CN110577991B

CN110577991B - Diagnosis box for non-obstructive azoospermia

Info

Publication number: CN110577991B
Application number: CN201910897201.9A
Authority: CN
Inventors: 韩飞; 刘晋祎; 曹佳; 孙磊; 刘文斌; 张中豪; 陈卿
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2022-05-27
Anticipated expiration: 2039-09-23
Also published as: CN110577991A

Abstract

In the application, the methylation state of the SOX30 gene, the expression level of mRNA and encoded protein in normal control OA and NOA puncture testis tissues are detected, and the detection result shows that the SOX30 gene is subjected to specific hypermethylation and expression silencing in the NOA puncture testis tissues. Based on the detection result, the diagnosis box for the non-obstructive azoospermia is provided, and can help clinical NOA patients to make an effective treatment scheme, so that accuracy and treatment progress are improved, and meanwhile, the diagnosis box has important significance in better understanding of NOA pathogenic reasons, making risk assessment and early diagnosis.

Description

Diagnosis box for non-obstructive azoospermia

Technical Field

The application relates to the technical field of molecular biology, in particular to a diagnosis kit for non-obstructive azoospermia.

Background

Infertility, as an important disease affecting human proliferation, has become a serious medical health problem and a serious social problem worldwide and is receiving wide attention from people. The World Health Organization (WHO) predicts that infertility will become the third largest disease after tumor and cardiovascular disease in diseases affecting human life and Health. According to the latest statistical data of WHO, the number of couples suffering from infertility in the world is about 8000 ten thousand pairs, wherein male patients account for about 50 percent, and the proportion of infertility caused by male reasons is in an increasing situation year by year. In China, the male sterility problem is particularly prominent along with the opening of the national policy of 'two children'. The etiology of male infertility is very complex, and the pathological mechanism is not clear. Although some male infertility is caused by known causes such as Obstructive Azoospermia (OA), cryptorchism, varicocele, and the like, most male infertility is diagnosed as Non-obstructive azoospermia (NOA) of unknown cause.

At present, the diagnosis procedure for NOA patients is complex, semen routine analysis, semen biochemistry, blood hormone analysis, testis morphology analysis, testis puncture pathology analysis and the like need to be carried out, the detection processes are time-consuming and labor-consuming, and the illness state and treatment progress of the patients are seriously influenced. Therefore, a diagnostic kit that can rapidly and accurately diagnose NOA disease is in great demand.

Disclosure of Invention

The application provides a diagnosis box for non-obstructive azoospermia to solve the problems that the existing NOA detection process is time-consuming and labor-consuming, and the illness state and the treatment progress of a patient are seriously influenced.

The application provides a diagnostic kit for non-obstructive azoospermia, which comprises a reagent A for detecting the methylation state of the SOX30 gene,

the reagent A comprises a reagent A1 and a reagent A2, wherein the reagent A1 is used for amplifying a methylation product, and the reagent A1 comprises a pair of methylation primers, and the primer sequences of the methylation primers are respectively as follows:

SOX30F-M:TGG GTA GTA GTT ATG GAG TTT TTT TC，

SOX30R-M:CTC TAA CTT CAC CTA CAA CAA CCG；

the reagent A2 is used for amplifying an unmethylated product, and the reagent A2 comprises a pair of unmethylated primers, wherein the primer sequences of the unmethylated primers are respectively:

SOX30F-U:TGG GTA GTA GTT ATG GAG TTT TTT TT，

SOX30R-U:CTC TAA CTT CAC CTA CAA CAA CCA C。

optionally, the diagnostic kit further comprises a reagent B, wherein the reagent B is a primer pair for detecting the expression level of the gene SOX30, the primer pair is used for RT-PCR for amplifying a SOX30 specific fragment, and the primer sequences of the primer pair are respectively as follows:

SOX30F:GAT GTC CCG CTC ACC GTG TTG C,

SOX30R:GAC AGG GCT TGG GCT CTG GAC T。

optionally, the diagnostic kit further comprises a reagent C, wherein the reagent C is a reagent for detecting the content of the SOX30 protein.

Alternatively, the reagent C is a monoclonal antibody to SOX30 protein.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the identification of SOX30 as an important methylated gene in NOA by methylation sequencing, wherein left to right represents the entire chromosome narrowing to chromosome five and the relocation to the SOX30 gene;

FIG. 2 is a graph of the methylation of SOX30 in OA and NOA-punctured testis tissue, wherein U represents the unmethylated product (shown as the band in the graph) and M represents the methylated product (shown as the band in the graph);

FIG. 3 shows the mRNA expression of SOX30 in OA and NOA-punctured testis tissue, where ACTIN stands for β -ACTIN;

FIG. 4 shows the protein expression of SOX30 in OA and NOA punctured testis tissue, wherein the brown-yellow color represents SOX30 expression staining.

