CN114164254A

CN114164254A - Application of proteasome in improving success rate of in vitro fertilization

Info

Publication number: CN114164254A
Application number: CN202111543582.4A
Authority: CN
Inventors: 陈小攀; 王成路; 王方玉; 舒崇医; 舒静; 高方; 郑珉; 杨雷香; 陈林洁
Original assignee: Zhejiang Provincial Peoples Hospital
Current assignee: Zhejiang Provincial Peoples Hospital
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-03-11

Abstract

The invention discloses an application of proteasome in improving the success rate of in-vitro fertilization, which belongs to the technical field of medicine, the success rate of in-vitro fertilization is evaluated through proteasome activity in sperms, and the proteasome activity among different sperm quality index groups is found to have no significant difference through measuring the proteasome activity among different age groups, different sperm activity groups, different DNA fragment groups, different morphological rates and different head teratocardia, but the higher the activity expression of the sperm proteasome is, the higher the fertilization rate and the 2PN rate of conventional in-vitro fertilization are. Therefore, the invention can be used for detecting the sperm proteasome activity of patients with unexplained infertility or low fertilization rate in conventional in vitro fertilization rate, and provides a basis for clinically selecting conventional in vitro fertilization or ICSI treatment.

Description

Application of proteasome in improving success rate of in vitro fertilization

Technical Field

The invention relates to the technical field of medicine, in particular to application of proteasome in improving success rate of in vitro fertilization.

Background

The 26S proteasome is the end point of the ubiquitin-proteasome pathway, a complex multi-subunit complex, consisting of 19S Regulatory Particles (RP) responsible for recognition and unfolding of ubiquitination substrates and a 20S catalytic Core (CP) responsible for protein degradation. 19S RP can be biochemically divided into two parts, a "base" and a "lid", where the base contains 6 ATPase subunits and 4 non-ATPase subunits, where the non-ATPase subunits provide binding sites for ubiquitin and ubiquitin-like proteins and the ATPase subunits have activity to activate substrate protein unfolding. The cap part is only composed of non-ATPase subunits, can recognize protein substrates combined by polyubiquitin, has deubiquitinating activity, can release ubiquitin, and transfers the substrate proteins into core protease for degradation. 20S CP is a highly conserved barrel complex, consisting of four stacked concentric rings of heptamers. Wherein 2 outer rings are composed of 7 alpha-type subunits, 2 inner rings are composed of 7 beta-type subunits, the outer rings are guarded at the entrance and exit to ensure that only protein entering the interior of the barreled structure is hydrolyzed, two inner rings form an inner chamber, active sites responsible for proteolysis are arranged in the inner chamber, only 3 subunits (beta 1, beta 2 and beta 5) in the 7 beta-type subunits retain complete protein degradation active sites, therefore, each 20S CP has 6 proteolytic active sites, which has glutamyl-like peptide hydrolase activity, tryptase activity and chymotrypsin-like activity and can break most peptide bonds.

Spermatogenesis is a complex and ordered series of processes in the male mammal, starting with spermatogonial stem cells, undergoing spermatogonial cells, primary spermatocytes, secondary spermatocytes, globular spermatocytes. During which biological processes such as mitosis, meiosis, etc. occur. Then the spherical spermatids undergo the deformation processes of nucleus condensation, acrosome formation, flagella formation and the like to become elongated spermatids, and further evolve into mature sperms. The 26S proteasome plays an important role in spermatogenesis and deformation. It is proved by research that three kinds of protease activities in the 20S CP inner ring beta subunit can be detected on the surface of the plasma membrane of the human sperm, and the occurrence of sperm differentiation can be blocked by applying a proteasome inhibitor. And the generation and deformation of the sperms are not perfect processes, abnormal sperms are generated in the process, and in order to reduce the number of the abnormal sperms, the in vivo protease can play a role of similar quality control on the spermatogenesis process by degrading the abnormal sperms. A large amount of ubiquitinated proteins and 26S proteasomes exist in the human epididymis, which indicates that a process of degrading proteins through a ubiquitin-proteasome pathway exists in the epididymis, so that abnormal sperms are eliminated when epididymis spermatogenesis is finished. In the mammalian testis, defective sperm such as bipitch, twin-tailed sperm, cytoplasmic remnant sperm, etc. will be ubiquitinated and subsequently degraded by proteasomes.

