Application of cathepsin L and product for predicting ovulation

Abstract

The invention relates to the technical field of biology, in particular to application of cathepsin L and a product for predicting ovulation. According to the present invention, the first research results show that whether ovulation can be accurately determined according to the CTSL concentration in the female mammal body fluid such as urine and / or blood, the ovulation time can be accurately predicted, the accuracy is significantly higher than the luteinizing hormone (LH) determination method, and the method can be widely used for mammal breeding and human ovulation monitoring, fertility control or contraception.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to the application of cathepsin L and products for predicting ovulation. BACKGROUND

[0002] In the process of intensive management of livestock, the integration of embryo engineering technology for batch production and regular insemination of female livestock to improve production efficiency is based on the breakthrough of reproductive regulation theory. Practice has proved that correctly mastering the estrus and ovulation rules of cows, and timely mating at the best mating period are important measures to improve the pregnancy rate of cows. For the breeding of some high-value pets (horses, dogs, cats, etc.), veterinary personnel often need to make accurate judgments on the estrus rules of females through non-invasive means to implement artificial insemination to ensure the success rate of breeding or reduce disease transmission, etc. to meet the needs of users. Therefore, by strengthening basic research, enriching and revealing the molecular regulation mechanism of ovulation in female mammals, it is helpful to find key target molecules for controlling ovulation, and to develop more non-invasive detection methods and detection reagents, etc. based on this, and to promote the improvement of the reproductive efficiency of livestock and pets. It can be seen that how to quickly and accurately determine the accurate estrus time of female livestock (and pets) is the key to improving the pregnancy rate and shortening the interval between two pregnancies of female livestock to improve the production efficiency of animals.

[0003] However, the accuracy and reliability of the existing various estrus identification technical solutions are dependent on the proficiency of the technician and the degree of mastery of the condition of each female. The estrus of the female is often irregular due to the influence of the age, nutrition, parity, health condition and complex physicochemical and biological environmental factors of the female, which leads to the deficiency of the existing ovulation determination methods. For example, the release of LH in the blood is in pulses, and the appearance of the peak has a typical window period. The matching degree between the determination result and the peak period of the release is often poor, which leads to unstable determination result and inaccurate feedback information. In the existing research on mammals, the mature oocyte discharged by the animal after estrus maintains the fertilization ability in the oviduct for a shorter time than the sperm. If the vaginal mucus resistance determination method is used to determine ovulation, the defects are that the animal needs to be fully restrained, the insertion depth of the monitoring probe depends on experience, and the calibration of the probe is affected by the different ion concentrations in the tap water in different regions, which leads to inaccurate results. At the same time, the repeated insertion of the probe into the reproductive tract of the animal can cause inflammation of the reproductive tract and cross infection and transmission of diseases between animals, thereby affecting the health and reproduction of the animals. For example, the result of progesterone determination is relatively accurate for dogs, but it is delayed to a certain extent from the ovulation time. Since the appearance of the progesterone determination result represents that the animal has ovulated, this to a certain extent delays the accurate determination of the ovulation time, and it is possible that the oocyte has aged when artificial insemination or mating is performed, which affects the quality of early embryo development and even the success rate of embryo implantation.

[0004] The current method for determining ovulation in females is limited to determining the change in the level of luteinizing hormone (LH) in the blood before ovulation. The peak of LH appears a certain period of time before the mature follicle ovulates. The problem is that the concentration of LH in the serum is relatively low (ng level) and has a pulse release feature, which leads to the need to improve the accuracy of the results obtained by the existing determination method.

[0005] Therefore, the development of a new, more sensitive, cheaper and more convenient estrus identification method and product will help to improve the management level and production efficiency of the breeding females in the livestock farm, make full use of the reproductive resources of the breeding females, improve the efficiency of batch production of livestock while reducing production costs, promote the green and efficient development of the breeding enterprises, and fully ensure the improvement of the production efficiency of livestock and the supply of animal protein. SUMMARY

[0006] Therefore, the development of a new, more sensitive, cheaper and more convenient estrus identification method and product will help to improve the management level and production efficiency of the breeding females in the livestock farm, make full use of the reproductive resources of the breeding females, improve the efficiency of batch production of livestock while reducing production costs, promote the green and efficient development of the breeding enterprises, and fully ensure the improvement of the production efficiency of livestock and the supply of animal protein.

