. The gluconeogenic substrate is an embodiment of pyruvic acid at a concentration of about 0.25 to 0.50 mM, such as, for example, about 0.15 to 0.66 mM, such as about 0.25 to 0.40 mM or about 0.30 mM. Any one of 99 to 103 methods.

105. Glycolytic energy sources are, for example, about 0.6 mM to 10.0 mM, 1.0 to 7.0 mM, 2.5 to 7.0 mM, 3.5 to 6.5 mM or 5 mM, for example at least about 1, 2. The method of any one of embodiments 99-104, which is glucose at a concentration of 3, or 4 mM.

106. The first method according to any one of embodiments 99 to 105, wherein the first energy source is a glycolytic energy source such as glucose.

107. The second energy source is any one of embodiments 99-105, which is a glycolytic energy source such as glucose.

108. The first method of any one of embodiments 99-105, wherein the first energy source is a gluconeogenic substrate such as pyruvic acid.

109. The method of any one of embodiments 99-105, wherein the second energy source is a gluconeogenic substrate such as pyruvic acid.

110. The method of any one of embodiments 99-109, wherein the sperm is a mammalian sperm (eg, bovine, sheep, pig, horse, cat, dog or primate sperm, eg, human sperm).

111. During energy depletion, it is carried out at volumetric osmolal concentrations in the range of about 200-280 mOsm, eg about 220-260, 225-255, 230-250 mOsm, optionally with an effective amount of first and / or second energy sources. The method of any one of embodiments 99-110, wherein when added, the volume osmolal concentration increases to at least about 270, 275, 280, 285, 290 or 295 mOsm.

112. Further comprising one or more quantitative assessments of sperm motility or quality by measuring, for example, CASA or DNA fragmentation (eg, by TUNEL), lipid peroxidation, reactive oxygen species or combinations thereof. Any one of the forms 99-111.

113. Prior to treatment, sperm are recovered from cold storage, any one of the methods of the aforementioned embodiment.

114. Prior to treatment, sperm are recovered from non-cold storage, any one of embodiments 99-112.

115. The method of any one of embodiments 99-114, wherein the sperm are pooled from two or more doses of ejaculate (eg, 2, 3, 4, 5, 6 doses or more).

116. The sperm are obtained, for example, from any one of embodiments 99-115, obtained from a low fertility male or an oligospermia male in which the number of sperms per milliliter is less than about 15 million.

117. Prior to energy depletion, sperm are concentrated (or isolated) from semen by, for example, density gradient centrifugation, swim-up or microfluidics, any one of embodiments 99-116.

118. The method of any one of embodiments 99-117, further comprising donating sperm to the female reproductive tract, optionally providing an effective amount of a second energy source to the female reproductive tract.

119. The method of any one of embodiments 99-118, carried out in vitro.

120. 119. The method of embodiment 119, further comprising contacting a sperm with improved function with an egg under conditions that enhance fertilization.

121. The method comprising providing the sperm prepared by any one of the methods 99 to 117 with a means of reaching the egg (including, for example, by ICSI) for a time sufficient to fertilize the egg. Fertilization method.

122. At least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 of superactivated sperm and / or intermediate motile sperm as compared to a suitable control. % Increase or greater, about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4 One method of any one of embodiments 99-121, wherein there is an increase, including about 1.2 times, such as, or greater than, a 5-fold increase.

123. It further comprises providing the sperm with one or more components upstream or downstream of glycolysis such as NADH, NAD +, citric acid, AMP or ADP in combination with at least a first or second energy source. , Any one method of embodiments 99-122.

124. A super-activated sperm preparation solution containing at least 5% of super-activated sperm, optionally not preselected based on super-activation, optionally super-activated sperm and / or intermediate. Sperm account for 10, 15, 20, 25, 30, 35, 40, 45, 50% or greater of intracellular RNA concentrations (such as small non-coding RNAs, including microRNA) compared to suitable controls (such as small non-coding RNA containing microRNA). For example, a sperm preparation solution having a reduction of 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more).

125. a. Concentrating sperm from the semen of a male subject, such as a male with normal sperm, a male with low fertility or a male with oligospermia, such as a male with low fertility (including oligospermia).
b. Incubating sperm under energy depletion for a period of time suitable for sperm augmentation,
c. A first energy source that is selected from an effective amount of glycolytic energy source or an effective amount of gluconeogenic substrate for the enhanced sperm, but not both an effective amount of glycolytic energy source and a gluconeogenic substrate. A sperm preparation solution prepared by providing.

126. To the effective amount of the second energy source and the egg so that the preparation of embodiment 125 provides both an effective amount of both a gluconeogenic substrate and a glycolytic energy source for a time sufficient to fertilize the egg. Fertilization methods, including providing a means of reaching.

127. The method of embodiment 126, which is performed extracorporeally.

128. The method of embodiment 126, performed in the body in the female reproductive tract (vaginal or uterus).

129. The preparation of embodiment 125, wherein the sperm are from a subject with oligospermia or a subject with low fertility (eg, low sperm motility).

130. The preparation solution of embodiment 125, which is prepared by any one of the methods 99 to 120.

131. i) Sperm-enhancing solutions that induce energy depletion upon contact with sperm,
ii) A solution that provides a first energy source that is selected from an effective amount of glycolytic energy source or an effective amount of gluconeogenic substrate, but not both an effective amount of glycolytic energy source and a gluconeogenic substrate. , And iii) Products containing solutions that provide an effective amount of a second energy source.

132. The product of embodiment 131, further comprising a sperm isolation matrix.

133. The product of embodiment 132, wherein the sperm isolation matrix is silanized silica, optionally in a medium in which the silanized silica is substantially free of any glycolytic energy source or gluconeogenic substrate.

134. Contains buffer and has a weakly acidic pH and about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340, 350, 360, 370 or 380. A sperm storage medium having, for example, a weight osmolal concentration of about 350, which does not contain a significant amount of egg yolk or, in some embodiments, does not contain any egg yolk.

135. The sperm storage medium of embodiment 134, further comprising a carbon source and optionally the carbon source being selected from glucose, fructose, mannose, sucrose or a combination thereof.

136. The sperm storage medium of embodiment 135, wherein the carbon source is glucose.

137. Glucose is present at a concentration of about 0.1-0.4M, such as about 0.2-0.4M, for example at a concentration of about 0.30 to 0.36M or about 0.33M, sperm storage of embodiment 136. Culture medium.

138. The buffer is an amphoteric ion buffer and the buffer concentration is about 1-100 mM, eg 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, 5-15 mM, eg about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, eg, about 10 mM, sperm storage of any one of embodiments 134-137. Culture medium.

139. The buffer is the sperm storage medium of embodiment 138, which is 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS) or a combination thereof. ..

140. A sperm storage medium according to any one of embodiments 134-139, further comprising an antibiotic.

141. The sperm storage medium of embodiment 140, wherein the antibiotic is an aminoglycoside.

142. The antibiotic is gentamicin, the sperm storage medium of embodiment 140 or 141.

143. Gentamicin is the sperm storage medium of embodiment 142, which is present at a concentration of about 5-20 μg / ml, for example about 10 μg / ml.

144. A sperm storage medium according to any one of embodiments 131 to 143, further comprising serum albumin.

145. Serum albumin is bovine serum albumin (BSA) or human serum albumin (HSA) or a combination thereof, and more specifically, serum albumin is about 1.5-4.5% (W / V), eg, about. The sperm storage medium of embodiment 144, which is present at a concentration of 2-4%, 2.5-3.5% or 3%.

146. A sperm storage medium according to any one of embodiments 134-145, having a weight osmolal concentration of about 340-360 mOsm / kg.

147. The sperm storage medium of any one of embodiments 134-146 having a pH of about 6-7, eg 6.6-6.9.

148. The sperm storage medium of any one of embodiments 134-147 further comprising one or more of sterols, antioxidants or anti-inflammatory agents.

149. A significant sperm storage medium containing an amphoteric ion buffer and a pH of about 6.6 to 6.9, a concentration of glucose of about 0.25 to 0.36 M, and a weight osmolal concentration of about 320 to 380 mOsm / kg. A sperm storage medium that does not contain an amount of egg yolk or, in some embodiments, no egg yolk.

150. The sperm storage medium of embodiment 149, further comprising an antibiotic and optionally the antibiotic being gentamicin.

151. The pH is about 6.8, the glucose concentration is about 0.330 mM, the osmolality by weight is about 350 mOsm / kg, and the serum albumin is BSA and / or HSA, optionally. BSA and / or HSA is the sperm storage medium of embodiment 149 or 150, which is present at a concentration of about 2-4% (W / V).

152. The buffer is the sperm storage medium of embodiment 151, which is HEPES, MOPS or a combination thereof.

153. The sperm storage medium according to any one of embodiments 149 to 152, which is optionally provided as a sterilized preparation in a closed sterilized container.

154. Sperm storage of embodiment 153, lyophilized.

155. The sperm storage medium of embodiment 153, which is a liquid preparation.

156. Sperm stored in storage medium at about 4 ° C. for up to 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer are suitable controls when transferred to fertility acquisition medium. The sperm storage medium of any one of embodiments 149-155, which maintains at least about 40, 45, 50, 55, 60, 65, 70, 75, 80% or more motile sperm relative to sperm.

157. Sperm stored in storage medium at about 4 ° C. for 7 days are at least about 40, 45, 50, 55, 60, 65, 70, 75 compared to suitable control sperm when transferred to fertility acquisition medium. , 80% or more of motile sperm, any one of embodiments 149-156.

158. Sperm stored in storage medium at about 4 ° C. for 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer are suitable control sperms for transfer to fertility acquisition medium. A sperm storage medium according to any one of embodiments 149-157, having at least about 75% motile sperm as compared to.

159. Sperm stored in storage medium at about 4 ° C. for 7 days were 1, 2, 5, 10, 15 or 1, 2, 5, 10, 15 or 1, 2, 5, 10, 15 or more when transferred to fertility acquisition medium compared to pre-storage control sperm in storage medium. A sperm storage medium according to any one of embodiments 149-158, which has a motile sperm rate reduced by 20% or less.

160. Sperm stored in the medium
Reduction of TUNEL staining by at least 30, 40, 50, 55, 60, 65, 70, 80, 85, 90, 95% or more compared to cold stored cells,
Reduction of lipid peroxidation by at least 30, 40, 50, 55, 60, 65, 70, 80, 85, 90, 95% or more compared to cryopreserved cells,
Reduction of at least 30, 40, 50, 55, 60, 65, 70, 80, 85, 90, 95% or more reactive oxygen species, or one, two or all three compared to cryopreserved cells A sperm storage medium according to any one of embodiments 156 to 159, which exhibits one or more of the above-mentioned combinations including.

161. Having a pH of about 6.7 to 6.9, amphoteric ion buffer, glucose at a concentration of about 0.25 to 0.35 M, antibiotics, weight osmol concentration of about 340-360 mOsm / kg, about 2-4. Sperm that is essentially a sterile liquid sperm storage medium consisting of% (W / V) concentration of BSA or HSA and stored in this storage medium at 4 ° C. for up to 12 days is transferred to the fertility acquisition medium. A sterile liquid sperm storage medium that maintains at least 60% motile sperm compared to suitable control sperm upon transfer and, optionally, contains no egg yolk.

162. A composition comprising any one of the sperm storage media of the above embodiments, further comprising live sperm, optionally sperm from semen (eg, density gradient centrifugation, swim-up, filtration or filtration). A composition that is concentrated (by microfluidics).

163. The composition of embodiment 162, wherein the sperm are mammalian sperms, such as primate sperms such as cows, sheep, horses, pigs, rabbits, cats, dogs or humans.

164. The composition of embodiment 163, wherein the mammalian sperm is from a subject having a reduced sperm count, eg, less than about 15 million sperms per milliliter.

165. When stored at about 4 ° C. for up to 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer, at least about about compared to suitable control sperm for transfer to fertility acquisition medium. 40, 45, 50, 55, 60, 65, 70, 75, 80% or more sperm are motivated, the composition of any one of embodiments 162-164.

166. A composition comprising (i) sperm, such as human sperm, and (ii) buffer, having a pH of 5-7 (eg, 6-7 or 6.6-6.9) and about 300-400 mOsm / kg. The medium has a weight osmolal concentration of (eg, about 300-380, 320-370, 330-370, 340-360, eg, about 320, 330, 340, 350, 360, 370 or 380, eg, about 350). , An optional composition that does not contain a significant amount of egg yolk or, in some embodiments, does not contain any egg yolk.

167. The composition of embodiment 166, wherein the non-sperm portion of the composition is the sperm storage medium of any one of embodiments 134-161.

168. A composition comprising human sperm and a liquid sperm storage medium, the liquid sperm storage medium having a pH of about 6.7 to 6.9 and an amphoteric ion buffer, about 0.25 to 0.35 M. Consistently consisting of a concentration of glucose, a weight osmolar concentration of about 340-360 mOsm / kg, an antibiotic, a BSA or HSA at a concentration of about 2-4% (W / V), when stored at 4 ° C. for up to 12 days. A composition in which at least 60% of spermatozoa have motility and, optionally, the medium does not contain egg yolk, as compared to suitable control spermatozoa at the time of transfer to a fertility acquisition medium.

169. The composition of any one of embodiments 134-168, wherein the sperm are pooled from two or more doses of ejaculate (eg, 2, 3, 4, 5, 6 doses or more).

170. A method for storing sperm, which comprises contacting the sperm with the medium of any one of embodiments 134-161.

171. A method of fertilization comprising introducing the composition of any one of embodiments 162-169 into the reproductive organs of a female recipient (eg, vagina or uterus), optionally to the reproductive organs of the female recipient. Prior to introduction, the sperm are isolated from the composition and placed in a fertility-acquiring medium.

172. A fertilization method comprising contacting an egg with any one of the compositions of embodiments 162-169 (including, for example, by injection, such as by ISCI), optionally before contacting the egg. A method in which sperm are isolated from the composition and placed in a fertilizing medium.

173. (A) Incubating mammalian sperm for a period suitable for producing enhanced mammalian sperm under energy depletion, and (b) an effective amount of the enhanced mammalian sperm from step (a). A sperm preparation solution prepared by sequentially providing an energy source of 1 and a second energy source, wherein the sperm of step (b) contains an epigenetic profile different from that of a suitable control sperm.

174. Suitable control spermatozoa are untreated mammalian spermatozoa, enhanced mammalian spermatozoa of step (a), which are provided independently with an effective amount of a first or second energy source, an effective amount of a first energy source. Sperm of embodiment 173, which is the enhanced mammalian sperm of step (a) or sperm treated with standard capacitation medium (C-HTF), which is simultaneously provided with an energy source and a second energy source of Preparation solution.

175. Different epigenetic profiles include altered levels of DNA methylation, DNA acetylation, RNA methylation, protein (eg, histone) methylation, protein (eg, histone) acetylation or combinations thereof. Any one of 174 sperm preparation solutions.

176. The sperm preparation solution of any one of embodiments 173 to 175, wherein the sperm of step (b) further comprises reduced RNA levels as compared to a suitable control sperm.

177. Reduced RNA levels include a reduction in non-coding RNA (ncRNA), the sperm preparation of embodiment 176.

178. The non-coding RNA is a sperm preparation solution of embodiment 177, which is miRNA.

179. A method of producing offspring with improved fitness,
(A) Incubating a sperm sample under energy depletion for a time suitable for producing enhanced sperm.
(B) providing an effective amount of a first energy source for the enriched sperm, and (c) then providing an effective amount of a second energy source for the sperm from step (b).
(D) Fertilizing the sperm from step (c) into an egg to generate an embryo, and (e) growing the embryo in a female subject to produce offspring with improved fitness. , Improved fitness, including reduced risk of developing pathology, methods.

180. The method of embodiment 179, wherein the offspring with improved fitness do not develop the condition.

181. The method of any one of embodiments 179-180, wherein the sperm of step (c) comprises an epigenetic profile different from the preferred control sperm.

182. The method of any one of embodiments 179-181, wherein the sperm of step (c) comprises reduced intracellular RNA levels as compared to a suitable control sperm.

183. The method of any one of embodiments 179-182, wherein the condition is obesity or an obesity-related disorder (eg, type 2 diabetes, cardiovascular disease, respiratory disease, infertility or cancer).

184. The method of any one of embodiments 179-183, wherein the first energy source is a glycolytic energy source and the second energy source is a gluconeogenic substrate.

185. The method of any one of embodiments 179-183, wherein the first energy source is a gluconeogenic substrate and the second energy source is a glycolytic energy source.

It is a bar graph of the ratio of superactive and intermediate motile sperm under control and starvation (glucose, pyruvate and lactic acid free) conditions. Both glucose and pyruvic acid (simultaneous starvation / rescue), glucose only (starvation / glucose rescue), pyruvate only (starvation / pyruvic acid only), 1 In the bar graph of the ratio of superactive and intermediate motile sperm after addition of hourly glucose + 15 minutes pyruvic acid (starvation / glucose rescue + 15 minutes pyruvate) or 1 hour pyruvate + 15 minutes glucose (starvation / pyruvate rescue + 15 minutes glucose) be. It is explanatory drawing of sperm isolation by the density gradient method combined with the specific exemplary embodiment of the method provided by this invention. It is explanatory drawing of sperm isolation by the swim-up method combined with the specific exemplary embodiment of the method provided by this invention. It is a bar graph of the ratio of intermediate motile sperm in 7 different donors ± SEM. N = 20, ^* : p <0.05 relative to the control as determined by t-test. Semen samples were obtained from healthy volunteers. It is a bar graph of the ratio of superactive motile sperm in 7 different donors ± SEM. N = 20, ^* : p <0.05 relative to the control as determined by t-test. Semen samples were obtained from healthy volunteers. It is a bar graph of the ratio of intermediate motile sperm ± SEM. N = 5, ^* : p <0.05 relative to the control as determined by t-test. Semen samples were obtained from a man seeking treatment for infertility. It is a bar graph of the ratio of superactive motility sperm ± SEM. N = 5, ^* : p <0.05 relative to the control as determined by t-test. Semen samples were obtained from a man seeking treatment for infertility. Common practices for IVF and IUI will be described. IVF sperm treatment (left) generally consists of a separation step, such as density gradient centrifugation (shown here), followed by a wash step. A swim-up method (not shown) may be used as an alternative separation means. IUI sperm processing (right) requires only a wash step, but some clinics may include an initial separation step by either density gradient centrifugation (shown) or swim-up (not shown). prefer. Provided is an overview of sperm processing for IVF using the exemplary kits of the present invention. In one exemplary IVF application, a kit for nutrient-free density gradient centrifugation can be utilized with a kit for nutrient-free washing, incubation and sequential nutrient addition. This allows for the preparation of sperm preparations by nutrient-free separation, washing and incubation, followed by stepwise re-addition of glucose and pyruvic acid. After the final glucose incubation, the sample is centrifuged and resuspended in an appropriate volume of fertilization medium (which may be, for example, the clinic's preferred fertilization medium) prior to coincubation with oocytes. An overview of sperm processing for IUI using the exemplary kit of the present invention is provided. In one exemplary IUI application, a kit for nutrient-free density gradient centrifugation can be utilized with a kit for nutrient-free washing, incubation and mononutrient addition. This makes it possible to prepare sperm preparations by nutrient-free separation, washing and incubation, followed by stepwise re-addition of pyruvic acid. For IUI, glucose is not reintroduced prior to insemination due to the presence of glucose in the uterus. Shows the effect of the hunger-rescue protocol. First, the starvation-rescue protocol of incubating human sperm in the absence of glucose and pyruvate was compared to common sperm preparations. Shortly after reintroduction of glucose and pyruvate (C), a higher proportion of sperm exhibited intermediate and hyperactivated motility phenotypes (insets show typical trajectories of each). N = 20 (samples from 7 individuals). ^* P = 0.0084. The number of two-cell stage embryos and blastocyst stage embryos obtained is shown. N = 4 (C57BL / 6J mouse: low fertility mouse strain), ^* P <0.005, ^** P = 0.17. Transplantation of these embryos into females also resulted in a more than 3-fold increase in live birth rate. Sperm using nutrient-free sHTF (shown here as "sHTF medium" or "sHTF wash buffer") for swim-up protocol sperm separation, which is a component of the IVF and IUI kits. Provides an overview of the process. Following common practice techniques, pipette carefully form a layer of semen under sHTF medium and incubate to allow semen to swim out and allow upward motile sperm to enter the upper medium. After incubation, the upper sHTF medium containing motile sperm is carefully transferred to the washing step of the IVF kit or IUI kit to complete sperm preparation. It is a line graph of the ratio of the motile sperm recovered after storing at 4 degreeC. Sperm stored in either the test storage medium or EFM was collected for the time indicated on the x-axis, and the motility after acquisition of fertility was measured. Data are shown as a percentage of total motile sperm (acquired immediately after sample processing) at time 0. It is a line graph of the ratio of motile sperm recovered after storing at 4 degreeC. Sperms stored in the test storage medium or the refrigerated medium were collected at 4 ° C. for the time indicated on the x-axis, and the motility after acquisition of fertility was measured. Data are shown as a percentage of total motile sperm (acquired immediately after sample processing) at time 0. It is a bar graph of the ratio of sperms having DNA fragmentation as determined by TUNEL staining after storage in a test medium (preservation medium) at 4 ° C. for 7 days or after cryopreservation. FIG. 5 is a scatter plot of the percentage of motile sperm recovered after storage at 4 ° C. for 7 days in a test medium (preservation medium) compared with cryopreservation (CRYO). Sperm were collected and evaluated for motility after acquisition of fertility. Data are shown as a percentage of total motile sperm at time 0, with each data point representing an individual measurement.

About 50% of couples who have difficulty getting pregnant are due to male factors. Low sperm count is a widely recognized cause of male infertility. The World Health Organization defines low sperm count (oligospermia) as less than 15 million sperms per milliliter (Cooper et al., Human Reproduction Update, 16 (3), 231-245, 2009). Other causes of male infertility or hypofertility include hypomotility or morphological abnormalities. An important aspect of reproductive support is maximizing the function of male gametes (sperm) and promoting maximal fertilization. Prior to fertilization, sperm must undergo a series of changes that allow the egg to be fertilized, a process called capacitation. The in vitro fertilization medium contains three components (albumin, calcium and bicarbonate) and induces sperm capacitation. Sperm first swim straight in a nearly symmetrical flagellar movement. After various species-dependent times, this linear sperm motility replaces the in-situ helical movement known as "superactivation". Although there are methods of activating sperm, such methods fail to achieve maximum sperm activation and therefore do not adequately address the effects of male factors on infertility. Therefore, there is a need for media, compositions and methods for improving sperm function, eg, thereby promoting reproductive support.

The present disclosure provides, among other things, methods of storing sperm, methods of improving sperm function, methods of enhancing fertilization, kits for carrying out such methods, sperm preparation solutions and products. The present invention is based, at least in part, on Applicant's surprising finding that gradual reintroduction of different energy sources after a starvation period results in superior sperm activation.

Definitions To facilitate the understanding of this disclosure, many terms and phrases are defined below.