Fertilization is the fusion of male and female gametes to produce a single diploid cell, the zygote. This process is a complex and continuous process comprising a series of steps: capacitation, passage of sperm through the cumulus cell layer, sperm binding and penetrating the zona pellucida, acrosome reaction generation, sperm-egg binding and fusion, etc. Studies have shown that proteasomes in porcine sperm are gradually released during in vitro capacitation as the integrity of the acrosomal outer membrane and matrix is destroyed, suggesting that this process plays an important role in subsequent fertilization. The capacitated sperm has the function of penetrating the zona pellucida of the oocyte, and the process depends on the tail swing of the sperm and the hydrolysis of proteasome. In mammals, the sperm proteasome is present in the sperm acrosome and on the plasma membrane surface, and may play a dual role during the sperm-zona pellucida interaction: after capacitation, before acrosome exocytosis, proteasomes positioned on the surface of an acrosome membrane of a sperm degrade proteins on the acrosome membrane to trigger acrosome exocytosis; secondly, after acrosome exocytosis, proteasomes located in sperm acrosome are used as a medium of the acrosome exocytosis, so that zona pellucida matrix is promoted to be locally degraded, and fertilization gaps are formed. The proteasome inhibitor can block sperms of pigs, mice and invertebrates from penetrating the zona pellucida of oocytes, can also block the acrosome reaction induced by progesterone, and can obviously reduce the IVF fertilization rate of cattle. In the sperm cell fusion stage, the proteasome, which is located near the sperm centromere, becomes part of the proteolytic apparatus and contributes to sperm centromere release during fertilization. After fertilization, proteasomes located near the centromere enter the oocyte, activating the maternal proteasome in the oocyte to drive degradation of protein substrates in fertilized eggs through the ubiquitin-proteasome pathway. And after fertilization, the oocyte self proteasome can recognize and degrade the homoplasmic mitochondria marked by the polyubiquitin chains, thereby ensuring maternal inheritance of mitochondrial genes.

The male sterility refers to normal and regular sexual life after marriage of couples in the childbearing age, and the couple is not pregnant under the condition that the female has the pregnancy capability after one year without any contraceptive measures. According to related reports, the quality of semen of men is reduced, which is one of the important reasons for infertility, and reproductive disorders exist in about 15% of women of reproductive age worldwide, of which about 50% are caused by male factors. The reasons for this are complex, and mainly include spermatogenesis disorder, semen abnormality, sperm-egg binding disorder, systemic factors, etc. Currently, the quality assessment of semen in clinic is usually obtained by analyzing semen routine, sperm DNA fragmentation rate, sperm normal morphology rate, seminal plasma zinc concentration and the like. However, because these assessment methods have their own limitations and the variability of semen specimens themselves, the clinical use of these methods for detecting semen quality cannot fully reveal the cause of male sterility and assess male fertility. It has also been found that: poor-quality sperms in separating liquid of sperms obtained by gradient centrifugation show inherent proteasome activity defects, and the proteasome activity of the sperms is in positive correlation with the sperm motility and normal forms.

In Vitro Fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are currently the most commonly used assisted reproductive technologies, and are one of the main approaches to male infertility, but currently, an effective method for evaluating the fertilization rate of in vitro fertilization is lacking.

Disclosure of Invention

The invention aims to provide application of proteasomes in improving the success rate of in vitro fertilization, so as to solve the problems in the prior art, and tests prove the relationship between sperm proteasome activity and semen quality parameters, evaluate sperm quality and the success rate of in vitro fertilization, and provide reference and basis for clinical selection of conventional in vitro fertilization therapy and ICSI therapy.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of proteasome in improving the success rate of in vitro fertilization, and the success rate of in vitro fertilization is improved by improving the activity of sperm proteasome.

The invention also provides application of the protease activity in evaluating the success rate of in vitro fertilization.

Further, the in vitro fertilization success rate takes the proteasome activity in the sperm as an evaluation index; the proteasome activity assay comprising the steps of:

(1) collecting a fresh semen sample, processing the semen sample by adopting a gradient centrifugation method, and separating to obtain a sperm sample;

(2) mixing the sperm samples evenly, diluting the sperm samples with PBS solution and counting, and counting to 2-10 after dilution⁶The sperm sample/mL is evenly mixed with the protease detection reagent;

(3) diluting a standard 20S proteasome reagent according to a proportion, and uniformly mixing the diluted 20S proteasome and a protease detection reagent;

(4) detecting the fluorescence intensity of the protease in the sperm and the standard substance respectively by using a multifunctional microplate reader for the mixed solution treated in the step (2) and the standard substance treated in the step (3);

(5) and (3) making a standard curve according to the fluorescence intensity value of the standard substance, and calculating the proteasome activity in the sperm sample according to a formula obtained by the standard curve.

Further, in the step (1), the step of processing the semen sample by the gradient centrifugation method specifically comprises the following steps:

s1: adding the liquefied semen into the top of the gradient separation liquid, centrifuging for 10-20min at 400g of 300-;

s2: adding the sperm precipitate into Fertilization Medium culture solution, fully and uniformly mixing, and centrifuging for 3-8min at 400g of 300-;

s3: centrifuging at S2, removing supernatant, adding Fertilization Medium culture solution to adjust sperm concentration to 20 × 10⁶In 6% CO/mL₂And culturing at 37 ℃ for later use.

