Application of spermidine in preparation of medicine for improving ovary iron overload

Abstract

The invention discloses application of spermidine in preparation of a medicine for improving ovarian iron overload, and relates to the technical field of biological medicine. The concentration of the spermidine in the medicine is 3mmol / L, and the spermidine with the concentration of 3mmol / L can improve ovarian endocrine dysfunction caused by iron overload, increase the number of follicles at all levels, reduce the number of atresia follicles, promote follicle development and regulate the expression of antioxidant and ferroptosis related proteins. According to the invention, spermidine is utilized, so that a new treatment thought is provided for follicular dysplasia caused by environmental factors, a new way is opened up for preventing and treating ovarian function impairment caused by iron overload, improving the reproductive performance of mammals and promoting overall health, and the problems of follicular atresia and ovarian function decline caused by environmental pollution are effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of spermidine in preparing a medicine for improving ovarian iron overload. Background Art

[0002] Iron is a transition metal and an essential trace element in almost all organisms. It is also an important component of hemoglobin and plays a variety of functions in the body. Iron overload can cause oxidative stress damage and physiological dysfunction in cells and tissues, ultimately leading to cell ferroptosis. Ferroptosis is an iron-dependent non-apoptotic cell death. It is essentially a cell death caused by the failure of membrane lipid repair enzyme activity, disordered lipid peroxide metabolism in cells, and lethal accumulation of iron-dependent lipid reactive oxygen free radicals, which leads to the destruction of intracellular redox balance. So far, it has been found that ferroptosis exists in a variety of reproductive pathological processes and germ cell aging, and participates in the regulation of granulosa cell oxidative stress and metabolism, oocyte development, early embryonic development, etc., reducing the reproductive function of female animals. In addition, iron overload-induced ferroptosis is one of the main factors for follicular atresia and decreased reproductive performance in female animals, which seriously restricts the development of female animals' reproductive potential.

[0003] Spermidine is a polycation widely present in organisms and has multiple biochemical activities, including regulation of gene expression, ion channels, mitochondrial Ca 2+ Transport, autophagy induction, programmed cell death, etc. Science reports that dietary intake of exogenous spermidine is an effective reactive oxygen scavenger. Studies have shown that spermidine is the key to the realization of reproductive functions in various mammals, and is closely related to oogonium formation, embryogenesis, implantation, and placenta formation. In the physiological process of women, autophagy, as an important factor in regulating ovarian function, participates in folliculogenesis, ovulation, and corpus luteum development, and improves fertility and reproductive health. The key to the survival of germ cells during development is autophagy, which occurs before the development of the primordial follicle pool. Spermidine is used as a natural activator of autophagy as a food supplement to maintain mitochondrial function. It is a natural endogenous polyamine synthesized from putrescine diamine. In addition, spermidine has the effects of anti-oxidative stress, improving mitochondrial function and regulating cell apoptosis. Spermidine induces protective autophagy in female germline stem cells in vitro and improves germline stem cell aging. In female animals, the reduction of spermidine levels in the ovaries leads to follicle development stagnation. In short, spermidine has a very important potential in improving animal reproductive performance. Currently, there is no research related to the effect of spermidine on ovarian follicle development caused by iron overload in the prior art. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of spermidine in the preparation of a drug for improving ovarian iron overload, which effectively solves the problems of follicular atresia and ovarian function decline caused by environmental pollution.

[0005] To achieve the above object, the technical solution adopted by the present invention to solve the technical problem is: to provide the use of spermidine in the preparation of a drug for improving ovarian iron overload.

[0006] Furthermore, the concentration of spermidine in the drug is 2.5-3.5 mmol / L.

[0007] Furthermore, the concentration of spermidine in the drug is 3 mmol / L.

[0008] Furthermore, the drug is administered orally.

[0009] Furthermore, iron overload is iron overload caused by excessive intake of exogenous iron.

[0010] Furthermore, the application is at least one of the following (1)-(4):

[0011] (1) Improve ovarian function;

[0012] (2) Increase the number of follicles at all levels;

[0013] (3) reduce the number of atretic follicles;

[0014] (4) Promote follicle development.

