Method for improving quality of aged oocytes

Abstract

The invention provides a method for improving the quality of aged oocytes, and belongs to the technical field of biology. According to the method for improving the quality of the aged oocytes, LED light is used for irradiating the aged oocytes, the aged oocytes comprise in-vitro aged oocytes and in-vivo reproductive aged oocytes, and the LED light is red light and infrared light. According to the LED illumination method provided by the invention, the conditions of abnormal distribution of mitochondria, reduction of mitochondrial activity and reduction of ATP (adenosine triphosphate) level in in-vitro aged oocytes are effectively improved, the conditions of excessive accumulation of reactive oxygen species (ROS) in the aged oocytes, spindle abnormality, reduction of fertilization ability and the like are improved, and LED light has the advantages of lower energy consumption, longer service life, simplicity in operation, low cost and the like. And the cost is low.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for improving in vivo reproductive aging and the quality of in vitro aged oocytes. Background Art

[0002] As is well known, high-quality oocytes are a prerequisite for successful fertilization and subsequent embryonic development. However, in most mammals, the main feature of female reproductive aging is a significant decline in the number and quality of follicles and oocytes. Low-quality oocytes are a common and insurmountable problem for older women and are also the main cause of unsatisfactory reproductive outcomes.

[0003] Existing studies have shown that in in vivo reproductive aging and in vitro aged oocytes, the function of mitochondria is severely impaired, including abnormal distribution of mitochondria, reduced ATP production, and decreased membrane potential. Therefore, in order to improve the fertilization efficiency of assisted reproduction and the subsequent embryo quality, it is very important to activate the mitochondrial activity of aged oocytes.

[0004] Near-infrared laser as low-level laser therapy (LLLT) has been widely used in regenerative medicine (such as wound healing, tissue regeneration). Previous research reports have stated that laser or LED irradiation in the red light to near-infrared wavelength range can activate mitochondrial activity. Lasers and ordinary light-emitting diode (LED) lights have significant differences in many aspects such as light source principle, light wave characteristics, directivity, penetrability, energy, and application. Compared with lasers, LED lights have lower energy consumption, longer lifespan, and are simple to manufacture and low in cost. However, there is currently no report on the method of improving in vivo reproductive aging and in vitro aged oocytes by LED light irradiation and the exploration of its internal mechanism. Therefore, it is very necessary to explore the method of improving aged oocytes by LED light irradiation. Summary of the Invention

[0005] The present invention provides a method for improving in vivo reproductive aging and in vitro aged oocytes, which solves the problems such as poor quality of aged oocytes and decreased fertilization ability.

[0006] A method for improving the quality of aged oocytes, wherein the method is to irradiate the aged oocytes with LED light.

[0007] Further, the LED light is red light and infrared light.

[0008] Further, the aged oocytes are in vitro aged oocytes.

[0009] Further, the aged oocytes are in vivo reproductive aged oocytes.

[0010] Furthermore, the wavelengths of the red light and the infrared light are 810 nm to 950 nm.

[0011] Furthermore, the power density of the light irradiation is 20 mW / cm 2 .

[0012] Furthermore, the total energy density of the light irradiation reaches 1.5 J / cm 2 or 5 J / cm 2 .

[0013] Furthermore, the light irradiation time is 75 s or 250 s.

[0014] Furthermore, the in vitro aged oocytes are cultured in a single drop of culture medium.

[0015] Furthermore, the volume of the single drop of culture medium is 20 μL and the diameter is 4 mm.

[0016] Beneficial Effects

[0017] 1. The LED light irradiation method provided by the present invention effectively improves the abnormal distribution of mitochondria, the decrease in mitochondrial activity, and the level of ATP production in in vitro aged oocytes;

[0018] 2. The LED light irradiation method provided by the present invention effectively improves the excessive accumulation of reactive oxygen species (ROS) in in vitro aged oocytes;

[0019] 3. The LED light irradiation method provided by the present invention effectively improves the abnormal spindle in in vitro aged oocytes;

[0020] 4. The LED light irradiation method provided by the present invention effectively improves the fertilization ability of in vitro aged oocytes;

[0021] 5. The LED light irradiation method provided by the present invention significantly reverses the abnormal transcriptome of in vitro aged oocytes;

[0022] 6. The LED light irradiation method provided by the present invention can improve the quality of in vivo reproductive aged oocytes and improve the fertilization ability.