Further, after the semen sample is processed by the gradient centrifugation method, the average value of the sperm motility is 90.8 +/-8.81%.

Further, in the step (2), the count after dilution is 2-10⁶the/mL sperm sample was mixed with the protease detection reagent at an equal volume ratio.

Further, in the step (3), the standard 20S proteosome reagent is 2500ng/ml, 100ng/ml, 500ng/ml, 250ng/ml and 125ng/ml after being diluted according to the proportion.

Further, in the step (4), the excitation wavelength for measuring the fluorescence intensity is 380nm, and the emission wavelength is 460 nm.

The invention also provides application of the reagent for detecting proteasome activity in preparing a kit for evaluating the success rate of in vitro fertilization.

Further, the kit comprises a proteasome buffer solution, a fluorescein detection reagent and Suc-LLVY-Glo^TMSubstrate and 20S Proteasome standard.

The invention discloses the following technical effects:

the invention evaluates the correlation between proteasome activity in sperms and the conventional in vitro fertilization and ICSI after fertilization through experiments, and shows that: the proteasome activity is obviously related to the fertilization rate and the 2PN rate of the conventional in vitro fertilization (p is less than 0.05). The proteasome activity has no obvious correlation with the conventional in vitro fertilization cleavage rate, normal cleavage rate and excellent embryo rate (p is more than 0.05). The proteasome activity has no obvious correlation with the fertilization rate, 2PN rate, cleavage rate, normal cleavage rate and excellent embryo rate after ICSI (p is more than 0.05). Patients with conventional in vitro fertilization sperm proteasome activity slightly higher than that of the ICSI method were analyzed by mean analysis of conventional in vitro fertilization and ICSI sperm proteasome activity (681.0 ± 57.13, 644.6 ± 84.53, respectively).

The invention shows that the activity of sperm proteasome has no significant difference among different age groups, different activity groups, different DNA fragment rates, different morphological rates and different head deformity rate of male patients. However, the higher the expression of the sperm proteasome activity, the higher the fertilization rate of the conventional in vitro fertilization and the 2PN rate. The activity of sperm proteasome has no obvious correlation with the conventional in vitro fertilization cleavage rate, normal cleavage rate and excellent embryo rate, and has no obvious correlation with the fertilization rate, 2PN rate, cleavage rate, normal cleavage rate and excellent embryo rate after ICSI. Therefore, for patients with sterility with unknown reasons or low fertilization rate of conventional in vitro fertilization, the detection of the sperm proteasome activity can be considered, and the basis is provided for clinical selection of conventional in vitro fertilization or ICSI treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a standard curve constructed from standard 20S Proteasome;

FIG. 2 shows comparison of proteasome activity between age groups;

FIG. 3 is a comparison of proteasome activity between groups of asthenospermia;

FIG. 4 is a comparison of proteasome activity between normal morphometric groups;

FIG. 5 shows comparison of proteasome activity between groups of head deformities;

FIG. 6 shows a comparison of proteasome activity between sperm DNA fragmentation rates;

FIG. 7 is a comparison of proteasome activity between normal and abnormal sperm groups;

FIG. 8 is a graph showing the relationship between IVF fertilization rate and proteasome activity;

FIG. 9 is a graph of IVF 2PN rate vs proteasome activity;

FIG. 10 is a graph of IVF cleavage rate vs. proteasome activity;

FIG. 11 is a graph showing the relationship between normal cleavage rate and proteasome activity for IVF;

FIG. 12 is a graph showing the relationship between IVF excellent embryo rate and proteasome activity;

FIG. 13 is a graph showing the relationship between ICSI fertilization rate and proteasome activity;

FIG. 14 is a graph of ICSI 2PN rate vs. proteasome activity;

FIG. 15 is a graph showing the relationship between ICSI cleavage rate and proteasome activity;

FIG. 16 is a graph showing the relationship between ICSI normal cleavage rate and proteasome activity;

FIG. 17 shows the relationship between ICSI excellent embryo rate and proteasome activity;

FIG. 18 is a graph showing the effect of protease inhibition on sperm capacitation.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Materials and methods

1. Primary test reagents

2. Preparation of the principal reagents

1. Preparation of protease detection reagent

(ii) purchase of Proteasome-Glo from Promega^TMChymotrypsin-Like Assay kit detects Chymotrypsin activity.

The kit comprises: 10ml proteasome buffer solution, 1 bottle of flask fluorescein detection reagent and 50. mu.l Suc-LLVY-Glo^TMA substrate.

And mixing 10ml of proteasome buffer solution with 1 bottle of bottom fluorescein detection reagent uniformly to obtain 10ml of proteasome fluorescein detection buffer solution. The mixture was dispensed into 20 portions in 500. mu.l portions using 1.5ml brown EP tubes and stored in a refrigerator at-20 ℃.