The terms "improved", "improved", "improved" or "enhanced" or "enhanced" are all used herein generally to mean improvement. For the avoidance of doubt, the terms "improved," "improved," or "enhanced" are at least 5%, eg, at least 10%, improved when compared to a suitable control, eg, compared to a suitable control. At least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or At least about 90% improvement, or less than 100% improvement, or any improvement of 10-100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, as compared to a suitable control. Or at least about 5 times, or at least about 10 times, or any improvement between 2 to 10 times, or more. The improvement can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably male infertility due to a given disease (eg, sperm dysfunction or oligospermia). It is to reach a level that is recognized as within the range of normal sperm from male mammalian subjects without disease.

The terms "reduce," "reduce," "reduce," "reduce," or "reduce," or "inhibit" are all used herein generally to mean reduction. For example, "decrease," "reduce," "reduce," or "inhibit" is at least a 5%, eg, 10% reduction, eg, at least when compared to a suitable control. A decrease of about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or 100. It means a reduction of% or less (eg, a level that is absent or undetectable when compared to a suitable control), or any reduction that is between 10 and 100%. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more than the normal range for an individual without a given disease.

As used herein, the term "effective amount" refers to the detectable function of mammalian sperm (eg, sperm motility, curve velocity, head amplitude, autophagy, sperm fertility acquisition, superactivated sperm). Sufficient first or second energy source or second energy to cause changes in proportion, proportion of intermediate motile sperm and proportion of superactivated and intermediate motile sperm, ability to fertilize eggs and embryonic development) Means the total amount of one or more active ingredients of the source. When applied to an individual energy source administered alone, the term refers to that energy source alone. When applied to a combination, the term refers to a first and second source of energy that produces an effect, whether administered in combination, sequentially, or simultaneously. Refers to the amount of combination.

The term "effective amount" includes, within its meaning, an amount of energy source (eg, a gluconeogenic substrate or glycolytic energy source) sufficient to provide the desired effect. When relevant to the present disclosure, the desired effect can be one or more sperm function enhancements or fertilization enhancements. The exact amount required depends on factors such as the age and general condition of the male subject from which the mammalian sperm is obtained from the treated mammalian sperm species, eg, when the sperm is obtained from a poorly fertilizing mammalian subject. Will change. Therefore, it is not possible to specify a strict "effective amount". However, in any case, a suitable "effective amount" can be determined by one of ordinary skill in the art using experiments that are only routine.

The term "spermatozoon" refers to live germ cells from male mammals. The term "spermatozoa" refers to multiple surviving male germ cells. Plurals and singulars are synonymous unless otherwise required by the context. The term "sperm" is used as an abbreviation, which refers to at least one sperm.

As used herein, the term "ability to fertilize an egg" means that sperm (eg, mammalian sperm) invade unfertilized eggs (egg cells), resulting in a combination of their genetic materials, and as a result. Refers to the ability to form a zygote. In the context of the present disclosure, the "ability to fertilize an egg" can be the ability to fertilize an egg in vitro and / or in-vivo. In some embodiments, the ability to fertilize in vitro includes spontaneous pregnancy, intravaginal fertilization, intracytoplasmic sperm injection or fertilization by intracytoplasmic sperm injection (ICSI).

The term "embryonic" is used herein to refer to diploid pluripotent cells, eg, conjugates formed during fertilization of unfertilized eggs by mammalian sperm to form fertilized eggs, followed by cell division 2 Refers to both embryos that develop beyond the cell stage (eg, 4-cell stage, 16-cell stage, 32-cell stage, vesicle stage (with differentiated trophozoites and internal cell clusters) or development to offspring). Is used.

As used herein, the term "developmental ability" refers to the ability or potential of an embryo to grow or develop. These terms describe the ability or potential of an embryo to reach at least the two-cell stage of development, the blastocyst stage of development, implant in the uterus and develop into a complete offspring, or be born alive. Can point. The term "offspring" as used herein refers to a progeny of a parent, where the offspring is a prenatal foetation or newborn.

The term "blastocyst" refers to an embryo 5 or 6 days after fertilization and is filled with the inner cell mass from which the entire embryo is derived, the outer cell layer called the ectoderm, and the body fluid that contains the inner cell mass. It has a blastocyst cavity. The vegetative ectoderm is a precursor of the placenta. The blastocyst is surrounded by a zona pellucida, which subsequently sheds when the blastocyst "hatches". The zona pellucida, which is composed of the coat of glycoprotein, surrounds the oocyte from the 1-cell stage to the blastocyst stage of the developmental stage. Before the embryo attaches and implants, the zona pellucida sheds from the embryo by a number of mechanisms, including proteolysis. The zona pellucida first serves to prevent two or more sperms from invading the oocyte, and then later prevents the embryo from prematurely adhering before reaching the uterus.

As used herein, the term "concentrated" means that the concentration of the target species is substantially higher than the naturally occurring level of the species in the unconcentrated end product or preparation. A composition or fraction or preparation in which the target species is partially purified.

The term "assisted reproductive technology", or "ART", or "assisted fertilization" has its general meaning in the art and is used to achieve pregnancy by artificial or partially artificial means. Refers to the method used. Assisted reproductive technology includes, but is not limited to, classical in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), intracytoplasmic sperm injection (IUI), and intracytoplasmic sperm injection.

The term "intrauterine insemination" or "IUI" refers to the direct intrauterine injection of sperm or spermatozoa into the uterus.

The term "in vitro fertilization" or "IVF" refers to a method of fertilizing an oocyte with sperm outside the body in vitro. IVF is the primary treatment for unsuccessful pregnancy in the body in infertility.

The term "intracytoplasmic sperm injection" or "ICSI" refers to an in vitro fertilization procedure in which a single sperm is injected directly into the cytoplasm of an egg. This procedure is most commonly used to overcome male infertility factors, but it can also be used when oocytes are less susceptible to sperm invasion and sometimes as an in vitro fertilization method. ..

Some numbers disclosed throughout are expressed, for example, as "X is at least or at least about 100 or 200 [or any number]". This number includes the number itself and all of the following:
i. X is at least 100
ii. X is at least 200
iii. X is at least about 100, and iv. X is at least about 200.

All of these different combinations are conceived by the numbers disclosed throughout. All of the numbers disclosed, whether it refers to the administration of a therapeutic agent or to the number of days, months, years, weights, dosages, etc., unless otherwise specifically indicated. Should be interpreted as.

The range disclosed throughout may be expressed, for example, as "X is administered on days 1-2 or 2-3 [or any numerical range] or about that number of days." This range includes the number itself (eg, the endpoints of the range) and all of the following:
i. X was administered on the 1st and 2nd days,
ii. X was administered on the 2nd to 3rd days,
iii. X was administered about 1 to 2 days,
iv. X was administered about 2 to 3 days,
v. X was administered from the 1st day to the 2nd day,
vi. X was administered from the 2nd day to the 3rd day,
vii. X was administered from about day 1 to about day 2, and vivii. X is administered from about 2 days to about 3 days.

All of these different combinations are contemplated, to the extent disclosed throughout. All of the disclosed scope, whether it refers to the administration of a therapeutic agent or to the number of days, months, years, weights, dosages, etc., is this unless specifically indicated otherwise. Should be interpreted as.

Sperm Function In some embodiments, the present specification provides methods for improving sperm function. The method involves incubating mammalian sperm under energy depletion for a time suitable for enriching mammalian sperm, to enriched mammalian sperm in an effective amount, (i) a gluconeogenic energy source, or ( ii) To provide a first energy source selected from the gluconeogenic substrate, and then to the mammalian sperm from step (b), an effective amount of (i) glycolytic energy source, or (ii) sugar. To provide a second energy source selected from the gluconeogenic substrate, the energy source provided is one that is not selected as the first energy source, comprising providing, thereby being a suitable control sperm. Induces improved sperm function compared to. In some embodiments, the method is performed in vitro. In some embodiments, the provision of a second energy source is performed in the body, eg, by intracervical or endometrial insemination of sperm that have been previously incubated under energy depletion and provided with a first energy source. Will be done. Improved sperm function includes improved motility (Goodson et al., 2017, Biol. Reprod. 97: 698-708; doi: 10.1093 / biore / iox120), improved autophagy, improved fertility acquisition and improved fertility rates, such as development up to at least 2 cell stages, blastocyst development or life. One or more of the births of the offspring can be mentioned. Thus, in some embodiments, sperm function includes sperm motility, curve velocity, head amplitude, autophagy, sperm capacitation, superactivated sperm ratio, intermediate motility sperm ratio and superactivated sperm. And the proportion of intermediate motile sperm, the ability to fertilize eggs, the development of embryos. In some embodiments, embryos generated by sperm with improved function have improved fitness, improved implantation, at least two-cell stage development, blastocyst stage development, progeny-ie, live. Includes one or more traits selected from the offspring of improved fitness or improved fitness (eg, improved fitness, including reduced risk of developing the condition) to the birth of the offspring.

In some embodiments, the first and second energy sources are provided sequentially (eg, providing a first energy source, followed by a second energy source). In some embodiments, the first and second energy sources are provided simultaneously. An improvement in one or more sperm functions as contemplated herein corresponds to an improvement in one or more sperm function as compared to a suitable control sperm. In some embodiments, one or more sperm functions are at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or 100. It can be improved by%, 200%, 300% or more. In some embodiments, one or more sperm functions may be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%.

The present specification provides methods for improving sperm function and preparation of sperm containing improved function as compared to suitable control sperm. When relevant to the present disclosure, sperm "activity" and / or "function" are, for example, sperm motility, sperm tropism (ie, the property of a sperm to move towards or away from a particular stimulus). ) And the ability to fertilize an egg, including physiological processes. The terms "activity" and / or "function" refer to the processes that occur before and during fertilization and / or interaction with the egg (or its membrane / layer) (such processes include, for example, acquisition of sperm fertility and sperm fertility). (Acrosome activity can be mentioned) and / or post-fertilization processes of the egg, such as embryo formation, may be further included. In some embodiments, embryos exhibit the ability to improve survival (prolongation), improve implantation and / or develop to the two-cell stage, blastocyst stage, or even offspring to result in offspring.

Exemplary methods for measuring improvement in sperm function can be assessed by motility, mucus permeability, oocyte fertilization or subsequent embryonic development. Methods of determining sperm function are well known in the art and are described, for example, in SS. Vasan Indian J Urol. See 2011 Jan-Mar; 27 (1): 41-48.

Sperm motility For sperm motility, those skilled in the art are not only concerned with the term "motility" for general motility, but also for example sperm cell motility and / or any of the moving sperm cells in a given population. It will be appreciated that it can also be applied to other aspects of athletic performance, such as any increase or decrease in proportion. Therefore, the methods disclosed herein not only improve sperm motility, but also the rate of sperm cell motility and / or the proportion of moving cells in any given sperm population. It can also be used to improve.

Sperm motility is expressed as total motile sperm rate or rate of motile sperm. These measurements can be made by a variety of testing methods, but conveniently one of the two is tested. A phase-contrast microscope is used to either make a subjective visual measurement when the sperm are placed on a hemocytometer or a microscope slide, or a computer-aided semen analyzer is used. Under a phase-contrast microscope, motile sperm count and total sperm count are counted and the velocity is assessed as fast, moderate or slow. The second method of assessing sperm motility is by using a computer-aided semen analyzer (Hamilton Horn, Beverly, Mass.), In which the motility characteristics of individual sperm cells in the sample are objective. Is determined. Briefly, the sperm sample is placed in a slide or chamber designed for the analyzer. An analyzer tracks individual sperm cells and determines sperm motility and velocity. The data are expressed as kinetic rates and measurements for path speed and trajectory speed are also available.

Thus, the term "motility" includes the proportion of motile sperm, which is described in Cooper et al. As discussed in Human Reproduction update, Vol 16, No. 3 pp 231-245, 2010, all except the category designated as "incapacitated" by the World Health Organization (WHO), regardless of speed or direction. It can be the percentage of total sperm count evaluated to fit into the motility category. By manual counting, sperm cells are classified into four categories using qualitative and subjective selection criteria (immobility, local motility, non-linear motility and linear motility).

The term "motility" refers to the proportion of motile sperm, i.e. the proportion of total sperm evaluated to exhibit straight-line motility, superactivated motility and / or intermediate motility in a population based on computer-aided sperm analysis. Include (eg, when assessed by CASAnova; see Goodson et al, 2017, Biol. Report. 97: 698-708).

The methods disclosed herein are head rotations compared to the proportion of sperms capable of straight-line movement, that is, sperms that are inherently non-straight-lined, that is, sperms that move but do not move forward. However, it is possible to increase the proportion of sperm that exhibit a linear movement from one point to another that is less than 90 degrees. In some embodiments, the methods disclosed herein can increase the proportion of intermediate motile sperm. Intermediate motility Sperm are similar to straight-line active motility, but are characterized by more variability from the pathway and movements that exhibit about 90 degrees of sperm head rotation, such as swinging movements. Be done. In some embodiments, improved motility includes an increase in the proportion of activated superactive sperm, also known as superactivated sperm. Superactivated sperm motility is characterized by sperm with flagella with asymmetric whipping patterns of high amplitude. Superactivated motility is characterized by active motility, often with seemingly random fluctuations without a well-defined forward pathway and rotation of the sperm head greater than 90 degrees. Super-activated sperm motility is more active and shorter than straight-line motility. Biologically, super-activated sperm motility is important for allowing sperm to pass through the egg coat prior to fertilization of the mature egg. In some embodiments, the methods disclosed herein can improve the proportion of superactivated sperm and intermediate motile sperm in a given sperm population.

It must be understood that other standardized measures of sperm motility parameters can also be used. Other sperm motility measures include "velocity" and "linearity", which can be evaluated using an automated semen analyzer. In some embodiments, the methods disclosed herein are mean path velocity (VAP), linear velocity (VSL), curve velocity (VCL), head amplitude (ALH) and head frequency (BCF). Alternatively, sperm function can be improved, including improvements in sperm motility parameters, including other parameters known to those skilled in the art. Curved velocity (VCL) is a measure of the velocity of movement of the center of gravity of the sperm head over a given period of time. The average pathway velocity (VAP) is the velocity along the average pathway of sperm. Linear velocity (VSL) is the linear or linear velocity of a cell. Straightness (LIN) is the ratio of VSL to VCL and is expressed as a percentage. The head amplitude (ALH) of the sperm head is calculated from the amplitude of its lateral deviation with respect to the cell's path axis or mean pathway. Methods for measuring sperm motility by CASA are well known in the art, see, for example, International Publication No. 2012061578A2 pamphlet. Improving sperm motility, as contemplated herein, corresponds to improving sperm motility as compared to a suitable control sperm.

In some embodiments, sperm with improved motility are provided, in which the sperm are incubated under energy depletion conditions to enhance the sperm, followed by the enhanced sperm as a first energy source and a second. Is a product of methods involving providing an energy source for. In some embodiments, the first and second energy sources are provided simultaneously. In some embodiments, the first and second energy sources are provided sequentially. In some embodiments, the improvement in sperm motility is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99 as compared to a suitable control sperm. Can be over%. In some embodiments, the improvement in sperm motility is 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%, It can be 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, and at least 10,000-fold. In some embodiments, sperm motility can 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 can be an increase in the proportion of superactivated sperm. In some embodiments, an improvement in sperm function or sperm motility can be an increase in the proportion of intermediate motility sperm. In some embodiments, improved sperm function or improved sperm motility can be an increase in the proportion of straight-moving sperm. In some embodiments, improved sperm function or improved sperm motility can be an increase in the proportion of superactivated sperm and intermediate motility sperm. In some embodiments, the level of superactivated sperm, straight-moving sperm, intermediate-motile sperm or a combination thereof is prepared by super-activated sperm, straight-moving sperm, intermediate motile sperm or a combination thereof. At least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0% of all sperm in. 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%, Improve to account for 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more. Improved sperm motility points to improved sperm function.

Sperm Capacitation In some embodiments, improved sperm function includes improved sperm capacitation. “Capacitation of sperm” refers to sperm capable of acrosome open secretion, binding of unfertilized eggs to the zona pellucida, and invasion through them. Completion of fertility manifests itself in the ability of sperm to bind to the zona pellucida and elicit a ligand-induced acrosome reaction. Methods for determining sperm fertility acquisition are known in the art, for example, the most common sperm-zona pellucida binding tests currently in use are the translucent zona pellucida test (or HZA) and the competitive zona pellucida. It is a binding test. The zona pellucida test measures the ability of sperm to acquire fertility and bind to oocytes. Incubate sperm with dead oocytes surrounded by the zona pellucida, the acellular oocyte coat of the oocytes. Sperm that have acquired fertility bind to the zona pellucida, and the number of sperm bonds is counted under a microscope. This number correlates with the number of sperms that have normally acquired fertility in the sample and the fertility of the sperm sample. For example, Cross NL et al. Gamete Res. See 1986; 15: 213-26.

In some embodiments, sperm with improved fertility acquisition is provided by incubating the sperm under energy depletion conditions to enhance the sperm, followed by a first energy source and a first energy source for the enhanced sperm. It is the product of a method comprising providing two energy sources simultaneously or sequentially. In some embodiments, the improvement in sperm fertility acquisition is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or compared to a suitable control sperm. It can be over 99%. In some embodiments, the improvement in sperm fertility acquisition is 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 fertility acquisition can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, sperm fertility acquisition can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the fertilization capacity acquisition level 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 improve so that it can occupy more than that. Improved acquisition of sperm fertility points to improved sperm function.

Fertilization Ability In some embodiments, sperm function includes the ability of a sperm to fertilize an egg. The fertility of sperm can be determined, for example, by a sperm invasion test. Sperm invasion testing (SPA) utilizes golden hamster eggs, which are unusual in that removal of their zona pellucida results in the loss of any species specificity for egg invasion. By performing this test, the ability of sperm to invade oocytes is determined (Rogers et al., Fert. Star. 32: 664, 1979). Briefly, commercially available zona pellucida-free hamster oocytes can be used (Fertility Technologies, Natick, Mass.). For sperm of any species, hamster oocytes are suitable for this test. Incubate sperm with hamster oocytes for 3 hours. After incubation, the oocytes are stained with acetolacmoid or a uniform stain and the number of sperms invading each oocyte is counted under a microscope. Another parameter of sperm fertility is the ability to invade cervical mucus. This penetration test can be performed either in vitro or in vivo. Briefly, in vitro, a commercially available kit containing cervical mucus (Tru-Trax, Fertility Technologies, Natick, Mass.), Typically bovine cervical mucus, is prepared. Place the sperm at one end of the runway and determine the distance the sperm have invaded the mucus after a given time. Instead, sperm mucus invasion can be measured in the female body. In certain embodiments, sperm with improved fertility are provided, in which the sperm are incubated under energy depletion conditions to enhance the sperm, and then the enhanced sperm are subjected to a first energy source and a second energy source. It is a product of methods involving providing sources simultaneously or sequentially. In some embodiments, the improvement in fertility is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% compared to a suitable control sperm. Can be super. In some embodiments, the improvement in fertility is 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. It can be 90% or at least 95%. In some embodiments, the improvement in fertility can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, fertility can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the fertility level is such that the number of sperm capable of fertilizing the egg is at least about 5%, 5.5%, 6.0%, 6.5% of the total sperm in the preparation. , 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5% 1,12.0%, 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. Improved fertility points to improved sperm function and improved fertilization.

Embryo development In some embodiments, sperm function involves embryonic development. In some embodiments, enhancing fertilization by providing a means of reaching an egg to a sperm with enhanced function prepared by the methods herein, where enhancing fertilization, is described. May include embryonic development. In some embodiments, sperm with improved function prepared by the methods herein provide a means of reaching the egg in vitro, so that the embryo develops in vitro. In some embodiments, sperm with enhanced function prepared by the methods disclosed herein are provided with a means of reaching the egg in the body by the sperm IUI, thus the embryo is in the body. appear. In some embodiments, sperm incubated under energy-deficient conditions and provided with a first energy source are fertilized in the reproductive tract of a female subject, thus providing a second energy source and to the egg. Providing a means of reaching the embryo will be done in the body. In some embodiments, when embryos are developed in vitro, the embryos can be cryopreserved for later use or further in vitro cultured to allow embryo development. In some embodiments, embryos develop to at least 2 cell stages prior to cryopreservation and / or injection into female subjects. In some embodiments, embryos develop in vitro to the developmental stage after the two-cell stage prior to further treatment. In some embodiments, embryos develop in vitro to the blastocyst stage prior to further treatment (eg, cryopreservation or injection into a female subject for development to complete offspring). A variety of suitable media are available for in vitro incubation and culturing of embryos during assisted reproductive technology (ART) procedures, the types and compositions of which are well known to those of skill in the art. Preferably, the culture medium contains at least water, salts, nutrients, essential amino acids, vitamins and hormones and may also contain one or more growth factors. A variety of suitable culture media such as Earl's medium, Ham's F10 medium and Human Fallopian Tube Solution (HTF) medium are commercially available. The disclosure also contemplates in vitro co-culture of embryos on a "feeder cell" layer by methods known in the art. Examples of "feeder cells" suitable for co-culture include bovine oviduct cells or human oviduct epithelial cells.

Those skilled in the art will appreciate that the benefits provided by sperm with enhanced function prepared by the methods disclosed herein are not limited to improved fertilization. Rather, the methods and preparations of the invention are equally applicable as treatments that promote fertilization, whether embryos are produced in vitro by assisted reproductive technology (ART) or in the reproductive tract of animals. be. The methods of the present invention are applicable for improving fertilization, embryo viability, embryo implantation and pregnancy rate in reproductive-assisted or other reproductive-free pregnancies. The embodiments of the present disclosure also provide a method of improving the fertilizing ability of male animal sperm.

In the context of this specification, the terms "improved viability embryo" and "prolonged viability embryo" are compared to the viability of one or more embryos generated by a suitable control sperm. Means the enhancement or enhancement of viability of one or more embryos generated by mammalian sperm of the methods and preparations disclosed herein, eg, mammalian sperm having one or more enhanced sperm function. In some embodiments, embryos are developed by assisted reproductive technology, such as IVF or ICSI. In some embodiments, embryos develop in the body by artificial insemination in the reproductive tract of female mammal subjects.

For the purposes of the present disclosure, embryo viability can be reflected in a number of indicators. For example, improved embryo viability can result in increased post-implantation embryo implantation rates, lower pre-implantation and post-implantation embryo mortality rates, increased clinical pregnancy rates or increased fertility rates. Accordingly, the present disclosure also relates to methods of preventing embryonic apoptosis or growth retardation and improving pregnancy rates in animals. Embryo viability can refer to the viability of an embryo in vitro or in vivo.

In some embodiments, sperm capable of developing embryos with improved viability are provided, in which sperm are incubated under energy depletion conditions to enhance sperm, followed by enhanced sperm. It is a product of a method comprising providing one energy source and a second energy source simultaneously or sequentially. In some embodiments, fertilization is enhanced by providing sperm with improved function with a means of reaching the egg. In some embodiments, enhancing fertilization involves the development of one or more embryos with improved viability. In some embodiments, the improvement in viability of embryos developed upon arrival at the egg by sperms prepared by the methods herein is approximately 10%, 20% compared to embryos produced by suitable control sperms. , 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 99%. In some embodiments, the improvement in embryo viability is 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%, It can be at least 90% or at least 95%. In some embodiments, the improvement in embryo viability can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, embryo viability can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the level of sperm capable of developing viable embryos is at least about 5%, 5.5%, 6.0%, 6.5 of all sperm in the preparation. %, 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. Embryo development with improved viability points to improved sperm function and / or improved fertilization.