[0023] 7. The LED light used in the present invention has the advantages of low energy consumption, long lifespan, simple operation, and low cost. Description of the Drawings

[0024] Figure 1 To study the transcriptomes of in vitro aged and senescent (in vivo reproductive aging, hereinafter referred to as aging) oocytes using a public database, where Figure 1 A is a heat map showing the gene expression profiles of in vitro aged oocytes and fresh oocytes (log2FC > 1 or < -1, Padj < 0.05); Figure 1B is a volcano plot showing differentially expressed genes (DEGs) in in vitro aged oocytes compared to fresh oocytes, with blue for downregulation and red for upregulation; Figure 1 C is a graph showing the results of GO enrichment analysis of DEGs in fresh and in vitro aged oocytes; Figure 1 D is a heatmap showing gene expression in senescent and young oocytes (log2FC > 1 or < -1, Padj < 0.05); Figure 1 E is a volcano plot showing the results of differentially expressed genes in senescent oocytes compared to young oocytes, with blue for downregulation and red for upregulation; Figure 1 F is a graph showing the results of GO enrichment analysis of DEGs in senescent and young oocytes.

[0025] Figure 2 shows the effects of different irradiation protocols on the mitochondrial membrane potential levels of POA oocytes, where Figure 2 A of is a representative image of POA oocytes. From left to right in the first row are normal and activated, and from left to right in the second row are degenerated and fragmented. The scale bar is 50 μm; Figure 2 B of is a pie chart showing the proportion of abnormal oocytes in oocytes after 24 hours of culture (n = 130, i.e., the number of cells is 130); Figure 2 C of is a timeline for obtaining oocytes in different groups; Figure 2 D of is the detection of mitochondrial membrane potential in fresh, POA, and irradiated POA oocytes by JC-1 staining. The scale bar is 50 μm; Figure 2 E of is to calculate the ratio of red to green fluorescence intensities in fresh (n = 15), POA (n = 15), and irradiated POA (n = 15 for each of the 6 light irradiation protocols) oocytes. The data are presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.001, ****p < 0.0001.

[0026] Figure 3 is a diagram of the device for the light irradiation experiment using an LED device.

[0027] Figure 4 shows the results of the effects of near-infrared light on the morphology, mitochondrial distribution, and function of POA oocytes, where Figure 4 A of is a graph of the results of oocytes in different treatment groups after 6 hours and 24 hours of in vitro culture. The scale bar is 100 μm; Figure 4 B of is a graph of the results of analyzing the proportion of abnormal oocytes (n = 20 for each group); Figure 4 C of is a representative image of mitochondrial distribution in fresh, POA, and irradiated POA oocytes. The oocytes are stained with MitoTracker, and green shows mitochondria. The scale bar is 20 μm; Figure 4Figure D shows the abnormal rate results of mitochondrial distribution in fresh (n = 29), POA (n = 15), and irradiated POA (n = 14 for each group) oocytes; Figure 4 Figure E shows the results of measuring ATP levels in fresh oocytes (n = 20), POA oocytes (n = 20), and irradiated POA oocytes (n = 20); Figure 4 Figure F shows the results of detecting the expression of Drp1 in fresh oocytes (n = 35), POA oocytes (n = 35), and irradiated POA oocytes (n = 35) by RT-PCR; Figure 4 Figure G shows the results of detecting the expression of Mfn1 in fresh oocytes (n = 35), POA oocytes (n = 35), and irradiated POA oocytes (n = 35) by RT-PCR; Figure 4 Figure H shows the results of detecting the expression of Opa1 in fresh oocytes (n = 35), POA oocytes (n = 35), and irradiated POA oocytes (n = 35) by RT-PCR; POA + 810nm: POA oocytes irradiated with 810nm / 1.5*cm -2 POA oocytes irradiated with 950nm / 1.5*cm -2 POA oocytes; Data are presented as the mean percentage (mean ± SEM) of at least three independent experiments, *p < 0.05, **p < 0.01.