Fourthly, 50 mu l of Suc-LLVY-Glo^TMThe substrate was dispensed into a PCR tube in 20 portions in 2.5. mu.l portions, and stored in a refrigerator at-20 ℃.

When using, the proteasome fluorescein detection buffer solution is mixed with Suc-LLVY-Glo^TMThe substrate was equilibrated to room temperature, according to 200: 1, and equilibrating for 30 minutes at room temperature.

2. Preparation of standards

The 20S Proteasome reagent from Lifesensors was purchased. The resulting mixture was divided into 50 portions of 1. mu.g portions using a PCR tube and stored in a refrigerator at-80 ℃. When in use, the mixture is balanced to room temperature, the existing 20S Proteasome concentration is 1mg/ml, and the mixture is prepared into a concentration gradient of a standard product according to the following steps:

1 μ l of 1mg/ml 20S Proteasome is added into 9 μ l PBS solution to prepare 0.1mg/ml 20S Proteasome.

② 2 mul of 20S Proteasome with 0.1mg/ml is put into 78 mul PBS to prepare the 20S Proteasome with the concentration of 2500 ng/ml.

③ 1 ul of 20S Proteasome with 0.1mg/ml is put into 99 ul of PBS to prepare the 20S Proteasome with the concentration of 1000 ng/ml.

Fourthly, 1 mul of 20S Proteasome with 0.1mg/ml is put into 199 mul of PBS to prepare the 20S Proteasome with the concentration of 500 ng/ml.

Fifthly, 1 mul of 20S Proteasome with 0.1mg/ml is put into 399 mul of PBS to prepare the 20S Proteasome with the concentration of 250 ng/ml.

Sixthly, 1 mu l of 20S Proteasome with the concentration of 125ng/ml is put into 799 mu l of PBS to prepare the 20S Proteasome with the concentration of 125 ng/ml.

And seventhly, finally taking a concentration gradient as follows: 2500ng/ml, 100ng/ml, 500ng/ml, 250ng/ml, 125 ng/ml.

3. Study object

The relationship between the activity of sperm proteasome and the semen quality parameters was studied: 115 cases of men who performed conventional in vitro fertilization or ICSI assisted pregnancy in my hospital on months 07-2021, 2020 were selected as subjects. And (3) inclusion standard: the concentration of the sperms after density gradient centrifugation is more than 2 multiplied by 10⁶Perml, microscopically observing the number of leucocytes in the semen which is less than 1 × 10⁶And/ml. Exclusion criteria: those with chromosomal abnormalities, those with a history of inherited family diseases, those with acute reproductive system infections. Age of the patient: the minimum is 25 years old and the maximum is 57 years old, and the average age is (36).17 ± 6.29) years old.

Study of relationship between sperm proteasome activity and embryo outcome in vitro fertilization: males who performed conventional in vitro fertilization or ICSI assisted pregnancy at my hospital on months 7-2021, 2020 were selected as subjects. And (3) inclusion standard:

the male patient: the concentration of the sperms after density gradient centrifugation is more than 2 multiplied by 10⁶Perml, microscopically observing the number of leucocytes in the semen which is less than 1 × 10⁶And/ml. ② female patients: for those without chromosome abnormality and hereditary family history, the number of ova is not less than 5, and the MII rate of oocytes is not less than 70%. Exclusion criteria: the male patient: those with chromosomal abnormalities, those with a history of inherited family diseases, those with acute reproductive system infections. ② female patients: morphological abnormality of ovum. The final enrollment experimental specimens were 66 cases, where male patients were aged: the minimum is 25 years old, the maximum is 47 years old, and the average age is (33.62 + -4.99) years old. Age of female patients: the minimum is 24 years old, the maximum is 42 years old, and the average age is (31.86 +/-4.29) years old.

4. Test method

Collecting and checking semen samples: taking semen specimen, treating male sexual desire for 2-7 days, evacuating bladder before semen, retaining semen in a prepared sterile cup by masturbation, and placing the specimen in 37 deg.C incubator until completely liquefied. A portion of the semen was taken for routine examination. The examination results were referred to the world health organization manual fifth edition of the human semen examination and processing laboratory by using a computer assisted sperm quality analysis system (CASA). The rest sperm examination results (such as sperm DNA fragmentation rate, sperm normal morphology rate and the like) are statistically collated with the recent examination results of the patient in the hospital.

