阅读说明：本技术 一种根据精浆mRNA预测无精子症者睾丸中有无精子的方法 (Method for predicting sperms in testis of azoospermia patient according to seminal plasma mRNA ) 是由 闫威 唐运革 于 2020-05-08 设计创作，主要内容包括：本发明公开了一种根据精浆mRNA预测无精子症者睾丸中有无精子的方法,以睾丸活检为“黄金标准”,将无精子症患者精液标本分为睾丸有精子组和睾丸无精子组,应用深度测序技术确定两组精液exRNA表达谱差异,然后与现有生精细胞转录组数据库进行比较,寻找具有判定生精细胞种类的,差异表达的exRNA,用于判定无精子症患者睾丸内是否存在单倍体精子细胞。本发明的方案在临床中的应用将极大提高无精子症患者取精成功率,也有助于指导无精子症患者选择经济,无痛苦,有效的治疗方法来解决其不育的难题。(The invention discloses a method for predicting the existence of sperms in the testis of a patient without spermatids according to seminal plasma mRNA, which takes testis biopsy as 'gold standard', divides a semen sample of the patient without spermatids into a testis spermatid group and a testis spermatid group, determines the difference of expression profiles of two groups of semen exRNA by applying a deep sequencing technology, compares the difference with the existing spermatid transcriptome database, and searches for the exRNA which has the function of judging the types of the spermatids and is differentially expressed, and is used for judging whether haploid spermatids exist in the testis of the patient without spermatids. The application of the scheme of the invention in clinic can greatly improve the sperm collection success rate of patients with azoospermia, and is also helpful for guiding the patients with azoospermia to select an economical, painless and effective treatment method to solve the problem of infertility.)

1. A method for predicting the presence or absence of sperm in the testis of a patient with azoospermia based on seminal plasma mRNA, comprising the steps of:

(1) collecting and processing a semen sample: collecting semen of patients with azoospermia, and freezing and storing with liquid nitrogen; azoospermia is defined as taking semen every 4-5 weeks, centrifuging the semen for three times, and then performing microscopic examination to find no semen; performing low-speed centrifugation twice on the collected semen of the patient without spermatozoon, and then using the supernatant for RNA sequencing;

(2) isolation and sequencing of RNA: the mirVana miRNA separation kit can be used for separating large-fragment RNA and small-fragment RNA, and the quality, size distribution and quantity of the RNA are analyzed by using Bioanalyzer 2100(Agilent) after separation;

(3) multi-point puncture biopsy of testis: obtaining mature sperms in the testis of a patient without spermatozoon disease by adopting a testis multi-point puncture method through a testis sperma suction technique, and finding the mature sperms under an inverted microscope; then dividing the sample into a microscopic group and an azoospermia group by taking the pathological histological examination as a gold standard;

(4) differential expression profiling of seminal plasma mRNA in azoospermia patients: the method comprises the steps of (1) carrying out pathological detection on whether haploid spermatids exist in the testis of a patient with azoospermia by using testis multipoint biopsy, and dividing a sample of the patient with azoospermia into a testis spermatid group and a testis azoospermia group; unfreezing and centrifuging the two groups of semen at a low speed, separating semen supernatant from sperm cells, centrifuging the semen supernatant at a low speed once again, and sequencing the obtained supernatant to obtain a pure semen RNA sequence; then comparing with the RNA sequencing result of each patient and the existing RNA library to obtain differentially expressed RNA with prediction value;

(5) bioinformatics analysis: annotating endogenous and exogenous mRNA using TCF annotation pathways; annotating mRNA using the lncRNA annotation pathway;

(6) and (3) verifying the sensitivity and specificity of the specific RNA prediction index: detecting the RNA expression level with prediction value of each group by using real-time PCR; applying SPSS software, carrying out variance analysis, Kruskal-Wallis H test and Mann-whitney U test on experimental data, taking alpha as 0.05 as a test standard, and evaluating the sensitivity and specificity of a detection index by calculating integral analysis under an ROC curve by adopting a subject working characteristic curve method;

(7) screening a plurality of genes which can be used for predicting whether haploid sperms exist in the testis from a sequencing result; and (3) comparing whether the semen of the azoospermia patient contains corresponding genes or not by sequencing specific samples to predict whether haploid sperms exist in the testis or not.