[0028] Figure 5 Regarding the effect of near-infrared light on the quality of POA oocytes, where Figure 5 Figure A shows representative images of the levels of reactive oxygen species (ROS) detected by DCFH staining in fresh, POA, and irradiated POA oocytes, with a scale bar of 100μm; where Figure 5 Figure B shows the statistical results of the fluorescence intensity of measuring ROS signals in fresh (n = 5), POA (n = 6), and irradiated POA (n = 5 for each group) oocytes; Figure 5 Figure C shows representative images of the metaphase II spindle morphology and chromosome alignment in fresh, POA, and irradiated POA oocytes, with a scale bar of 20μm; Figure 4 Figure D shows the results of the abnormal rate of abnormal spindles in metaphase II recorded in fresh (n = 10), POA (n = 10), and irradiated POA (n = 10 for each group) oocytes; Figure 5 Figure E shows representative images of early embryos developed from fresh, POA, and irradiated POA cells, with a scale bar of 100μm; Figure 5 Figure F shows the results of recording the pronuclear formation rate (fertilization rate) of fresh (n = 161), POA (n = 169), and irradiated POA (n = 166, 162) groups; Figure 5G is the result graph of the 2-cell (2C) formation rate of the fresh (n = 147), POA (n = 36), and irradiated POA (n = 53, 44) groups; POA + 810nm is the POA oocytes irradiated with 810nm / 1.5*cm -2 ; POA + 950nm is the POA oocytes irradiated with 950nm / 1.5*cm -2 . The data are presented as the mean percentage (mean ± SEM) of at least three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0029] Figure 6 shows the effect of near-infrared light on the transcriptome profile of POA oocytes, where Figure 6 A is the result graph of the PCA analysis of the transcriptome profile, and different colors represent different treatment groups; Figure 6 B is the heatmap showing the comparison results of gene expression in fresh, POA, and irradiated POA oocytes. POA is compared with fresh (log2FC > 1 or < -1, Padj < 0.05) (with the fresh group as the control), and irradiated POA is compared with POA (log2FC > 1 or < 1, Pvalue < 0.05) (with the POA group as the control); Figure 6 C is the volcano plot showing that there are differences in the gene expression screened in irradiated POA oocytes compared with POA oocytes. Some high DEGs are listed, with downregulation in blue and upregulation in orange; Figure 6 D is the Venn diagram showing the overlap of DEGs between 810nm and 950nm irradiations; Figure 6 E is the result graph of the GO enrichment analysis of different gene clusters; POA + 810nm is the POA oocytes irradiated with 810nm / 1.5*cm -2 ; POA + 950nm is the POA oocytes irradiated with 950nm / 1.5*cm -2 .

[0030] Figure 7 shows the result graph of the effect of near-infrared light on reproductive senescent oocytes, where Figure 7 A is the result graph of detecting the mitochondrial membrane potential of young, senescent, and irradiated senescent oocytes by JC-1 staining, and the scale bar is 50μm; Figure 7 B is the result graph of calculating the ratio of red to green fluorescence intensity in young (n = 13), senescent (n = 10), and irradiated senescent (n = 15, 11) oocytes; Figure 7C shows representative images of metaphase II spindle morphology and chromosome alignment in young oocytes, senescent oocytes, and irradiated senescent oocytes. Oocytes were immunostained with an a-tubulin-FITC antibody to visualize the spindle and Hoechst stained to visualize the chromosomes. Scale bar is 20 μm; Figure 7 D shows the results of the abnormal spindle abnormality rate recorded at metaphase II in young (n = 10), senescent (n = 10), and irradiated senescent (n = 10 per group) oocytes; Figure 6 E shows the developmental rates (fertilization rates) of the young (n = 53), senescent (n = 36), and irradiated senescent (41, 39) groups; Aged + 810 nm indicates irradiated senescent oocytes with 810 nm / 1.5*cm -2 Aged + 950 nm indicates irradiated senescent oocytes with 950 nm / 1.5*cm -2 Data are presented as the mean percentage (mean ± SEM) of at least three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001. Detailed implementation