Detailed Description

The SOX family is a type of developmental nuclear transcription factor, and is a gene superfamily consisting of male sex determining gene SRY (sexing region of Chr Y) related genes, and the members of the gene superfamily have highly conserved DNA binding domains (HMG-box). The current research shows that the SOX gene is widely involved in the processes of embryonic development, sex determination, organogenesis, tumorigenesis and the like. SOX30(SRY box containing gene 30) is the only member of the H subfamily of the SOX gene family, which has a very important role in spermatogenesis.

Obtaining a clinical sample: 15 cases of normal spermatogenic OA puncture testicle tissues of the large-plateau hospital and 58 cases of NOA puncture testicle tissues with detailed pathological information are selected as liquid nitrogen preservation tissues and paraffin specimen tissues, wherein white slices of the paraffin specimen are cut and stored in a refrigerator at 4 ℃. Wherein, the OA inclusion standard is that the tissue morphology is normal, the testicle tissue is punctured to be spermatogenic and normal, and a child can be born by an assisted reproduction means (all detection indexes are the same as or similar to those of normal people). NOA inclusion criteria were determined for patients with NOA for each index, including morphological, biochemical and pathological tests. In addition, all OA and NOA patients received no adjuvant treatment with hyperacidin, chemotherapy and radiation prior to biopsy.

Test 1

In this test, the methylation state of SOX30 gene in normal control OA and NOA-punctured testis tissue was examined, and NOA disease was diagnosed based on the methylation state of SOX30 gene, and the detailed examination procedure is as follows.

1) Purification and extraction of genomic DNA

DNA of the tissue sample is purified and extracted by using a DNA Purification and extraction Kit (Wizard Genomic DNA Purification Kit, A1125) from Promega corporation, the specific operation of which is a technique commonly used in the art and will not be described herein.

2) Methylation modification of tissue DNA

The specific operation of the method, which is a technique commonly used in the art, is to perform Methylation modification on the tissue sample DNA by using a Methylation-modification Kit (EZ DNA Methylation-Gold Kit, D5005) of Zymo-Research corporation, and is not described herein again.

3) Methylation analysis (MSP) in modified DNA

During methylation analysis, the MSP primer sequence is shown in Table 1.

Table 1 shows MSP primer sequences

In Table 1, U denotes an unmethylated primer and M denotes a methylated primer.

In the methylation analysis process, the MSP analysis reaction system is as follows: deionized water 7.5. mu.L, 2xTaq mixture 10. mu.L, upstream primer/F0.5. mu.L, downstream primer/R0.5. mu.L, modified DNA 1.5. mu.L.

In the methylation analysis process, the MSP analysis reaction conditions are as follows: 95 ℃ for 3min, (95 ℃ for 35s, 60 ℃ for 35s, 72 ℃ for 1min) x38, 72 ℃ for 10min, 12 ℃ for 2 h.

In the methylation analysis process, the electrophoresis and statistics process of the MSP product is as follows: PCR amplification products for MSP analysis were electrophoresed on 2.5% EB-containing agarose gel, photographed and statistically analyzed.

4) Results of methylation analysis

The 5 cases of OA patients puncturing testis tissues and 5 cases of NOA patients puncturing testis tissues, of which the indexes are close to the normal human testis form and pathology, are selected to be respectively subjected to whole genome methylation sequencing, and comparative analysis and chromosome walking analysis show that the SOX30 Gene (Pubmed, Gene ID number: NM-178424) presents the most obvious hypermethylation state in the NOA puncturing testis tissues, as shown in figure 1. The analysis result shows that the methylation of the SOX30 gene in the NOA puncture testis tissue is a specific characteristic event.

In summary, in this experiment, MSP (Methylation-Specific PCR) was performed in randomly selected OA and NOA patients by designing SOX30 Methylation primers (primer sequences are shown in Table 1) by online methprimer based on the SOX30 Methylation sequencing position and puncturing testis tissue by OA and NOA patients (reaction system, conditions and electrophoresis statistics are as described above). The results in FIG. 2 show that SOX30 was significantly methylated in NOA-spiked testis tissue, but not in OA-spiked testis tissue. By analyzing the methylation rate of SOX30 in the tissues of the punctured testis of 15 control OA patients and 58 NOA patients, the result shown in Table 2 is the SOX30 methylation rate analysis table, and as shown in Table 2, SOX30 is methylated only in 6.67% (1/15) of OA, but is methylated in as much as 91.38% (53/58) of NOA, and the methylation rate is significantly different between OA and NOA. The data in table 2 further illustrate that SOX30 methylation is a specific important characteristic event in NOAs.