Typically, the cleavage stage of the embryo occurs in the first 3 days of culture. Embryos generated in vitro are transferred to female subjects by embryo transfer. "Embryo transfer" is a procedure in which one or more embryos and / or blastocysts are placed in the uterus or in the Falloppiox duct. In the conventional IVF method, embryos are transplanted into the uterine cavity 2 days after fertilization when each embryo is in the 4-cell stage, 3 days after fertilization when the embryo is in the 8-cell stage, or 5 days after fertilization when the embryo is in the blastocyst stage. Will be done. It has been recognized that it would be desirable to use blastocyst stage embryos when they reach days 5-7 of culture. According to the present disclosure, embryo transfer is possible at any time along a series of embryo / blastocyst development. When the blastocoel is clearly visible and occupies more than 50% of the embryo's volume through visual observation, such as by using microscopy, the blastocyst or embryo is ready to be transplanted into the uterus. Be considered. In the internal environment, this stage can usually be achieved 4-5 days after fertilization, shortly after the embryo passes through the Fallopian tubes and reaches the uterus. The developmental stage of the embryo can be determined by visual observation of the embryo using a microscope (eg, Nikon Eclipse TE 2000-S microscope), and the embryo has a specific fixed physical or morphology prior to transplantation into the uterus. It will exhibit scientific features at the same time. The maturation state of the blastocyst is described in Gardener et al. , 1998 will be determined as Range II AB ~ VI AA.

According to the methods disclosed herein, embryos with improved 2-cell stage developmental stage, blastocyst stage developmental stage, or offspring development rate and offspring birth rate are generated. In some embodiments, sperm capable of developing embryos capable of developing through normal developmental stages (eg, two-cell stage, scutellum stage, progeny development and offspring birth) are provided. , This is the product of a method comprising incubating sperm under energy depletion conditions to enhance sperm, and then providing the enhanced sperm with a first and second energy sources simultaneously or sequentially. be. In some embodiments, fertilization is enhanced by providing sperm with improved function with a means of reaching the egg. In some embodiments, enhancing fertilization is the development of embryos with increased ability to develop through normal developmental stages (eg, 2-cell stage, blastocyst stage, progeny development and offspring birth). including. In some embodiments, an increase in the proportion of embryos that develop through normal developmental stages, generated by method-prepared sperm, is approximately 10%, 20%, compared to embryos developed by suitable control sperm. It can be 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 99%. In some embodiments, the increase in the proportion of embryos that develop through the normal developmental stage is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, It can be at least 70%, at least 80%, at least 90% or at least 95%. In some embodiments, the increase in the proportion of embryos that develop through normal developmental stages can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, the proportion of embryos that develop through normal developmental stages can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the level of sperm capable of developing an embryo capable of developing through the normal developmental stages is at least about 5%, 5.5%, and 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. Development of embryos capable of developing through one or more normal developmental stages indicates improved sperm function and / or improved fertilization.

In the body, the embryo attaches to or implants on the wall of the uterus, creates a placenta, and develops into fetal offspring during pregnancy until delivery. A test to determine if one or more embryos have implanted in the endometrium, i.e., if the procedure succeeded in initiating pregnancy, is two weeks after transplantation, eg b-hCG (human chorionic gonadotropin). It is performed using blood tests related to the above and other techniques generally known in the art. Zer et al. U.S. Pat. No. 4,315,908 describes a method for detecting urinary hCG by radioimmunoassay. O'Connor et al. U.S. Pat. No. 8,163,508 provides a method and kit for predicting pregnancy by the hCG method by determining the amount of hCG of an early pregnancy-related isoform in a sample. Such diagnostic methods and other methods are useful within the scope of the present disclosure.

In some embodiments, sperm having the ability to develop embryos with improved implantation or improved pregnancy rates are provided by incubating the sperm under energy depletion conditions to enhance the sperm, followed by , A product of a method comprising providing a first energy source and a second energy source to the enriched sperm simultaneously or sequentially. In some embodiments, fertilization is enhanced by providing sperm with improved function with a means of reaching the egg. In some embodiments, enhancing fertilization involves the development of embryos with improved implantation rates or improved pregnancy rates. In some embodiments, an increase in the implantation rate of embryos or embryos at the time of implantation of embryos produced by sperm prepared by the methods herein is about as compared to embryos produced by suitable control sperm. It can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 99%. In some embodiments, the improvement in embryo implantation rate or pregnancy rate is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, It can be at least 80%, at least 90% or at least 95%. In some embodiments, the improvement in embryo implantation rate or pregnancy rate can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, the implantation rate or pregnancy rate can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the level of sperm capable of developing embryos with improved implantation or improved pregnancy rates is at least about 5%, 5.5%, 6 of all sperm in the preparation. 0.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11 0.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16 0.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more. Embryo development with improved implantation (ie improved implantation rate) or improved pregnancy rate at implantation points to improved sperm function and / or improved fertilization.

Autophagy In some embodiments, improved sperm function includes an improvement in autophagy. Methods of determining improvement in autophagy are known in the art. For example, an improvement in autophagy can be determined by an improvement in one or more of the autophagy marker proteins. Detection of improvement in marker protein can be performed by conventional methods such as immunoblotting. Non-limiting examples of autophagy marker proteins include Atg 5, Atg 16, p62, LC3-II, AMPK, m-TOR and Beclin 1. LC3-II is widely used in the study of autophagy and is considered to be a mammalian autophagosome marker. The LC3-II / LC3-I ratio can be used as a determinant of autophagy improvement. Increased levels of one or more autophagy marker proteins (eg, Atg 5, Atg 16, p62 and LC3-II, AMPK, m-TOR and Beclin 1) and / or improved LC3-II / LC3-I ratios , Can indicate an improvement in sperm function. In some embodiments, improved autophagy can be dictated by lowering cellular RNA levels, such as small non-coding RNAs, including microRNAs.

In some embodiments, autophagy-enhanced sperm are provided by incubating the sperm under energy depletion conditions to enhance the sperm, followed by a first energy source and a second source of enhanced sperm. Is a product of methods involving the simultaneous or sequential provision of energy sources for. In some embodiments, the improvement in autophagy is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% compared to a suitable control sperm. Can be super. In some embodiments, the improvement in sperm autophagy is 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%, It can be at least 90% or at least 95%. In some embodiments, the improvement in sperm autophagy can be at least 10-fold, at least 100-fold, at least 1,000-fold, and at least 10,000-fold. In some embodiments, sperm autophagy can be improved by 10% to 200%, 25% to 150%, 50% to 100% or 70% to 90%. In some embodiments, the sperm autophagy level is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0% of the total sperm in the preparation with improved autophagy. %, 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 Improve to occupy more than that. Improved sperm autophagy points to improved sperm function.

Starvation "Energy depletion" suppresses cellular energy production, whether by depletion, reduction (until less effective), or removal of such energy sources or inhibition of enzymatic or uptake transport mechanisms. Or it means to limit. In some embodiments, energy depletion inhibits one or more of glycolysis, gluconeogenesis, the Krebs cycle or oxidative phosphorylation, and in detailed embodiments, energy depletion comprises glycolytic energy depletion. Is done. Exemplary glycolytic energy depletion conditions include glucose in the medium of sperm (other embodiments include combinations thereof with mannose, fructose, dextrose, sucrose and glucose). ) To remove substantially all glycolytic sugars, or to reduce the concentration of glycolytic sugars, or to use inhibitors of glycolytic, glycolytic or glycolytic sugar uptake transporters. Includes use. In a preferred embodiment, the energy depletion is glucose energy depletion (including starvation), since the main energy source for sperm is glucose, which is a gluconeogenic substrate (eg, pyruvic acid or some embodiments). Lactate (which can be converted to pyruvate by lactic acid dehydrogenase) and Krebs circuit substrate (acetyl-CoA, citric acid, isocitrate, α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, malic acid And oxaloacetate) depletion (including starvation).

In some embodiments, energy depletion is, for example, about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, Includes low glucose concentrations of less than 0.05, 0.04, 0.03 mM or lower, such as about 0.02 or less than 0.01 mM, for example less than about 0.01 mM. In some embodiments, energy depletion means a condition that is substantially glucose-free. The present invention provides a method involving the stepwise provision of effective amounts of first and second energy sources, and those skilled in the art will appreciate an ineffective amount of saccharification in some embodiments within the scope of the present invention. It will be appreciated that the system energy source is energy depletion, for example in such an embodiment the aforementioned low glucose concentration can be used as energy depletion.

In some embodiments, the energy depletion is, for example, about 0.15, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, Includes low pyruvate concentrations below 0.02, 0.01, 0.005, 0.003, 0.002 mM or less. In some embodiments, energy depletion means a condition that is substantially pyruvate-free. As pointed out above and exemplified for glycolytic energy sources in glucose, those skilled in the art will appreciate that in some embodiments within the scope of the present invention an ineffective amount of gluconeogenic substrate is energy depletion, eg. It will also be appreciated that in such embodiments the aforementioned low pyruvate concentration can be used as energy depletion.

In some detailed embodiments, energy depletion is a condition that is substantially free of carbon sources, such as low glucose and low pyruvate concentrations, such as substantially glucose-free and substantially pyruvate-free conditions. including.

In some embodiments, the energy depletion is at least about 10, 20, 30, 40, 50, 60 minutes, eg at least about 30, 40, 45, 50, 55, 60, 90, 120, 150 or 180 minutes or 1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9, Over 9.5 or 10 hours.

Energy depletion according to the present invention enhances sperm. “Strengthening” or “strengthening” sperm is defined as suitable induction, for example, when energy depletion is removed or undone and the sperm are incubated under fertility acquisition conditions or gradual energy reintroduction, for example. One or more of increased proportions of superactivated spermatozoa, intermediate spermatozoa or straight-moving spermatozoa (or increased proportions of two (such as superactivated and intermediate) or all three combinations) and / or increased curve velocity It means conditioning sperm so that it recovers its motility rapidly.

Gradual energy reintroduction After sufficient energy depletion for sperm enrichment, the enriched sperm are provided with an effective amount of a first energy source and then an effective amount of a second energy source. In some embodiments, the time between providing an effective amount of the first energy source and providing an effective amount of the second energy source after sperm enrichment is at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, for example at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 minutes, for example at least about 5 to 15 minutes. In some embodiments, the time between providing an effective amount of the first energy source and providing an effective amount of the second energy source after sperm enrichment is longer, eg, at least 2. 3, 4 or 5 hours, etc. or more.

In some embodiments, the gluconeogenic substrate is, for example, about 0.15 to 0.66 mM, such as about 0.25 to 0.50 mM, such as about 0.25 to 0.40 mM, or about 0.30 mM. Concentration of pyruvic acid. The aforementioned concentration is, for example, an exemplary effective amount of the gluconeogenic substrate when provided as either the first or second energy source in the method provided by the present invention. Those skilled in the art will recognize other effective amounts of gluconeogenic substrates based on their ability to improve sperm function, following the teachings of the present invention. In some embodiments, the first energy source is a gluconeogenic substrate such as pyruvic acid. In some embodiments, the second energy source is a gluconeogenic substrate such as pyruvic acid.

In some embodiments, the glycolytic energy source is, for example, about 0.6 mM to 10.0 mM, 1.0 to 7.0 mM, 2.5 to 7.0 mM, 3.5 to 6.5 mM or 5 mM. For example, glucose at a concentration of at least about 1, 2, 3 or 4 mM. The aforementioned concentration is, for example, an exemplary effective amount of glycolytic energy source when provided as either the first or second energy source in the method provided by the present invention. Those skilled in the art will recognize other effective amounts of glycolytic energy sources based on their ability to improve sperm function according to the teachings of the present invention. In some embodiments, the first energy source is a glycolytic energy source such as glucose. In some embodiments, the second energy source is a glycolytic energy source such as glucose. In some embodiments, the first energy source is a glycolytic energy source such as glucose, while the second energy source is a gluconeogenic substrate such as pyruvic acid.

An additional condition adjusted in some embodiments of the methods provided by the present invention is volume osmolality (mOsm) or weight osmolality (mOsm / kg). In some embodiments, the method comprises volumetric osmolal concentrations ranging from about 200 to 280 mOsm (mOsm / kg), eg, about 220 to 260, 225 to 255, 230 to 250 mOsm (mOsm / kg) during energy depletion. Or by weight osmolality), and optionally adding a first or second energy source, the volume osmolality (or weight osmolality) is at least about 270,275,280, 285,290 or 295mOsm (or weight osmolality). It increases to mOsm / kg).

Suitable gluconeogenic substrates for use in the methods of the present disclosure are, but are not limited to, pyruvic acid, lactic acid, succinic acid, citric acid, fumaric acid, malic acid, aspartic acid, glycerol, acetyl-CoA, isocitric acid, α-. Physiologically acceptable derivatives, salts, esters, polymers or α-keto analogs of ketoglutaric acid, succinyl-CoA, oxaloacetate or gluconeogenic substrates can be mentioned. Any gluconeogenic amino acid or its physiologically acceptable derivative, salt, ester, or polymer, or α-keto analog is also suitable as a gluconeogenic substrate. Non-limiting examples of glycosylated amino acids include alanine, arginine, asparagine, cystine, glutamine, glycine, histidine, hydroxyproline, methionine, proline, serine, threonine and valine. Non-limiting examples of pharmaceutically acceptable salts of pyruvate are lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate and zinc pyruvate. In some embodiments, the pyruvate is sodium pyruvate. Non-limiting examples of lactic acid salts include sodium lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate and manganese lactate. The gluconeogenic substrate of the method disclosed herein can be any one of the gluconeogenic substrates listed above.

Glycolytic energy sources suitable for use in the methods of the present disclosure include, but are not limited to, glycolytic carbon sources. Non-limiting examples of oligosaccharide-based energy sources useful in the methods disclosed herein include monosaccharides (such as fructose, glucose, galactose and mannose) and disaccharides (sucrose, lactose, maltose and trehalose) and Examples include polysaccharides, galactose, oligosaccharides and their polymers.

In some embodiments of the methods provided by the present invention, additional components are provided for sperm. For example, in combination with at least the first or second energy source, other components upstream and downstream of glycolysis are added, such as NADH, NAD +, citric acid, AMP, ADP or a combination thereof.

Some embodiments of the methods provided by the present invention include evaluation of sperm. For example, in some embodiments, the method comprises, for example, one or more quantitative assessments of sperm motility by CASA and / or DNA fragmentation (eg, by TUNEL), lipid peroxidation, reactive oxygen species or these. Includes measuring sperm quality, such as a combination of.

The methods provided by the present invention provide improved sperm function. In some embodiments, it is compared to a suitable control sperm. In some embodiments, a suitable control is sperm in standard capacitation medium (C-HTF) without a starvation step, while in some embodiments a suitable control is after 3 hours of starvation. -For example, after reintroduction of an effective amount of energy source that does not phase out starvation and reintroduction of energy source-sperm in standard capacitation medium (C-HTF).

Mammalian sperm The methods disclosed herein include enhancing the function of one or more sperms. The present disclosure also relates to promoting fertilization. Sperm preparations with improved function are also provided. As used herein, sperm can be from vertebrates, preferably mammals. Therefore, the sperm of the present disclosure can be mammalian sperm.

Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including wild animals, agricultural animals, competition animals and pets. Rodents include, for example, mice, rats, woodchuck, ferrets, rabbits and hamsters. Livestock and hunting animals include, for example, cows, horses, pigs, deer, bison, squirrels, cat species such as domestic cats and canines such as dogs, foxes, wolves and birds such as chickens and emu. , Octopus and fish, such as trout, catfish and salmon. Mammalian sperm are ungulates of the order Artiodactyla (eg, pigs, peccaries, hippopotamus, camels, llamas, deer (rats), deer, giraffes, pronghorns, leiodes, canines (goat-antelope)). Ungulates such as (including sheep, goats, etc.) or cows) or Eventoed ungulates (eg, horses, tapirs and sai), non-human primates (eg, monkeys, chimpanzees, etc.) It can be from non-human mammals, including ungulates, spider monkeys and ungulates, such as ungulates, canines (eg, dogs) or cats. Mammalian sperm can be from members of the Laurasiatheria. For Laurasiatheria, Waddell et al. , "Towards Reserving the International Relationships of Placental Mammals". A group of mammals as described in Systematic Biology 48 (1): 1-5 (1999) can be mentioned. Members of the Laurasiatheria include the Eulipotyphla (Harines, Togarinezumi and Mogra), the Perissodactyla (Pai, horse and tapir), the Carnivora (Carnivora). , Carnivora (Cateratiodactyla) (Eulipotyphla and whales), Carnivora (Carnivora) and Pangolin (Pangolin). Members of the Laurasiatheria can be ungulates, such as odd-toed ungulates or even-toed ungulates. Ungulates can be pigs. Mammalian sperm can be from members of the Carnivora, such as cats or dogs. In some embodiments, the mammalian sperm are human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm. In some embodiments, the mammalian sperm is a human sperm.

In some embodiments, the mammalian sperm are from healthy male mammals. In some embodiments, mammalian sperm are from males suffering from sperm dysfunction, such as low sperm count, decreased sperm motility and sperm morphological abnormalities. In some embodiments, the mammalian sperm can be from a low fertility male or an oligospermia male. Mammalian sperm can be, for example, from males suffering from sperm depletion, malformed sperm, asthenozoospermia or oligospermia / asthenozoospermia / malformed sperm. Oligospermia refers to a condition characterized by a sperm concentration of less than 20 million / ml. Asthenozoospermia refers to a condition characterized by decreased sperm motility. Azoospermia refers to a condition characterized by the presence of morphologically abnormal sperm. Oligospermia / asthenozoospermia / malformed spermia refers to conditions including oligospermia (low sperm count), asthenozoospermia (impaired sperm motility), and malformed spermia (abnormal sperm shape). In some embodiments, the sperm count is, for example, about 20 million, 19 million, 18 million, 18 million, 16 million, 15 million, 14 million, 13 million, 12 million, 11 million, 1000 per milliliter. Low fertilization human male or sperm with less than 10,000, 9 million, 8 million, 7 million, 6 million, 5 million, 4 million, 3 million, 2 million sperms, eg less than 15 million sperms per milliliter Obtained from a human male with oligospermia.

In some embodiments, sperm are concentrated (or isolated) from semen prior to energy depletion. Any sperm enrichment or isolation method can be used in accordance with the present invention, including density gradient centrifugation, swim-up, microfluidics or combinations thereof.

In the methods provided by the present invention, either fresh sperm or sperm from storage stock can be used. For example, in some embodiments, sperm are recovered from cold storage prior to treatment. In some embodiments, sperm are recovered from non-cold storage prior to treatment. In some embodiments, sperm are provided on storage medium prior to treatment. In some embodiments, sperm in storage medium are stored in cold conditions prior to incubation under energy depleted conditions. Under some conditions, sperm in storage medium are stored in non-cold conditions prior to incubation under energy depleted conditions.

In some embodiments, the storage medium comprises a weakly acidic pH and contains about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340. , 350, 360, 370 or 380, eg, a buffer solution having a weight osmolal concentration of about 350 mOsm / kg. In some embodiments, the weight osmolal concentration of the storage medium provided by the present invention is approximately 320-340 mOsm / kg. "Weakly acidic" pH means less than 7 and more than 5. In some embodiments, the weakly acidic pH is about 6-7, such as about 6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9. Or higher and lower than 7.0 (such as 6.99), such as about 6.5-6.99, for example about 6.7-6.9, for example about 6.8. In some embodiments, the storage medium has a pH of about 6.7-6.9, an amphoteric ion buffer, a concentration of glucose at about 0.25 to 0.35 M, antibiotics, about 340-360 mOsm. A buffer solution containing (or essentially becoming) BSA or HSA at a weight osmolal concentration of / kg and a concentration of about 2-4% (W / V), where the longest at 4 ° C. in this storage medium. Sperm stored for 12 days retains at least 60% of motile sperm compared to suitable control sperm upon transfer to fertility acquisition medium, and optionally the medium does not contain egg yolk.

In the method provided by the present invention, various amounts of sperm can be used, including a single divided amount of ejaculated liquid or the total amount of ejaculated liquid. In some embodiments, sperm are pooled from two or more doses of ejaculate (eg, 2, 3, 4, 5, 6 doses or more).

Conservation Medium In one aspect, the present invention provides a sperm storage medium. These storage media provided by the present invention advantageously produce progeny with the methods provided by the present invention (eg, inducing improved sperm function, enhancing fertility, improving adaptability). Etc.), and in some embodiments it can be carried out using the various kits provided by the present invention, thus in certain embodiments the sperm preparation solution provided by the present invention is prepared. It can be incorporated into use in additional methods provided by the present invention, such as methods to be performed and / or fertilization methods including reproductive assist methods. These storage media provided by the present invention can advantageously be incorporated into the kits provided by the present invention, which, in some embodiments, the various methods provided by the present invention. Thus, in certain embodiments, it is useful in practicing the method by which the sperm preparation solution provided by the present invention will be made and / or enhances fertility, which induces improved sperm function. , Further methods provided by the present invention, such as producing offspring with improved adaptability, may be useful in fertilization methods including reproductive support methods. In some embodiments, sperm are recovered from the storage medium prior to implementation of the methods disclosed herein. In some embodiments, recovery of sperm from storage medium includes, for example, concentration, washing or dilution of sperm samples using the kit or components thereof as disclosed herein. In some embodiments, the sperm or sperm preparation of the present disclosure is the method described herein, eg, after implementation of the method performed using the kits disclosed herein. It can be stored in a storage medium.

The storage medium provided by the present invention contains a weakly acidic pH and contains about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340. Includes a buffer solution having a weight osmolal concentration of 350, 360, 370 or 380, eg, about 350 mOsm / kg. In certain other embodiments, the weight osmolal concentration of the storage medium provided by the present invention is approximately 320-340 mOsm / kg. "Weakly acidic" pH means less than 7 and more than 5. In some embodiments, the weakly acidic pH is about 6-7, such as about 6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9. Or higher and lower than 7.0 (such as 6.99), such as about 6.5-6.99, for example about 6.7-6.9, for example about 6.8. These storage media, including the embodiments described below, are referred to as "one or more storage media provided by the present invention" or "one or more sperm storage media provided by the present invention". Will be done.

