Application of orientin in improvement of quality of frozen sperms and in-vitro cryopreservation of sperms

Abstract

The invention discloses application of orientin in improvement of frozen sperm quality and in-vitro cryopreservation of sperms. The invention further discloses a sperm cryopreservation solution, and the sperm cryopreservation solution contains orientin. Orientin is found to have a good protective effect in the sperm freezing process; through multiple mechanisms of reducing oxidative stress, improving mitochondrial functions, improving sperm ultrastructures and the like, the activity and quality of sperms are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of sperm preservation, and in particular to an application of orientin in improving the quality of frozen sperm and cryopreserving sperm in vitro. Background Art

[0002] Male infertility refers to the inability of a couple to conceive, despite regular, normal, unprotected intercourse for a period of one year or longer. Currently, approximately 17.5% of couples worldwide face infertility issues, with approximately 50% of cases related to male reproductive dysfunction. Autologous sperm freezing has become an important clinical treatment for male infertility, particularly for men undergoing assisted reproductive treatment, cancer, or other reasons requiring sperm preservation. Using ultra-low temperature freezing technology, a patient's sperm or testicular tissue can be cryopreserved in liquid nitrogen at -196°C, ensuring sufficient sperm for future fertility.

[0003] Currently, sperm is typically frozen using permeable cryoprotectants such as glycerol and dimethyl sulfoxide, as well as non-permeable cryoprotectants such as egg yolk and sucrose. These cryoprotectants have shown some success in improving sperm freezing and are widely used in clinical practice. However, the freezing process can induce cryo-damage, impairing sperm motility and DNA integrity, leading to reduced sperm quality. Therefore, there is a need to improve the quality of frozen sperm. Summary of the Invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes an application of orientin in improving the quality of frozen sperm and cryopreserving sperm in vitro. The present invention finds that orientin has a good protective effect during the sperm freezing process; it improves the motility and quality of sperm through multiple mechanisms such as reducing oxidative stress, improving mitochondrial function, and improving sperm ultrastructure.

[0005] The present invention provides an application of orientin in improving the quality of frozen sperm and cryopreserving sperm in vitro.

[0006] Preferably, the application comprises: freezing and preserving semen containing sperm in a cryopreservation solution containing orientin.

[0007] Preferably, the concentration of orientin in the cryopreservation solution is 1×10 -8 -1×10 -6 mol / L.

[0008] Preferably, the volume ratio of semen to the cryopreservation solution containing orientin is 2:1-2.

[0009] Preferably, the sperm is human sperm.

[0010] Preferably, the improvement of the quality of frozen sperm includes: reducing sperm freezing damage, improving sperm motility, improving sperm membrane integrity, improving sperm DNA integrity, improving sperm mitochondrial function, and reducing oxidative stress response.

[0011] The invention also discloses a sperm freezing preservation solution, which contains orientin.

[0012] Preferably, the concentration of orientin is 1×10 -8 -1×10 -6 mol / L.

[0013] The above-mentioned sperm cryopreservation solution also contains other substances acceptable for sperm cryopreservation; preferably, the other substances acceptable for sperm cryopreservation can be commercially available sperm cryopreservation solutions, such as sperm cryopreservation solutions produced by ORIGIO a / s, FUJIFILM Irvine Scientific, Anhui Anke Bioengineering (Group) Co., Ltd., and Ruibo Biotechnology Co., Ltd.

[0014] The present invention selects 10 -6 mol / L, 10 -7 mol / L, 10 -8 The sperm were frozen at three different concentrations of orientin (10 mol / L) and the plasma membrane integrity, motility, ROS, MMP, DFI and ultrastructure of the sperm were detected and analyzed after freezing and thawing. -8 The sperm in the 100 mol / L concentration group showed significantly better indicators after freezing and thawing than those in the other groups, and the differences were statistically significant compared to the control group. This finding fully demonstrates that orientin can effectively improve the freezing effect of sperm and reduce the damage caused by the freezing process to sperm.