TABLE 2 SOX30 methylation incidence analysis table

Test 2

In this test, expression levels of mRNA and encoded protein in normal control OA and NOA-punctured testis tissues were detected, and NOA disease was diagnosed based on the expression level of SOX30, and the specific detection process is shown below.

1) Extraction of tissue total RNA

The extraction of total RNA of the tissue is carried out by using a classical RNA extraction reagent Trizol, and the extraction process is a common technology in the field and will not be described in detail herein.

2) Reverse transcription of tissue RNA into cDNA

The specific operation of Reverse Transcription of RNA treated with DNase I (Deoxyribonuclease I) into cDNA using a Reverse Transcription kit (GoScript Reverse Transcription System, A5001) from Promega is a common technique in the art and is not described herein.

3) Reverse transcription-PCR

RT-PCR primer sequences during reverse transcription-PCR (RT-PCR) are shown in Table 3.

TABLE 3 RT-PCR primer sequences

The RT-PCR system during reverse transcription-PCR (RT-PCR) is shown in Table 4.

TABLE 4 RT-PCR System

The RT-PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, (denaturation at 95 ℃ for 35s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 1.0min) x35, extension at 72 ℃ for 10min, and sample preservation at 12 ℃.

4) PCR product electrophoresis and statistics

The RT-PCR product was added to 3.0% agarose gel containing EB dye for electrophoresis, observation, photography and statistical analysis.

5) Immunohistochemical (IHC) staining

Conventional tissue fixing, embedding and slicing, baking, hydrating, antigen repairing, enzyme eliminating, serum sealing, antibody incubating, DAB coloring, differentiating and turning blue, sealing, microscopic examination, photographing and statistical analysis.

6) Results of analysis of SOX30 mRNA and protein expression levels

To determine whether SOX30 methylation affects its expression and the altered profile of SOX30 expression in NOA, this experiment examined the mRNA expression of SOX30 in the spiked testis tissue of randomly selected OA and NOA patients as described above by RT-PCR using Actin (Actin) as an internal reference for PCR, with the results shown in FIG. 3 for the silencing of SOX30 in methylated NOA patients and the normal expression of SOX30 in unmethylated OA patients. FIG. 4 shows the expression changes of SOX30 protein in OA and NOA patients analyzed by immunohistochemistry, and the results in FIG. 4 show that SOX30 protein is also silenced in NOA patients. The results of the above tests show that SOX30 methylation is closely related to the silencing of its expression.

To characterize the change in expression of SOX30 in NOA, this experiment analyzed the change in expression of SOX30 in the punctured testis tissue of the same 15 control OA and 58 NOA patients by IHC, and the results are shown in table 5, wherein SOX30 was positive in 15 control OA, while only 17.24% (10/58) of NOA was positive, and the positive rates of SOX30 expression in OA and NOA were significantly different. The results of the above tests show that inactivation of SOX30 expression is also a specific important characteristic event in NOA.

TABLE 5 expression of SOX30 protein in OA and NOA-punctured testis tissue

According to the detection results of the test 1 and the test 2, the methylation or expression inactivation of the SOX30 has important significance in the formation of the NOA and is a specific important characteristic event of the NOA. Therefore, whether the azoospermia patient belongs to the NOA patient or not can be judged rapidly by detecting the methylation or expression state of the SOX30 in the punctured testis of the azoospermia patient, and the clinical azoospermia patient is helped to make a treatment scheme faster and better, so that the accuracy is improved, and the early treatment is carried out.