"Sperm preservation" refers to maintaining sperm viability and function over time, for example, when assessed by the ability to restore motility under known conditions that induce sperm fertility acquisition. .. The storage medium provided by the present invention and the method comprising storing sperm in the storage medium provided by the present invention advantageously preserve such sperm function, suppress sperm damage and degradation, or. One or more of these combinations may be made. In certain embodiments, Molina et al. Capacitation is induced and measured using techniques known in the art, such as those disclosed in 2018 (doi.org/10.389/fcell.2018.00072)-Fertilization briefly. Capacitation was performed with modified human oviduct (mHTF) medium (HEPES 21 mM, NaCl 97.8 mM, KCl 4.7 mM, KH ₂ PO ₄ 0.37 mM, supplemented with 5 mg / ml human serum albumin and 25 mM sodium bicarbonate. MgSO ₄ 0.2 mM, CaCl ₂ 2 mM, glucose 2.78 mm, 0.33 mM pyruvate, is carried out for 4 hours at 37 ° C. and 5% CO ₂ in lactate 21.4 mm). In some embodiments, sperm storage rapidly states the rest state (eg, within about 1, 2, 3, 4, 8, 12, 16, 20 or 24 hours after storage at 4 ° C., in some embodiments. In the embodiment, it is accompanied by induction within about 24 hours after storage at 4 ° C., during which time motile sperm are rapidly (eg, within about 1, 2, 3 or 4 hours) under the conditions for acquiring fertility (above). Or less, in some embodiments within about 30, 40, 50 or 60 minutes) ability to recover, eg 60, 65, 70, 75, 80, compared to the pre-storage motile sperm count, ie control. The ability to recover 85, 90, 95% or more, eg 96, 97, 98, 99% is retained. Thus, in some embodiments, provided by the present invention after rapid induction of a stationary state. The storage medium to be preserved was incubated in modified human oviduct (mHTF) medium supplemented with 5 mg / ml human serum albumin and 25 mM sodium bicarbonate at 37 ° C. and 5% CO ₂ , after sperm concentration and before quiescence. It provides a high percentage of motile sperm counts (eg, 60, 65, 70, 75, 80, 85, 90, 95% or more) compared to the control samples taken.

In certain embodiments, the storage medium provided by the present invention is egg yolk, added electrolytes (other than buffers, carbon sources, optionally serum albumin or optionally attributed to antibiotics), glycerol, lecithin or dextrose. Lacking one or more of (ie, 1, 2, 3, 4 or all 5) (completely or in significant amounts). In other embodiments, the storage medium may contain a cryoprotectant such as glycerol. In certain embodiments, the sperm storage media provided by the present invention include osmolytes (containing ionic components such as calcium), lipids, viscosity modifiers, sterols, antioxidants (such as trehalose), anti-inflammatory agents (eg, trehalose, etc.). , Doxycycline) and additional ingredients including the above combinations.

In some embodiments, the sperm storage medium provided by the present invention comprises a carbon source such as glucose, fructose, mannose, sucrose or a combination thereof (eg, 1, 2, 3 or all 4). In some embodiments, the carbon source comprises glucose. In certain embodiments, glucose is the major (> 50%) or substantially the only carbon source. In certain embodiments, in the storage medium provided by the present invention, glucose is about 0.1 to 0.7 M, for example about 0.2 to 0.5 M, for example about 0.25 to 0.36. 0.25 to 0.35, 0.30 to 0.36 or 0.3 to 0.33M or about 0.20, 0.25, 0.26, 0.27, 0.28, 0.29, 0 It exists at concentrations of .30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37M. In some embodiments, carbon sources such as glucose are the major sources (eg, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97) in the storage medium provided by the present invention. Osmolyte (> 50%, such as 98% or more), but the concentration of major osmolyte is such that the weight osmolyte concentration according to the present invention, eg, 300-400 mOsm / kg, is maintained. It can be easily adjusted.

The sperm storage provided by the present invention includes a buffer to facilitate the regulation of weakly acidic conditions. In some embodiments, the buffer is not a bicarbonate buffer. In certain embodiments, the buffer is an amphoteric ion such as 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS) or a combination thereof. It is a buffer. The concentration of one or more buffer components can vary, eg from about 1-100 mM, eg 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, 5-15 mM, eg about 2, 3, It can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, for example about 10 mM. In certain detailed embodiments, the buffer is 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS) or a combination thereof. .. In some embodiments, the buffer is HEPES. In another embodiment, it is MOPS. Still in other embodiments, the buffer is a mixture of HEPES and MOPS. In such embodiments, HEPES and MOPS are such as at a ratio of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 or 90:10, for example. It is used in a 50:50 mixture of HEPES and MOPS in which HEPES and MOPS are each present at 5 mM in 10 mM buffer.

The sperm storage medium provided by the present invention, in some embodiments, comprises an antibiotic. One exemplary class in which various antibiotics are suitable for use in the present invention is aminoglycosides such as gentamicin. In these detailed embodiments, gentamicin can be present in the storage medium provided by the present invention at a concentration of about 5-20 μg / ml, such as about 10 μg / ml.

An additional component of the preservation medium provided by the present invention is, in some embodiments, serum albumin. Although multiple serum albumin sources are useful according to the present invention, in some embodiments the serum albumin is bovine serum albumin (BSA), human serum albumin (HSA) or a combination thereof. In the presence of serum albumin, in certain embodiments, serum albumin (eg, BSA, HSA or combination) is about 1.5-4.5% (W / V), such as about 2-4%, about 2. It is present at a concentration of 5-3.5% or about 3%.

In certain embodiments, the sperm storage medium provided by the present invention is an amphoteric ion buffer (as described above) and glucose at a pH of about 6.6 to 6.9 and a concentration of about 0.25 to 0.36 M. And a weight osmolal concentration of about 320-380 mOsm / kg, where, in a detailed embodiment, the medium is egg yolk free. According to the teachings herein, these embodiments may further comprise antibiotics such as gentamicin. In some embodiments, the storage medium has a pH of about 6.8 (eg, using HEPES, MOPS or a combination thereof), a glucose concentration of about 0.330 mM, and a weight osmolal concentration of about 350 mOsm. / Kg, where serum albumin is BSA or HSA, optionally BSA or HSA is present at a concentration of about 2-4% (W / V).

As mentioned above, without being bound by theory, the sperm storage medium provided by the present invention advantageously maintains high levels of sperm function while minimizing sperm damage. In certain embodiments of the sperm storage medium provided by the present invention, it is stored in the storage medium at about 4 ° C. for up to 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer. Sperm were at least about 40, 45, 50, 55, 60, 65, 70 compared to suitable controls, such as the total number of motile sperm present in the sample after incubation under capacitation conditions and prior to storage and refrigeration. , 75, 80% or more motile sperm are maintained. For example, in certain embodiments, sperm stored in the storage medium provided by the present invention at about 4 ° C. for 7 days will be at least about 40 compared to suitable control sperm when transferred to the fertility acquisition medium. It has 45, 50, 55, 60, 65, 70, 75, 80% or more motile sperm. In some embodiments, sperm stored in the storage medium provided by the present invention at about 4 ° C. for 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer It has at least about 75% of motile spermatozoa compared to suitable control spermatozoa at the time of transfer to the fertility acquisition medium. In some embodiments, sperm stored in the storage medium provided by the present invention at about 4 ° C. for 7 days is 1%, 2.5%, compared to the pre-storage motile sperm rate in the storage medium. It has a lower motile sperm rate of 10%, 15% or 20% or more.

In addition to preserving sperm function, in certain embodiments, the quality of sperm stored in the storage medium provided by the present invention compared to control refrigerated or cryopreserved samples is such that after 7 days of incubation, eg TUNEL staining. It is clear from the decrease in lipid peroxidation and the decrease in reactive oxygen species, or a combination thereof. For example, in some embodiments, the sperm stored in the storage medium provided by the present invention is, for example, either the medium provided by the present invention or a cryopreservation medium (tyB containing gentamicin and 12% glycerol). After 7 days of storage and thawing / recovery in, at least: 30, 40, 50, 55, 60, 65, 70, 80, 85, 90, 95% or more reduction in TUNEL staining compared to cold stored cells. Present. In certain embodiments, TUNEL staining is performed by Simone et al. (Hum Report. 2014 May; 29 (5): 904-17. An additional assay by Gorgzyca et al. Int JOncol 1 (6): 639-48 (1992) may be used. In certain embodiments, the sperm stored in the storage medium provided by the present invention is, for example, 7 in either the medium provided by the present invention or the cryopreservation medium (TYB containing gentamicin and 12% glycerol). After days of storage and thawing / recovery, at least: 30, 40, 50, 55, 60, 65, 70, 80, 85, 90 compared to cold stored cells as measured by flow cytometry using BODICY C11. , 95% or more. In certain embodiments, lipid peroxidation is Naguib, Anal Biochem. 265 (2): 290-8 (1998) or Pap et al., FEBS Lett. .453 (3): Determined by the method of 278-82 (1999). In certain embodiments, the sperm stored in the storage medium provided by the present invention is, for example, the medium provided by the present invention. Or at least: 30, 40, 50, 55, 60, 65, 70, compared to cells stored cold after 7 days of storage and thawing / recovery in either cryopreservation medium (TYB containing gentamicin and 12% glycerol). It exhibits a reduction of 80, 85, 90, 95% or more of active oxygen species.

In certain embodiments, any medium provided by the present invention is provided as a sterile formulation. Such sterile product may be in a closed sterile container. In certain embodiments, the sterilized formulation is a liquid formulation. In other embodiments, the medium is lyophilized. As those skilled in the art will understand, the lyophilized preparation is substantially water-free, but the medium provided by the present invention, especially the pH and weight according to the present invention, when the preparation is reconstituted with sterile distilled water, for example. It is formulated to have an osmol concentration and other component concentrations.

Thus, in certain exemplary embodiments, the present invention has a pH of about 6.7 to 6.9, an amphoteric ion buffer, glucose at a concentration of about 0.25 to 0.35 M, about 340-360 mOsm. Provided is a sperm storage medium which is a sterile solution containing (or becoming essentially) BSA or HSA at a weight osmolal concentration of / kg, antibiotics, and a concentration of about 2-4% (W / V), wherein the sperm storage medium is provided. Sperm stored in this storage medium at 4 ° C. for up to 14 days maintain at least 60% of motile sperm at a suitable control, time 0, upon transfer to capacitation conditions. Although exemplified herein in detail, the components of such a detailed storage medium provided by the present invention are such that the storage medium provided by the present invention in a particular embodiment does not contain egg yolk. It can vary, as illustrated above, including optionally lacking (or possibly containing) the other components pointed out above.

A relevant aspect of the invention is a composition comprising the preservation medium provided by the invention, along with additional ingredients, including live sperm, in some embodiments. In some embodiments, sperm are concentrated (or isolated) from semen. Various sperm enrichment or isolation means are possible, including by centrifugation (such as density gradient centrifugation), swim-up, filtration, microfluidics or a combination thereof. In certain embodiments, the surviving sperm are mammalian sperm, such as bovine, sheep, horse, pig, rabbit, cat, dog or primate sperm. In certain embodiments, the sperm are from humans. In some embodiments, the human subject exhibits reduced sperm count, eg oligospermia, eg, sperm count of about 20 million, 19 million, 18 million, 18 million, 16 million, 15 million per milliliter. 14 million, 13 million, 12 million, 11 million, 10 million, 9 million, 8 million, 7 million, 6 million, 5 million, 4 million, 3 million, less than 2 million sperms, for example 15 million per milliliter Less than 5 sperms. In some embodiments, sperm are from two or more doses of ejaculate, eg, 2, 3, 4, 5, 6 doses or more.

Thus, according to the preservation medium provided by the present invention, in certain exemplary embodiments, the invention comprises a composition comprising a liquid sperm preservation medium with additional components, including viable sperm, in some embodiments. Provided, this liquid sperm storage medium has a pH of about 6.7 to 6.9, an amphoteric ion buffer, glucose at a concentration of about 0.25 to 0.35 M, and a weight of about 340-360 mOsm / kg. Essentially from osmolar, antibiotics, BSA or HSA at a concentration of about 2-4% (W / V), where stored at 4 ° C. for up to 12 days, upon transfer to fertility acquisition medium. At least 60% of spermatozoa are motile compared to suitable control spermatozoa. Although exemplified herein in detail, in some embodiments the components of the composition comprising a liquid sperm storage medium along with additional components comprising live sperm are for the storage medium provided by the present invention. It can vary as illustrated. Thus, in certain embodiments, the composition comprising the sperm storage medium may (or may) be free of egg yolk and optionally lack the other components pointed out for the storage medium listed above.

Given the storage medium provided herein and the composition comprising the storage medium, the present invention also provides a relevant aspect of the method of storing sperm. Such a method involves contacting sperm, including enriched or isolated sperm, with the storage medium provided by the present invention. As mentioned above, a variety of sperms can be used, including human sperms such as sperms from human males with oligospermia. Sperm can be pooled from multiple doses of ejaculate. In certain embodiments, the method of storing sperm is such that the composition containing the storage medium and sperm is stored at, for example, about 4 ° C. for a period of, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or Storage beyond that, meanwhile, in certain embodiments, at least 40, 45, 50, 55, 60, 65, 70, 75, 80% or more compared to a suitable control during capacitation. More than motile sperm are retained, accompanied by storage.

Preservation provided by the present invention, in some embodiments, prior to contacting the sperm with the egg, eg, in vitro, or by introducing the sperm into the reproductive organs of a female recipient. Sperm can be isolated from the medium. In some embodiments, after isolation of sperm from storage medium, sperm can be placed in fertility acquisition medium.

Methods for Obtaining Sperm Samples Various methods for collecting live sperm are known. Such methods include, for example, masturbation in a sterile container, a gloved hand method, the use of artificial vagina and electric ejaculation. Animal semen can be collected using artificial vagina, by electric ejaculators, or by hand-massaging the ampulla of the animal. Intratesticular sperm and epididymal head using various methodologies, such as puncturing the testis or epididymis using surgical techniques or removing the testis or epididymis to collect sperm in the surrounding medium. It can also be collected directly from any part of the male testis, including sperm obtained from the body or tail. Sperm are preferably harvested or rapidly transferred to an adiabatic vessel to avoid abrupt temperature changes from physiological temperatures (typically about 35 ° C to about 39 ° C). Ejaculate typically contains approximately 500-15 billion sperms per milliliter, depending on the species and detailed animal. This number can be low when obtained from low fertility males or males suffering from sperm dysfunction.

Sperm can be a sample freshly taken from a source animal (eg, a mammal) or a pre-thawed or cryopreserved sample. At the time of collection or subsequently, the collected sperm may be any of a number of different buffers compatible with the sperm, such as TCA, HEPES, PBS, or US Patent Application Publication No. 2005/0003472. The content may be combined with any of the other buffers disclosed herein) by reference herein. For example, bovine semen samples, typically containing about 500 to about 10 billion sperm cells per milliliter, are taken directly from the source mammal into a buffered vessel to form a sperm suspension. obtain. The sperm suspension may also contain various other additives to maintain sperm survival. Exemplary additives include protein sources, antibiotics, growth factors and compositions that regulate intracellular and / or extracellular oxidation / reduction reactions. Examples of each of these additives are, for example, US Patent Application Publication No. 20070092860A1 and 2005024805A1 (the contents of each of which are incorporated herein by reference). As demonstrated in the disclosure of, it is well known in the art. Alternatively, the semen sample can be collected in an empty container and then contacted with a buffer within minutes to hours of collection to form a sperm suspension. In some embodiments, sperm cells can be harvested directly into a container containing energy depleted medium (eg, HTF medium lacking glucose, pyruvate and lactic acid) for incubation under energy depletion. In some embodiments, sperm cells can be harvested in an empty container and subsequently incubated under energy depleted conditions.

In some embodiments, sperm harvesting comprises washing sperm cells prior to performing the methods disclosed herein. In general, washing involves centrifuging the semen or thawed sperm sample in a diluting wash medium, which allows the collection of sperm-rich pellets. After or instead of the sperm washing step, a special isolation procedure for motile sperm from the sample can be performed.

In some embodiments, sperm are isolated from semen prior to use in the methods disclosed herein. In some embodiments, sperm with improved function can be further concentrated from sperm prepared by the methods disclosed herein (eg, enrichment of sperm with improved motility). In general, sperm are either by swimming motile sperm and separating them from dead sperm, non-motile sperm and debris (sperm swim-up method), by centrifuging sperm in a density gradient, or by centrifuging sperm and debris. Is isolated or concentrated by passing sperm through a column to which the sperm are bound. Isolation (or concentration) of sperm from semen is performed by a method selected from washing and spinning methods, sedimentation methods, direct swim-up methods, pellet and swim-up methods and suspended density gradient methods. These methods are well known in the art. These have been conventionally used in assisted reproductive technology, and have been used in "A extbook of Invitro Fertilization and Assisted Reproduction, The Bourn Hall parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon Parthenon It is described in detail in. In some embodiments, sperm prepared by the methods disclosed herein are motile sperm by isolation procedures such as sedimentation, direct swim-up, pellet and swim-up and suspended density gradient methods. It can be further concentrated.

The direct swim-up method implies self-selection of motile sperm, essentially superimposing an aliquot of the medium on a semen sample or sperm preparation solution disclosed herein and keeping it constant at room temperature. Including leaving it to stand for a period of time. Motile sperm cells migrate to the upper layer (medium) and can be recovered from there. The method may also include one or more centrifugation steps. The advantages of sperm selected by the "sperm-up method" are that the motile cells present in the sample are isolated and concentrated and that the proportion of morphologically normal sperm is improved.

This method varies depending on the amount of motile cells in the sample and can be combined with further isolation / isolation techniques. For example, the swim-up procedure can be performed by overlaying a layer of 1 ml albumin-containing medium on top of 1 ml of underlying semen fluid in vitro. After incubation in air at 37 ° C. or in 5% CO2 for 1 hour, the medium of the upper phase in which sperm with better motility characteristics migrate is collected. This technique may include or be combined with a centrifugal step, such as a Percoll gradient centrifuge. In a typical application, a solution containing sperm is overlaid on a gradient material, preferably 30-90% Percoll mixed with 0.05% pectin, and then centrifuged to concentrate for improved function. Sperm is collected. Separated, isolated or concentrated sperm can then be cryopreserved until used in the methods disclosed herein or, for example, further processed. For sperm preparations prepared by the methods herein, it can be used for IVF, ICSI or artificial insemination after the concentration step, or can be cryopreserved, for example for later use. Thus, for any of these isolation or concentration methods, the sample is semen, partially purified sperm, purified sperm or sperm with enhanced function prepared by the methods herein. obtain. In some embodiments, the proportion of motile cells is untreated after isolation or concentration of sperm using isolation methods such as direct swim-up, pellet and swim-up and floating density gradient methods. At least 10%, at least 20%, at least 50%, at least 75%, at least 80% or about 100% improvement compared to the semen sample or unconcentrated sperm preparation.

In some embodiments, after isolation, concentration and washing, the sperm pellet contains storage medium, HTF medium for culture, medium for energy depletion conditions (eg, HTF lacking glucose, lactic acid and pyruvic acid). Including, it can be resuspended in a medium suitable for further treatment. When it comes to sperm with enhanced function prepared by the methods disclosed herein, the sperm preparation medium is a storage medium, HTF medium for culture, for insemination, fertilization as described herein. It can be resuspended in media for feasibility testing, in vitro fertilization, freezing, in utero insemination, cervical cap insemination, etc. Sperm can be added to the medium, or the medium can be added to the sperm. The medium can be an equilibrium salt solution that may contain an amphoteric ion buffer such as TES, HEPES, PIPES or another buffer such as sodium bicarbonate. Generally, the medium for diluting sperm or the medium for culturing sperm, oocytes, embryos or embryonic stem cells is M199, synthetic oviduct fluid, PBS, BO, Test-egg yolk, Tyrode fluid, HBSS, Ham F10, Equilibrium salt solutions such as HTF, B2 of Menezo, B3 of Menezo, F12 of Ham, DMEM, TALP, Earl buffer salt, CZB, KSOM, BWW medium and embryo medium (PETS, Canton, Tex.). In some embodiments, TALP or HTF is used as the sperm culture medium and CZB is used as the embryo culture medium. The sperms or embryos of the present disclosure can be stored in a low temperature medium containing a cryoprotectant.

In some embodiments, sperm are donated or harvested to storage medium prior to incubation in energy depleted conditions. In some embodiments, the storage medium comprises a weakly acidic pH and contains about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340. , 350, 360, 370 or 380, eg, a buffer solution having a weight osmolal concentration of about 350 mOsm / kg. In some embodiments, the weight osmolal concentration of the storage medium provided by the present invention is approximately 320-340 mOsm / kg. "Weakly acidic" pH means less than 7 and more than 5. In some embodiments, the weakly acidic pH is about 6-7, such as about 6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9. Or higher and lower than 7.0 (such as 6.99), such as about 6.5-6.99, for example about 6.7-6.9, for example about 6.8. In some embodiments, the storage medium has a pH of about 6.7-6.9, an amphoteric ion buffer, a concentration of glucose at about 0.25 to 0.35 M, antibiotics, about 340-360 mOsm. A buffer solution containing (or essentially becoming) BSA or HSA at a weight osmolal concentration of / kg and a concentration of about 2-4% (W / V), where the longest at 4 ° C. in this storage medium. Sperm stored for 12 days retains at least 60% of motile sperm compared to suitable control sperm upon transfer to fertility acquisition medium, and optionally the medium does not contain egg yolk. In some embodiments, sperm are placed in storage medium for about 2 hours, about 3 hours, about 4 hours, about 24 hours, about 10 days, about 1 month, about 6 months prior to incubation in energy depleted conditions. , Stored for about 10 months, for about 1 year or longer. In some embodiments, sperm in storage medium are washed, centrifuged and pelleted and resuspended in energy depleted medium. In some embodiments, sperm with enhanced function prepared by the methods disclosed herein are further stored in the storage medium described above.

Suitable Control Sperm A suitable control sperm is a sperm incubated under control conditions, ie, in a control buffer such as human oviduct (“HTF”) medium or modified HTF medium, under non-energy depletion conditions. obtain. HTF contains a sodium bicarbonate buffer system and can be used in applications that require a carbon dioxide atmosphere during incubation. The modified HTF comprises a combination buffer of sodium bicarbonate and HEPES ([4-2 (2-hydroxyethyl) -1-piperazine ethanesulfonic acid]). Preferable examples of HTF medium or modified HTF medium include those commercially available from Irvine Scientific, Santa Ana, California. In some embodiments, incubating under energy depleted conditions can be incubating HTF medium depleted of glucose, lactic acid and pyruvic acid. Sperm can be incubated for a period sufficient to bring about measurable changes in sperm motility (or other properties), and in a specific embodiment of this 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 partial range or value thereof. In some embodiments, suitable control sperm are incubated under energy depletion conditions, followed by a first energy source (eg, selected from gluconeogenic or glycolytic substrates) or a second energy source (eg, selected from gluconeogenic or glycolytic substrates). For example, sperm selected from gluconeogenic or glycolytic substrates, but not the same as the first energy source). In some embodiments, a suitable control sperm is a sperm that is incubated under energy depletion conditions and subsequently processed independently by a gluconeogenic or glycolytic substrate. In some embodiments, a preferred control is sperm that are incubated under energy depletion conditions and subsequently processed simultaneously by a first and second energy source. In some embodiments, a suitable control sperm is a sperm that is incubated under energy depletion conditions and subsequently processed simultaneously with a gluconeogenic substrate and a glycolytic substrate. In some embodiments, a suitable control sperm is untreated sperm. 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. A suitable control can be sperm incubated under control conditions, i.e. in control buffer. Control conditions may include, for example, incubating sperm under standard capacitation conditions. "Standard Capacitation Conditions" as used herein refers to incubating sperm in standard capacitation medium.