[0015] A drug for improving ovarian iron overload comprises spermidine and pharmaceutically acceptable excipients.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention provides the use of spermidine in the preparation of a drug for improving ovarian iron overload. Spermidine at a concentration of 3 mmol / L can alleviate the problem of follicular hypoplasia in mice caused by iron overload, and improve ovarian endocrine function, which is specifically manifested in a significant increase in the number of follicles at all levels, a significant decrease in atretic follicles, relief of excessive autophagy in the iron-overloaded ovaries, and differential expression of antioxidant and ferroptosis-related proteins (FHC / GPX4 / ACSL4).

[0018] 2. The present invention provides a method for improving follicular development and ovarian endocrine function in iron-overloaded ovaries, provides a reference for the diagnosis and treatment of patients with ovarian dysfunction caused by environmental factors in clinical practice, and has important theoretical guiding significance for the treatment of female reproductive diseases caused by iron overload. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the animal model constructed for the present invention;

[0020] Figure 2 Representative images of the ovarian morphology of mice in each group;

[0021] Figure 3 Results of body weight, food intake and ovarian index of mice;

[0022] Figure 4 The results are as follows: The effect of spermidine on serum and ovarian iron levels in mice;

[0023] Figure 5 The results of vaginal smear test for estrous cycle in mice;

[0024] Figure 6 Representative images of HE staining of mouse vaginal smears;

[0025] Figure 7 This is a graph showing the regularity of the estrous cycle in mice;

[0026] Figure 8 The results are as follows: The effects of spermidine on serum sex hormones in iron-overloaded mice;

[0027] Fig. 9 This is the HE staining picture of mouse ovarian tissue section;

[0028] Fig.10 The results of the follicle count analysis were:

[0029] Fig.11 This is the ultrastructure of mouse ovarian granulosa cells;

[0030] Fig.12 The results are as follows: The effects of spermidine on the antioxidant enzyme activities in the ovaries of iron-overloaded mice;

[0031] Fig.13 This is a diagram showing the results of Western blotting of proteins related to anti-oxidation, ferroptosis and autophagy in mouse ovaries;

[0032] Fig.14 This is a graph showing the quantification results of proteins related to antioxidant, ferroptosis and autophagy in mouse ovaries. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0034] Example 1

[0035] 1. Preparation of experimental animals and feeding management

[0036] 125 7-week-old female specific pathogen-free ICR mice weighing 20.0-21.5 g were purchased from Chengdu Enswell Biotechnology Co., Ltd. All animals were given free access to food and water in the animal room of the Animal Management Center of Sichuan Agricultural University. The ambient temperature was 22±2°C, the relative humidity was 60%-70%, and the light-dark cycle was 12 h per day. All animal experimental protocols were approved by the Animal Care and Use Committee of Sichuan Agricultural University.

[0037] After 125 female mice were adaptively fed for 1 week, they were randomly divided into 5 groups (n=25 / group): control group (0.045g / kg), medium iron concentration group (MFe group, 0.55g / kg), high iron concentration group (HFe group, 1.350g / kg), high iron concentration + 3mmol / L spermidine treatment group (3SH group), high iron concentration + 6mmol / L spermidine treatment group (6SH group). The mice were fed with different concentrations of ferrous sulfate diets, spermidine dissolved in sterile water for drinking water, and the control group was fed with the same amount of sterile water for 8 weeks. The weight and food intake of mice were recorded. The estrus cycle was recorded by vaginal smear from the 7th week. After feeding, blood was collected through the orbital venous plexus under ether anesthesia, and the serum was separated and stored for later use. After blood collection, the mice were killed by cervical dislocation, and the ovarian tissue was collected and weighed. One side of the ovary was fixed with a paraformaldehyde aqueous solution for making slices, and the other side of the ovary was frozen in liquid nitrogen for later use. Schematic diagram of the animal model Figure 1 shown.

[0038] 2. Prepare the main reagents and instruments for the experiment

[0039] The main reagents used in the experiment are shown in Table 1.

[0040] Table 1 Main reagents

[0041]

[0042]

[0043] The main instruments used in the experiment are shown in Table 2.