② a semen density gradient centrifugation laboratory processing method: 1) the 15ml conical bottom tubes were labeled with the name of the female. 2) Draw 1mL of supernatant (50% Isolate) and add to the bottom of a 15mL conical tube; then, 1mL of the lower layer of the separation solution (90% Isolate) was carefully pipetted into the bottom of the tube, taking care to keep the separation from the upper layer clearly. 3) About 1.2-1.5 mL of liquefied semen is carefully added from the top of the prepared gradient separation liquid. Centrifuge at 350g for 15min to separate the layers. 4) 1 new 15ml conical test tube is labeled with the name of the female. Taking 1ml of Fertilization Medium culture solution to a new pipetteSucking bottom layer sperm precipitate (volume is not more than 0.1mL) in tube, adding into the bottom layer sperm precipitate, mixing well, centrifuging at 350g for 5 min. 5) The supernatant was discarded, and the resulting mixture was centrifuged at 350g for 5min in 1mL of Fertilization Medium. 6) Discarding supernatant, adding appropriate amount of Fertilization Medium culture solution to adjust sperm concentration to 20 × 10⁶and/mL. 7) Loosening cover, placing in 6% CO₂And culturing at 37 ℃ in an incubator for later use. 8) The average value of the sperm motility observed after the density gradient centrifugation is 90.8 +/-8.81 percent, and the influence of the poor sperm motility after the density gradient centrifugation on the proteasome activity can be eliminated.

③ detecting the activity of sperm proteasome: 1) mixing the sperm sample after density gradient centrifugation, placing 10 μ l sperm on a sperm counting plate, counting with an upright microscope, gradually diluting the sperm concentration to 2-4 × 10 with PBS solution⁶And/ml. 2) 50 μ l of the counted sperm sample and 50 μ l of the protease detection reagent were added to a 96-well white plate and mixed well in a shaker for 5 minutes. 3) The 20S Proteasome reagent is diluted according to the proportion, 50 mul of diluted 20S Proteasome and 50 mul of protease detection reagent are added into a 96 white board, and the mixture is fully mixed for 5 minutes in a shaking table. 4) And (3) putting the uniformly mixed sample into a multifunctional enzyme labeling instrument to detect the fluorescence intensity (excitation wavelength is 380nm, and emission wavelength is 460nm) of proteasomes in the sperms and the standard products. 5) Making a standard curve according to the luminous value of the standard product, and making a standard curve R²Not less than 0.98 (if the standard curve R²If the value is less than 0.98, the experimental result is abandoned). The proteasome activity in the corresponding sperm was calculated according to the formula obtained for the standard curve (see FIG. 1 for the standard curve).

Judging the maturity of the ootheca complex (OCCC): OCCC needs to be observed after recovery to understand oocyte maturation. Errors due to oocyte abnormality in the experiment were excluded. 1) Stage I, prophase I, which is an immature stage, has no first polar body, and surrounding cells tightly wrap cumulus cells without loosening. Sometimes a large nucleus, the GV stage oocyte, is visible, the zona pellucida is obscured, in vitro culture usually takes more than 24 hours, and maturation rates are low. 2) And in the stage II, the stage I is between the prophase I and the metaphase II, the stage is free of germinal vesicle, a large number of cumulus cells are tightly wrapped at the periphery without a first polar body (MI-stage oocyte), a layer of compact radial corona cells surrounds the oocyte, the zona pellucida is unclear, and the culture is required for 6-12 hours. 3) Stage III, metaphase II, where the first polar body has been expelled (MII stage oocytes), the most common and most susceptible to successful insemination mature oocytes, radial corona cells in radial arrangement, cumulus cells in loose form, and clear zona pellucida. In the above test 2), the MII rate of the oocyte is ensured to be more than or equal to 70%.

Conventional in vitro fertilization: 1) OCCC was grown for 4-6 hours maturation and transferred to Fertilization Medium insemination dishes (2-3/dish). 2) About 2000 prepared sperms are added and placed into an incubator for cultivation. 3) Culturing for l 6-20 hours, taking out the culture dish, and blowing the oocyte/fertilized egg by a capillary glass tube with the diameter of 150 mu m until the granular cells fall off. The oocytes/zygotes were transferred to embryo culture dishes for incubation.

Sixthly, injecting by using single sperm in the egg cytoplasm: 1) installing the micro-injection needle and debugging the system. 2) ICSI patient information was checked prior to treatment. An appropriate amount of sperm was added to the PVP droplet on the left of the ICSI dish. 3) Washing the needle, repeatedly blowing and sucking the injection needle in PVP liquid drops, so that the harmful substances possibly remaining on the wall of the needle tube can be easily controlled and removed. 4) Sucking sperm in an ICSI dish, selecting sperm with good vitality and appearance form, cutting the tail of the sperm by using an injection needle, braking the sperm, paying attention to gentle operation and avoiding damaging the neck and the middle section of the sperm. 5) The braked sperms are sucked into the injection needle from the tail part and repeatedly enter and exit for 2 times to ensure that the sperms smoothly run in the ICSI needle. 6) 4-8 of the digested eggs were transferred to a pre-equilibrated G-MOPS medium droplet on the right of the ICSI dish. 7) The oocyte is fixed by an ovum holding needle, the polar body is positioned at 12:00 or 6:00, and the injection needle is positioned at 3:00, so that the spindle body is prevented from being damaged. 8) During injection, the oocyte should be sucked back to ensure that the sperm is injected after the membrane is broken. 9) After injection, the oocytes are washed for 4-5 times in FM culture solution and transferred to an insemination culture dish for incubation.