Sperm Preparation Solution In some embodiments, the present invention is superactive, in which activated sperm (eg, after starvation, followed by introduction of effective amounts of both first and second energy sources). Preparation of sperm (sperm enriched with chemical and intermediate sperm), partially activated sperm (sperm placed in starvation and contacted only with an effective amount of the first energy source) or enriched sperm Etc., provide a sperm preparation solution. Collectively, these are "sperm preparations provided by the present invention" or "preparations provided by the present invention". In some embodiments, the invention presents at least 5% of superactivated sperm, eg 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, for example A super-activated sperm preparation solution containing about 5-20, 8.5-20, 10-20 or 12.5-20% super-activated sperm is provided. In some embodiments, the preparation is at least about 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28 or 30%, for example about 20.5 to about 30%, about 22.5 to about 30% intermediate exercise It also contains Noh sperm. Therefore, in some embodiments, the total proportion of superactivated sperm and intermediate motile sperm is at least 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, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25, 26, 27, 28, 29, 30, 35 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% or more, such as about 10-50, 30.5-50, 32.5-50. As will be appreciated by those skilled in the art, sperm can be separated based on a superactivated (and / or intermediate) phenotype, but in some embodiments the aforementioned proportions are unactivated preparations. Sorted on the basis of fluid and then on the basis of superactivation (however, in some embodiments, the sperm preparation fluid draws sperm from other sperm components, including, for example, some irregular sperm. Can be pretreated for separation or other concentration). In some embodiments, super-activated (or intermediate or super-activated and intermediate-motivated) sperm in the preparations have intracellular RNA concentrations (small non-coding RNAs, including microRNAs) as compared to suitable controls. (Code RNA, etc.) is 10, 15, 20, 25, 30, 35, 40, 45, 50% or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more). Beyond that) it is declining. In some embodiments, sperm in the preparation provided by the present invention will have changes in sperm head morphology (eg, as assessed by either bulk mean metric or percentage within a category), tail motility (eg,). It is characterized by an increase in amplitude) or a combination thereof.

In some embodiments, the present disclosure provides sperm preparations containing epigenetic profiles that differ from suitable control sperms.

An "epigenetic profile" is defined as a DNA, RNA or protein modification (eg, methylation or acetylation) that does not involve a change in nucleotide sequence. Non-limiting examples of modifications include methylation and / or acetylation, and / or binding of non-coding RNAs, and / or histone modifications. Non-coding RNA (eg, miRNA, piRNA, snoRNA, endosiRNA) binding involves gene signaling of spliced intron or exon RNA and generation of single- or double-stranded RNA entities, including RNAi-like entities. .. An "epigenetic profile" can include protein modifications such as protein methylation, protein acetylation, and can include histone modifications such as acetylation, methylation and other changes.

In some embodiments, sperm treated by the methods disclosed herein exhibit a different or altered epigenetic profile as compared to a suitable control (eg, a suitable control sperm). As used herein, the term "different epigenetic profile" refers to a change in the pattern of one or more modifications to DNA, RNA and / or protein. In some embodiments, pattern changes include the presence of modifications (eg, methylation or acetylation) at specific sites on sperm DNA, RNA or protein compared to those of suitable control sperm. .. In some embodiments, pattern changes include the absence of modification (eg, methylation or acetylation) at specific sites on sperm DNA, RNA or protein as compared to those of suitable control sperm. .. In some embodiments, pattern changes include altered levels of one or more modifications. In some embodiments, pattern changes include an increase in the level of one or more modifications. In some embodiments, pattern changes include a decrease in the level of one or more modifications. For example, altered levels of one or more modifications may include an increase or decrease in methylation levels, acetylation levels, and the like. In some embodiments, different epigenetic profiles include altered DNA methylation and / or DNA acetylation levels. In some embodiments, different epigenetic profiles include the presence and / or absence of methylation at specific DNA sites. In some embodiments, different epigenetic profiles include the presence and / or absence of acetylation at a particular DNA site. Methods for measuring epigenetic changes are well known in the art. For example, Stephens K. et al. E. et al. , Biol Res Nurs. 2013 Oct; 15 (4): 373-381 and DeAngelis T. et al. J. Mol Biotechnol. See 2008 Feb; 38 (2): 179-183, which are incorporated herein by reference in their entirety.

In some embodiments, the epigenetic profile that differs from the control sperm in the sperm sample is associated with physiological conditions, traits, phenotypes or conditions. For example, different epigenetic profiles may include the absence of a condition such as obesity (or obesity-related disorders such as cancer or diabetes), or the presence of desirable traits such as increased milk production, or decreased fertility. It may be related to the absence of unwanted traits. Thus, sperm processed by the methods disclosed herein, whether male or female, may be useful in the production of offspring with improved fitness compared to their parents.

In some embodiments, the present invention provides a sperm preparation solution prepared by any one of the methods provided by the present invention.

In some embodiments, the present invention produces sperm from the semen of a male subject, such as a male with normal sperm, a male with low fertility or a male with hyposperm, such as a male with low fertility (including sperm hypoplasia). Concentration, incubating sperm under energy depletion for a time suitable for sperm augmentation and selecting from an effective amount of glycolytic energy source or an effective amount of glycosogenic substrate, provided with an effective amount. Provided is a sperm preparation solution prepared by providing sperm with a first energy source, which is neither a glycolytic energy source nor a glycanogenic substrate.

For any of the preparations provided by the present invention, sperm can be from any male subject, such as mammals, and in some embodiments humans. In some embodiments, the human is a male with normal sperm, or in other embodiments, the male is subject to oligospermia or low fertility (eg, low sperm motility).

In some embodiments, the sperm preparations described herein can be stored in the sperm storage medium provided by the present invention. In some embodiments, the sperm preparations provided herein are various methods provided by the present invention (eg, enhance sperm function, enhance fertilization, etc.) and in some embodiments. In embodiments, it can be carried out using the various kits provided by the present invention, and thus in certain embodiments, by the method by which the sperm preparation solution provided by the present invention will be made. / Or can be prepared by a further method provided by the present invention, such as a fertilization method including a reproductive assist method.

Fertilization Enhancement A sperm preparation with enhanced function prepared by the methods disclosed herein may be useful in enhancing fertilization. Therefore, the present disclosure also relates to methods of promoting fertilization. The method enhances one or more sperm function by incubating sperm under energy depletion conditions to enhance sperm and sequentially providing the enhanced sperm with a first and second energy source. It also includes providing sperm with improved function a means of reaching the egg under conditions that promote fertilization. Sperm preparations with improved function include IVF, ICSI, artificial insemination (eg, intracytoplasmic sperm injection) in humans, and the biomedical research industry of animal models of human diseases (infertility, sperm dysfunction), as well as breeding and breeding. It can be applied in the agricultural industry. Can sperm with improved function prepared by the methods disclosed herein enhance in vitro fertilization, ICSI by being provided with a means of reaching unfertilized eggs of the same species as sperm? Or, for example, it can be used for artificial insemination including intracytoplasmic sperm injection of female subjects of the same species as sperm.

Internal fertilization A sperm with improved function, prepared by the methods disclosed herein, provides a means of reaching a sperm with improved function, eg, an egg in the reproductive tract of a female subject of the same species as the sperm. By providing, it may be useful to promote fertilization in the body. Internal fertilization can be performed by artificial insemination of sperm, such as endometrial or intrauterine insemination. Standard artificial insemination and endometrial insemination and other methods are well known to those of skill in the art. In some embodiments, fertilization of the egg is enhanced by providing the sperm with improved function with a means of reaching the egg in the reproductive tract of the female subject by endometrial insemination of the sperm. In other embodiments, intrauterine insemination of mammalian spermatozoa incubated under energy depleted conditions and provided with a first energy source in vitro provides a means of reaching the egg and a second energy source in the body. Can be provided. The sperm to be injected can be used while being retained in a suitable liquid. The liquid used for this purpose can usually be the liquid used as a medium for artificial insemination.

In Vitro Fertilization The methods and sperm preparations disclosed herein are useful for assisted reproductive technology fertilization, such as enhancing embryonic viability after ART, specifically IVF. Other suitable ART techniques to which this disclosure is applicable include, but are not limited to, zygote intrafallus transplantation (GIFT), zygote intrafallus transplantation (ZIFT), blastocyst transplantation (BT), intracytoplasmic sperm sperm. Other forms of embryos, including injection methods (ICSI), mating, embryo and cell cryopreservation, in vitro preparation of embryos for embryo biopsy and embryo formation by nuclear transplantation and production of transgenic and genetically modified strains. Microoperation can be mentioned. This is also applicable to the production of embryonic stem cell lines.

In some embodiments, sperm with enhanced function prepared by the methods disclosed herein are known to those skilled in the art and microinjection methods, including, for example, intracytoplasmic sperm injection (ICSI). It can be used for in vitro fertilization of eggs, such as by other methods. Typically, in IVF, cells are grown to the blastocyst stage after fertilization and then transplanted. The methods disclosed herein improve embryo formation with longer survival and improved ability to develop during the two-cell and blastocyst stages. Therefore, the sperm preparations disclosed herein may be useful in in vitro fertilization procedures, including, for example, ICSI.

The methods of the present disclosure include providing a means of reaching an egg to sperm prepared by the methods herein to promote in vitro fertilization. Providing sperm with a means of reaching the egg in vitro can be done in a suitable medium. The medium used for this purpose can be a medium normally used as a medium for in vitro fertilization, such as HTF medium. The temperature condition for providing the means of arrival can be the general temperature used for in vitro fertilization, eg, the average body temperature of mammals or a temperature close to it. The time to provide the means of arrival can generally be any time required for in vitro fertilization, but is not particularly limited and is preferably 6 to 24 hours. In vitro fertilization rate can be determined by incubating one or more sperms with mature oocytes for about 24 hours. The oocytes are then stained with a 1% acetoorcein stain to determine fertilization rate, or split by placing in culture to count the number of embryos formed. Oocytes can be matured in vitro in M199 medium containing 50 μg luteinizing hormone / ml (Blackett and Zuelke, Theriogenology 39: 43, 1993).

Fertilization Uses These methods and sperm preparations disclosed herein are generally applicable to many species, including humans, cattle, dogs, horses, pigs, sheep, birds, rodents and the like. Although this method is always useful when fertilization is desired, it has special uses for improving fertility in animals and humans presenting with fertilization dysfunction. Such dysfunctions include low sperm count, decreased sperm motility and sperm morphological abnormalities. Therefore, the methods disclosed herein may be useful in infertility clinics to prepare sperm with improved function prior to their use in in vitro fertilization or endometrial insemination. The methods described herein can be used for artificial insemination, IVF or ICSI improvement in exotic and / or endangered species. Thus, this method is used in conservation programs to promote fertilization of captive animals in zoos and to improve the reproduction of endangered animals in the world. Can be found. For example, the methods and sperm preparations of the present disclosure can be used to improve fertility and pregnancy rates for seeds of agricultural value in the livestock industry and in seeds bred for protection.

In addition, the methods and compositions of the present invention are useful, for example, in artificial insemination procedures in commercial breeding. The method can be performed with sperm from domesticated animals, especially livestock and sperm from wild animals (eg, endangered species). For example, as disclosed herein, embodiments of the methods and compositions disclosed herein are found to be applicable to cattle breeding. The method and preparations include offspring with one or more favorable traits or traits by introducing a particular genetically determined trait into livestock, such as offspring of a particular gender, offspring with increased milk production. It can be useful for artificial insemination in the livestock industry, where it is desirable to influence performance towards offspring for high quality meat production. By using the methods described herein, the pregnancy rate will be improved. Mammalian sperm are frequently damaged by freezing and thawing, resulting in reduced fertility. Sperm prepared by the methods disclosed herein can enhance the increase in pregnancy rate per sexual cycle when used for insemination by improving the performance of surviving sperm, thus ensuring conception. The number of cycles required for pregnancy can be reduced, thus reducing the overall cost of artificial insemination.

Semen from animals with highly desirable traits can be used to fertilize more females, because fewer cycles are required to ensure conception in any one female. Because. For such applications, semen is obtained from males with the desired properties. To influence the sex performance of the resulting offspring, sperm preparations are sorted into X and Y chromosome-bearing cells and / or sperm with one or more enhanced sperm function disclosed herein. Can be concentrated with respect to. For sperm, for example, Johnson et al. A flow cytometer / cell sorter can be used to sort into X-chromosome and Y-chromosome-supported sperm-enriched populations by commonly used methods as described in US Pat. No. 5,135,759. Sperm prepared by the methods disclosed herein can be sorted into a population that includes a constant X-chromosome-supported or Y-chromosome-supported sperm cell rate. For example, sperm in one of these populations are at least about 65% X-chromosome-bearing or Y-chromosome-bearing sperm cells, at least about 70% X-chromosome-bearing or Y-chromosome-bearing sperm cells, and at least about 75% X-chromosome-bearing. Or Y chromosome-bearing sperm cells, at least about 80% X-chromosome-bearing or Y-chromosome-bearing sperm cells, at least about 85% X-chromosome-bearing or Y-chromosome-bearing sperm cells, at least about 90% X-chromosome-bearing or Y-chromosome-bearing sperm It may contain cells or at least about 95% X-chromosome-bearing or Y-chromosome-bearing sperm cells. In some embodiments, sorting can be performed prior to preparation of sperm with enhanced function as disclosed herein. In some embodiments, sorting can be performed prior to providing sperm with improved function with a means of reaching the egg for fertilization, as in the case of IVF, ICSI or AI.

The methods and preparations provided by the present invention can be used in co-fertilization such as IVF, including those by ICSI (intracytoplasmic sperm injection). In some embodiments, any of the methods provided by the present invention can include the step of donating sperm to the female reproductive tract, optionally the female reproductive tract being an effective amount of a second source of energy. Will be provided. In some embodiments, the sperm preparation solution provided by the present invention (having improved sperm function) can provide a means of reaching the egg for a time sufficient to fertilize the egg. The egg can be in vitro (eg, IVF with ICSI) or, in some embodiments, in the female reproductive tract. Such a method, in some embodiments, involves implantation of a fertilized egg in a subsequent female egg carrier.

In some embodiments, the invention provides an effective amount of gluconeogenesis over a period of time sufficient to fertilize an egg in the preparation provided by the invention that is not in contact with an effective amount of a second energy source. Fertilization methods are provided that include providing an effective amount of a second energy source and a means of reaching the egg, such as providing both a substrate and a gluconeogenic energy source. In some embodiments, the method is performed in vitro. In other embodiments, the method is performed in the body in the female reproductive tract (vaginal or uterus).

In some embodiments, the present invention provides a fertilization method comprising providing a preparation of sperm having a different epigenetic profile. In some embodiments, the epigenetic profile that differs from the control sperm in the sperm sample is associated with physiological conditions, traits, phenotypes or conditions. For example, different epigenetic profiles may include the absence of conditions such as obesity (or obesity-related disorders such as cancer or diabetes), or the presence of desirable traits such as increased milk production, or decreased fertility. It may be related to the absence of unwanted traits. Therefore, sperm treated by the methods disclosed herein may be useful in the production of offspring with improved fitness compared to male parental subjects. In one aspect, the present specification is a method of producing offspring with improved adaptability as compared to a male parent subject, wherein the sperm sample from the male parent is processed by the method disclosed herein. , And methods are provided that include fertilizing the treated sperm into an egg to develop an embryo, and growing the embryo in a female subject to produce offspring with improved adaptability. The term "offspring with improved fitness" refers to offspring that exhibit the desired changes or improvements in physiological conditions, traits, phenotypes or conditions compared to the parent subject. For example, desirable changes may include the absence of conditions such as obesity (or obesity-related disorders such as cancer, cardiovascular disease, infertility). For example, improvement may include the presence of desirable traits such as increased milk production.

Products In some embodiments, the invention also provides products and kits suitable for, for example, performing any of the methods provided by the present invention or preparing any of the preparations provided by the present invention. For example, in some embodiments, the invention is selected from a sperm-enhancing solution that induces energy depletion upon contact with sperm, an effective amount of glycolytic energy source, or an effective amount of gluconeogenic substrate, provided that it is effective. Provided are products containing a first solution that provides a first energy source and a second solution that provides an effective amount of a second energy source, which is neither an amount of glycolytic energy source nor a gluconeogenic substrate. .. In some embodiments, the product further comprises means for isolating or concentrating sperm, such as a sperm isolation matrix, in some embodiments. In some embodiments, the sperm isolation matrix is silanized silica, optionally in a medium that is substantially free of any glycolytic energy source or gluconeogenic substrate. In some embodiments, the kit includes instructions for performing the methods disclosed herein. The kit may also include wash medium, storage medium, culture medium (eg, HTF), diluent and the like. The kit may further include adjuvants, reagents and buffers as needed.

In certain embodiments, all components and reagents of the kits disclosed herein meet at least the United States Pharmacopeia (USP) monograph grade purity for that component. A USP monograph may not be available for some ingredients, so in certain embodiments a suitable pharmaceutical grade reference standard purity for that ingredient is used. In some embodiments, the purity of the components of the kit is about 95% pure. Ingredients and reagents have a purity of at least about 95% in more detail, at least about 98% in more detail, and at least about 99% in more detail. In some embodiments, the components and reagents of the kit are substantially sterile, substantially pyrogen-free, or substantially sterile and substantially pyrogen-free.

In some embodiments, the components contained in the kit's reagents are substantially pure. As used herein, substantially pure only appears to be free of impurities that are readily detectable when determined by any suitable method, such as column chromatography, gel electrophoresis or HPLC. Means that there is sufficient uniformity in. The term "substantially pure" means a formulation in which at least 60% by weight (dry weight) is the component of interest (eg, glucose or pyruvic acid). In a detailed embodiment, the formulation comprises at least 75% by weight, more specifically at least 90% by weight, and even more specifically at least 99% by weight. When a formulation comprises two or more target ingredients, a "substantially pure" formulation means a formulation in which the total weight (dry weight) of all target ingredients is at least 60% of the total dry weight. do. Similarly, for such formulations containing two or more objectives, the total weight of the two or more objectives is at least 75%, more specifically at least 90%, and even more detailed of the total dry weight of the formulation. Is at least 99%. In some embodiments, the disclosure also provides products and kits suitable for, for example, practicing any of the methods provided by the present invention or preparing any of the preparations provided by the present invention.

For example, in some embodiments, the disclosure provides a kit comprising a first container comprising a sperm-enhancing energy depletion composition that induces energy depletion upon contact with sperm to produce enhanced mammalian sperm. do.

In some embodiments, the sperm-enhancing energy depletion composition is, for example, about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07. , 0.06, 0.05, 0.04, less than 0.03 mM glucose or lower, eg, less than about 0.02 or 0.01 mM, eg less than about 0.01 mM low glucose concentration. In some embodiments, the sperm-enhancing energy depletion composition is substantially glucose-free. In some embodiments, the energy depletion composition for sperm enhancement is, for example, about 0.15, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04. , 0.03, 0.02, 0.01, 0.005, 0.003, 0.002 mM or less containing low pyruvate concentrations. In some embodiments, the sperm-enhancing energy depletion composition is substantially pyruvate-free.

In some embodiments, the sperm-enhancing energy depletion composition is substantially free of carbon sources, such as low glucose and low pyruvate concentrations, eg, substantially glucose-free and substantially pyruvate-free. Is. In some embodiments, the sperm-enhancing energy depletion composition comprises a buffer. In some embodiments, the buffer is HEPES, MOPS or a combination thereof. In some embodiments, the sperm-enhancing energy depletion composition comprises HEPES at a concentration of about 1 mM-50 mM, 2-40 mM, 3-30 mM, 5-20 mM, 7-15 mM or 7.5-12.5 mM. .. In some embodiments, HEPES has a concentration of about 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45 or 50 mM. .. In some embodiments, HEPES is at a concentration of at least about 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45 or 50 mM. be. In some embodiments, HEPES has a concentration of 10 mM.

In some embodiments, the sperm-enhancing energy depletion composition further comprises serum albumin, such as human serum albumin, fetal bovine serum or bovine serum albumin. In some embodiments, the sperm-enhancing energy depletion composition comprises, for example, human serum albumin (HSA) at concentrations of about 1-10 mg / ml, 2-8 mg / ml or 3-7 mg / ml. In some embodiments, the HSA is at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / ml. In some embodiments, the HSA is at a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / ml. In some embodiments, the HSA is at a concentration of 4 mg / ml. In some embodiments, serum albumin, such as human serum albumin, is provided in a separate solution, such as a concentrated stock solution, and is diluted to one or more of the compositions in the kit provided by the present invention.

In some embodiments, the sperm-enhancing energy depletion composition further comprises an antibiotic. In some embodiments, the antibiotic is about 1-20 μg / ml, 2-18 μg / ml, 4-16 μg / ml, 6-14 μg / ml or 8-12 μg / ml in the sperm-enhancing energy depletion composition. Present at the concentration of. In some embodiments, the antibiotic is at a concentration of about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 μg / ml. In some embodiments, the antibiotic is at a concentration of at least about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 μg / ml. In some embodiments, the antibiotic is at a concentration of 10 μg / ml. In some embodiments, the antibiotic is gentamicin or penicillin.

In some embodiments, the sperm for enhancing energy depletion compositions further comprise one or more salts, for example _{NaCl, KCl, CaCl 2, KH} 2 PO 4, MgSO 4 · 7H 2 O, and NaHCO _3.

In some embodiments, NaCl is present at concentrations of about 50-150 mM, 60-140 mM, 70-130 mM, 80-120 mM or 90-100 mM. In some embodiments, NaCl is about 50, 60, 70, 80, 90, 95, 96, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6. , 97.8, 97.9, 98, 98.5, 99, 99.5, 100, 110, 120, 130, 140 or 150 mM. In some embodiments, NaCl is at least about 50, 60, 70, 80, 90, 95, 96, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97. It exists at concentrations of 6, 97.8, 97.9, 98, 98.5, 99, 99.5, 100, 110, 120, 130, 140 or 150 mM. In some embodiments, NaCl is present at a concentration of 97.8 mM.

In some embodiments, KCl is about 1-10 mM, 1.5-9.5 mM, 2-9 mM, 2.5-8.5 mM, 3-8 mM, 3.5-7.5 mM, 4-7 mM. , 4.5-6.5 mM, 4.5-6 mM or 4.5-5 mM. In some embodiments, KCl is about 1, 2, 3, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. It is present at a concentration of 4.9, 5, 5.5, 6, 7, 8, 9 or 10 mM. In some embodiments, KCl is at least about 1, 2, 3, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4. It is present at a concentration of 8, 4.9, 5, 5.5, 6, 7, 8, 9 or 10 mM. In some embodiments, KCl is present at a concentration of 4.7 mM.

In some embodiments, CaCl ₂ is about 1-5 mM, 1.1-4.5 mM, 1.2-4 mM, 1.3-3.5 mM, 1.4-3 mM or 1.5-2. It is present at a concentration of 5 mM. In some embodiments, CaCl ₂ is about 1.0, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, It is present at a concentration of 3, 3.5, 4, 4.5 or 5 mM. In some embodiments, CaCl ₂ is at least about 1.0, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8. It is present at a concentration of 3, 3.5, 4, 4.5 or 5. In some embodiments, CaCl ₂ is present at a concentration of 2 mM.