[0007] The application provides the use of cathepsin L (CTSL) as a marker in predicting ovulation in mammals.

[0008] The application also provides use of a reagent for detecting CTSL in predicting ovulation of a mammal or preparing a product for predicting ovulation of a mammal.

[0009] The application provides use of a reagent for detecting CTSL in predicting ovulation or preparing a product for predicting ovulation.

[0010] In the above use, the CTSL is cathepsin L in urine or serum.

[0011] In the above use, the mammal is a human or another mammal; the other mammals include but are not limited to mice, cats, pigs, dogs, cows, sheep, horses, etc.

[0012] In some specific embodiments, the mammal is a mouse, and the gene ID of the CTSL is 13039.

[0013] According to mouse spatial transcriptome sequencing data (https: / / www.gencod.es / ), Figure 1 analysis of the detection results of a plurality of molecules related to ovulation in the data shows that only CTSL is a key molecule whose change rule in blood and urine is consistent with its change in the ovary, and further verification shows that the concentration of CTSL in urine and serum of a female mammal (such as a mouse) can accurately determine the time to ovulation. CTSL can be used as a more efficient and accurate ovulation marker molecule, and determination of the concentration of CTSL in the urine or blood of a female can be used to determine whether she is in the periovulatory period. Based on the present application, methods and products for improving the reproductive efficiency of livestock and pets, the success rate of human reproduction, and the effect of contraception can be developed.

[0014] Compared with the secretion of traditional luteinizing hormone (LH) in the body, which is affected by its physiological pulse release, the determination method has obvious shortcomings. For example, it is reported that it completely depends on the naked eye judgment of the observer, which is too subjective and has a large error; or this method needs to be combined with B-ultrasound examination and other auxiliary means to achieve relatively accurate judgment. The concentration of CTSL in the present application does not have the defect of obvious fluctuation of the concentration in the blood caused by pulse secretion, and has the advantages of wider detection window and concentration change with corresponding physiological regularity of the ovulation process, which makes it provide more stable and accurate prediction results in actual application. For example, in mouse experiments, it is found that the change of the concentration of CTSL can not only accurately reflect the time node of ovulation, but also further distinguish different stages of the periovulatory period, thereby providing a scientific basis for accurate prediction.

[0015] Other molecules related to follicle maturation, such as metalloproteinase ADAMTS-1, MMP-2, MMP9, etc., although closely related to ovulation, need to be accurately determined by collecting follicular fluid or cells from the pre-ovulatory follicle developing in the ovary from the abdominal cavity by surgery, which is very difficult to operate in practice, has very high cost, damages the quality of follicular development, and has a great risk of failure of oocyte maturation; at the same time, there is no report on the concentration change in blood or urine during ovulation, and whether the concentration change has a stable and unified rule is unknown, so it is impossible to accurately judge the ovulation process (such as whether the follicle is mature, whether ovulation is about to occur) by the concentration change trend. The high difficulty of such sampling, the "weak significance" and "irregularity" of the concentration change, make it impossible to provide accurate and repeatable judgment basis for ovulation prediction, so it is difficult to be used as a marker for ovulation prediction.

[0016] The present application also provides products for predicting ovulation, including products for detecting the level of cathepsin L in mammalian tissues. Further, the products include products for quantitatively detecting CTSL or ELISA kits, sensors and / or test strips, protein immunoblotting detection reagents, etc. for semi-quantitatively detecting CTSL. The detection principles of the ELISA kit and the detection paper include but are not limited to direct method, indirect method, competition method and sandwich method, etc.

[0017] In some embodiments of the present application, the product for predicting ovulation is an ELISA kit, which includes a solid support coated with CTSL protein or coated with CTSL antibody, and at least one of the following reagents:

[0018] Washing solution, color developing solution, enzyme-labeled reagent, enzyme-labeled coated plate, sample diluent, standard, standard diluent.

[0019] The present application also provides a method for predicting ovulation, taking serum or urine of a human or other mammal as a sample to be tested, measuring the concentration of CTSL twice at an interval of 4 hours (ng or pg level according to different species), and judging whether the sample to be tested is before or after ovulation; the judging method includes:

[0020] If the concentration of CTSL in the two samples shows an upward trend, it is determined that the sample to be tested is before ovulation; otherwise, it is after ovulation.