And seventhly, interpretation of fertilization results: 1) observation of pronucleus: normally, 2 clear Pronucleus (PN) can be observed in normal fertilized eggs under an inverted microscope at 16-20 hours after insemination, and abnormal fertilization of 1PN or more than or equal to 3PN is observed. 2) Embryo cleavage was observed on days 2 and 3 of in vitro culture after fertilization. (see Table 1 for the criteria for excellent embryo assessment). 3) Fertilization rate is the number of fertilized eggs/obtained eggs multiplied by 100%; 2PN rate is 2PN forming number/egg obtaining number multiplied by 100%; the cleavage rate is the number of cleavage embryos/fertilized eggs multiplied by 100%; normal cleavage rate is equal to normal cleavage embryo number/fertilized egg number × 100%; the excellent embryo rate is equal to the number of excellent embryos/the number of normal fertilized eggs multiplied by 100 percent.

TABLE 1 cleavage embryo ranking (based on the ASEBIR consensus, the 1 and 2 embryos in the Table are excellent embryos)

5. Statistical method

Statistical analysis of the experimental data obtained using Prism 6.0 software and using mean ± standard error

And (4) showing. And (3) judging that all variables conform to normal distribution by adopting a single sample K-S test, so that One-way ANOVA variance analysis is adopted for the comparison among multiple groups, and a t test is adopted for the comparison between two groups. The correlation between each parameter and proteasome activity was analyzed using the Pearson correlation test, as P<0.05 is that the difference and the correlation have statistical significance.

Second, results and analysis

Relationship between sperm proteasome activity and semen quality parameter

1. Comparison of proteasome Activity between age groups

The average values of the proteasome activities of the sperm divided into two groups of the male patients with the age of less than or equal to 35 years old and more than 35 years old are respectively (663.3 +/-50.93) and (676.3 +/-62.95), and the proteasome activities between the two groups have no significant difference (p is more than 0.05), which is shown in figure 2.

2. Comparison of proteasome Activity between groups of different sperm motility

The specimen is divided into a weak sperm group and a non-weak sperm group according to the sperm PR less than 32 percent and not less than 32 percent, the average values of the sperm proteasome activities of the two groups are respectively (735.8 +/-106.7) and (652.4 +/-41.87), and the proteasome activities between the two groups have no significant difference (p is more than 0.05), which is shown in figure 3.

3. Comparison of Histone proteasome Activity for different sperm morphological rates

The specimen is divided into a teratogen group and a non-teratogen group according to the sperm normal morphology rate of less than 4 percent and the sperm normal morphology rate of more than or equal to 4 percent, the average values of the sperm proteasome activity of the two groups are respectively (673.4 +/-50.30) and (673.3 +/-66.21), and the proteasome activity between the two groups has no significant difference (p is more than 0.05), which is shown in figure 4.

4. Comparison of proteasome Activity between different sperm head malformation rates

The sperm head deformity rate is divided into three groups of 90-94%, 95-96% and 97-100%, the average value of the proteasome activity of each group of sperm is (713.4 + -81.55), (611.3 + -58.04) and (622.2 + -62.73), the proteasome activity between the groups has no significant difference (p is more than 0.05), but the proteasome activity of the two groups of the sperm head deformity rate of 95-96% and 97-100% is lower than that of the group of the sperm head deformity rate of 90-94%, as shown in figure 5.

5. Comparison of proteasome Activity between different sperm DNA fragmentation rates

The sperm DNA fragmentation rate is divided into three groups of less than 10%, 10% -25% and more than 25%, the average value of the activity of the sperm proteasome of each group is respectively (678.8 +/-59.81), (708.4 +/-60.16) and (461.6 +/-85.36), and no significant difference (p >0.05) exists among the groups, as shown in figure 6.

6. Comparison of proteasome Activity between Normal and abnormal sperm

The sperm are divided into two groups of normal sperm and abnormal sperm according to semen convention, sperm morphology rate and sperm DNA fragmentation rate, the average values of the proteasome activity of the sperm between the two groups are respectively (702.7 +/-75.43) and (649.4 +/-45.07), and the proteasome activity between the two groups has no significant difference (p is more than 0.05), which is shown in figure 7.