[0044] Table 2 Main instruments

[0045]

[0046] Example 2 Effect of spermidine on body weight and ovarian index of mice

[0047] 1. Quantitative detection of iron content in feed

[0048] Refer to the synthetic feed formula for rodent experimental animals recommended by the Association of Official Analytical Chemists (AOAC), and adjust the formula appropriately and reasonably, remove the iron in the inorganic salt in the original formula, and add different doses of ferrous sulfate to the feed. The quantitative detection of trace element iron content in feed refers to the national GB / T13885-2017 "Determination of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc content in feed by atomic absorption spectrometry". The steps are as follows: weigh about 0.2-0.3g of feed sample, put it into an ordinary tetrafluoroethylene digestion tank, add 10mL nitric acid-perchloric acid mixed acid (nitric acid: perchloric acid = 4:1) and hydrolyze at room temperature overnight; place it on a hot plate at 160℃ for 2h, add 8mL nitric acid and 2mL perchloric acid after cooling, continue heating to digest into colorless, evaporate most of the acid, and the acid remover concentrates the volume of the acid to nearly 1mL and then cools to room temperature, and then transfers it to a 50mL volumetric flask. According to the estimated lower limit of the content of the measured elements in the feed, 0.5% nitric acid was added to the volume and mixed for testing, and a blank test was performed at the same time. The national standard sample (standard value: 1000 μg / mL) was purchased from the National Nonferrous Metals and Electronic Materials Analysis and Testing Center (iron: GSB 04-1726-2004). An atomic absorption spectrometer (CONTRAA 700; Analytik Jena AG, Jena, Germany) was used for detection.

[0049] The iron ion content in ferrous sulfate feeds with different concentrations was detected by flame atomic absorption spectrometry. The results are shown in Table 3. According to the national trace element detection standard, the error was <15%, and the iron content in the feed met the requirements of this experiment.

[0050] Table 3 Quantitative detection results of iron ion content in mouse feed

[0051]

[0052] 2. Effects of spermidine on body weight and ovarian index of mice

[0053] The mice were fed with different concentrations of ferrous sulfate diets and spermidine dissolved in sterile water for drinking. The control group was fed with the same amount of sterile water for 8 weeks. The body weight and food intake of mice in the control group (0.045 g / kg), medium iron concentration group (MFe group, 0.55 g / kg), high iron concentration group (HFe group, 1.350 g / kg), high iron concentration + 3 mmol / L spermidine treatment group (3SH group) and high iron concentration + 6 mmol / L spermidine treatment group (6SH group) were recorded. The changes in the body weight and food intake of mice and the results of ovarian index were as follows: Figure 2 As shown, Figure 2 In the figure, A is the weight change of mice, B is the food intake change of mice, and C is the ovarian index result of mice.

[0054] After feeding, blood was collected through the orbital venous plexus under ether anesthesia, and the serum was separated and stored for later use. After blood collection, the mice were killed by cervical dislocation, and the ovarian tissue was collected and weighed. Representative images of the ovarian morphology of mice in each group are shown in the figure. Figure 3 As shown, Figure 3 In the figure, from left to right are the results of the control group, MFe group, HFe group, 3SH group, and 6SH group.

[0055] Depend on Figure 2-Figure 3 It can be seen that after 8 weeks of feeding with different concentrations of ferrous sulfate feed, all mice survived. Compared with the control group, the weight of mice in the medium concentration iron group (0.55g / kg) increased steadily, with no significant effect on body weight (P>0.05); with the increase of ferrous sulfate treatment time, the weight of mice gradually decreased. After the end of the experiment, the weight of mice in the HFe group (1.35g / kg) was lower than that in the Control group (P<0.01). 3mmol / L and 6mmol / L spermidine intervention significantly increased the weight gain of iron-overloaded mice (P>0.05). The results showed that high concentrations of iron caused the weight of mice to decrease, and spermidine could increase the weight gain of iron-overloaded mice. Compared with the control group, the ovaries of mice in the HFe group became darker in color and decreased in size, while the ovarian morphology of the MFe group did not change significantly. The ovarian index of mice in the HFe group was significantly lower than that of the control group and the MFe group (P<0.05). The ovarian index of the 3SH group was significantly increased compared with the HFe group, and the ovarian index of the 6SH group showed an upward trend, but there was no significant statistical difference (P>0.05).