[0031] Example 1.

[0032] I. Bioinformatics analysis to explore whether oocyte aging and in vitro aging status are related to the mitochondrial pathway.

[0033] To dissect the potential mechanisms underlying the decline in the quality of in vitro aged oocytes, we accessed the public RNA-seq database (PRJNA978320) of in vitro aged and fresh oocytes, converted it to count data, and performed differential gene analysis. The screening criteria were set as log2FC > 1 or < -1, Padj < 0.05 to screen out differentially expressed genes between the two groups. The transcriptome profiles of in vitro aged oocytes and fresh oocytes were analyzed using volcano plots and heatmaps for the differentially expressed genes between the two groups. As Figure 1 shown in A and B, the transcriptome profiles of in vitro aged oocytes and fresh oocytes were significantly different. There were 1480 downregulated differentially expressed genes (DEGs) and 1479 upregulated differentially expressed genes (DEGs) in in vitro aged oocytes.

[0034] Subsequently, we performed GO pathway enrichment analysis on the above differentially expressed genes (DEGs) and found that these genes were significantly enriched in mitochondrial-related pathways. This indicates that the decline in the quality of aged oocytes is related to changes in mitochondrial function, as Figure 1 shown in C.

[0035] To further explain the potential mechanism of the decline in the quality of aging oocytes, we accessed the public RNA-seq database (GSE239551) of oocytes from the aging group and the young group, performed differential expression gene pathway enrichment analysis, and found that it was also significantly enriched in mitochondrial-related pathways, such as Figure 1 D, E, and F of

[0036] Therefore, both oocyte aging and in vitro aging status are related to the mitochondrial pathway.

[0037] Second, screen and determine the wavelength and energy of LED light that can effectively improve mitochondrial activity.

[0038] Under normal circumstances, mouse oocytes at the MⅡ stage (metaphase of the second meiosis, capable of fertilization) are characterized by clear cytoplasm, a tight zona pellucida, and a clear first polar body. In in vitro aged mouse oocytes, various morphological defects will occur, including spontaneous parthenogenetic activation, degeneration, fragmentation, etc. of the oocytes. Therefore, these morphological defects are used as indicators to evaluate the aging of in vitro aged mouse oocytes.

[0039] Mouse (6 - 8 weeks) MⅡ stage oocytes cultured in KSOM medium for 24 hours are generally referred to as post-ovulatory in vitro aged oocytes (hereinafter referred to as POA oocytes). After culturing mouse (6 - 8 weeks) MⅡ stage oocytes in KSOM medium for 24 hours, the status of the oocytes was counted ( Figure 2 A of Figure 2 B of

[0040] Six different red light and infrared light irradiation schemes were designed, namely: 630nm / 1.5J*cm -2 、810nm / 1.5J*cm -2 、950nm / 1.5J*cm -2 、630nm / 5J*cm -2 、810nm / 5J*cm -2 and 950nm / 5J*cm -2 。The oocytes were divided into three groups: fresh group, POA group, and irradiated POA group. The operation procedures for obtaining the three groups of oocytes are as shown in Figure 2 C of On the first day, 6 - 8 week old mice were superovulated and MⅡ oocytes were collected, and then POA group oocytes were obtained after culturing for 24 hours; the above MⅡ oocytes were cultured for 18 - 21 hours and then irradiated with LED light (red light and infrared light), and then cultured for another 24 hours to obtain irradiated POA group oocytes; on the second day, 6 - 8 week old mice were superovulated and MⅡ oocytes were collected to obtain fresh group oocytes.