2. The method according to claim 1, wherein the step (3) of performing a testicular multiple biopsy for the presence or absence of sperm in the testicle of azoospermia based on seminal plasma mRNA comprises the steps of:

taking the patient in a supine position, disinfecting the operation area by a conventional method, laying a sterile hole towel, and carrying out nerve block anesthesia in the spermatic cord of 1% lidocaine;

a disposable 20ml medicine dissolving empty needle (a needle head with a side hole) is used as a puncture needle; fixing the testicle on the side of the left hand three fingertips to cling to the subcutaneous part of the scrotum, taking the middle upper part of the testicle at the puncture part, puncturing the scrotum by 1% lidocaine local anesthesia at the puncture point, utilizing a No. 5 half needle injector to puncture the scrotum until the white membrane of the testicle makes an access, holding a hollow needle for drug dissolution for puncture by the right hand to puncture into the parenchyma of the testicle along the access through the skin, repeatedly pulling the needle plug for a plurality of times, then pulling the needle plug to the position of 20ml to form negative pressure, and pulling out the needle head under the state of the.

Dividing the sucked testicular tissue into two parts, placing one part into a small culture dish containing HTF liquid, separating, scratching, shredding and extruding the testicular tissue by using a 1ml syringe needle to release spermatids and sperms to prepare cell suspension, and searching for mature sperms under an inverted microscope; if sperm exist, the operation is ended;

if the testicular tissue at the side of the operation is spermadless, puncture at the same side for the 2 nd time can be carried out, if the spermadless still exists, the opposite side TESE is carried out according to whether mature spermadless is found under an inverted microscope;

after the operation, the other part of the testis tissue fixed by the Bouin liquid is sent to the pathology, the routine HE staining is carried out, the pathological histological examination is carried out by a pathologist, and the specimen is divided into a group with a microscope and a group without a sperm by taking the pathological histological examination as a gold standard.

3. The method of claim 1 for predicting the presence or absence of sperm in the testis of a patient with azoospermia based on seminal plasma mRNA, wherein: in the step (4), because the frozen semen contains exogenous RNA pollution such as a protective agent and the like, the frozen protective agent needs to be sequenced separately, the obtained sequence is removed from the experimental result, and then the pure semen RNA sequence can be obtained.

4. The method of claim 1 for predicting the presence or absence of sperm in the testis of a patient with azoospermia based on seminal plasma mRNA, wherein: in the step (4), the low-speed centrifugation refers to 2000g centrifugation.

5. The method according to claim 1, wherein in the step (7), the prediction rule of the related genes is as follows:

(1) the expression of TDRP7-2 and PRM2 mRNA in the seminal plasma is reduced, so that the pathological types of normal and spermatogenic block can be distinguished;

(2) the expression of SMCP and ACSBG2-2 mRNA in the seminal plasma is reduced, so that normal and obstruction, non-obstructive azoospermia pathological types can be distinguished;

(3) decreased ARID5B-3 and FRS2-2 mRNA expression in the seminal plasma can distinguish between normal and supportive cytopathic types of cell syndrome alone.

Technical Field

The invention relates to the technical field of medical detection, in particular to a method for predicting the existence of sperms in testes of a patient without spermacrasia according to seminal plasma mRNA (messenger ribonucleic acid).

Background

About 10% -20% of male infertility patients are azoospermia, which is further classified into Obstructive Azoospermia (OA) and non-obstructive azoospermia (NOA). If there is sperm in testis of azoospermia patient, sperm can be obtained by external techniques such as testicular sperm aspiration (TESE) and assisted reproduction technique, so that offspring with self blood relationship can be obtained, nearly 100% of OA patients can obtain sperm by TESE, while only about 50% -60% of NOA patients can obtain sperm. However, on the day of ova aspiration by the patient's partner, some of the azoospermia patients may suffer the embarrassment of not being able to aspirate sperm, which is as high as about 30% of the cases in NOA, when the female partner has received expensive ovulation-promoting treatment and may have undergone ova aspiration, and thus, effective assessment of male during the time the NOA couple enters the assisted reproductive cycle is of great clinical significance. For patients with azoospermia, the choice of having their own children is limited, mainly by two routes: (1) intracytoplasmic injection of sperm (ICSI) or IUI by sperm supply; (2) haploid spermatids are searched in the testis by surgical operation, and if the haploid spermatids can be found, offspring of the self blood relationship can be obtained through ICSI. Despite the great invasiveness and uncertainty of surgery, most patients prefer to have sperm harvested in this manner because offspring of their consanguineous relationship are available. Currently, there is no way to determine whether haploid spermatids are present in the testis of a patient without spermatozoa prior to surgical exploration. We have been thinking about whether we can use the semen or the components of seminal plasma as a biological marker to determine if haploid spermatids are present in the testis of a patient with azoospermia? If a method for judging whether haploid spermatids exist in the testis through semen/plasma analysis can be established, the operation pain and the economic burden of a patient can be reduced, unnecessary operation exploration can be avoided, and the diagnosis and treatment efficiency is improved. For example, for patients who have no haploid spermatids in their testis at all, the patient should be informed that a testicular biopsy is unnecessary and may choose for artificial insemination to address infertility. Therefore, it is a research topic with great clinical value.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for predicting the presence or absence of sperms in the testis of a patient with azoospermia according to seminal plasma mRNA, taking testis biopsy as "gold standard", dividing a semen sample of a patient with azoospermia into a testis spermatic group and a testis azoospermia group, determining the difference of expression profiles of two groups of spermatic exRNA by using a deep sequencing technology, comparing the difference with the existing spermatic cell transcriptome database, and searching for the exRNA with difference expression for judging the types of spermatic cells, wherein the difference expression exRNA is used for judging whether haploid spermatids exist in the testis of the patient with azoospermia. The success of the invention and the future clinical application can greatly improve the sperm-taking success rate of the azoospermia patients, and is also helpful for guiding the azoospermia patients to select an economical, painless and effective treatment method to solve the problem of infertility.