[0056] Example 3 Detection of iron content in mouse serum and ovaries

[0057] The detection of serum iron ion content in mice was carried out according to the manufacturer's commercial kit instructions, serum iron determination kit (A039-1-1, Nanjing Jiancheng). The detection of ovarian tissue iron ion content was carried out according to the instructions of Nanjing Jiancheng tissue iron determination kit (Nanjing Jiancheng, A039-2-1).

[0058] Flame atomic absorption spectrometry was used to detect serum iron ion levels, and colorimetry was used to detect iron ion levels in ovarian tissue. Figure 4 As shown, Figure 4 In the figure, A is the serum iron level, and B is the iron level in ovarian tissue.

[0059] Depend on Figure 4 It can be seen that compared with the control group, there was no significant change in serum and ovarian iron content in the MFe group (P>0.05), and the serum and ovarian iron content in the HFe group was significantly increased (P<0.01); compared with the HFe group, 3mmol / L spermidine combined treatment significantly reduced the high iron-induced increase in serum and ovarian iron ion levels, and 6mmol / L spermidine combined treatment significantly inhibited the ovarian iron ion level.

[0060] Example 4 Effect of spermidine on ovarian endocrine function in iron-overloaded mice

[0061] The endocrine function of the mouse ovary can be reflected by evaluating the estrous cycle and sex hormone levels.

[0062] 1. Detection of estrus cycle

[0063] The estrus cycle was observed by vaginal smear from the 7th week and continued for 3 weeks. Ten mice were selected from each group for estrus cycle monitoring. Each mouse was housed individually, and vaginal smears were taken from the mice at the same time every morning to detect the estrus cycle. A complete estrus cycle of normal female mice is 4-5 days, with four stages: proestrus, estrus, metestrus and diestrus. Figure 5 As shown, Figure 5 In the figure, each row from top to bottom represents the estrus cycle of 3 replicate mice in each group, where ordinate 1 represents proestrus, 2 represents estrus, 3 represents metestrus, and 4 represents diestrus (n=10). Representative images of mouse vaginal smears stained with HE are shown in Figure 1. Figure 6 As shown, Figure 6 In the figure, from top to bottom and from left to right, they are HE-stained images of proestrus, estrus, metestrus, and diestrus. Scale bar, 50 μm. Figure 7 As shown, *P<0.05, **P<0.01.

[0064] Depend on Figure 5-Figure 7 It can be seen that 92.5% of the mice in the control group had regular estrus cycles, and 83.7% of the mice in the MFe group had regular estrus cycles compared with the control group, with no significant changes (P>0.05). Only 35.6% of the mice in the HFe group had regular estrus cycles, which was significantly lower than the control group and MFe group (P<0.05). 63.1% of the mice in the 3SH group had regular estrus cycles, and the proportion of estrus cycles was significantly higher than that in the HFe group; 45.5% of the mice in the 6SH group had regular estrus cycles, which had an upward trend compared with the HFe group, but there was no statistical difference (P>0.05).

[0065] 2. ELISA determination of serum hormone levels

[0066] According to the instructions of the mouse estradiol ELISA kit (Mlbio, ml001962, Shanghai, China) and the progesterone ELISA kit (Mlbio, ml057778, Shanghai, China), serum samples of each group of mice were collected. Then, 80 μL of HRP detection antibody was added to the wells of the sample to be tested and the wells of the standard, incubated at 37°C for 1 hour, covered with washing solution, shaken for 30 seconds, and washed 5 times. 50 μL of substrate A and B were added to each well, incubated at 37°C for 10 minutes, and the OD value was detected at 450nm. The actual concentration of the sample was calculated according to the standard curve. The sensitivity of estradiol and progesterone detection was 0.1 pmol / L and 0.1 ng / mL, respectively. The intra-batch / inter-batch coefficients of variation were less than 10% and 15%. All samples with estradiol levels greater than 64 pmol / L were diluted before repeated measurements to obtain results within the maximum accuracy range. The results are shown in the figure. Figure 8 As shown, Figure 8 In the figure, A is the serum estradiol level of mice, and B is the serum progesterone level of mice.