In some embodiments, KH ₂ PO ₄ is about 0.1-0.6 mM, 0.15-0.55 mM, 0.2-0.5 mM, 0.25-0.45 mM or 0.3-. It is present at a concentration of 0.4 mM. In some embodiments, KH ₂ PO ₄ is about 0.1, 0.15, 0.2, 0.22, 0.25, 0.26, 0.3, 0.32, 0.34, It is present at concentrations of 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.45, 0.5 or 0.6 mM. In some embodiments, KH ₂ PO ₄ is at least about 0.1, 0.15, 0.2, 0.22, 0.25, 0.26, 0.3, 0.32, 0.34. , 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.45, 0.5 or 0.6 mM. In some embodiments, KH ₂ PO ₄ is present at a concentration of 0.37 mM.

In some embodiments, NaHCO ₃ is present at concentrations of about 10-50 mM, 12-45 mM or 15-30 mM. _{In some embodiments, NaHCO 3} is present at a concentration of about 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45 or 50 mM. _{In some embodiments, NaHCO 3} is present at a concentration of at least about 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45 or 50 mM. In some embodiments, NaHCO ₃ is present at a concentration of 20 mM.

In some embodiments, sulfonyl ₄ . 7H ₂ O is present at concentrations of about 0.1 to 0.5 mM, 0.12 to 0.45 mM, 0.14 to 0.4 mM, 0.16 to 0.35 or 0.18 to 0.3 mM. .. In some embodiments, sulfonyl ₄ . 7H ₂ O is about 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, It is present at concentrations of 0.35, 0.4, 0.45 or 0.5 mM. In some embodiments, sulfonyl ₄ . 7H ₂ O is at least about 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3 , 0.35, 0.4, 0.45 or 0.5 mM. In some embodiments, sulfonyl ₄ . 7H ₂ O is present at a concentration of 0.2 mM.

In some embodiments, the energy depletion composition for sperm enhancement is, for example, from about 0.0001 to 0.001%, 0.0002 to 0.009%, 0.0003 to 0.0008%, 0.0004 to. It contains a pH indicator having a concentration of 0.0007% or 0.0005 to 0.00065%, such as phenol red. In some embodiments, phenol red is about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008. It is present at a concentration of%, 0.0009% or 0.001%. In some embodiments, phenol red is at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0. It is present in concentrations of 0008%, 0.0009% or 0.001%. In some embodiments, phenol red is present at a concentration of 0.0006%.

In some embodiments, the sperm-enhancing energy depletion composition comprises a weakly acidic pH and comprises from about 200 to 280 mOsm (mOsm / kg), such as from about 220 to 260, 225 to 255, 230 to 250 mOsm (mOsm / kg). ), And optionally the addition of a first or second energy source, the volume osmolality (or weight osmolality) is at least about 270, 275, 280, 285, 290. Or it increases to 295 mOsm (mOsm / kg). "Weakly acidic" pH means less than 7 and more than 5. In some embodiments, the weakly acidic pH is about 6-7, such as about 6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9. Or higher and lower than 7.0 (such as 6.99), such as about 6.5-6.99, for example about 6.7-6.9, for example about 6.8.

In some embodiments, the sperm-enhancing energy depletion composition is a nutrient-free synthetic human oviductal fluid. In some embodiments, the nutrient-free synthetic human oviduct fluid is, for example, a concentration of 97.8 mM NaCl, for example 4.7 mM KCl, for example 2 mM CaCl ₂ , for example 0.37 mM. KH ₂ PO ₄ , eg, silicate at a concentration of 0.2 mM ₄ . 7H ₂ O, including for example, 4 mg / ml concentration of HSA, for example, 10 [mu] g / ml of gentamicin concentration, for example, phenol red HEPES and for example 0.0006% concentration of 10mM concentration. In some embodiments, the nutrient-free synthetic human oviduct fluid is, for example, a concentration of 97.8 mM NaCl, for example 4.7 mM KCl, for example 2 mM CaCl ₂ , for example 0.37 mM. KH ₂ PO ₄ , eg, silicate at a concentration of 0.2 mM ₄ . 7H ₂ O, consists essentially of example 4 mg / ml concentration of HSA, for example, 10 [mu] g / ml of gentamicin concentration, for example from phenol red HEPES and for example 0.0006% concentration of 10mM concentration. In some embodiments, the nutrient-free synthetic human oviduct fluid is, for example, a concentration of 97.8 mM NaCl, for example 4.7 mM KCl, for example 2 mM CaCl ₂ , for example 0.37 mM. KH ₂ PO ₄ , eg, silicate at a concentration of 0.2 mM ₄ . 7H ₂ O, for example 4 mg / ml concentration of HSA, for example, 10 [mu] g / ml of gentamicin concentration of, for example, phenol red HEPES and for example 0.0006% concentration of 10mM concentration. In some embodiments, the nutrient-free synthetic human oviduct fluid is substantially free of carbon sources, such as low glucose concentration, low lactate concentration and low pyruvate concentration, eg, substantially glucose free, substantially free of glucose. It is lactic acid-free and substantially pyruvic acid-free.

In some embodiments, the kit is selected from a glycolytic energy source or a gluconeogenic substrate, but comprises a second energy source that is not both a glycolytic energy source and a gluconeogenic substrate. It further comprises a second container containing the composition of. In some embodiments, the kit further comprises a third container comprising a third composition comprising a second energy source selected from glycolytic energy sources or gluconeogenic substrates, the first selected. The second energy source is one that is not selected as the first energy source.

In some embodiments, the glycolytic energy source is glucose. In some embodiments, the gluconeogenic substrate is pyruvic acid. In some embodiments, the kit comprises only a first solution containing a first energy source. In some embodiments, the first energy source is a gluconeogenic substrate (eg, pyruvic acid). In some embodiments, the first energy source is a glycolytic energy source (eg, glucose). In some embodiments, the kit comprises both a first solution and a second solution, wherein, for example, the first energy source is glucose and the second energy source is pyruvic acid. Is. In some embodiments, the kit comprises both a first solution and a second solution, wherein, for example, the first energy source is pyruvic acid and the second energy source is glucose. Is. In some embodiments, the glycolytic energy source is, for example, about 100 mM to 1 M, 200 to 900 mM, 300 to 800 mM, 400 to 600 mM or 500 mM, for example at least about 100, 200, 300, 400, 500, 600, It is glucose at a concentration of 700, 800, 900 mM or 1 M. In some embodiments, the gluconeogenic substrate is, for example, pyruvic acid at a concentration of about 10-50 mM, 15-45 mM, 20-40 mM or 25-35 mM. In some embodiments, pyruvic acid is about 10, 15, 20, 25, 30, 35, 40, 45 or 50 mM, eg, about 31, 32, 33, 34, 35, 36, 37, 38, 39 or It has a concentration of 40 mM. In some embodiments, pyruvic acid is at a concentration of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM.

In some embodiments, the kit further comprises means for concentrating or isolating sperm, such as a microfluidic device, density gradient solution or sperm isolation matrix. In some embodiments, the sperm isolation matrix is silanized silica, optionally in a medium that is substantially free of any glycolytic energy source or gluconeogenic substrate. In some embodiments, the sperm-enhancing energy depletion composition comprises silica silane. In some embodiments, the silanized silica is suspended in a suitable diluent, such as a nutrient-free synthetic human oviduct.

In some embodiments, the kit includes instructions for performing the methods disclosed herein. The kit may also include wash medium, storage medium, culture medium (eg, HTF), diluent and the like. The kit may further include adjuvants, reagents and buffers as needed. If desired, other additives (eg, amino acids (eg, glutamic acid) or free radical scavengers) may be present. In addition, hormones or other proteins can be added. Such hormones and proteins include luteinizing hormone, estrogen, progesterone, follicle-stimulating hormone, human chorionic gonadotropin, growth factors, follicular fluid and obidactin, albumin and amino acids. Typically, glycerol is added in an amount of 3% to 15% and other suitable concentrations can be readily determined by methods known in the art. Other agents are typically added at concentrations in the range of about 0.1% to 5%. Proteins such as skim milk, gelatin, casein or obidactin can also be added.

Other kits according to the present invention may include, for example, one or more (one, two or all three) reagents that do not require antibiotics (or provide antibiotics other than gentamicin). And / or those with or without phenol red are included. It will be readily appreciated that kits that replace components as a whole according to the parameters described by the present disclosure are part of the present invention. Substantially similar kits are specifically envisioned, where a "substantially similar" kit is one or more of the components (ie, 1, 2, 3, 4, 5, 6, 7, 8 or 9 components, if applicable), with the molar concentrations described in these detailed embodiments, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12, Includes 13, 14 or 15% different. Both liquid solutions (eg, those containing purified water, eg with pH adjustment with HCl and / or NaOH) and lyophilized compositions are included in these detailed examples.

The kit may also include means of transport, packaging or containers that are partitioned to accommodate one or more containers such as vials, tubes, etc., each of which is described herein. Includes one of the distinct elements, such as a sperm-enhancing composition, a second composition comprising a first energy source, and a third composition comprising a second energy source. Suitable containers include, for example, bottles, vials, syringes and test tubes. Such containers can be made of a variety of materials, such as glass or plastic. The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles and any packaging material suitable for use in the methods disclosed herein. The kit typically includes a label with the contents and / or instructions for use and a package insert that accompanies the instructions for use. A set of instructions may also be included.

The kits disclosed herein may be useful in a variety of applications, including, but not limited to, sperm processing for IVF and IUI. The kits of the present disclosure are useful in practicing the methods disclosed herein. The present specification discloses a kit containing an energy depleting composition for sperm enhancement, for example, a nutrient-free synthetic HTF. In some embodiments, the kits of the present disclosure include silane-coated silica diluted in nutrient-free synthetic HTF. Such kits may be useful in the process of separating sperm from sperm samples, for example by density gradient or swim-up methods. The kits of the present disclosure include a sperm-enhancing energy depletion composition (eg, including nutrient-free synthetic HTF) and a second composition comprising a first energy source (eg, glucose). One exemplary use of such a kit is the preparation of sperm for IUI. Sperm separated by the density gradient method or the swim-up method can be washed or diluted using a sperm-enhancing energy-depleting composition, such as a nutrient-free synthetic HTF. Preparing sperm for IUI by providing an effective amount of a first energy source (eg, a gluconeogenic substrate or glycolytic energy source) to sperm incubated with a sperm-enhancing energy depletion composition for a suitable period of time. Can be done. In some embodiments, sperm incubated with a sperm-enhancing energy depletion composition for a suitable period of time are provided with an effective amount of a first energy source, such as a gluconeogenic substrate such as pyruvic acid or a salt thereof, for IUI. Sperm can be prepared. In another embodiment, in addition to the second composition comprising a first energy source, the kit disclosed herein is a third container comprising a third composition comprising a second energy source. A third container containing the above can be included. In some embodiments, the first energy source is a gluconeogenic substrate (eg, pyruvic acid), and the second energy source is a glycolytic energy source (eg, glucose). In other embodiments, the first energy source is a glycolytic energy source (eg, glucose), and the second energy source is a gluconeogenic substrate (eg, pyruvic acid). Such kits can be useful, for example, for IVF. A further effective amount of the first energy source is provided to the sperm incubated with the sperm-enhancing energy depletion composition (including, for example, nutrient-free synthetic HTF) for a suitable time, and the effective amount of the second energy source is sequentially or simultaneously provided. Can provide an energy source for the preparation of sperm for IVF.

The components of the kit provide an initial incubation of sperm in a nutrient-free synthetic HTF that does not contain a glycolytic energy source (eg, glucose) or a gluconeogenic substrate (eg, pyruvate), and then later glycolytic. The simultaneous or sequential addition of energy sources or gluconeogenic substrates will improve sperm function. Gluconeogenic substrate means a non-carbohydrate carbon source used in the gluconeogenic process. The gluconeogenic substrate serves as a substrate for the gluconeogenic pathway and further promotes gluconeogenesis. Suitable gluconeogenic substrates for use in the kits of the present disclosure are, but are not limited to, pyruvic acid, lactic acid, succinic acid, citric acid, fumaric acid, malic acid, aspartic acid, glycerol, acetyl-CoA, isocitric acid, α- Physiologically acceptable derivatives, salts, esters, polymers or α-keto analogs of ketoglutaric acid, succinyl-CoA, oxaloacetate or gluconeogenic substrates can be mentioned. Any gluconeogenic amino acid or its physiologically acceptable derivative, salt, ester, or polymer, or α-keto analog is also suitable as a gluconeogenic substrate. Non-limiting examples of glycosylated amino acids include alanine, arginine, asparagine, cystine, glutamine, glycine, histidine, hydroxyproline, methionine, proline, serine, threonine and valine. Non-limiting examples of pharmaceutically acceptable salts of pyruvate are lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate and zinc pyruvate. In some embodiments, the pyruvate is sodium pyruvate. Non-limiting examples of lactic acid salts include sodium lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate and manganese lactate. The pyruvate component of the kit can be replaced with any of the other gluconeogenic substrates listed above.

Glycolytic energy sources include glycolytic carbon sources. Non-limiting examples of glycolytic energy sources include monosaccharides (such as fructose, glucose, galactose and mannose) and disaccharides (sucrose, lactose, maltose and trehalose) and polysaccharides, galactose, oligosaccharides and their polymers. Can be mentioned. The glucose component of the kit can be replaced with any of the other glycolytic energy sources listed above.

In some embodiments, the kit comprises additional components, such as NADH, NAD +, citric acid, AMP, ADP or a combination thereof, in combination with at least a first or second energy source. , Other components upstream and downstream of glycolysis are added.

Physiologically acceptable is understood to mean non-toxic to sperm, oocytes or embryos, as well as improving their function and survival during in vitro and in vivo handling and manipulation.

Non-limiting uses of the sperm-enhancing energy depletion composition of the kit include, for example, use in sperm isolation using the swim-up method, use as a diluent, eg, silane-coated silica for density gradients. Use for dilution of silica and use for washing sperm. Sperm samples can be treated, for example, by separation or washing with a sperm-enhancing energy depletion composition and then used in a variety of diagnostic or research protocols, including infertility testing and sperm toxicity testing. Examples of infertility tests include tests for sperm motility, viable sperm rate, sperm count, membrane function, invasion rate and in vitro fertilization rate. Protocols available in the art that are suitable for detailed sperm cell types and detailed diagnostic or research suitability can be used. In some embodiments, incubation of sperm with a sperm-enhancing energy-depleting composition results in sperm augmentation. In some embodiments, providing an effective amount of the first energy source to the enhanced sperm improves sperm function and prepares sperm for IUI. In some embodiments, providing an effective amount of a first energy source to the enhanced sperm and simultaneously or sequentially providing an effective amount of a second energy source improves sperm function and for IVF. Sperm is prepared.

In some embodiments, the compositions and solutions of the kit are provided in prefilled tubes in a predetermined volume. This product may also be provided in solution in a dispenser for a particular application. In one embodiment, a centrifuge tube is provided. The kit can be stored refrigerated or at room temperature.

In some embodiments, the kits described herein include storage media provided by the present invention. The kit provided by the present invention is a method of the present invention (eg, inducing improved sperm function, enhancing fertilization, producing progeny with improved adaptability, etc.) and is part of the practice. In the embodiment, it can be carried out using various kits provided by the present invention, and thus, in a specific embodiment, it is useful for carrying out the method in which the fertilization preparation solution provided by the present invention will be prepared. And / or useful in additional methods provided by the present invention, such as fertilization methods including reproductive assist methods. In some embodiments, the kits provided herein are useful in the preparation of the sperm preparations of the present disclosure.

The present disclosure will be described in more detail with reference to the following specific examples. The following examples are provided for purposes of illustration and are not intended to limit the invention in any way. One of ordinary skill in the art will readily recognize a variety of non-deterministic parameters that can be modified or modified to provide an alternative embodiment of the invention. All patents, patent applications and publications listed herein are incorporated herein by reference in their entirety.

Example 1: Material and Method Medium The medium for acquiring human sperm fertility was 97.8 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.37 mM KH2PO4, 0.2 mM 00734, equilibrated with 5% CO2. PH 7 containing 7H2O, 25.1 mM NaHCO3-, 0.33 mM Na pyruvate, 2.78 mM glucose, 21.4 mM lactic acid and 5 mg / mL human serum albumin (HSA), 10 μg / mL gentamicin and 0.0006% phenol red. It was a human oviductal fluid (complete HTF or C-HTF) medium of .4. For sperm starvation treatment, glucose, lactic acid and pyruvic acid were removed from the above HTF medium (F-HTF, test medium).

Semen Samples Semen samples were obtained by masturbating in sterile containers from healthy men or men seeking treatment for infertility. The ejaculate was liquefied for up to 2 hours prior to the treatment of this experiment. After liquefaction, the volume of ejaculate was equally divided for treatment with F-HTF (test) conditions or C-HTF (control conditions). Semen samples were treated by either density gradient centrifugation or direct swim-up to collect live sperm cells.

Sperm Treatment Density Gradient Centrifugation After liquefaction, the total volume of each ejaculate was equally divided into two different gradient conditions. Test samples were prepared using a 45-90% Percoll (Sigma, P-1644) gradient in phosphate buffered saline. Control samples were prepared using the Isolate gradient in human oviduct fluid (Irvine Scientific, Santa Ana, CA; 99264). Both samples were centrifuged at 500 xg for 20 minutes. After centrifugation, the supernatant was removed and the pellet was washed with 10 ml medium. The test sample was washed with F-HTF, and the control sample was washed with C-HTF.

Sperm swim-up method After liquefaction, the total volume of each ejaculate was divided into a test sample and a control sample as described above. The test sample was gently layered with 2.5 ml of F-HTF. The control sample was layered with C-HTF medium. The tube was carefully tilted at a 45 ° angle and incubated at 37 ° C. for 1 hour at _{5% CO 2.} The supernatant was carefully collected and washed with F-HTF, and the control sample was washed with C-HTF.

Analysis of sperm motility Load a sperm suspension (6 μl) of test sperm and control sperm onto one pre-warmed chamber slide (depth, 20 μm) (Leja slide, Spectrum Technologies) and microscopically stage at 37 ° C. I put it in. Sperm motility was examined using a CEROS II computer-aided semen analysis (CASA) system (Hamilton Horne Research, Beverly, MA). Trajectory for 1 second was captured using the following settings: 60 frames per second, 60 frames acquired, minimum contrast = 80, minimum size = 3 pixels, default cell size = 6 pixels, default cell strength = 160, slow cell movement Low VAP cutoff = 10 μm / s, low VSL cutoff = 0 μm / s, minimum intensity gate = 0.18, maximum intensity gate = 1.21, minimum size gate = 0.56 pixels, counting as capable , Maximum size gate = 2.63 pixels, minimum extension gate = 0 pixels and maximum extension gate = 99 pixels. Raw data were selected and analyzed using the CASAnova parameter (Goodson et al., 2018, supra). In each experiment, at least 20 microscopic fields of view corresponding to at least 500 sperms were analyzed.

Example 2: Experimental Results This example shows that reintroduction of energy sources in sequence after nutrient depletion improves sperm superactivation.

As shown in FIG. 1, incubation of sperm in glucose, pyruvic acid and lactate-free medium for 3 hours reduced motility. Rescue of sperm motility was tested with different energy substrates. When sperm were starved for 3 hours and rescued with complete HTF, sperm superactivation and intermediate motility were increased compared to control treatment (Fig. 2). In contrast, sperm treated with glucose (5 mM) or pyruvate (0.33 mM) alone did not improve sperm hyperactivation compared to controls (FIG. 2). Reintroduction of pyruvate alone had no effect on sperm motility from starvation, however, reintroduction of glucose alone restored motility to control levels (Fig. 2). Is suggested to be the major source of energy required for sperm superactivation. Surprisingly, when pyruvate was added to glucose-treated sperm or when glucose was added to pyruvate-treated sperm, compared to control conditions or when both pyruvate and glucose were simultaneously reintroduced into sperm. Caused a significant increase in superactivated motor activity.

Example 3: Enhanced activation The volume osmolal concentration of C-HTF is about 290 mOsm, where F-HTF is about 243 mOsm. Efficiently with sperm, such as trehalose, dextran or other long-chain sugars, to show that hypotonic conditions stress the sperm and thus cause an increase in sperm motility and function when restored. In the presence of non-metabolized carbon sources, sperm are incubated under different conditions of hypotonic, isotonic or hypertonic, and the effect on motility during incubation under hypotonic conditions and after returning to isotonic conditions Observe by CASA analysis. This includes adjusting the concentration of various ions such as calcium, sodium and potassium during the enhancement phase and determining motility after returning to C-HTF. In addition, the gradual process of ions to mimic the ion cycle that occurs in the female reproductive tract during natural pregnancy for the effects of increasing or decreasing concentrations of ions such as calcium, sodium and potassium during both the enhancement and rescue stages. Test as well as addition. In addition to motility, the calcium ion flux is evaluated. These manipulations, alone or in combination with the described glucose and pyruvate manipulations, increase the proportion of sperm that achieve hyperactivity or intermediate motility.

Human sperm exhibit reduced motility during the starvation phase of these processes, but sperm do not stop moving altogether and cells utilize internal energy sources such as glycogen, or lipids, proteins or It is suggested that it decomposes cellular components such as RNA. Total lipid content, RNA content and protein content are assessed for sperm exposed to the starvation stage. Proteomics, metabolomics and lipidomics analysis are performed after the starvation phase, after the addition of the first energy source and after the addition of the second energy source, to reveal intracellular changes associated with sperm motility status. Total RNA (including certain fractions, such as small non-coding RNA such as mRNA or microRNA) in sperm treated under control conditions and sperm treated under test conditions as shown in Example 2 is measured. The results of this analysis indicate that RNA is being used as an energy source by sperm.

Nutrient depletion and reintroduction can also alter sperm DNA, RNA and protein methylation and acetylation patterns in a manner that improves sperm fitness and / or progeny health and fitness. DNA methylation analysis can be performed by bisulfite sequencing of DNA from either bulk cell or single cell sperm. Changes in sperm DNA methylation after nutrient depletion and after reintroduction of each nutrient will be evaluated.

Gradual introduction of upstream carbon sources of glycolysis (glucose, mannose, fructose, dextrose or sucrose, etc.) and downstream metabolites (pyruvic acid, lactic acid, succinic acid, citric acid, fumaric acid, malic acid, etc.) from AMP The rate of conversion to ATP is altered, which improves sperm motility and function compared to simultaneous addition. After starvation, after the introduction of the first energy source and after the introduction of the second energy source, ATP and AMP levels in sperm are measured. Gradual introduction of nutrients after starvation improves the conversion of AMP to ATP.

Example 4
This example provides additional evidence that sperm activation upon stepwise reintroduction of the energy source.