[0021] After determining that the sample to be tested is in the pre-ovulation stage by the above method, the time of ovulation of the sample to be tested can also be predicted according to any one of the two CTSL concentrations. The specific prediction method is as follows (taking a mouse as an example):

[0022] The judging method also includes predicting the ovulation according to any one of the two CTSL concentrations:

[0023] When the to-be-tested sample is a urine sample, the judging method further comprises predicting ovulation according to the CTSL concentration at any one time:

[0024] When the CTSL concentration in the urine is 475 pg / mL≤CTSL concentration<520 pg / mL, it is indicated that ovulation will occur after 7-9 hours;

[0025] When the CTSL concentration in the urine is 520 pg / mL≤CTSL concentration<615 pg / mL, it is indicated that ovulation is occurring;

[0026] When the to-be-tested sample is a serum sample, the judging method further comprises predicting ovulation according to the CTSL concentration at any one time:

[0027] When the CTSL concentration in the serum is 400 pg / mL≤CTSL concentration<500 pg / mL, it is indicated that ovulation will occur after 11-13 hours, specifically, ovulation will occur after 11 hours, 12 hours or 13 hours;

[0028] When the CTSL concentration in the serum is 500 pg / mL≤CTSL concentration<600 pg / mL, it is indicated that ovulation will occur after 7-9 hours, specifically, ovulation will occur after 7 hours, 8 hours or 9 hours.

[0029] The application further provides a method for improving the reproduction rate of mammals, comprising: detecting and predicting the ovulation period of the mammals by using the method for predicting the ovulation of mammals, and mating the female mammals with male animals on the predicted ovulation day.

[0030] The application further provides a method for assisting human reproduction, comprising: determining the content of CTSL in a female patient and / or a serum sample, predicting the ovulation of the female, determining the ovulation day, and guiding the patient to complete fertilization or artificial insemination through assisted reproductive means on the ovulation day and / or 1-2 days before and after the ovulation day.

[0031] The application further provides a method for artificial contraception, comprising: taking serum or urine of a human or a mammal as a to-be-tested sample, determining the CTSL concentration twice at an interval of 4 hours, if the CTSL concentration shows an upward trend both times, it is indicated that the animal is in the pre-ovulation stage; otherwise, it is in the post-ovulation stage, the ovulation day of the to-be-tested sample is determined according to the CTSL concentration at any one time, and the fertility plan or contraception is realized.

[0032] The application first discovers that the CTSL concentration in the urine and / or serum of a female mammal can be used to accurately determine whether ovulation occurs and accurately predict the ovulation time, and can be widely used in the breeding of mammals and the ovulation monitoring, fertility control or contraception of humans. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 ​For spatial transcriptome sequencing data, which shows a plurality of molecules specifically highly expressed near the ovulatory follicle; subsequent detection results of a plurality of ovulation-related molecules therein show that cathepsin L (CTSL) is the first discovered key molecule directly related to the ovulation event, and the change rule in blood and urine is consistent with that in the ovary;

[0034] Figure 2 For the expression pattern and hormone responsiveness of CTSL in mouse ovarian tissue; wherein A is the immunofluorescence detection result: after the mice were treated with PMSG and HCG, the mouse ovaries were collected at each time point for immunofluorescence staining of CTSL and DDX4, a marker for oocyte cytoplasm; B is the immunoblotting result: after the mice were treated with PMSG and HCG, the mouse ovaries were collected at each time point to extract proteins for immunoblotting to detect the relative expression of pro-CTSL and mature CTSL; C is the relative protein expression of CTSL to GADPH gene in different groups. P represents the injection of a substitute for follicle-stimulating hormone FSH (pregnant mare serum gonadotropin PMSG, referred to as P), of which 0 hours before injection is represented as P0; 48 hours after injection of PMSG is represented as P48; H represents the injection of a substitute for LH hormone (human chorionic gonadotropin hCG, referred to as H) after 48 hours of PMSG injection, of which 4 hours after hCG injection is represented as H4; 12 hours after hCG injection is represented as H12;