[0067] Depend on Figure 8 It can be seen that compared with the control group, the ovarian E2 and P levels in the HFe group were significantly decreased (P<0.01), the estradiol and progesterone levels in the 3SH group were significantly higher than those in the HFe group (P<0.05), the estradiol level in the 6SH group was significantly higher than that in the HFe group, and the progesterone level showed an upward trend but no statistical difference (P>0.05). In summary, 3mmol / L spermidine intervention significantly promoted the secretion of estradiol and progesterone, and partially improved the endocrine dysfunction of the ovary caused by iron overload.

[0068] Example 5 Effect of spermidine on ovarian tissue morphology in high-iron mice

[0069] To investigate the effect of spermidine on the morphology of ovarian tissue in high-iron mice, each ovarian sample of the mouse was fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, and embedded in paraffin. These samples were then cut into 4 μm slices at intervals of 5 μm, and slices were taken every 80 μm until the last slice. HE staining was performed, and the follicles were counted under a high-power microscope (×100). The HE staining results are shown in the figure. Fig. 9 As shown, Fig. 9 In the figure, CL indicates corpus luteum, AF indicates atretic follicle; green arrows point to growing follicles, black arrows point to primordial follicles, scale bar, 100 μm (upper) or 50 μm (lower). Fig.10 As shown, Fig.10 In the figure, A to D are the counting results of primordial follicles, primary follicles, secondary follicles and atretic follicles respectively (n=4 in each group).

[0070] Depend on Fig. 9 and Fig.10It can be seen that the number of secondary follicles in the MFe group was significantly reduced compared with the control group (P<0.05); the number of ovarian follicles in the HFe group was significantly reduced, which was manifested by a decrease in the number of primordial, primary, and secondary follicles, a decrease in the number of corpora lutea, and an increase in the number of atretic follicles (P<0.05). The number of primordial, primary, and secondary follicles in the 3SH group was significantly increased compared with the HFe group, and the number of atretic follicles was significantly decreased (P<0.01). The number of primordial follicles and corpora lutea in the 6SH group was significantly increased, and the number of atretic follicles was decreased (P<0.05).

[0071] Example 6 Transmission electron microscopy observation of cell ultrastructure

[0072] Ovarian tissue was collected and fixed with 3% glutaraldehyde (Sigma-Aldrich, 49626) at 4°C for 24 h. It was then dehydrated with a gradient ethanol series, embedded in epoxy resin, and then sliced ​​into 60 nm sections. Uranyl acetate and lead citrate were stained at room temperature for 15 min, and images were observed and collected using a transmission electron microscope (JEM-1400FLASH, JEOL, Japan). The ultrastructure of ovarian granulosa cells is shown in Figure 2. Fig.11 As shown, Fig.11 In the figure, white arrows indicate autophagosomes, white arrows indicate autolysosomes, and black arrows indicate mitochondria, which are characteristic of primary ferroptosis (n=3).

[0073] Depend on Fig.11 It can be seen that compared with the control group, the mitochondrial autophagy of ovarian granulosa cells in the MFe group increased; the granulosa cells in the HFe group had shrunken mitochondria with ruptured membranes; the mitochondrial autophagy of granulosa cells in the 3SH group increased, and the mitochondrial morphology of cells in the 6SH group was significantly improved compared with that in the HFe group, indicating that spermidine can improve high-iron-induced mitochondrial damage and morphological changes in ovarian granulosa cells.

[0074] Example 7 Determination of antioxidant capacity of ovarian tissue

[0075] To analyze the effect of spermidine on the activity of antioxidant enzymes in ovarian tissue, approximately 4 ovaries (0.02 g) were weighed and homogenized (2500 r, 10 min), and the supernatant was collected. SOD, MDA, and CAT activities were measured using kits from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China) according to the instructions. Fig.12 As shown, Fig.12 In the figure, A to C are the measurement results of SOD, MDA and CAT respectively.