[0015] The freezing and thawing process can significantly reduce the vitality and quality of sperm. The present invention has found that orientin can reduce freezing damage, improve sperm motility, membrane integrity, DNA integrity and mitochondrial function, reduce oxidative stress response, and improve the quality of sperm. -8 Adding orientin to sperm cryoprotectant at a concentration of 1.5 mol / L significantly improved sperm quality. This suggests that orientin has a good protective effect during sperm freezing; it improves sperm motility and quality through multiple mechanisms, including reducing oxidative stress, enhancing mitochondrial function, and improving sperm ultrastructure. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The sperm morphology pictures of each group before and after freezing, among which A is fresh sperm, B is the control group, C is 10 -6 mol / L group, D is 10-7 mol / L group, E is 10 -8 mol / L group.

[0017] Figure 2 The following are the percentages of normal sperm morphology in each group before and after cryopreservation, where Fresh refers to fresh sperm and Control refers to the control group.

[0018] Figure 3 These are typical images of sperm plasma membrane eosin-nigrosine staining after cryopreservation, where ▲ represents sperm with intact plasma membrane and → represents sperm with damaged plasma membrane.

[0019] Figure 4 These are the results of sperm plasma membrane integrity in each group after cryopreservation, where control is the control group.

[0020] Figure 5 The mitochondrial membrane potential results of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0021] Figure 6 These are the MMP results of sperm in each group after cryopreservation, where control is the control group.

[0022] Figure 7 The results of the active oxygen levels of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0023] Figure 8 These are the ROS levels in sperm of each group after cryopreservation, where control is the control group.

[0024] Figure 9 The results of sperm acrosome integrity in each group after cryopreservation, where A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0025] Figure 10 These are the reaction changes of the sperm acrosome in each group after cryopreservation, where control is the control group.

[0026] Figure 11 The DNA fragmentation results of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10-7 mol / L group, D is 10 -8 mol / L group.

[0027] Figure 12 The DFI changes of sperm in each group after cryopreservation, among which control is the control group.

[0028] Figure 13 The electron microscope images of the sperm ultrastructure before and after cryopreservation, where A is fresh semen before cryopreservation, B is 10 - 8 mol / L orientin-treated group, and C was the control group. DETAILED DESCRIPTION

[0029] Hereinafter, the technical solutions of the present invention will be described in detail through specific embodiments. However, it should be clearly stated that these embodiments are provided for illustration only and are not to be construed as limiting the scope of the present invention.

[0030] The statistical analysis of the experimental data in the following examples was performed using SPSS 25.0 software. For quantitative data that conformed to the normal distribution, mean ± standard deviation was used to express them. The differences between the two groups of data were compared by paired sample t-test. Multiple groups of data were compared using one-way analysis of variance, followed by pairwise comparisons using the Bonferroni correction method. The statistical significance level was set at α = 0.05 (two-sided), and the difference was considered statistically significant when the P value was less than 0.05.

[0031] Example 1 (Collecting semen samples, freezing and thawing)

[0032] Semen sample collection:

[0033] Multiple male research subjects were randomly screened, and men with the following conditions were excluded: 1. Judging from their medical history, men with varicocele, endocrine diseases, a history of vasectomy or varicocelectomy, cryptorchidism, or genital infections will be excluded from this analysis; 2. Men with a history of long-term illness, smoking, drinking, taking medications, taking vitamins (such as vitamin C, ascorbic acid and tocopherol), or antioxidant supplements within 3 months before the start of the study. Then, semen samples of the remaining male research subjects were collected, and the semen volume, sperm concentration, motility, etc. were measured according to the requirements of the WHO "Laboratory Manual for the Examination and Processing of Human Semen" (Sixth Edition). 105 semen samples with good semen parameters were selected, and samples containing more white blood cells in the semen were eliminated. The final selected semen samples had a liquefaction time of ≤30min, a semen volume ≥2ml, and a semen concentration ≥40×10 6 / ml, sperm motility ≥40%; and the semen sample was divided into 4 parts.

[0034] Preparation of sperm cryopreservation solution: Orientin was added to sperm cryopreservation solution (manufactured by ORIGIO a / s) to prepare 10 -6 mol / L, 10 -7 mol / L, 10 -8 mol / L orientin-containing sperm cryoprotectant, denoted as 10 - 6 mol / L group, 10 -7 mol / L group, 10 -8 mol / L group; and sperm cryopreservation solution without orientin was used as the control group.