[0041] Red light and infrared light irradiation experiments are as follows Figure 3 shown. Three groups of oocytes were cultured in a single drop of culture medium (20 μL, 4 mm in diameter). Considering the non-uniformity from the LED light source, we controlled the size of the culture drop to minimize the intensity variation to <10%. And paraffin oil was covered on each drop to prevent osmotic pressure changes caused by evaporation.

[0042] In this study, an LED light irradiation device was used, which emits red light and near-infrared light of different wavelengths. The light beam irradiates the oocytes from the top of the culture dish. The parameters on the device were adjusted. The specific parameters are shown in Table 1. By adjusting the parameters of the LED light irradiation device, light of different wavelengths was irradiated on the oocytes at a power density of 20 mW / cm 2 for 75 s or 250 s, so that the total energy density reached 1.5 J / cm 2 or 5 J / cm 2 .

[0043] Table 1

[0044]

[0045] After in vitro aging of MⅡ-phase mouse oocytes, their mitochondrial activity and function will be damaged. The changes in their activity and function can be reflected by the changes in the mitochondrial membrane potential. That is, when the membrane potential decreases, it represents a decrease in its activity. Therefore, we performed JC-1 staining on three groups of oocytes to reflect the mitochondrial membrane potential. By observing the ratio of their red and green fluorescence, mitochondria with high membrane potential showed red fluorescence, and mitochondria with low membrane potential showed green fluorescence, to determine the wavelength and energy of the LED light that can effectively improve in vitro aged oocytes.

[0046] As Figure 2 shown in D and E, compared with the fresh group, the membrane potential of the POA group did show a certain decrease, indicating that the activity and function of mitochondria were damaged to a certain extent. And when irradiating POA oocytes with 810 nm / 1.5 J*cm -2 or 950 nm / 1.5 J*cm -2 , it can effectively improve the decrease in mitochondrial membrane potential caused by in vitro aging, thereby delaying the decline of mitochondrial function in in vitro aged mouse oocytes. Therefore, in subsequent experiments, these two irradiation schemes were selected for further analysis.

[0047] Example 2. Evaluate the improvement effect of different irradiation schemes on oocytes.

[0048] I. The proportion of morphologically defective cells in the total number of cells.

[0049] After culturing fresh group oocytes for 18 - 21 hours, red light and infrared light irradiation were carried out. The irradiation protocol was to use 810nm / 1.5J*cm - 2 or 950nm / 1.5J*cm - 2, and observe whether infrared light irradiation can rescue morphological defects caused by in vitro aging, such as spontaneous parthenogenetic activation, degeneration, fragmentation, etc.

[0050] The results are as Figure 4 shown in A and B of, the proportion of cells with morphological defects caused by in vitro aging in the total number of cells cannot be effectively reduced by red light and infrared light irradiation.

[0051] II. Mitochondrial distribution in different oocytes.

[0052] The distribution pattern of mitochondria was observed by MitoTracker staining. In the fresh group, the mitochondrial distribution characteristics were cumulative distribution around the chromosomes and uniform distribution in the cytoplasm. However, in the POA group, a large number of mitochondria partially or completely lost the cumulative distribution around the chromosomes and showed a missing or aggregated distribution in the cytoplasm ( Figure 4 shown in C and D of). Quantitatively, nearly 40% of POA oocytes showed mislocalization of mitochondria. This abnormal distribution of mitochondria indicates that mitochondrial dynamics are impaired in aged oocytes, leading to impaired mitochondrial function.

[0053] To verify the improvement effect of the two irradiation protocols, fresh group oocytes were cultured for 18 - 21 hours and then irradiated with red light and infrared light (810nm / 1.5J*cm - 2 or 950nm / 1.5J*cm - 2), and the results are as Figure 4 shown in C and D of, both irradiation protocols can improve the abnormal distribution of mitochondria to a certain extent, and the improvement effect of the 950nm / 1.5J*cm - 2 irradiation protocol is more significant.