(II) correlation between sperm proteasome activity and embryo fate in vitro fertilization

1. Correlation of sperm proteasome activity with embryo fate after conventional in vitro fertilization

The final inclusion of experimental samples was 45 cases according to the inclusion and exclusion criteria. Wherein the mean age of the male is (33.76 + -5.35) years, and the mean age of the female is (31.69 + -3.90) years. Proteasome activity correlated significantly with fertilization rates of conventional in vitro fertilization, where r is 0.4499(p < 0.05), see fig. 8. Proteasome activity showed significant correlation with 2PN rates for conventional in vitro fertilization, where r is 0.4125 and p < 0.05, see fig. 9. The proteasome activity has no obvious correlation with the conventional in vitro fertilization cleavage rate, normal cleavage rate and excellent embryo rate, and p is more than 0.05, which is shown in figure 10, figure 11 and figure 12.

2. Correlation of sperm proteasome Activity with ICSI post-embryonic fate

The final test sample was 21 cases according to the inclusion and exclusion criteria. Wherein the mean age of the male is (33.33 + -4.22) years, and the mean age of the female is (32.24 + -5.11) years. Proteasome activity has no obvious correlation with fertilization rate, 2PN rate, cleavage rate, normal cleavage rate and excellent embryo rate after ICSI, p is more than 0.05, and the results are shown in figure 13, figure 14, figure 15, figure 16 and figure 17.

Some studies have found differences in proteasome activity between different semen parameters by healthy donors, but have not evaluated proteasome and semen-related parameters and fertilization in infertile male sperm. The invention evaluates the relationship between related parameters of proteasome activity and semen quality in sperms through the test to obtain: the activity of sperm proteasome has no significant difference among different age groups, different activity groups, different DNA fragment rates, different morphological rates and different head deformity rate of male patients. But the sperm head deformity rate is divided into three groups of 90-94%, 95-96% and 97-100%, although no significant difference (p >0.05) exists between the three groups, the sperm head deformity rate is 90-94%, and the activity of the proteasome is obviously higher than that of the two groups of the sperm head deformity rate of 95-96% and 97-100%. Considering that the abnormal sperm head often causes acrosome defect and reduces the activity of proteasome in sperm probably because the proteasome exists on the surface and in the sperm acrosome membrane in sperm, which may be related to the abnormal sperm morphological rate and low in vitro fertilization rate or failure of fertilization.

By assessing the correlation of proteasome activity in sperm with conventional in vitro fertilization and ICSI post fertilization events: the proteasome activity is obviously related to the fertilization rate and the 2PN rate of the conventional in vitro fertilization (p is less than 0.05). The proteasome activity has no obvious correlation with the conventional in vitro fertilization cleavage rate, normal cleavage rate and excellent embryo rate (p is more than 0.05). The proteasome activity has no obvious correlation with the fertilization rate, 2PN rate, cleavage rate, normal cleavage rate and excellent embryo rate after ICSI (p is more than 0.05). Patients with conventional in vitro fertilization sperm proteasome activity slightly higher than that of the ICSI method were analyzed by mean analysis of conventional in vitro fertilization and ICSI sperm proteasome activity (681.0 ± 57.13, 644.6 ± 84.53, respectively). The reasons for this may include the following: the conventional in vitro fertilization is a process of combining sperms and oocytes naturally in vitro, culturing the combined sperms and oocytes to cleavage stage embryos or blasts, and then transplanting the embryos back to mothers for implantation. The process still comprises the processes of sperm in vitro capacitation, sperm crossing a cumulus cell layer, sperm adhesion, recognition and crossing an egg zona pellucida, acrosome reaction generation, sperm-egg combination and fusion and the like. Wherein proteasomes are present around the nucleus, within the acrosome, and within the acrosome and at the junction between the acrosome and the posterior head and neck of the mature sperm. Proteasomes in sperm play an important role in conventional in vitro fertilization, affecting the fertilization rate and 2PN rate of conventional in vitro fertilization. ICSI is a method of forming fertilized egg by injecting sperm directly into oocyte cytoplasm by means of microscopic technology, and the process only needs to select one normal sperm to inject oocyte cytoplasm, during which the oocyte does not generate natural activation process, and the process of natural acrosome reaction and sperm penetrating oocyte zona pellucida is bypassed. The proteasome contained in the sperm may have impaired effects on acrosome reaction and digestion of oocyte zona pellucida during this process. And thirdly, although the higher the proteasome activity is, the higher the fertilization rate and the 2PN rate of the conventional in vitro fertilization are, the proteasome activity has no obvious correlation with the cleavage rate, the normal cleavage rate and the excellent embryo rate after the conventional in vitro fertilization, probably because in the subsequent development of the embryo, the sperm carrying paternal genetic materials and a central body has the capacity of further cleavage after the fertilization of the ovum and developing into a normal embryo.