[0035] Semen cryopreservation: Semen samples were thoroughly mixed with sperm cryopreservation solution of each group at a volume ratio of 2:1 in a cryopreservation tube. After equilibration at room temperature for 5 minutes, the samples were placed 5 cm above the liquid nitrogen surface for fumigation for 15 minutes. After fumigation, the samples were transferred to liquid nitrogen for storage for at least 2 weeks.

[0036] Semen Thawing: Remove each frozen semen vial from the liquid nitrogen tank and unscrew the cap to release the air inside the vial to prevent rupture or damage due to excessive pressure. Thaw the vial in a 37.5°C water bath for 5-10 minutes before performing sperm quality analysis or functional testing. The results are shown in Examples 2-9.

[0037] Example 2

[0038] Effects of orientin on sperm motility after cryopreservation

[0039] The sperm motility of each group was tested before and after freezing. The results are shown in Table 1, where A is fresh sperm, used to evaluate the effect of freezing treatment itself on sperm motility, B is the control group, and CE is the orientin intervention group, which are 10 - 6 mol / L group, 10 -7 mol / L group, 10 -8 mol / L group.

[0040] Table 1 Effects of orientin on sperm motility parameters after cryopreservation

[0041]

[0042] *: P < 0.05 compared with the fresh group after thawing;

[0043] #: P<0.05 compared with the control group in the experimental groups supplemented with orientin.

[0044] As can be seen from Table 1, the sperm motility of each group decreased after cryopreservation, and the sperm motility of the group supplemented with orientin was higher than that of the control group, with 10-8 mol / L group was significantly improved (P<0.05).

[0045] Example 3

[0046] Effects of orientin on sperm morphology after cryopreservation

[0047] The sperm morphology of each group was tested before and after freezing. Figure 1-2 shown.

[0048] Figure 1 The sperm morphology pictures of each group before and after freezing, among which A is fresh sperm, B is the control group, C is 10 -6 mol / L group, D is 10 -7 mol / L group, E is 10 -8 mol / L group.

[0049] Figure 2 The following are the percentages of normal sperm morphology in each group before and after cryopreservation, where Fresh refers to fresh sperm and Control refers to the control group.

[0050] Depend on Figure 1-2 As shown, the percentage of normal sperm morphology was 15.62±2.64% in fresh sperm and 14.06±2.3% in control group. -6 mol / L orientin group was 14.83±2.21%, 10 -7 mol / L orientin group was 14.60±2.11%, 10 - 8 mol / L orientin group was 14.64±2.13%, and there was no significant difference among the groups (P>0.05).

[0051] Example 4

[0052] Effects of orientin on the integrity of sperm plasma membrane after cryopreservation

[0053] Eosin-aniline black staining is a preliminary screening tool for evaluating the integrity of sperm plasma membranes and can quickly determine the survival rate of sperm after freezing damage. When using eosin staining solution for staining, the cell membrane of dead sperm allows the dye to penetrate and stain, appearing red; while live sperm show a phenomenon of rejecting the dye because the cell membrane has a selective permeability barrier function, preventing the dye from entering and appearing colorless. The plasma membrane integrity of each group of sperm after freezing was tested using eosin-aniline black staining method, and the results are as follows Figure 3-4 shown.

[0054] Figure 3 These are typical images of sperm plasma membrane eosin-nigrosine staining after cryopreservation, where ▲ represents sperm with intact plasma membrane and → represents sperm with damaged plasma membrane.

[0055] Figure 4 These are the results of sperm plasma membrane integrity in each group after cryopreservation, where control is the control group.

[0056] Depend on Figure 3-4 It can be seen that the control group, 10 -6 mol / L orientin group, 10 -7 mol / L orientin group, 10 -8 The sperm survival rates of the orientin group were 48.28±6.47%, 49.32±6.14%, 56.20±6.65% and 57.08±6.64% respectively; compared with the control group, the sperm survival rates of the orientin group were 10 -7 mol / L orientin group, 10 -8 The sperm survival rate in the 100 mol / L orientin group was significantly increased (P<0.05).