[0076] Depend on Fig.12It can be seen that the activities of SOD and CAT enzymes in ovarian tissue of HFe group were significantly lower than those of control group (P<0.05), and the MDA content was significantly increased (P<0.01); the CAT activity of ovaries in 3SH group and 6SH group were significantly increased compared with HFe group, and the MDA content was significantly decreased (P<0.05); the SOD activity in 6SH group was significantly higher than that in HFe group, and the SOD activity in 3SH group showed an upward trend but there was no statistical difference (P>0.05).

[0077] Example 8 Western blotting detection

[0078] The ovaries were homogenized and lysed on ice using RIPA buffer (Biyuntian, P0013B) and centrifuged at 4°C and 12000r for 15min. The supernatant was collected as the total protein sample. The protein concentration of the obtained samples was determined using the BCA protein concentration assay kit, and after dilution to a consistent concentration, an appropriate volume of loading buffer was added for denaturation at 100°C for 10min. The proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was blocked in TBST containing 5% skim milk powder and incubated with the primary antibody (1:1000) at 4°C overnight, followed by incubation with HRP-labeled secondary antibody (1:1000) at room temperature for 2h. Visualization was performed using an ECL detection kit (Baoguang Bio, BG0015, Chongqing, China). The protein bands were visualized using an automated gel imaging system (GelView 5000ProⅡ, Guangzhou, China). ImageJ2x (ImageJ2x software, USA) was used to determine significant differences in protein expression. The protein bands are shown in Figure 2. Fig.13 The quantitative results are shown in Fig.14 As shown, Fig.14 In the figure, from top to bottom and from left to right, they are the quantification results of LC3B / β-actin, P62 / β-actin, Nrf2 / β-actin, p-Nrf2 / β-actin, FHC / β-actin, HO-1 / β-actin, ACSL4 / β-actin, and GPX4 / β-actin.

[0079] Depend on Fig.13 and Fig.14It can be seen that compared with the control group, the ferroptosis-related protein FHC / GPX4 in the MFe group was significantly downregulated, and there was no significant difference in ACSL4; the protein levels of FHC / GPX4 and ACSL4 in the HFe group were significantly decreased; the endogenous antioxidant pathway Nrf2 / p-Nrf2 in the MFe and HFe groups was significantly decreased, the HO-1 level in the MFe group was decreased, and the HO-1 level in the HFe group was significantly increased; there was no significant difference in the ratio of autophagy protein LC3BⅡ / Ⅰ in the MFe group, and P62 was significantly upregulated; LC3BⅡ / Ⅰ in the HFe group was significantly increased, while P62 was significantly upregulated. In the 3SH group, Nrf2 / p-Nrf2 and FHC / GPX4 were significantly upregulated compared with the HFe group, the LC3BⅡ / Ⅰ ratio and P62 level were decreased, the excessive cellular autophagy level was inhibited, and the HO-1 and ACSL4 levels did not change significantly. In the 6SH group, the LC3BⅡ / Ⅰ and P62, GPX4, HO-1 and ACSL4 levels were significantly decreased compared with the HFe group, while there was no significant statistical difference in the Nrf2 / p-Nrf2 and FHC levels.

[0080] In summary, spermidine can alleviate the follicular dysplasia in mice caused by iron overload, which is manifested by an increase in the number of follicles at all levels, a decrease in atretic follicles, and upregulation of anti-ferroptosis and antioxidant-related proteins.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of spermidine in the preparation of drugs for improving ovarian iron overload.

2. The use according to claim 1, characterized in that The concentration of spermidine in the medicine is 2.5-3.5 mmol / L.

3. The use according to claim 1, characterized in that The drug is administered orally.

4. The use according to claim 1, characterized in that The iron overload is iron overload caused by excessive intake of exogenous iron.

5. The use according to claim 1, characterized in that The application is at least one of the following (1)-(4): (1) Improve ovarian function; (2) Increase the number of follicles at all levels; (3) reduce the number of atretic follicles; (4) Promote follicle development.

6. A drug for improving ovarian iron overload, characterized in that: The invention comprises the spermidine as claimed in claim 1 and pharmaceutically acceptable excipients.

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