Therefore, the invention discloses the following technical scheme:

a method for predicting the presence or absence of sperm in the testis of a patient with azoospermia based on seminal plasma mRNA, comprising the steps of:

(1) collecting and processing a semen sample: collecting semen of patients with azoospermia, and freezing and storing with liquid nitrogen; azoospermia is defined as taking semen every 4-5 weeks, and no sperm is found in microscopic examination after three times of semen centrifugation; performing low-speed centrifugation twice on the collected semen of the patient without spermatozoon, and then using the supernatant for RNA sequencing;

(2) isolation and sequencing of RNA: the mirVana miRNA separation kit can be used for separating large-fragment RNA and small-fragment RNA, and the quality, size distribution and quantity of the RNA are analyzed by using Bioanalyzer 2100(Agilent) after separation;

(3) multi-point puncture biopsy of testis: obtaining mature sperms in the testis of a patient without spermatozoon disease by adopting a testis multi-point puncture method through a testis sperma suction technique, and finding the mature sperms under an inverted microscope; then dividing the sample into a microscopic group and an azoospermia group by taking the pathological histological examination as a gold standard;

(4) differential expression profiling of seminal plasma mRNA in azoospermia patients: the method comprises the steps of (1) carrying out pathological detection on whether haploid spermatids exist in the testis of a patient with azoospermia by using testis multipoint biopsy, and dividing a sample of the patient with azoospermia into a testis spermatid group and a testis azoospermia group; unfreezing and centrifuging the two groups of semen at a low speed, separating semen supernatant from semen cells, centrifuging the semen supernatant at a low speed once again, and sequencing the obtained supernatant to obtain a pure semen RNA sequence; then comparing with the RNA sequencing result of each patient and the existing RNA library to obtain differentially expressed RNA with prediction value;

(5) bioinformatics analysis: endogenous and exogenous mRNA were annotated using TCF (Tophat, Cufflink and fusion map) annotation pathway; annotating mRNA using the lncRNA annotation pathway;

(6) and (3) verifying the sensitivity and specificity of the specific RNA prediction index: detecting the RNA expression level with prediction value of each group by using real-time PCR; applying SPSS software, carrying out variance analysis, Kruskal-Wallis H test and Mann-whitney U test on experimental data, taking alpha as 0.05 as a test standard, and evaluating the sensitivity and specificity of a detection index by calculating integral analysis below an ROC curve by adopting a subject working characteristic curve method;

(7) screening a plurality of genes which can be used for predicting whether haploid sperms exist in the testis from a sequencing result; and (3) comparing whether the semen of the azoospermia patient contains corresponding genes or not by sequencing specific samples to predict whether haploid sperms exist in the testis or not.

Preferably, in the step (3), the testicular multipoint needle biopsy comprises the following specific steps:

taking the patient in the supine position, disinfecting the operation area by a conventional method, laying a sterile hole towel, and carrying out nerve block anesthesia in the spermatic cord by 1% lidocaine;

a disposable 20ml medicine dissolving empty needle (a needle head with a side hole) is used as a puncture needle; fixing the testicle on the side of the left hand three fingertips to cling to the subcutaneous part of the scrotum, taking the middle upper part of the testicle at the puncture part, puncturing the scrotum by using 1% lidocaine local anesthesia at the puncture point, utilizing a No. 5 half needle injector to puncture the scrotum until the white membrane of the testicle makes an access, holding a medicine dissolving empty needle for puncture by a right hand to puncture into the parenchyma of the testicle along the access through the skin, repeatedly drawing a needle plug for a plurality of times, then drawing the needle plug to the position of 20ml to form negative pressure, and pulling out the needle head under the state of keeping the.