[0035] Figure 3 For the concentration of CTSL in the urine of mice at different time periods during the periovulatory period; when the mouse is in the H4 period, the concentration of CTSL in the urine reaches 475 pg / mL, indicating that ovulation will occur in 8 hours; when the mouse is in the H12 period, the concentration of CTSL in the urine reaches 520 pg / mL, indicating that the mouse is ovulating; the meanings of P and H are the same as Figure 1 ;

[0036] Figure 4 For the concentration of CTSL in the serum of mice at different time periods during the periovulatory period; when the mouse is in the P48 period, the concentration of CTSL in the serum reaches 400 pg / mL, indicating that ovulation will occur in 12 hours; when the mouse is in the H4 period, the concentration of CTSL in the urine reaches 500 pg / mL, indicating that the mouse will ovulate in 8 hours; the meanings of P and H are the same as Figure 1 ;

[0037] Figure 5Fig. 2 is a white light microscope image of the ovary of a mouse at different time periods around ovulation; the red arrow indicates that the cumulus oocyte complex (COC) is being discharged; the results show that H12 is the time point of ovulation; the local morphology and tissue structure of the ovulation follicle on the surface of the ovary from ovulation to ovulation provided by the figure provides evidence for determining the specific ovulation process;

[0038] Figure 6 Fig. 3 is a hematoxylin staining image of the ovary of a mouse at different time periods around ovulation (using the unilateral ovary of 3 different mice as representative structures); in the H12 group, the red arrow indicates that the follicle is being ovulated in the ovary 12 hours after hCG treatment; in the H18 group, the red font CL indicates that the corpus luteum (the tissue after the follicle ovulates) exists in the ovary 18 hours after hCG treatment;

[0039] Figure 7 Fig. 4 is the concentration of CTSL in the serum of a sheep at different time periods around ovulation; the meanings represented by P and H are the same as in Figure 1 Fig. 5 is the concentration of CTSL in the serum of a cow at different time periods around ovulation.

[0040] Figure 8 Fig. 5 is the concentration of CTSL in the serum of a cow at different time periods around ovulation. DETAILED DESCRIPTION

[0041] The present application provides the application of cathepsin L and the product for predicting ovulation. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0042] In this paper, the description of "including", "containing" and "having" includes a closed technical solution consisting of the listed features, and also includes an open technical solution containing the listed features.

[0043] In this paper, the term "and / or" used includes any and all combinations of one or more related listed items.

[0044] In this paper, if no special instructions are given, the above numerical interval is regarded as continuous, and includes the minimum value and the maximum value of the range, and each point value between the minimum value and the maximum value. Further, when a range is referred to as an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. Unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0045] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0046] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0047] The application is further described below in conjunction with examples:

[0048] Example 1 Detection of expression pattern and hormone responsiveness of CTSL in mouse ovary

[0049] Experimental method: After the mice were treated with PMSG and HCG, the mouse ovaries were collected at each time point for immunofluorescence staining of CTSL and DDX4, a marker of oocyte cytoplasm; B is the result of Western blotting: After the mice were treated with PMSG and HCG, the mouse ovaries were collected at each time point for Western blotting to detect the relative expression of precursor CTSL and mature CTSL; C is the relative protein expression of CTSL to GADPH gene in different groups. P represents the injection of a substitute for follicle-stimulating hormone FSH (pregnant mare serum gonadotropin PMSG, referred to as P), of which 0 hours before injection is represented as P0; 48 hours after injection of PMSG is represented as P48. H represents the injection of a substitute for LH hormone (human chorionic gonadotropin hCG, referred to as H) after 48 hours of PMSG injection, of which 4 hours after hCG injection is represented as H4; 12 hours after hCG injection is represented as H12.

[0050] The results of immunofluorescence analysis are shown in Figure 2 .

[0051] Result analysis: The immunofluorescence results show that (Fig. Figure 2 A), with the stimulation of the gonadotropin (hCG) peak, the expression of CTSL showed a time-dependent response. Among them, the expression of CTSL in follicular granulosa cells increased in turn at the three detected time points of P48, H4 and H14. Correspondingly, according to the results of Western blotting and analysis (Fig. Figure 2 B and C), both the inactive CTSL precursor and the active mature protein were significantly up-regulated after hormone treatment, and reached the highest value at H4. This result shows that the sharp increase in the expression of CTSL in the ovary after the arrival of the gonadotropin peak is positively correlated with the time of ovulation, i.e. the closer to the time of ovulation, the higher the expression.