[0054] III. ATP levels in different oocytes.

[0055] The most important function of mitochondria is to produce ATP for cell development. By measuring the ATP content in fresh group, POA group, and irradiated POA group oocytes, the results are as Figure 4 shown in E of. Compared with fresh oocytes, the ATP level of POA oocytes decreased significantly, but after treatment with infrared light irradiation (810nm / 1.5J*cm - 2 or 950nm / 1.5J*cm - 2), the ATP level had a certain recovery, and the recovery effect was significant.

[0056] The reason for the decrease in ATP levels in POA oocytes is that mitochondrial aggregation caused by impaired mitochondrial dynamics ultimately damages mitochondrial function, resulting in reduced ATP production.

[0057] IV. Regulate the expression of genes related to mitochondrial fusion and fission.

[0058] The expression levels of Drp1, Mfn1, and Opa1 in oocytes of the fresh group, POA group, and irradiated POA (810 nm / 1.5 J*cm - 2; 950 nm / 1.5 J*cm - 2) were detected by RT-PCR. Opa1 and Mfn1 are genes that regulate mitochondrial fusion, and Drp1 is a gene that regulates mitochondrial fission. The results are as Figure 4 shown in Figures F, G, and H. The relative expression levels of all three genes in the POA group decreased significantly, indicating that the above three genes are misexpressed in aged oocytes. However, after treatment with red light and infrared light irradiation, all three genes were restored to some extent. After treatment with red light and infrared light irradiation at 810 nm / 1.5 J*cm - 2, the relative expression level of the gene Drp1 was comparable to that of fresh oocytes; after treatment with infrared light irradiation at 810 nm / 1.5 J*cm - 2 or 950 nm / 1.5 J*cm - 2, the relative expression level of the gene Mfn1 was comparable to that of fresh oocytes; after treatment with red light and infrared light irradiation at 810 nm / 1.5 J*cm - 2, the relative expression level of the gene Opa1 was restored to a level comparable to that of fresh oocytes, and after treatment with red light and infrared light irradiation at 950 nm / 1.5 J*cm - 2, its relative expression level also had a significant recovery. Therefore, treatment with red light and infrared light irradiation can indeed improve the decline in mitochondrial activity in POA oocytes.

[0059] V. The accumulation of ROS in oocytes.

[0060] The aging of mouse oocytes is closely related to the occurrence of oxidative stress, which refers to a state of imbalance between oxidation and antioxidant action in the body. After mouse oocytes age, excessive electron leakage occurs in their mitochondrial respiratory chain, and these leaked electrons combine with oxygen molecules in mouse oocytes, thereby generating excessive reactive oxygen species (ROS). Although under normal circumstances, mouse oocytes have a certain ability to scavenge ROS, POA oocytes cannot effectively scavenge excessive oxygen free radicals due to impaired antioxidant capacity. Excessive ROS accumulate continuously in mouse oocytes, damaging proteins, DNA, and lipids in mouse oocytes, thereby leading to a decline in the quality of mouse oocytes and having an adverse impact on the quality of mouse oocytes. Therefore, reducing the level of ROS in mouse oocytes and avoiding the occurrence of oxidative stress are of great significance for delaying the aging of mouse oocytes. Thus, we observed whether the above two irradiation regimens could effectively improve the excessive accumulation of ROS in POA oocytes by DCFH staining for ROS. As Figure 5 shown in A and B, excessive accumulation of ROS indeed occurs in POA oocytes, and when irradiated with red light and infrared light (810 nm / 1.5 J*cm - 2 or 950 nm / 1.5 J*cm - 2), this adverse accumulation can be significantly rescued.

[0061] VI. Abnormalities of spindles in oocytes.