Dividing the sucked testicular tissue into two parts, putting one part into a small culture dish containing HTF liquid, separating, scratching, shredding and extruding the testicular tissue by using a 1ml syringe needle to release spermatids and sperms to prepare cell suspension, and searching for mature sperms under an inverted microscope; if sperm exist, the operation is ended;

if the testicular tissue at the side of the operation is spermadless, puncture at the same side for the 2 nd time can be carried out, if the spermadless still exists, the opposite side TESE is carried out according to whether mature spermadless is found under an inverted microscope;

after the operation, the other part of the testis tissue fixed by the Bouin liquid is sent to the pathology, the routine HE staining is carried out, the pathological histological examination is carried out by a medical doctor in a pathology department, and the specimen is divided into a group with a mirror and a group without a sperm by taking the pathological histological examination as a gold standard.

Preferably, in the step (4), due to exogenous RNA contamination such as protective agent contained in the frozen semen, the frozen protective agent needs to be sequenced separately, and the obtained sequence is removed from the experimental result, so as to obtain a pure semen RNA sequence.

Preferably, in the step (4), the low-speed centrifugation refers to 2000g centrifugation.

Preferably, in the step (7), the prediction rule of the related gene is:

(1) the expression of TDRP7-2 and PRM2 mRNA in the seminal plasma is reduced, so that the pathological types of normal and spermatogenic block can be distinguished;

(2) the expression of SMCP and ACSBG2-2 mRNA in the seminal plasma is reduced, so that normal and obstruction, non-obstructive azoospermia pathological types can be distinguished;

(3) decreased ARID5B-3 and FRS2-2 mRNA expression in the seminal plasma can distinguish between normal and supportive cytopathic types of cell syndrome alone.

The invention screens 17 genes which can be used for predicting whether haploid sperms exist in the testis from a sequencing result. Wherein PRM2, ODF1, ACSBG2 and SMCP are specifically expressed in sperm cells; genes such as FRS2, TBX3, CDKN1B, CSDE1, AR, ARID5B, DLD, TDRD7, UBE3A and CAT are mainly expressed in spermatogonium and spermatocyte; genes such as UBA52, POLR2L, RPL36 are mainly expressed in supporting cells. The clinical group of the experimental setup was: a normal control group, a spermatogenic arrest group, a group with spermatozoa by puncture and a group with only supporting cells; with RPL41 as the reference gene, the results of further validation of 17 candidate genes are shown in the figure: genes that differ among groups include genes such as AR, TDRP7, ARID5B, SMCP, PRM2, FRS2, and ACSBG 2. Wherein the expression of the SMCP and ACSBG2 genes can be detected only in a normal control group; the PRM2 gene is only expressed in the normal control group and the group punctured with sperm; the expression level of the AR gene is significantly different between the normal control group and the punctured sperm group (P <0.05), the TDRP7 gene is significantly different between the support cell group only and the sperm production blocking group compared with the normal control group (P <0.05), and the ARID5B gene is significantly different between the normal control group and the support cell group (P < 0.05).

The method can predict the spermatogenic function of the testis of the patient without spermatozoon by obtaining a plurality of seminal plasma mRNA: (1) the expression of TDRP7-2 and PRM2 mRNA in the seminal plasma is reduced, so that the pathological types of normal and spermatogenic block can be distinguished; (2) the expression of SMCP and ACSBG2-2 mRNA in the seminal plasma is reduced, so that normal and obstructive and non-obstructive azoospermia pathological types can be distinguished; (3) decreased ARID5B-3 and FRS2-2 mRNA expression in the seminal plasma can distinguish between normal and supportive cytopathic types of cell syndrome alone. The achievement is expected to be transformed into clinic to improve the semen collection success rate of the azoospermia patient, and is also helpful for guiding the azoospermia patient to select an economic and effective treatment method to solve the infertility problem.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly described below.

FIG. 1 is a major analysis of the azoospermia transcriptome for different types (TESA, MA, SOS and CTR);

FIG. 2 shows the difference between the normal control group and the extracellular mRNA of TESA, MA and SOS type azoospermia;

FIG. 3 shows the difference between SOS and TESA, MA type azoospermia extracellular mRNA;

FIG. 4 is a heat map of the expression of different types of azoospermia extracellular mRNA;

FIG. 5 shows the results of gene screening.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The method of carrying out the process, in which the specific conditions are not specified in the examples, is generally carried out in a conventional manner.