A male sperm sample seeking treatment for infertility was obtained from a fertility clinic. These samples contained normal fertilizing sperm and low fertilizing sperm. To improve sperm quality, samples were prepared by density gradient centrifugation as described in Example 1. After liquefaction, the total volume of each ejaculate was equally divided and subjected to two different density gradient conditions. The test sample was prepared using a 45-90% Percoll (Sigma, P-1644) gradient diluted in a nutrient-deficient phosphate buffered saline solution having a final pH of 7.4 (Sigma, P-1644). F-PERCOLL). Control samples were prepared using a 45-90% Percoll gradient diluted in phosphate buffered saline containing nutrients such as (lactic acid, glucose and pyruvate) having a final pH of 7.4. (C-PERCOLL). Both samples were centrifuged at 500 xg for 20 minutes. After centrifugation, the supernatant was removed and the pellet was washed with 10 ml medium. The test sample was washed with F-HTF, and the control sample was washed with C-HTF.

Samples were treated with C-HTF medium as described in Example 1, or separated by density gradient in nutrient-free medium and washed with F-HTF. Sperm with F-HTF, A) 1 hour incubation in F-HTF, followed by 1 hour 15 minute incubation with glucose (5 mM), pyruvic acid (0.33 mM) and lactic acid, B) F-HTF 1 hour incubation in, 1 hour pyruvic acid addition, then 15 minutes glucose addition, or C) 1 hour incubation in F-HTF, 1 hour glucose addition, then 15 minutes pyruvic acid addition. Samples were analyzed by CASA as outlined in Example 1. The results are shown in FIGS. 5A and 5B and 6A and 6B. Each test condition increased the number of spermatozoa with intermediate and superactive motility compared to controls, and the highest activation levels were observed in treatments B and C.

To speed up starvation, sperm were separated by density gradient in nutrient-free medium and washed with 10 ml F-HTF. As depicted in FIG. 3, after incubating in F-HTF for 1 hour, sperm with a decrease in motility similar to the decrease in motility as seen in FIG. 1 were pyruvic acid (0.33 mM) or Primed with either glucose (5 mM) for 1 hour and then rescued with either (B) glucose (5 mM) or (C) pyruvic acid (0.33 mM) for 15 minutes. Similar to the results shown in FIG. 2, this fast / starvation protocol also significantly improved the sperm motility parameters shown in FIG.

Example 5
This example describes the use of sperm treated according to a particular embodiment of the invention to improve fertility in a human subject receiving IUI.

The subject is an adult female (eg, 18-35 years old) with no history of recurrent pregnancy loss and may or may not have tried IUI in the past. Subjects are treated with standard medical care (eg, clomid preparations, with Hcg-induced injection if indicated), semen diluted and centrifuged with either C-HTF or F-HTF, and cells are C-HTF or Randomly allocate to receive IUI of sperm prepared by collecting and resuspending in any of the F-HTFs. Alternatively, sperm can be collected by density gradient centrifugation and washing and resuspension of cells with C-HTF or F-HTF. Sperm are treated with F-HTF (eg, 1 hour), then either pyruvic acid or glucose is added, the sperm is incubated (eg, 1 hour), and then the sperm are fertilized by a woman. Used for. Sperm are treated with C-HTF (eg, 2 hours) and then the sperm are used to inseminate a woman. Monitor pregnancy with regular follow-up. Women who received sperm incubated in the absence of glucose (eg, 5 mM) or pyruvic acid (eg, 0.33 mM) followed by gradual addition of glucose or pyruvic acid had improved fertility, eg, improved. It is expected to exhibit the parameters of pregnancy rate, fetal heart rate (eg, at 7 weeks), continuation of pregnancy (eg, at 10 weeks) and / or birth rate.

Example 6
This example describes the use of sperm treated according to certain embodiments of the invention to improve fertility in human subjects undergoing IVF.

The subject is an adult female (eg, 18-37 years old) with no history of recurrent pregnancy loss and may or may not have tried IVF in the past. Subjects are treated with standard medical care (eg, with ovulation suppression followed by ovulation stimulation, and if indicated, human chorionic gonadotropin-induced injection) before egg collection. At the time of egg collection, the eggs of interest are randomly assigned to insemination with sperm treated with control or therapeutic conditions. In the control group, sperm are collected by density gradient centrifugation and resuspended in either sperm lavage medium, C-HTF or fertilization medium. Non-limiting examples of commercially available fertilization media include Global Total for fertilization (Origio), Continuous Single Culture (registered trademark) -NX Complete (Irvine), Systemy IVF fertilization medium (Cook Semi). ). In the treatment group, sperm are collected by density gradient centrifugation and washed and resuspended with F-HTF for an incubation time sufficient to augment the sperm (eg, 1 hour). After this incubation, either pyruvic acid (0.33 mM) or glucose (5 mM) is added and the sperm are incubated (eg, 1 hour). After this incubation, either glucose (5 mM) or pyruvic acid (0.33 mM) (the one not added in the first step) is added and the sperm are incubated (eg, for at least 15 minutes). In both the treatment and control groups, sperm will be incubated with the egg in vitro to monitor fertilization rate and embryonic development. Embryos (eg, on day 5) will be transplanted into women, and blood tests will determine pregnancy (eg, after 2 weeks). Monitor pregnancy with regular follow-up. After incubation in the absence of glucose and pyruvate, embryos generated with sperm supplemented with glucose and pyruvate in a stepwise manner have improved fertility parameters such as improved fertility, blastocyst development. Expected to be presented. After incubation in the absence of glucose and pyruvate, women who received embryos developed with sperm supplemented with glucose and pyruvate in stages had improved pregnancy rates, fetal heart rate (eg, 7 weeks). It is expected to exhibit (at time), continued pregnancy (eg, at 10 weeks) and / or birth rate.

Example 7 Kits The kits disclosed herein prepare sperm for IUI or IVF by ordering two nutrients in nutrient-free sHTF, resulting in intermediate and hyperactive motility. The proportion of sperm presenting is improved. The components of the kit provide an initial incubation of sperm in a nutrient-free synthetic HTF that does not contain glucose or pyruvic acid, after which glucose and pyruvic acid are added sequentially. This starvation-renutrition method produces a higher proportion of intermediate and superactivated sperm compared to standard sperm preparations (Fig. 10).

Sperm isolated from the upper body of mice of low fertility strains are incubated in a nutrient-free synthetic HTF that does not contain glucose or pyruvate, followed by sequential provision of glucose and pyruvate, which results in hyperactivity. The proportion of sperm exhibiting motility increased, followed by improved fertility, blastocyst development and offspring birth rate (Fig. 11). No abnormal motility phenotype as a result of this nutrient ordering was observed in mouse or human sperm, and no embryonic development or offspring abnormalities were observed in mice. Based on the results and the known association between sperm motility and fertilization, the use of the kits disclosed herein can improve the probability of pregnancy and birth for couples undergoing IVF and IUI. ..

The kits described herein are useful for the processing and preparation of sperm for IVF and IUI. The kit may be managed as a separate kit or combined. For example, the density gradient separation kit can be separated from the IVF or IUI kit, or the density gradient separation kit can be combined with the IVF or IUI kit. In embodiments of such combined kits, the IVF kit may include components from separate kits, i.e. components for density gradient separation and components for IVF. The kit for density gradients is used in the process of separating sperm from ejaculate by the density gradient method. The IVF kit consists of nutrient-free sHTF. This reagent is useful for washing sperm to prepare sperm for the fertilization step in IVF, and in sperm separation, (1) isolation of motile surviving sperm by swim-up method, or (2) density gradient separation. Can be used for any of the reagents in the kit. The density gradient separation kit contains silane-coated silica in nutrient-free sHTF.

The kit for IUI contains nutrient-free sHTF. This component can be used for sperm washing, sperm retention for IUI procedures, and in sperm separation, like IVF, (1) isolation of motile surviving sperm by swim-up method, or (2) kit. It can be used for any of the dilutions of the density gradient component. The density gradient reagent of the kit can be silane coated silica in nutrient-free sHTF. An exemplary composition of the kit is provided in Tables 1-5 below. These tables provide exemplary components of individual reagents and their concentrations.

The components of the kit provide an initial incubation of sperm in a nutrient-free synthetic HTF that does not contain a glycolytic energy source (eg, glucose) or a gluconeogenic substrate (eg, pyruvate), and then later glycolytic. The simultaneous or sequential addition of energy sources or gluconeogenic substrates will improve sperm function. Gluconeogenic substrate means a non-carbohydrate carbon source used in the gluconeogenic process. The gluconeogenic substrate serves as a substrate for the gluconeogenic pathway and further promotes gluconeogenesis. Suitable gluconeogenic substrates for use in the kits of the present disclosure are, but are not limited to, pyruvic acid, lactic acid, succinic acid, citric acid, fumaric acid, malic acid, aspartic acid, glycerol, acetyl-CoA, isocitric acid, α- Physiologically acceptable derivatives, salts, esters, polymers or α-keto analogs of ketoglutaric acid, succinyl-CoA, oxaloacetate or gluconeogenic substrates can be mentioned. Any gluconeogenic amino acid or its physiologically acceptable derivative, salt, ester, or polymer, or α-keto analog is also suitable as a gluconeogenic substrate. Non-limiting examples of glycosylated amino acids include alanine, arginine, asparagine, cystine, glutamine, glycine, histidine, hydroxyproline, methionine, proline, serine, threonine and valine. Non-limiting examples of pharmaceutically acceptable salts of pyruvate are lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate and zinc pyruvate. In some embodiments, the pyruvate is sodium pyruvate. Non-limiting examples of lactic acid salts include sodium lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate and manganese lactate. The pyruvate component of the kit can be replaced with any of the other gluconeogenic substrates listed above.

Glycolytic energy sources include glycolytic carbon sources. Non-limiting examples of glycolytic energy sources include monosaccharides (such as fructose, glucose, galactose and mannose) and disaccharides (sucrose, lactose, maltose and trehalose) and polysaccharides, galactose, oligosaccharides and their polymers. Can be mentioned. The glucose component of the kit can be replaced with any of the other glycolytic energy sources listed above.

In some embodiments, the kit comprises additional components, such as NADH, NAD +, citric acid, AMP, ADP or a combination thereof, in combination with at least a first or second energy source. , Other components upstream and downstream of glycolysis are added.

Physiologically acceptable is understood to mean non-toxic to sperm, oocytes or embryos, as well as improving their function and survival during in vitro and in vivo handling and manipulation.

Example 8 Density Gradient Kit The Density Gradient Kit is useful for separating motile sperm from ejaculatory fluid using density gradient centrifugation. It consists of a density gradient reagent, a suspension of silanized silica in nutrient-free sHTF. Density gradient separation kits include two or more concentrations of silanized silica, which are provided, for example, based on the user's preference, especially among IVF clinic technicians. In addition, the nutrient-free sHTF component of the IVF and IUI kits can be used to dilute the density gradient reagent, allowing the user to customize the concentration of silica silane.

The Density Gradient Kit also includes instructions for use, which can reflect clinical practice with current Density Gradients. Generally, a semen sample is added to the surface of the density gradient and centrifuged. Collect motile sperm with a pipette from the pellet at the bottom of the tube. Therefore, the sample is ready to be washed at any time.

Density gradient reagents lack nutrients that would later be added from the IVF kit or IUI kit during sperm washing.

The density gradient kit is designed to be nutrient-free for the convenience of users of IVF and IUI kits. If there was no nutrient-free density gradient, the technician would wash the sperm more times to prepare the sperm for the stepwise introduction of the nutrients provided using the IVF and IUI kits. It has to be, and more time and resources will be spent. The density gradient reagent has a pH of about 7.4.

Example 9 Kit for IVF The kit for IVF is useful for washing sperm, isolating motile surviving sperm by the swim-up method, and diluting the density gradient reagent. The kit prepares sperm for the fertilization step in IVF. The IVF kit contains three reagents: nutrient-free sHTF and a concentrated solution of pyruvic acid and glucose (Tables 1-5). This kit also includes instructions for use. Instructions for use include instructions for performing the washing step, as well as instructions for the timed sequential addition of pyruvate and glucose. The general use of this kit is described below.

Sperm Separation: Sperm-Up Method In many clinics, the density gradient centrifugation method is preferred over the swim-up method as the separation method. However, nutrient-free sHTF reagents can be used to isolate motile surviving sperm by the swim-up method to accommodate user preference (FIG. 12). First, a layer of semen sample is overlaid under a small amount of nutrient-free sHTF. The sperm are then given sufficient time to swim into the nutrient-free sHTF. Finally, the upper nutrient-free sHTF layer is collected and centrifuged.

Sperm Separation: Dilution of Density Gradient Reagent When used for dilution of Density Gradient Reagent, nutrient-free sHTF is added to the Density Gradient Reagent to achieve the desired concentration. The preferred concentration of silica silane in the density gradient medium can vary from clinic to clinic. This IVF kit allows the user to customize the density gradient reagent concentration to suit their needs.

Washing of sperm After sperm separation, pelletized sperm are washed once with nutrient-free sHTF and incubated for a short time. Glucose is added and sperm are incubated as detailed in the kit instructions. Then, as detailed in the kit instructions, pyruvic acid is added, the sperm are incubated, and then the sperm are centrifuged once at the end. The sample is ready to be resuspended in fertilization medium for co-incubation with oocytes.

The IVF kit prepares sperm for the fertilization step in IVF. The IVF kit contains three reagents that allow for the stepwise introduction of nutrients-nutrient-free sHTF, glucose-containing and pyruvate-containing reagents. Glucose and pyruvic acid are not premixed, but rather exist as sequential additions. Tables 2 and 5 provide exemplary compositions of glucose-containing reagents. Tables 2 and 4 provide exemplary compositions of reagents containing pyruvic acid.

Example 10 Kit for IUI The kit for IUI is used for washing sperm, isolating motile surviving sperm by the swim-up method, diluting density gradient reagents and retaining sperm for the IUI procedure. The IUI kit does not contain glucose, but is otherwise the same as the IVF kit. The instructions for use of the IVF and IUI kits are generally the same, the main difference being that the instructions for the IUI kit do not include the glucose addition step. The instructions end when the preparation for intrauterine insemination is ready. Glucose is present in the uterus at a concentration greater than or equal to the final glucose concentration in the nutrient-free sHTF-glucose-pyruvic acid mixture of the IVF kit. Therefore, sperm in the prepared sample are exposed to sufficient glucose to induce hyperactivation upon intrauterine injection.

The kit for IUI contains two reagents-nutrient-free sHTF and pyruvate-containing reagents-in vitro stepwise introduction of pyruvate (included as a separate reagent) and natural glucose (through intrauterine exposure). To enable. In the kit for IUI, glucose and pyruvate are initially absent from the medium. Pyruvic acid is added after the initial nutrient-free incubation. Glucose is not added in exobibo and is provided during intrauterine insemination of sperm. This has the added benefit that sperm hyperactivation caused by exposure to pyruvate synchronizes with the timing of intrauterine infusion. Tables 2 and 5 provide exemplary compositions of glucose-containing reagents. Tables 2 and 4 provide exemplary compositions of reagents containing pyruvic acid.

Example 11-Instructions for Use Density Gradient Kit The Density Gradient Kit is useful for separating motile sperm from ejaculatory fluid using density gradient centrifugation. It is instructed to be used in combination with an IVF kit or an IUI kit.

Kit for IVF The kit for IVF is useful for washing sperm, isolating motile surviving sperm by the swim-up method, and diluting the density gradient reagent in the density gradient separation kit.

The kit prepares sperm for the fertilization step in IVF. When preparing sperm using the IVF kit and performing density gradient centrifugation to separate the sperm from the semen, the density gradient kit can be used.

Kits for IUI Kits for IUI are useful for washing sperm, isolating motile surviving sperm by swim-up, diluting density gradient reagents in kits for density gradients, and retaining sperm for IUI procedures. A density gradient separation kit can be used when performing density gradient centrifugation to separate sperm from semen prior to sperm preparation using the IUI kit.

Example 12-Improvement of Progeny's Metabolism Adaptation Obesity is known worldwide for public health because of its association with many human diseases including type 2 diabetes, cardiovascular disease, respiratory disease, arthritis and cancer. The above concerns are growing. Most obesity cases, coupled with a genetic predisposition, result from an imbalance between energy intake and energy expenditure. In addition to the genetic inheritance of classical risk genes, epigenetic messages can be incorporated into sperm. This example reveals the effect of sperm subjected to the methods provided by the present invention on the metabolic fitness of offspring.

A dietary obesity model is used to test the effects of sperm starvation / rescue treatment from obese mice on fertility and offspring weight. Male C57BL / 6 mice are fed a normal solid diet or a high-fat diet. Sperm are harvested and under control conditions (C-HTF, control) or after the starvation / rescue procedure described in Example 2 (test with stepwise reintroduction of F-HTF, glucose and pyruvate). Acquire fertility. Sperm is analyzed between control and test conditions for motility, total RNA (and small non-coding RNA including microRNA) content and DNA, RNA and protein methylation. RNAseq is performed to determine changes in RNA, such as small molecule non-coding RNAs, including microRNAs. Bisulfite sequencing is performed to determine changes in DNA methylation. In vitro fertilization is performed using sperm from control and test conditions to determine fertilized egg count, blastocyst formation and live birth. Finally, bisulfite sequencing for RNA seq and DNA methylation is performed on 2-cell stage embryos from each experimental condition. In addition, the weight of offspring from each condition will be monitored for 12 months and regular blood chemistry analysis will be performed. Sperm subjected to test conditions show reduced RNA levels (including microRNAs) and / or altered DNA methylation compared to controls, and corresponding offspring show reduced obesity compared to controls.

For sperm obtained from obese or overweight men, the effects of nutrient depletion and reintroduction on RNA content and methylation and acetylation of DNA, RNA and proteins will be assessed. Sperm are isolated and exposed to control conditions or starvation / rescue procedures (tests with stepwise reintroduction of F-HTF, glucose and pyruvate). Samples will be analyzed for changes in RNA content, DNA, RNA and protein methylation and acetylation after starvation and reintroduction of each nutrient.

Example 13 Illustrative Storage Medium Samples and Methods Semen Samples and Sperm Preparations For this study, an arbitrary extraction donor from a cohort supplied semen samples. The sample was produced by masturbating into a sterile container and delivered to the laboratory within 1 hour of ejaculation. Fractionation of semen samples was achieved by density gradient centrifugation (Isolate, Irvine Scientific). Tarchala SM, et al. , 53rd Annual Meeting of the American Society for Reproductive Medicine, Cincinnati, Ohio; P-116, 1998. After centrifugation at 300 xg for 20 minutes, the sperm fraction and low density layer are removed, and a high density fraction mainly containing sperm with a high proportion of viability, motility and normal morphology is placed in a suitable storage medium. Washed twice.

Sperm storage Riel et al. Electrolyte-free medium (EFM) was prepared as described in (Biol Reprod. 2011 Sep; 85 (3): 536-47). The test storage medium or test medium consisted of 0.33 M glucose, 3% bovine serum albumin, 10 mM HEPES, 10 μg / ml gentamicin in sterile cell culture quality water adjusted to pH 6.8. The refrigerated medium (RM) consisted of TYB (TES Tris and egg yolk buffer) containing gentamicin (Sigma-Aldrich G1272). The washed sperm samples were resuspended in 0.5-1.0 mL of medium (EFM, RM or test storage medium) and stored in a cooling incubator (Benchmark Scientific) at 4 ° C. For cryopreservation, samples are mixed with freezing medium (TYB containing glycerol and gentamicin) (FM, Irvine Scientific, Catalog No. 90128) (1: 1 sample medium ratio) and slowly at 4 ° C. (0.5 ° C./ Minutes) Cooled and then frozen and stored in liquid nitrogen gas phase as recommended by the manufacturer.

Computer-aided sperm motility analysis (CASA)
Goodson et al. Computer-aided sperm motility analysis (CASA) of semen and stored sperm samples was obtained with the CEROS II system from Hamilton Horne according to the protocol recommended by (Biol Report 2017 Nov 1; 97 (5): 698-708). .. Relative distribution of arrested and motile spermatozoa and curve velocity (VCL) were determined with at least 500 spermatozoa. Sperm were recovered prior to CASA in modified human oviduct (mHTF) at 5 mg / mL human serum albumin, 25 mM sodium bicarbonate, pH 7.3, under known conditions to induce fertility acquisition. .. 6 μl of sperm suspension was loaded onto a disposable 20 μm chamber slide (Leja Products SC 20-01-02-B) and video for CASA was acquired (1 second, 60 frames per second).

Terminal deoxynucleotide transfer enzyme dUTP nick end label (TUNEL)
For 3 × 10 ⁶ cells, Simone et al. DNA fragmentation was measured using the APO-DIRECT kit (BD Biosciences 556381) according to the protocol recommended by (Hum Report. 2014 May; 29 (5): 904-17). The presence of free 3'-OH groups was measured with a CytoFLEX flow cytometer (Beckman Coulter Life Sciences).

Lipid Peroxidation Assay For 1 × 10 ⁶ cells, sperm peroxidation damage was evaluated using the lipid peroxidation sensor BODIPY C11 (Thermo Fisher Scientific D3861). The shift of the fluorescence peak was measured with a CytoFLEX flow cytometer compared to a positive control (sample incubated with 80 uM ferrous sulfate) and a negative control (without dye).

Data processing Flow cytometry Raw data was processed with FlowJo v10 (FlowJo, LLC). CASA and flow cytometry data were imported into Prism 8 (GraphPad) for statistical analysis.

Results The motility of human sperm from multiple n donors when stored in electrolyte-free medium (EFM), refrigerated medium (RM) or test storage medium at 4 ° C. for a short period of time was evaluated using CASA. .. After storage in the test storage medium for 7 days, up to 80% of the original sperm motility was preserved, which was stored in EFM (Fig. 13A, 75% in test medium, whereas in EFM). 35%) and storage in RM (Fig. 13B, 80% in test medium versus 60% in EFM) corresponded to a significant improvement. After storage in the test storage medium for 14 days, up to 50% of the original sperm motility was maintained.

Short-term storage in the test storage medium is comparable to cryopreservation in terms of preventing sperm DNA damage. Fresh human sperm samples were stored in test medium at 4 ° C. for 7 days, or diluted in Irvine freezing medium and frozen in liquid nitrogen. After 7 days, there was no significant difference in motility parameters (assessed by CASA) between spermatozoa stored in test medium and cryopreserved spermatozoa (FIG. 14A). However, TUNEL analysis revealed that spermatozoa stored in the test medium had significantly lower DNA fragmentation than spermatozoa frozen and thawed (Fig. 14B), at 4 ° C. for short-term storage. It is suggested that storage in the test medium (test storage medium) in the above is less harmful to sperm than cryopreservation.

For all numerical boundaries representing some parameters in the present application, such as "about", "at least", "less than" and "super", the representation inevitably includes any range bounded by the values described. Must be understood to be included in. Thus, for example, the expression "at least 1, 2, 3, 4 or 5" is, among other things, ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, 4-5, etc. are also expressed.

All publications, patents and patent applications referred to herein are to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Incorporated herein by reference in its entirety for all purposes. For example, all publications and patents referred to herein are described in those publications that may be used in connection with the methods, kits and compositions described herein. Incorporated herein by reference in its entirety for the purpose of explaining and disclosing the kits, compositions and methodologies in use. The references discussed herein are provided only for those disclosures prior to the filing date of the present application. Any matter herein is to be construed as acknowledging that the inventors described herein do not have the right to precede such disclosure on the grounds of prior invention or for any other reason. must not. In the event of any inconsistency between the literature incorporated by reference and the present disclosure, the present disclosure shall prevail.