[0062] We detected the distribution of spindles in fresh, POA, and irradiated POA group oocytes by α-Tublin FITC immunofluorescence staining. Spindles are closely related to their maturation ability, fertilization ability, and subsequent embryonic development. In POA oocytes, the spindle microtubules are asymmetrically arranged and entangled, and the chromosomes are inconsistently arranged, which is called spindle abnormality, as Figure 5 shown in C and D. The spindle abnormalities are significantly reduced by irradiation with red light and infrared light.

[0063] VII. Fertilization ability of oocytes.

[0064] The intracytoplasmic sperm injection (ICSI) technique was used to detect the effects of near-infrared light irradiation on the fertilization potential of oocytes and their subsequent development.

[0065] The results are as Figure 5 shown in E, F, and G. According to the decrease in the developmental rate of POA oocytes at the 2-cell (2C) stage, that is, the pronuclear (PN) formation rate (i.e., the fertilization rate) of the POA group was significantly lower than that of the fresh group. However, after near-infrared light irradiation, this low fertilization rate will be significantly improved, especially at 810 nm / 1.5 J*cm -2 Process it. And near-infrared light irradiation can also improve the developmental potential of the 2-cell (2C) stage development rate, although through 950 nm / 1.5 J*cm - The improvement in the developmental potential of the 2-cell (2C) stage development rate after near-infrared light irradiation is not significant. Here, the developmental rate after the 2C stage is not discussed because in vitro aging for 24 hours is devastating to the development after the 2C stage and even near-infrared light irradiation cannot reverse it. The statistical analysis of the ICSI development rate is shown in Table 2. The above results indicate that near-infrared light irradiation can reduce the fertilization ability of POA oocytes and promote their subsequent 2C embryo development.

[0066] Table 2

[0067] Group Sum PN(%) 2C(％) Blastocyst(%) Fresh 161 147(91.16±1.8)**** 119(81.23±3.5)**** 101(68.73±2.0) POA 24h 169 36(21.32±1.4) 3(8.68±5.0) 810 / 1.5 166 53(31.78±2.78)*** 13(24.88±6.0)** 950 / 1.5 162 44(27.08±2.5)* 6(13.71±4.4)

[0068] Note: 147(91.16±1.8)****, 147 is the number of cells, and 91.16±1.8 is the percentage.

[0069] VIII. Transcriptome analysis.

[0070] To further explore the potential mechanism of the effect of near-infrared light irradiation on the quality of POA oocytes, we performed transcriptome analysis on oocytes in the fresh, POA, and irradiated POA (810 nm / 1.5 J*cm - 2 or 950 nm / 1.5 J*cm - 2) groups and obtained 4532 DEGs.

[0071] The results of principal component analysis (PCA) are as Figure 6 shown in A, indicating that there are obvious differences in the transcriptome profiles between POA oocytes and fresh oocytes, and near-infrared light irradiation can appropriately reduce this difference.

[0072] Then differential expression analysis was performed between the POA group and the fresh group. The above 4532 DEGs (including 2105 down-regulated differentially expressed genes and 2427 up-regulated differentially expressed genes) were continued for differential expression analysis between the POA group and the irradiated POA group. As Figure 6 shown in B, it shows the expression of these DEGs in different groups, and these genes can be divided into four clusters according to their expression: "up-normal", "up-down", "down-normal", and "down-up". However, according to the filtering threshold we established, there will be no "up-up" and "down-down" clusters, which indicates that near-infrared light irradiation can effectively reduce the abnormal expression of DEGs in POA. As Figure 6 shown in C, near-infrared light irradiation has a significantly stronger up-regulation effect on down-regulated DEGs in POA than the down-regulation effect on up-regulated DEGs in POA. As Figure 6D display, 810 nm / 1.5 J*cm - 2 and 950 nm / 1.5 J*cm - 2 irradiations both upregulated 494 DEGs in POA oocytes (494 DEGs among the downregulated DEGs), indicating that these two irradiations have similar and comparable improvement effects on the abnormally downregulated transcriptome observed in POA. This also suggests the importance of genes abnormally downregulated in POA for oocyte quality.