[0057] Example 5

[0058] Effects of orientin on mitochondrial membrane potential of sperm after cryopreservation

[0059] The mitochondrial membrane potential of sperm in each group after cryopreservation was tested. Figure 5-6 shown.

[0060] Figure 5 The mitochondrial membrane potential results of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0061] Figure 6 These are the MMP results of sperm in each group after cryopreservation, where control is the control group.

[0062] Depend on Figure 5-6 It can be seen that: 10 -6 mol / L group, 10 -7 mol / L group, 10 -8 The membrane potential level of the 1.5 mol / L group was higher than that of the control group; as the concentration of orientin gradually decreased, the MMP level increased and the mitochondrial function became more stable.

[0063] Example 6

[0064] Effects of orientin on reactive oxygen species levels in sperm after cryopreservation

[0065] The reactive oxygen species levels of sperm in each group after cryopreservation were tested. Figure 7-8 shown.

[0066] Figure 7The results of the active oxygen levels of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0067] Figure 8 These are the ROS levels in sperm of each group after cryopreservation, where control is the control group.

[0068] Depend on Figure 7-8 It can be seen that: 10 -6 mol / L group, 10 -7 mol / L group, 10 -8 The ROS level in the mol / L group was lower than that in the control group (P<0.05).

[0069] Example 7

[0070] Effects of orientin on sperm acrosome after cryopreservation

[0071] PSA can bind to acrosomal membrane glycoproteins, and FITC is a marker used for immunofluorescence. Using the PSA-FITC combination, the sperm acrosome can be fluorescently labeled under fluorescence excitation, emitting green fluorescence, which can demonstrate the integrity of the sperm acrosome. The judgment criteria are: Acrosome integrity: more than 1 / 2 of the sperm head shows uniform bright fluorescence. Acrosome reaction: only the equatorial plate area has a fluorescent band or the acrosome region is completely devoid of fluorescence. Acrosome abnormality: all sperm except the two types of sperm mentioned above.

[0072] The sperm acrosomes of each group were tested after freezing. The results were as follows: Figure 9-10 shown.

[0073] Figure 9 The results of sperm acrosome integrity in each group after cryopreservation, A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0074] Figure 10 These are the reaction changes of the sperm acrosome in each group after cryopreservation, where control is the control group.

[0075] Depend on Figure 9-10 It can be seen that there was no significant difference in the acrosome integrity of sperm in each group after cryopreservation (P>0.05).

[0076] Example 8

[0077] Effect of orientin on sperm DNA fragmentation index after cryopreservation

[0078] The DNA fragmentation index of sperm in each group after freezing was tested, and the results were as follows: Figure 11-12 shown.

[0079] Figure 11 The DNA fragmentation results of sperm in each group after freezing, among which A is the control group, B is 10 -6 mol / L group, C is 10 -7 mol / L group, D is 10 -8 mol / L group.

[0080] Figure 12 The DFI changes of sperm in each group after cryopreservation, among which control is the control group.

[0081] Depend on Figure 11-12 It can be seen that: 10 -8 The DFI of the 10 mol / L orientin group was lower than that of the control group (P<0.05).

[0082] Example 9

[0083] Effects of orientin on sperm ultrastructure after cryopreservation

[0084] The ultrastructure of sperm before and after cryopreservation was tested. Figure 13 shown.

[0085] Figure 13 The electron microscope images of the sperm ultrastructure before and after cryopreservation, where A is fresh semen before cryopreservation, B is 10 - 8 mol / L orientin-treated group, and C was the control group.

[0086] Depend on Figure 13 It can be seen that in fresh semen before freezing, the chromatin density of the sperm head is uniform, the mitochondria are compactly arranged and orderly, and the vacuoles are small. In the cross-section of the sperm midsection, nine pairs of doublet microtubules and one pair of central microtubules can be clearly observed surrounding the outside of the axon, with mitochondria located on its periphery.

[0087] 10 -8 In the 100 mol / L orientin-treated group, the chromatin density in the sperm head remained uniform, and the gap between the plasma membrane and the nuclear membrane in the post-acrosomal region increased. The mitochondria were neatly arranged but slightly loose and slightly damaged. The sperm midpiece also showed a clear "9+2" structure, and the outer mitochondrial sheath was slightly swollen.