Based on the above detection results, the present application provides a diagnostic kit for non-obstructive azoospermia, comprising a reagent a for detecting the methylation state of the SOX30 gene, wherein the reagent a comprises a reagent a1 and a reagent a2, wherein the reagent a1 is used for amplifying a methylation product, the reagent a1 comprises a pair of methylation primers, and the primer sequences of the methylation primers are respectively:

SOX30F-M:TGG GTA GTA GTT ATG GAG TTT TTT TC，

SOX30R-M:CTC TAA CTT CAC CTA CAA CAA CCG；

the reagent A2 is used for amplifying unmethylated products, and the reagent A2 comprises a pair of unmethylated primers, wherein the primer sequences of the unmethylated primers are respectively:

SOX30F-U:TGG GTA GTA GTT ATG GAG TTT TTT TT，

SOX30R-U:CTC TAA CTT CAC CTA CAA CAA CCA C。

in this example, the diagnostic kit further comprises a reagent B, wherein the reagent B is a primer pair for detecting the expression level of the gene SOX30, and the primer sequences of the primer pair are respectively as follows:

SOX30F:GAT GTC CCG CTC ACC GTG TTG C,

SOX30R:GAC AGG GCT TGG GCT CTG GAC T。

in this example, the diagnostic kit further comprises a reagent C, which is a reagent for detecting the content of SOX30 protein.

In this example, reagent C is a monoclonal antibody to the SOX30 protein.

In order to facilitate a better understanding of the present technical solution by the person skilled in the art, the following further description is given in connection with the use of the diagnostic kit for non-obstructive azoospermia of the present application.

Performing genome DNA purification extraction and methylation modification on a patient puncture testicular tissue by a conventional technology, performing RT-PCR amplification on the patient puncture testicular tissue and a reagent A, wherein an amplification system and conditions are shown in test 1, performing electrophoresis in agarose gel and analyzing results, and judging that the patient is an NOA patient when a methylation product (reflected as a strip in the agarose gel electrophoresis) is amplified by the reagent A1; when only reagent A2 amplified non-methylated products (as reflected as bands in agarose gel electrophoresis), the patient was judged to be a non-NOA patient.

Of course, the patient may be punctured with testis tissue (fresh) by conventional techniques to extract total RNA, reverse transcribing into cDNA, and performing RT-PCR amplification with reagent B, where the amplification system and conditions are shown in test 2, and the result of electrophoresis analysis in agarose gel is judged that the patient is NOA patient when reagent B cannot amplify the product (the product is reflected as a band in agarose gel electrophoresis).

Of course, the patient may be subjected to immunohistochemical staining analysis by puncturing a paraffin section of the testis tissue, and when the expression of the reagent C is negative, the patient is judged to be NOA patient.

In addition, the reagent A, the reagent B and the reagent C are simultaneously detected when the patient punctures the testicle tissue, so that the accuracy of the NOA judgment result is greatly improved.

In the application, the methylation state of the SOX30 gene, the expression level of mRNA and encoded protein in normal control OA and NOA puncture testis tissues are detected, and the detection result shows that the SOX30 gene is subjected to specific hypermethylation and expression silencing in the NOA puncture testis tissues. Based on the above detection results, the present application provides a diagnostic kit for non-obstructive azoospermia, which is used for rapid diagnosis of NOA diseases, and can rapidly and accurately identify the NOA diseases by specifically detecting the methylation or expression change of the gene SOX30, i.e., detecting the methylation or expression level of the gene. The diagnosis box for the non-obstructive azoospermia can help clinical NOA patients to make an effective treatment scheme, so that accuracy and treatment progress are improved, and meanwhile, the diagnosis box has important significance in better understanding NOA pathogenic reasons, making risk assessment and early diagnosis.

It should be noted that hypermethylation in this application means that SOX30 methylation is higher in NOA-spiked testis than in OA-spiked testis; the expression silencing means that SOX30 mRNA and protein are not expressed or are far less expressed than OA puncture testis in NOA puncture testis.

The above-described embodiments of the present application do not limit the scope of the present application.

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Claims

1. A diagnostic kit for non-obstructive azoospermia, comprising a reagent A for detecting the methylation state of the SOX30 gene,

SOX30F-M:TGG GTA GTA GTT ATG GAG TTT TTT TC，

SOX30R-M:CTC TAA CTT CAC CTA CAA CAA CCG；

SOX30F-U:TGG GTA GTA GTT ATG GAG TTT TTT TT，

SOX30R-U:CTC TAA CTT CAC CTA CAA CAA CCA C。

2. the diagnostic kit for non-obstructive azoospermia according to claim 1, further comprising a reagent B, which is a primer pair for detecting the expression level of gene SOX30, the primer sequences of the primer pair are respectively:

SOX30F:GAT GTC CCG CTC ACC GTG TTG C,

SOX30R:GAC AGG GCT TGG GCT CTG GAC T。

3. the diagnostic kit for non-obstructive azoospermia according to claim 1, further comprising a reagent C, which is a reagent for detecting the content of SOX30 protein.

4. The diagnostic kit for non-obstructive azoospermia according to claim 3, wherein the reagent C is a monoclonal antibody to the SOX30 protein.