[0073] Based on Gene Ontology (GO) analysis of DEGs (Cluster “up-down” & “down-up”), the results are as Figure 6 shown in E. These genes that were abnormally expressed in POA and significantly recovered after light irradiation were significantly associated with mitochondrial-related pathways.

[0074] In particular, we noticed that these clusters were also significantly enriched in ribosome-related pathways, indicating that near-infrared light irradiation can also specifically improve the abnormal changes of ribosomes in POA, which has certain significance for our future exploration of the effects of near-infrared light irradiation on ribosome function.

[0075] In summary, these results indicate that near-infrared light irradiation can significantly reverse the abnormal transcriptome in POA.

[0076] IX. Near-infrared light irradiation can improve the quality of reproductively aged oocytes.

[0077] The above all explored in vitro aged oocytes. Here, we also verified whether these two light irradiation regimens can also improve in vivo reproductively aged oocytes, which is crucial for the improvement of assisted reproduction in the elderly population.

[0078] Young (6 - 8 weeks old) and old (6 - 8 months old) ICR female mice were superovulated to obtain oocytes in the young group and the senescent group.

[0079] As Figure 7 shown in A and B of, JC-1 staining was performed on young, senescent (in vivo reproductively aged) and irradiated senescent oocyte groups. The results showed that both of these two near-infrared light irradiation regimens could significantly improve the decline of mitochondrial membrane potential, suggesting that these two near-infrared light irradiations can also reverse the decline of mitochondrial activity in reproductively aged oocytes. As Figure 7 shown in C and D of, its effects on quality-related indicators, the distribution of spindles were examined. The results showed that near-infrared light irradiation can improve the abnormal distribution of spindles.

[0080] Finally, the ICSI developmental rates of different groups were examined. The results are as Figure 7E showed that these near-infrared light irradiations could not effectively improve the decline in the blastocyst development rate of oocytes from aged mice. This may be due to the premature timing of the irradiation, resulting in its beneficial effects not being able to affect the development at the blastocyst stage. These results indicated that near-infrared light irradiation could also activate the mitochondrial activity of reproductively aged oocytes and slightly improve their quality. The statistical analysis of the ICSI development rates in different experimental groups is shown in Table 3.

[0081] Table 3

[0082] Group Sum PN(%) 2C(％) Blastocyst(%) Young 53 46(87.22±4.3) 37(80.57±4.4) 28(60.62±3.1)* Aged 36 30(83.13±7.5) 23(77.04±8.5) 8(27.6±7.0) 810 / 1.5 41 35(85.50±4.8) 24(67.33±8.5) 12(32.3±16.0) 950 / 1.5 39 33(84.13±2.9) 24(74.47±9.28) 9(29.4±9.4)

[0083] Note: 46(87.22±4.3), 46 is the number of cells, and 87.22±4.3 is the percentage.

Claims

1. A method for improving the quality of aging oocytes, characterized in that: The method comprises irradiating aged oocytes with LED light.

2. The method according to claim 1, characterized in that The LED light is red light and infrared light.

3. The method according to claim 1, characterized in that The aged oocytes are in vitro aged oocytes.

4. The method according to claim 1, characterized in that: The aged oocytes are in vivo reproductive aged oocytes.

5. The method according to claim 1, characterized in that The wavelengths of the red light and the infrared light are 810nm to 950nm.

6. The method according to claim 1, characterized in that The power density of the illumination is 20 mW / cm 2 .

7. The method according to claim 1, characterized in that The total energy density of the illumination reaches 1.5 J / cm 2 or 5J / cm 2 .

8. The method according to claim 1, characterized in that The illumination time is 75s or 250s.

9. The method according to claim 1, characterized in that: The in vitro aged oocytes were cultured in a single drop of culture medium.

10. The method according to claim 9, characterized in that The volume of the single drop of culture medium is 20 μL and the diameter is 4 mm.

Images