[0088] In the control group, the vacuoles in the sperm head became larger, the plasma membrane was partially ruptured, the mitochondria were swollen, the vacuoles were larger, and some mitochondria were dispersed; the peripheral mitochondrial sheath was swollen.

[0089] As can be seen from Examples 1-9, the present invention adds orientin during the freezing process and finds that the ROS level of sperm is reduced, proving that orientin can alleviate oxidative stress and reduce sperm damage, thereby effectively protecting sperm quality.

[0090] Compared with the control group, the addition of 10 -8 mol / L orientin can reduce the DFI value, suggesting that orientin plays a positive role in protecting sperm DNA integrity. The improvement in DFI may be due to orientin's dual effects: on the one hand, it reduces oxidative damage by controlling ROS levels, potentially directly reducing DNA strand breaks; on the other hand, it may promote damage repair by regulating the activity of DNA repair enzymes (such as PARP and XRCC1). Increased DFI is significantly correlated with decreased fertilization rate and abnormal embryonic development. A lower DFI indicates greater genetic stability in sperm during fertilization, which has positive implications for the outcomes of assisted reproductive technology (ART).

[0091] The experimental group (i.e. 10 -6 mol / L group, 10 -7 mol / L group, 10 -8 mol / L group) added orientin during sperm freezing. Compared with the control group, the motility indicators such as PR, VCL / VSL and MMP levels of sperm increased after thawing. It is speculated that orientin may stabilize the mitochondrial membrane structure, inhibit excessive electron release of the electron transport chain, thereby reducing the generation of ROS, while increasing the activity of mitochondrial respiratory chain complexes I, II, III and IV, thereby enhancing ATP synthesis and thus enhancing sperm motility.

[0092] During the freezing process, ice crystal formation and changes in osmotic pressure can disrupt cell membrane fluidity, leading to irreversible damage such as acrosome rupture and mitochondrial swelling. Electron microscopy revealed that the addition of orientin improved sperm ultrastructure, particularly in terms of cell membrane and mitochondrial structure. Sperm in the experimental group generally maintained an intact plasma membrane-acrosomal complex, with the mitochondrial sheath arranged regularly but slightly loosely and showing minor damage. In contrast, samples from the control group showed decreased chromatin density in the sperm head, enlarged vacuoles, localized plasma membrane rupture, and mitochondrial vacuoles and swelling. Structural integrity is fundamental to sperm function, and this result suggests that orientin not only protects sperm by reducing oxidative stress and improving cellular function but also may further enhance sperm quality by preserving sperm ultrastructure.

[0093] In conclusion, the freezing and thawing process can significantly reduce the vitality and quality of sperm. Orientin can alleviate freezing damage, improve sperm motility, membrane integrity, DNA integrity and mitochondrial function, and reduce oxidative stress response. -8Adding orientin to sperm cryoprotectant at a concentration of 1.5 mol / L significantly improved sperm quality. This suggests that orientin has a good protective effect during sperm freezing; it improves sperm motility and quality through multiple mechanisms, including reducing oxidative stress, enhancing mitochondrial function, and improving sperm ultrastructure.

[0094] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. An application of orientin in improving the quality of frozen sperm and cryopreserving sperm in vitro.

2. The use according to claim 1, characterized in that The application comprises: freezing and preserving semen containing sperm in a freezing preservation solution containing orientin.

3. The use according to claim 2, characterized in that The concentration of orientin in the cryopreservation solution was 1×10 -8 -1×10 -6 mol / L.

4. The use according to any one of claims 1 to 3, characterized in that The volume ratio of semen to the freezing solution containing orientin is 2:1-2.

5. The use according to any one of claims 1 to 4, characterized in that The sperm is human sperm.

6. The use according to any one of claims 1 to 5, characterized in that Improving the quality of frozen sperm includes: reducing sperm freezing damage, improving sperm motility, improving sperm membrane integrity, improving sperm DNA integrity, improving sperm mitochondrial function, and reducing oxidative stress response.

7. A sperm cryopreservation solution, characterized in that: The sperm freezing preservation solution contains orientin.

8. The sperm cryopreservation solution according to claim 7, characterized in that: The concentration of orientin was 1×10 -8 -1×10 - 6 mol / L.

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