[0052] Example 2 Prediction of mouse ovulation

[0053] I. Materials:

[0054] The ELISA assay kit for determining the concentration of mouse CTSL comprises: washing liquid, color developing liquid, enzyme-labeled reagent, enzyme-labeled coating plate, sample diluent, standard, standard diluent.

[0055] Serum samples or urine samples of ICR mice (12-14 g) for 23 days.

[0056] II. Specific method

[0057] 1. Collect 10 μL of urine or serum of female mammals in a sterile tube, and determine the concentration of CTSL in the sample to be tested by using the mouse CTSL-ELISA kit according to the instructions. The sample to be tested is the serum and urine samples of mice at different time periods during the superovulation process, and there are 10 mouse samples at each time point.

[0058] 2. Determination criteria for mice entering the periovulatory period:

[0059] 1) When the urine is used as the test material: 475 pg / mL≤CTSL concentration<520 pg / mL, it is suggested that ovulation will occur after 7-9 hours; when 520 pg / mL≤CTSL concentration<615 pg / mL in the urine, it is suggested that ovulation is occurring.

[0060] 2) When the serum is used as the test material: when 400 pg / mL≤CTSL concentration<500 pg / mL in the serum, it is suggested that ovulation will occur after 11-13 hours; when 500 pg / mL≤CTSL concentration<600 pg / mL in the serum, it is suggested that ovulation will occur after 7-9 hours.

[0061] The results are shown in Figures 3-4 .

[0062] Meanwhile, the corresponding mice of each sample to be tested are subjected to the following experiments:

[0063] The ovaries of each mouse are observed under a microscope, and the ovaries are dyed with hematoxylin, and the results are shown in Figures 5-6 .

[0064] The concentration of CTSL in the serum or urine samples of mice at 4, 12, and 18 hours after hCG injection is determined according to the method of the present application, and the ovulation situation is predicted. The results are as follows: the samples at 4 hours after hCG injection (H4 group) are detected, and the concentration of CTSL in the urine and serum both suggests that ovulation will occur after about 7-9 hours; the predicted results of the samples at 12 hours after hCG injection (H12 group) are all that ovulation is occurring. The samples at 18 hours after hCG injection are all determined to be in the post-ovulation period, and the microscopic imaging results and the staining results of the ovaries of the corresponding mice are shown in Figures 5-6 .

[0065] From Figure 5 andFigure 6 It was found that the cumulus-oocyte complex (COC) in the ovaries of mice in the H12 stage was being released. Ovaries from mice corresponding to serum or urine samples in the H4 group were taken 8 hours later for testing, and the results showed that ovulation was occurring, consistent with the H12 group. A total of 20 samples were tested, including 10 serum samples and 10 urine samples from 10 mice. Of these, 16 samples, when ovulation was predicted based on CTSL concentration according to the method described in this invention, matched the actual ovulation detected by HE staining, with an accuracy rate of 80%.

[0066] Following the method described in Example 1, CTSL concentrations were measured in 10 serum samples and 10 urine samples from 10 mice in the P48 group. The predicted ovulation result for all 20 samples was 12 hours later (calculated from 48 hours after PMSG injection). Hematoxylin staining of the ovaries of these mice 12 hours later confirmed that ovulation was occurring. The results indicate that the predicted ovulation was completely consistent with the actual ovulation, with an accuracy rate of 100%.

[0067] The above demonstrates that the results of the ovulation prediction method of the present invention are consistent with the actual ovulation situation of the sample, and the results are accurate and reliable.

[0068] Example 3: Detection of CTSL concentration in serum of ewes and cows

[0069] Experimental methods: 10 μL of serum from ewes or cows was collected using sterile tubes. The concentration of CTSL in the test samples was determined using a CTSL-ELISA kit according to the instructions. The test samples were samples from different time points during the superovulation treatment process, with more than 10 samples at each time point. The treatment method was the same as in Example 1. The mice used were 23-day-old ICR mice (12-14g).