The headings used in this application are for convenience only and do not affect the interpretation of this application. Headings should never be used to limit the invention. For example, a method described under the heading Sperm Function cannot be performed as described under the heading Sperm Motility, or a reagent disclosed under the heading Conservation Medium. It should not be limited to the fact that it cannot be carried out in. Rather, the methods and reagents disclosed and described under the various headings are entirely replaceable, and one of ordinary skill in the art will choose disclosure from any portion of the description of the invention herein. , It is intended that it can be carried out in any combination as it may be in combination with any other portion of the description of the invention herein.

Each preferred feature of any of the embodiments provided by the present invention (eg, medium, composition, preparation and method) can be appropriately modified and applied to any other aspect of the invention, without limitation, dependent claims. Combinations and rearrangements of the individual features of the detailed embodiments and embodiments of the invention (eg, elements including a range of numbers and exemplary embodiments), including examples illustrated and practiced in the section. Include. For example, the detailed experimental parameters exemplified in the actual examples can be fragmentarily adapted to the claims of the present invention without departing from the present invention. For example, with respect to the disclosed materials, specific references to each of the various individual and combined combinations and rearrangements of those compounds may not be explicitly disclosed, but each is specific herein. Intended and described in. Therefore, if the classes of elements A, B and C are disclosed, and examples of combinations of elements D, E and F and elements AD are also disclosed, and therefore each is not described individually. Intended individually and collectively. Therefore, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE and CF are specifically contemplated and A, B And C; D, E and F; and should be considered disclosed from the disclosure of exemplary combinations AD. Similarly, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, subgroups of AE, BF and CE are specifically contemplated and disclosed from the disclosure of A, B and C; D, E and F; and exemplary combinations AD. Should be considered. This concept applies to all aspects of the present application, including the elements of the composition of a substance and the steps of the method of making or using the composition.

According to the teachings of the present specification, the above-described aspects of the present invention as recognized by those skilled in the art can be claimed in any combination or rearrangement to the extent that they are novel and non-obvious in view of the prior art. -Therefore, to the extent that certain elements are described in one or more references known to those of skill in the art, they are claimed from the present invention, especially by negative proviso or disclaimer of features or combinations of features. Can be excluded.

According to the teachings of the present specification, the above-described aspects of the present invention as recognized by those skilled in the art can be claimed in any combination or rearrangement to the extent that they are novel and non-obvious in view of the prior art. -Therefore, to the extent that certain elements are described in one or more references known to those of skill in the art, they are claimed from the present invention, especially by negative proviso or disclaimer of features or combinations of features. Can be excluded.
The present invention provides, for example, the following items.
(Item 1)
It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for enriching the mammalian sperm.
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced mammalian sperm from step (a), and
(C) To provide the mammalian sperm obtained from step (b) with a means of reaching an egg under conditions that promote fertilization.
The method, wherein the effective amount is sufficient to induce improved sperm function.
(Item 2)
One or more sperm functions selected from curve velocity, head amplitude, autophagy, sperm fertility acquisition, superactivated sperm ratio, intermediate motile sperm ratio and superactivated and intermediate motile sperm ratio The enhanced mammalian sperm are provided with only one of the first energy source and the second energy source, or are provided with the first energy source and the second energy source at the same time. The method according to item 1, which is improved as compared with the method.
(Item 3)
The first energy source is a glycolytic energy source, and the second energy source is a gluconeogenic substrate, or the first energy source is the gluconeogenic substrate, and said. The method according to item 1, wherein the second energy source is the glycolytic energy source, and the mammalian sperm in step (a) is a human sperm.
(Item 4)
The method according to item 3, which is carried out outside the body.
(Item 5)
The method of item 3, wherein step (c) is performed in the body in the reproductive tract of a female subject by artificial insemination of the mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI).
(Item 6)
Providing the second energy source in step (b) is the reproductive tract of a female subject by intrauterine insemination (IUI) of the enhanced mammalian sperm provided with an effective amount of the first energy source. The method according to item 1, which is carried out in the body in.
(Item 7)
The method according to item 6, wherein the first energy source is a gluconeogenic substrate which is pyruvic acid, and the second energy source is a glycolytic energy source.
(Item 8)
In step (c), the mammalian sperm from step (b) is incubated with the egg, or the mammalian sperm from step (b) is injected into the cytoplasm of the egg to enhance in vitro fertilization of the egg. The method according to item 4, which comprises causing the egg to grow.
(Item 9)
The method of item 1, wherein enhancing fertilization comprises the development of embryos that exhibit improved viability and / or improved implantation compared to embryos generated by suitable control sperm. ..
(Item 10)
The method of item 1, wherein enhancing fertilization comprises at least a two-cell stage of development, a blastocyst stage of development, or the development of an embryo that develops to offspring.
(Item 11)
The method of item 1, wherein the mammalian sperm of step (a) is from a subject with oligospermia or a subject with low fertility.
(Item 12)
The method of item 1, wherein the mammalian sperm of step (a) is a human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm.
(Item 13)
The method of item 12, wherein the mammalian sperm of step (a) is a human sperm.
(Item 14)
The method of item 1, wherein the mammalian sperm of step (a) is a sperm recovered from a non-cold or cold storage.
(Item 15)
The method of item 1, wherein the mammalian sperm of step (a) is provided as a pool of two or more doses of ejaculatory fluid.
(Item 16)
The method of item 1, wherein the mammalian sperm of step (a) is concentrated from semen by density gradient centrifugation, swim-up or microfluidics prior to step (a).
(Item 17)
The method of item 1, wherein the method is performed at a weight osmolal concentration in the range of 200 to 280 mOsm / kg.
(Item 18)
Step (b) further comprises providing the mammalian sperm with one or more components upstream or downstream of glycolysis in combination with at least the first or second energy source. The method according to 1.
(Item 19)
The first energy source is selected from (i) glucose or (ii) pyruvic acid, and the second energy source is selected from (i) glucose or (ii) pyruvic acid, said first. And the second energy source is different, the method of item 3.
(Item 20)
The method of item 1, wherein the incubation in step (a) under energy depletion conditions lasts for at least 10 minutes.
(Item 21)
A method of inducing improved sperm function
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for producing enhanced mammalian sperm.
(B) To provide the enhanced mammalian sperm from step (a) with an effective amount of a first energy source selected from (i) glycolytic energy sources or (ii) gluconeogenic substrates. ,as well as
(C) The mammalian sperm from step (b) is then provided with an effective amount of a second energy source selected from (i) the glycolytic energy source or (ii) the gluconeogenic substrate. That is, the second energy source provided is not selected in step (b) and is provided.
The method, wherein the effective amount is sufficient to induce improved sperm function.
(Item 22)
The improved sperm function is enhanced as compared to a suitable control sperm, the suitable control sperm independently providing untreated mammalian sperm, an effective amount of the glycolytic energy source or the gluconeogenic substrate. The enhanced mammalian sperm of step (a), the enhanced mammalian sperm of step (a) or the standard provided simultaneously with an effective amount of the glycolytic energy source and the gluconeogenic substrate. 21. The method of item 21, wherein the sperm are treated with a fertility acquisition medium (C-HTF).
(Item 23)
21. The method of item 21, which is performed extracorporeally.
(Item 24)
21. The method of item 21, wherein step (c) is performed in the body in the reproductive tract of a female subject by artificial insemination of the mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI).
(Item 25)
21. The method of item 21, wherein the improved sperm function comprises an improvement in motility as measured by computer-aided semen analysis (CASA).
(Item 26)
25. The method of item 25, wherein the improvement in motility includes an increase in the curvaceous rate of the mammalian sperm, an increase in the proportion of superactivated sperm, an increase in the proportion of intermediate motility sperm, or a combination thereof.
(Item 27)
21. The method of item 21, wherein the improved sperm function comprises improved sperm fertility acquisition as measured by a sperm-zona pellucida binding test.
(Item 28)
21. The method of item 21, wherein the improved sperm function comprises an increase in the ability of the mammalian sperm to fertilize an egg, as measured by a sperm invasion test.
(Item 29)
The improved sperm function has improved viability, improved implantation, at least two-cell stage development, blastocyst stage development, or development to offspring compared to embryos generated in suitable control sperm. 21. The method of item 21, comprising developing a capable embryo.
(Item 30)
21. The method of item 21, wherein the mammalian sperm is a human, non-human primate, pig, cow, horse, sheep, dog, cat or mouse sperm.
(Item 31)
30. The method of item 30, wherein the mammalian sperm are human sperms.
(Item 32)
31. The method of item 31, wherein the glycolytic energy source is glucose and the gluconeogenic substrate is pyruvic acid.
(Item 33)
A sperm preparation solution containing the mammalian sperm having improved sperm function prepared by the method according to item 21.
(Item 34)
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for producing enhanced mammalian sperm, and
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced mammalian sperm from step (a).
A sperm preparation solution containing an improved proportion of superactivated sperm prepared by the process of the above, wherein the effective amount is sufficient to induce an improvement in one or more sperm functions and has been improved. The sperm preparation solution containing a proportion of super-activated sperm
The enhanced mammalian sperm of step (a), the effective amount of the first energy, which is independently provided with the untreated mammalian sperm, the effective amount of the first energy source or the second energy source. The enhanced mammalian sperm of step (a) or the mammalian sperm treated with standard capacitation medium (C-HTF), which is simultaneously provided with a source and said second energy source.
A sperm preparation solution comprising one or more improvements in sperm function as compared to a suitable control sperm selected from.
(Item 35)
A method of inducing improved sperm function
(A) Providing mammalian sperm to a storage medium containing a buffer and having a pH of about 6-7 and a weight osmolal concentration of about 300-400 mOsm / kg.
(B) Incubating the mammalian sperm under energy-depleted conditions for a time suitable for enriching the mammalian sperm, and
(C) To provide an effective amount of (i) glycolytic energy source and / or (ii) gluconeogenic substrate to the enhanced mammalian sperm from step (b).
A method of inducing improved sperm function as compared to a suitable control sperm.
(Item 36)
It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy depletion conditions for a time suitable for enriching the sperm, wherein prior to the incubation, the mammalian sperm contain a buffer and has a weakly acidic pH. And incubating, stored in a storage medium having a weight osmolal concentration of about 300-400 mOsm / kg.
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced sperm from step (a).
(C) Independently providing an effective amount of the first or second energy source to the enhanced mammalian sperm of step (a), or an effective amount of the first energy source and said. Improving one or more of sperm function to levels above those obtained by simultaneously providing a second source of energy, and
(D) To provide the mammalian sperm having an improved function with a means of reaching an egg under conditions that promote fertilization.
How to include.
(Item 37)
a) A first container containing a first composition containing a buffered sperm-enhancing energy depletion composition, and
b) A second container containing a second composition containing at least a first energy source suitable for mammalian sperm
Is a kit that includes
Incubation of the mammalian sperm in the first composition for a suitable period of time yields enhanced mammalian sperm.
A kit that provides an effective amount of at least the first energy source to the enhanced mammalian sperm to improve the function of the enhanced mammalian sperm as compared to a suitable control.
(Item 38)
A super-activated sperm preparation solution containing at least 5% of super-activated sperm, optionally not preselected based on super-activation, optionally said super-activated sperm and / or Intermediate sperm have 10, 15, 20, 25, 30, 35, 40, 45, 50% or less of intracellular RNA concentration (such as low molecular weight non-coding RNA concentration including microRNA) compared to a suitable control. A super-activated sperm preparation solution having a reduction of more than (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more).
(Item 39)
a. Concentrating sperm from the semen of a male subject, such as a male with normal sperm, a male with low fertility or a male with oligospermia, such as a male with low fertility (including oligospermia).
b. Incubating the sperm under energy depletion for a time suitable for enriching the sperm,
c. A first energy selected from an effective amount of glycolytic energy source or an effective amount of gluconeogenic substrate for the enhanced sperm, but not both an effective amount of glycolytic energy source and a gluconeogenic substrate. Providing a source
Sperm preparation solution prepared by.
(Item 40)
Contains buffer and has a weakly acidic pH and about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340, 350, 360, 370 or 380. A sperm storage medium having, for example, a weight osmolal concentration of about 350, which does not contain a significant amount of egg yolk or, in some embodiments, does not contain any egg yolk.
(Item 41)
(A) Incubating mammalian sperm under energy depletion for a time suitable for producing enhanced mammalian sperm, and
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced mammalian sperm from step (a).
A sperm preparation solution prepared by, wherein the sperm of step (b) contains an epigenetic profile different from that of a suitable control sperm.
(Item 42)
A method of producing offspring with improved fitness,
(A) Incubating a sperm sample under energy depletion for a time suitable for producing enhanced sperm.
(B) To provide an effective amount of a first energy source for the enhanced sperm, and
(C) Then providing an effective amount of a second energy source to the sperm from step (b).
(D) Fertilizing the sperm from step (c) into an egg to develop an embryo, and
(E) Growing the embryo in a female subject to produce progeny with the improved fitness.
The improved fitness includes a reduced risk of developing a pathological condition, the method.

Claims (42)

It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for enriching the mammalian sperm.
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced mammalian sperm from step (a), and (c) the mammal obtained from step (b). A method comprising providing sperm with a means of reaching an egg under conditions that promote fertilization, wherein the effective amount is sufficient to induce improved sperm function. One or more sperm functions selected from curve velocity, head amplitude, autophagy, sperm fertility acquisition, superactivated sperm ratio, intermediate motile sperm ratio and superactivated and intermediate motile sperm ratio The enhanced mammalian sperm are provided with only one of the first energy source and the second energy source, or are provided with the first energy source and the second energy source at the same time. The method according to claim 1, which is improved as compared with the method. The first energy source is a glycolytic energy source, and the second energy source is a gluconeogenic substrate, or the first energy source is the gluconeogenic substrate, and said. The method according to claim 1, wherein the second energy source is the glycolytic energy source, and the mammalian sperm in step (a) is a human sperm. The method according to claim 3, which is carried out outside the body. The method according to claim 3, wherein step (c) is performed in the body in the reproductive tract of a female subject by artificial insemination of the mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI). .. Providing the second energy source in step (b) is the reproductive tract of a female subject by intrauterine insemination (IUI) of the enhanced mammalian sperm provided with an effective amount of the first energy source. The method according to claim 1, which is carried out in the body in the body. The method according to claim 6, wherein the first energy source is a gluconeogenic substrate which is pyruvic acid, and the second energy source is a glycolytic energy source. Step (c) enhances in vitro fertilization of the egg by incubating the mammalian sperm from step (b) with the egg or injecting the mammalian sperm from step (b) into the cytoplasm of the egg. The method according to claim 4, which comprises causing the egg to grow. 1. Method. The method of claim 1, wherein enhancing fertilization comprises at least a two-cell stage of development, a blastocyst stage of development, or the development of an embryo that develops to offspring. The method of claim 1, wherein the mammalian sperm of step (a) is from a subject with oligospermia or a subject with low fertility. The method of claim 1, wherein the mammalian sperm of step (a) is a human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm. 12. The method of claim 12, wherein the mammalian sperm of step (a) is a human sperm. The method of claim 1, wherein the mammalian sperm of step (a) is a sperm recovered from a non-cold or cold storage. The method of claim 1, wherein the mammalian sperm of step (a) is provided as a pool of two or more doses of ejaculatory fluid. The method of claim 1, wherein the mammalian sperm of step (a) is concentrated from semen by density gradient centrifugation, swim-up or microfluidics prior to step (a). The method of claim 1, wherein the method is performed at a weight osmolal concentration in the range of 200 to 280 mOsm / kg. (B) further comprises providing the mammalian sperm with one or more components upstream or downstream of glycolysis in combination with at least the first energy source or the second energy source. Item 1. The method according to Item 1. The first energy source is selected from (i) glucose or (ii) pyruvic acid, and the second energy source is selected from (i) glucose or (ii) pyruvic acid, said first. The method according to claim 3, wherein the second energy source and the second energy source are different. The method of claim 1, wherein the incubation in step (a) under energy depletion conditions lasts for at least 10 minutes. A method of inducing improved sperm function
(A) Incubating mammalian sperm under energy-depleted conditions for a time suitable for producing enhanced mammalian sperm.
(B) To provide the enhanced mammalian sperm from step (a) with an effective amount of a first energy source selected from (i) glycolytic energy sources or (ii) gluconeogenic substrates. And (c) the mammalian sperm from step (b) is then subjected to an effective amount of a second energy source selected from (i) the glycolytic energy source or (ii) the gluconeogenic substrate. The second energy source provided is not selected in step (b) and comprises providing, the effective amount of which is sufficient to induce improved sperm function. There is a way. The improved sperm function is enhanced as compared to a suitable control sperm, the suitable control sperm independently providing untreated mammalian sperm, an effective amount of the glycolytic energy source or the gluconeogenic substrate. The enhanced mammalian sperm of step (a), the enhanced mammalian sperm of step (a) or the standard provided simultaneously with an effective amount of the glycolytic energy source and the gluconeogenic substrate. 21. The method of claim 21, wherein the sperm are treated with a fertility acquisition medium (C-HTF). 21. The method of claim 21, which is performed extracorporeally. 21. The method of claim 21, wherein step (c) is performed in the body in the reproductive tract of a female subject by artificial insemination of the mammalian sperm from step (b) by intravaginal or intrauterine insemination (IUI). .. 21. The method of claim 21, wherein the improved sperm function comprises an improvement in motility as measured by computer-aided semen analysis (CASA). 25. The method of claim 25, wherein the improvement in motility includes an increase in the curvaceous rate of the mammalian sperm, an increase in the proportion of superactivated sperm, an increase in the proportion of intermediate motility sperm, or a combination thereof. 21. The method of claim 21, wherein the improved sperm function comprises improved sperm fertility gain as measured by a sperm-zona pellucida binding test. 21. The method of claim 21, wherein the improved sperm function comprises an increase in the ability of the mammalian sperm to fertilize an egg, as measured by a sperm invasion test. The improved sperm function has improved viability, improved implantation, at least two-cell stage development, blastocyst stage development, or development to offspring compared to embryos generated in suitable control sperm. 21. The method of claim 21, comprising developing a capable embryo. 21. The method of claim 21, wherein the mammalian sperm are human, non-human primate, pig, bovine, horse, sheep, dog, cat or mouse sperm. 30. The method of claim 30, wherein the mammalian sperm are human sperms. 31. The method of claim 31, wherein the glycolytic energy source is glucose and the gluconeogenic substrate is pyruvic acid. A sperm preparation solution containing the mammalian sperm having an improved sperm function, which is prepared by the method according to claim 21. (A) Incubating mammalian sperm under energy depletion conditions for a time suitable for producing enhanced mammalian sperm, and (b) an effective amount for said enhanced mammalian sperm from step (a). A sperm preparation solution containing an improved proportion of superactivated sperm prepared by the process of sequentially providing a first energy source and a second energy source, wherein the effective amount is one or more. The sperm preparation solution is an amount sufficient to induce an improvement in sperm function and contains an improved proportion of superactivated sperm.
The enhanced mammalian sperm of step (a), the effective amount of the first energy, which is independently provided with the untreated mammalian sperm, the effective amount of the first energy source or the second energy source. A suitable control selected from the enhanced mammalian sperm of step (a) or the mammalian sperm treated with standard capacitation medium (C-HTF), which is simultaneously provided with the source and the second energy source. A sperm preparation solution containing one or more improvements in sperm function compared to sperm. A method of inducing improved sperm function
(A) Providing mammalian sperm to a storage medium containing a buffer and having a pH of about 6-7 and a weight osmolal concentration of about 300-400 mOsm / kg.
(B) Incubating the mammalian sperm under energy-depleted conditions for a time suitable for enriching the mammalian sperm, and (c) an effective amount of the enhanced mammalian sperm from step (b). A method comprising providing (i) a glycolytic energy source and / or (ii) a gluconeogenic substrate, thereby inducing improved sperm function as compared to a suitable control sperm. It ’s a way to improve fertilization,
(A) Incubating mammalian sperm under energy depletion conditions for a time suitable for enriching the sperm, prior to the incubation, the mammalian sperm containing a buffer and having a weakly acidic pH. And incubating, stored in a storage medium having a weight osmolal concentration of about 300-400 mOsm / kg.
(B) To sequentially provide an effective amount of a first energy source and a second energy source to the enhanced sperm from step (a).
(C) Independently providing an effective amount of the first or second energy source to the enhanced mammalian sperm of step (a), or an effective amount of the first energy source and said. Under conditions that enhance one or more of sperm function to levels above those obtained by simultaneously providing a second energy source, and (d) enhance fertilization of said mammalian sperm with enhanced function. , A method involving providing a means of reaching the egg. a) a first container containing a first composition containing a buffered sperm-enhancing energy depleting composition, and b) a second containing a second composition containing at least a first energy source suitable for mammalian sperm. It is a kit that includes the container of
Incubation of the mammalian sperm in the first composition for a suitable period of time yields enhanced mammalian sperm.
A kit that provides an effective amount of at least the first energy source to the enhanced mammalian sperm to improve the function of the enhanced mammalian sperm as compared to a suitable control. A super-activated sperm preparation solution containing at least 5% of super-activated sperm, optionally not preselected based on super-activation, optionally said super-activated sperm and / or Intermediate sperm have 10, 15, 20, 25, 30, 35, 40, 45, 50% or less of intracellular RNA concentration (such as low molecular weight non-coding RNA concentration including microRNA) compared to a suitable control. A super-activated sperm preparation solution having a reduction of more than (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more). a. Concentrating sperm from the semen of a male subject, such as a male with normal sperm, a male with low fertility or a male with oligospermia, such as a male with low fertility (including oligospermia).
b. Incubating the sperm under energy depletion for a time suitable for enriching the sperm,
c. A first energy selected from an effective amount of glycolytic energy source or an effective amount of gluconeogenic substrate for the enhanced sperm, but not both an effective amount of glycolytic energy source and a gluconeogenic substrate. A sperm preparation solution prepared by providing a source. Contains buffer and has a weakly acidic pH and about 300-400 mOsm / kg, such as about 300-380, 320-370, 330-370, 340-360, such as about 320, 330, 340, 350, 360, 370 or 380. A sperm storage medium having, for example, a weight osmolal concentration of about 350, which does not contain a significant amount of egg yolk or, in some embodiments, does not contain any egg yolk. (A) Incubating mammalian sperm for a time suitable for producing enhanced mammalian sperm under energy depletion, and (b) an effective amount of said enhanced mammalian sperm from step (a). A sperm preparation solution prepared by sequentially providing a first energy source and a second energy source, wherein the sperm in step (b) contains an epigenetic profile different from that of a suitable control sperm. liquid. A method of producing offspring with improved fitness,
(A) Incubating a sperm sample under energy depletion for a time suitable for producing enhanced sperm.
(B) providing the enhanced sperm with an effective amount of a first energy source, and (c) then providing the sperm from step (b) with an effective amount of a second energy source.
(D) Fertilize the sperm from step (c) into an egg to develop an embryo, and (e) grow the embryo in a female subject to produce progeny with the improved fitness. The improved fitness includes a reduced risk of developing a pathological condition, including the method.

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