In summary, the present invention shows that there is no significant difference in sperm proteasome activity among different age groups, different motility groups, different DNA fragment rates, different morphological rates, and different head teratogenesis rates of male patients. However, the higher the expression of the sperm proteasome activity, the higher the fertilization rate of the conventional in vitro fertilization and the 2PN rate. The activity of sperm proteasome has no obvious correlation with the conventional in vitro fertilization cleavage rate, normal cleavage rate and excellent embryo rate, and has no obvious correlation with the fertilization rate, 2PN rate, cleavage rate, normal cleavage rate and excellent embryo rate after ICSI. Therefore, for patients with sterility with unknown reasons or low fertilization rate of conventional in vitro fertilization, the detection of the sperm proteasome activity can be considered, and the basis is provided for clinical selection of conventional in vitro fertilization or ICSI treatment.

Example 2

To elucidate whether sperm proteasomes affect fertilization rates by affecting sperm capacitation. This example directly evaluates this process using a proteasome inhibitor in combination with a CTC fluorescence assay.

The method comprises the following steps:

the basic culture solution of the control group is Tyrode culture solution. Experimental group capacitation medium 2.6% BSA (w/v) and 25mM sodium bicarbonate were added to the medium. Two inhibitor groups were supplemented with MG132 (final concentration 10. mu.M) and Epoxomicin (final concentration 10. mu.M) based on the capacitation medium of the groups, respectively. Sperm were incubated for 60 minutes prior to testing in all but the control group.

Sperm capacitation status was assessed using CTC fluorescence analysis methods. 750 μ M CTC were freshly prepared in a buffer of 130mM NaCl, 5mM cysteine and 20mM Tris-HCl, pH adjusted to 7.8 and stored in foil protected from light at 4 ℃ until use. Mu.l of Hoechst treated 5. mu.l sperm suspension was placed on a 37 ℃ slide and 5. mu.l of CTC stock was added rapidly followed by 0.5. mu.l of 2% glutaraldehyde over 30 seconds. After coverslipping, 200 viable cells were assessed for CTC staining pattern using phase contrast and fluorescence microscopy for alternate visualization. There are three main types of CTC fluorescence: f, the fluorescence of the whole head is uniform, and the sperm belongs to the non-capacitated sperm with complete acrosome; b, no fluorescence or a dark fluorescence band appears in the rear area of the acrosome, and the sperm belongs to complete capacitation sperms of the acrosome; AR, which has dim or no fluorescence at the sperm head, loses acrosome and is a sperm that has completed acrosome reactions.

As a result:

as shown in FIG. 18, the control group had a background proportion of cells of about 9.5. + -. 1.1% of the capacitated sperm. The proportion of capacitated sperm in the experimental group increased significantly (36.2 ± 2.2%) after 60 minutes incubation with capacitating fluid. When the protease inhibitor MG132 or Epomicin was added while incubating with capacitation solution for 60 minutes, the proportion of capacitated sperm was significantly decreased compared to the experimental group (MG132 group, 16.1. + -. 3.4%; Epomicin group, 15.7. + -. 3.0%). Indicating that proteasomes affect fertilization rates by affecting sperm capacitation.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The application of proteasome in improving the success rate of in vitro fertilization is characterized in that the success rate of in vitro fertilization is positively correlated with the activity of sperm proteasome, and the correlation coefficient r is 0.4499; the activity of sperm proteasome is high, and the success rate of in vitro fertilization and the 2PN rate of in vitro fertilization can be obviously improved.

2. Use of proteasome activity in assessing success rate of in vitro fertilization.

3. The use according to claim 2, wherein the success rate of in vitro fertilization is evaluated by proteasome activity in sperm; the proteasome activity assay comprising the steps of:

(2) mixing the sperm samples evenly, diluting the sperm samples with PBS solution and counting, taking the diluted sperm samples and counting to be 2-10⁶The sperm sample/mL is evenly mixed with the protease detection reagent;

4. Use according to claim 2, characterized in that the treatment of the semen sample by gradient centrifugation in step (1) comprises in particular the following steps:

5. The use according to claim 4, wherein the average sperm motility is 90.8 ± 8.81% after processing semen samples by gradient centrifugation.

6. The use according to claim 2, wherein in step (2), the count after dilution is 2-10⁶the/mL sperm sample was mixed with the protease detection reagent at an equal volume ratio.

7. The use of claim 2, wherein in step (3), the standard 20S proteasome reagent is diluted in proportion to 2500ng/ml, 100ng/ml, 500ng/ml, 250ng/ml and 125ng/ml respectively.

8. The use according to claim 2, wherein in step (4), the excitation wavelength for measuring the fluorescence intensity is 380nm and the emission wavelength is 460 nm.

9. The application of a reagent for detecting proteasome activity in preparing a kit for evaluating the success rate of in vitro fertilization is characterized in that the kit comprises a reagent for detecting proteasome activity, and the success rate of in vitro fertilization is evaluated through the proteasome activity.

10. The use of claim 9, wherein the kit comprises proteasome buffer, fluorescein detection reagent, Suc-LLVY-Glo^TMSubstrate and 20S Proteasome standard.