[0070] The results of CTSL concentrations in ewe serum and cow serum samples at different time points are shown in the figure. Figures 7-8 .

[0071] Depend on Figure 7It was observed that the concentration of CTSL in the blood of ewes increased in a time-dependent manner with superovulation treatment (4 ewes). At 0 hours of PMSG treatment and in the early stages (data shown in the figure is from 24 hours of PMSG treatment), the absolute concentration was relatively low. However, when the ewes were in the H12 stage of superovulation treatment (12 hours of hCG treatment), the serum CTSL concentration reached 1.15 ng / mL, indicating that ovulation was approximately 6 hours away. When the ewes were in the H18 stage, the serum CTSL concentration reached 1.75 ng / mL, indicating that ovulation was occurring. Laparoscopic recordings taken 12 hours after hCG treatment showed that ovulation had occurred, with clear vaginal mucus and vulvar redness and swelling. These results demonstrate that ovulation occurred when the CTSL concentration peaked, which is entirely consistent with the expected results according to the method of this invention.

[0072] Depend on Figure 8 It was found that the concentration of CTSL in the blood of cows during the periovulation period showed a time-dependent change (average of 5 cows). Specifically, the absolute value of its concentration was relatively low 12 hours before ovulation (H0). The serum CTSL concentration reached its peak when the cow ovulated (equivalent to H12 hours). After ovulation (H24 hours), the serum CTSL concentration decreased again. The results suggest that the time to ovulation can be estimated by measuring CTSL in the blood. Ultrasound examination combined with physical examination revealed that these cows were all in estrus, and the change in CTSL concentration over time was consistent with the ovulation phenotype. The results indicate that the peak CTSL concentration occurred when the cow ovulated, which is consistent with the expected results of the method of this invention.

[0073] The results showed that the concentration of CTSL in the serum of ewes and cows was also closely related to ovulation. Based on the concentration of CTSL in urine and / or blood, ovulation could be accurately determined and the timing of ovulation could be accurately predicted. This indicates that CTSL can be used as a biomarker to predict ovulation in mammals (including mice, cattle, and sheep).

[0074] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of CTSL protein as a marker in predicting ovulation in a mammal. The mammal is a mouse, a cow or a sheep.

2. Use of a reagent for detecting CTSL protein level in predicting ovulation in a mammal.

3. Use according to claim 1 or 2, characterized in that, The CTSL protein is a CTSL protein in urine.

4. Use according to claim 1 or 2, characterized in that, The CTSL protein is a CTSL protein in blood.

5. A product for predicting ovulation, characterized in that The product comprises a reagent for detecting CTSL protein level in a mammal.

6. The product of claim 5, wherein, The product comprises a test strip or an ELISA kit.

7. The product of claim 6, wherein, The ELISA kit comprises a solid carrier coated with CTSL protein or coated with CTSL antibody and at least one of the following components: washing solution, color developing solution, enzyme-labeled reagent, enzyme-labeled coated plate, sample diluent, standard, standard diluent.

8. A method of predicting ovulation in a mammal, characterized in that, The sample is serum or urine of a mammal, and the CTSL concentration is determined twice at an interval of 4 hours to determine whether the sample is before or after ovulation; the determination method comprises: If the CTSL concentration of the two times is increasing, it is determined that the sample is before ovulation; otherwise, it is after ovulation.

9. The method of claim 8, wherein, The mammal is a mouse, and the determination method further comprises predicting ovulation according to any one of the CTSL concentrations; The method for predicting ovulation comprises: When the CTSL concentration in urine is 475 pg / mL≤CTSL concentration<520 pg / mL, it is prompted that ovulation will occur in 7-9 hours; when the CTSL concentration in urine is 520 pg / mL≤CTSL concentration<615 pg / mL, it is prompted that ovulation is occurring.

10. The method of claim 8, wherein, The determination method further comprises predicting ovulation according to any one of the CTSL concentrations; The method for predicting ovulation comprises: When the CTSL concentration in serum is 400 pg / mL≤CTSL concentration<500 pg / mL, it is prompted that ovulation will occur in 11-13 hours; when the CTSL concentration in serum is 500 pg / mL≤CTSL concentration<600 pg / mL, it is prompted that ovulation will occur in 7-9 hours.

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