Application of sperm-specific miRNAs in the diagnosis of in vitro fertilization in assisted reproduction

Abstract

The present invention discloses the application of sperm-specific miRNAs in the preparation of a diagnostic reagent or diagnostic kit for in vitro fertilization in assisted reproduction. The sperm-specific miRNAs are one or more of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p or hsa-miR-29a-3p. The present invention also discloses the application of sperm-specific miRNAs in the preparation of a molecular marker for predicting or evaluating sperm fertility during in vitro fertilization in assisted reproduction. The sperm-specific miRNAs of the present invention can be used as sperm-specific molecular markers, diagnostic reagents or diagnostic kits for in vitro fertilization in assisted reproduction, etc., to predict sperm fertility, thereby providing a reliable scientific basis and ideas for personalized treatment for the treatment of male infertility and clinical diagnosis in in vitro fertilization in assisted reproduction.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of sperm-specific miRNAs in the diagnosis of in vitro fertilization for assisted reproduction. Background Art

[0002] MicroRNAs (miRNAs) are a class of small, single-stranded non-coding small RNAs (sncRNAs) with a length of 20 - 23 nt. They can mediate gene silencing at the post-transcriptional level by incomplete pairing with the 3' UTR of target mRNAs, thereby achieving translational inhibition or degradation of target mRNAs. Currently, miRNAs are a class of sncRNAs that have been studied extensively. Many studies have confirmed that miRNAs can serve as potential biomarkers for sperm quality prediction and diagnosis. The expression of miRNAs in the testes of patients with non-obstructive azoospermia (NOA) is altered, indicating that miRNAs may play a role in spermatogenesis. Microarray detection of sperm from patients with asthenozoospermia or oligoasthenozoospermia revealed changes in the expression of many miRNAs. Further bioinformatics analysis showed that they may be involved in the process of spermatogenesis. In addition, studies have also found that the expression of three miRNAs (miR-141, miR-429, miR-7-1-3p) changes in the seminal plasma of azoospermia patients, and they may be involved in the methylation-miRNA-gene regulatory network related to NOA. These results suggest that miRNAs play an important role in the male reproductive system, but there is currently no evaluation and prediction of normal sperm by miRNA.

[0003] Existing technical means and methods for predicting sperm fertility during in vitro fertilization (IVF) in assisted reproduction mainly focus on detecting semen parameters, semen ROS values, sperm DNA fragmentation rates, etc. However, the existing methods only detect sperm-related parameters and are not associated with embryo quality, and cannot comprehensively, truly, and accurately evaluate sperm fertility. Therefore, the existing methods only make judgments on sperm quality, but the evaluation of sperm fertility is insufficient. The success rate of IVF in clinical practice is only about 30%, and the cost is high (the cost of one IVF in a reproductive center is about 30,000 yuan). Summary of the Invention

[0004] In this invention, 87 semen samples from normal men among IVF patients were selected. After optimizing the semen samples and detecting semen quality, normal sperm samples meeting the standards were selected. According to the corresponding good quality embryo rate (GQE), they were divided into H-GQE (GQE≥75%, high, H-GQE, n = 23; GQE≤25%, low, L-GQE, n = 64). Then sperm RNA was extracted, and finally the expression levels of miRNAs were detected by small RNA high-throughput sequencing. After small RNA high-throughput sequencing, a total of 389 miRNAs were detected. After analyzing the expression differences between the two groups, a total of 4 differentially expressed miRNAs were obtained. Finally, through data analysis, it was found that the discrimination efficiency of these 4 miRNAs for 87 samples could reach 71.54%.

[0005] Therefore, the primary objective of this invention is to provide the application of sperm-specific miRNAs in the preparation of diagnostic reagents for in vitro fertilization in assisted reproduction. The second objective of this invention is to provide the application of sperm-specific miRNAs in the preparation of diagnostic kits for in vitro fertilization in assisted reproduction. The third objective of this invention is to provide the application of sperm-specific miRNAs in the preparation of molecular markers for predicting or evaluating sperm fertility during in vitro fertilization in assisted reproduction.

[0006] To achieve the above objectives, this invention adopts the following technical solutions:

[0007] As the first aspect of this invention, the application of sperm-specific miRNAs in the preparation of diagnostic reagents for in vitro fertilization in assisted reproduction, wherein the sperm-specific miRNAs are one or several of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p or hsa-miR-29a-3p.

[0008] According to this invention, the diagnostic reagent is used to detect the specific low expression or specific high expression of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p and / or hsa-miR-29a-3p in a biological sample with a low good quality embryo rate.

[0009] As the second aspect of this invention, the application of sperm-specific miRNAs in the preparation of diagnostic kits for in vitro fertilization in assisted reproduction, wherein the sperm-specific miRNAs are one or several of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p or hsa-miR-29a-3p.

[0010] According to the present invention, the diagnostic kit comprises reagents for detecting the specific low expression or specific high expression of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p and / or hsa-miR-29a-3p in a biological sample with a low high-quality embryo rate.

[0011] As a third aspect of the present invention, there is provided an application of sperm-specific miRNAs in the preparation of molecular markers for predicting or evaluating sperm fertility during in vitro fertilization (IVF) assisted reproduction, wherein the sperm-specific miRNAs are one or more of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p or hsa-miR-29a-3p.

[0012] The beneficial effects of the present invention are as follows: hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p or hsa-miR-29a-3p can be used as sperm-specific molecular markers for predicting sperm fertility, thereby providing a reliable scientific basis and ideas for personalized treatment for the treatment of male infertility and clinical diagnosis in IVF. Specifically, it is embodied in:

[0013] 1. Since sperm-specific miRNAs can be specifically low-expressed or specifically high-expressed in L-GQE, it is possible to detect by preparing monitoring reagents, diagnostic reagents for in vitro fertilization assisted reproduction or molecular markers for predicting or evaluating sperm fertility during in vitro fertilization assisted reproduction, in combination with high-throughput sequencing methods, and the detection results are more real, comprehensive and reliable;

[0014] 2. Sperm-specific miRNAs, as monitoring reagents, diagnostic reagents for in vitro fertilization assisted reproduction or molecular markers for predicting or evaluating sperm fertility during in vitro fertilization assisted reproduction, can detect the miRNA expression level in sperm and predict sperm fertility from the perspective of embryo quality, rather than just sperm quality. The possibility for patients to obtain a high high-quality embryo rate can reach 71.54%. Therefore, the above-mentioned monitoring reagents, diagnostic reagents or molecular markers can early detect potential male infertility targets and perform personalized intervention and treatment, and ultimately improve the fertility outcome of IVF;

[0015] 3. Sperm-specific miRNAs, as monitoring reagents, diagnostic reagents for in vitro fertilization assisted reproduction or molecular markers for predicting or evaluating sperm fertility during in vitro fertilization assisted reproduction, can detect sperm samples at the beginning of IVF treatment and perform corresponding personalized treatment and intervention, which can reduce the diagnosis and treatment costs of patients. Description of the Drawings

[0016] Figure 1 It is the operation flowchart of Example 1.

[0017] Figure 2 It is the graph of the high-throughput sequencing results of miRNAs in Example 1.

[0018] Figure 3 It is the graph of the results of high-throughput sequencing of specifically expressed miRNAs in Example 1.

[0019] Figure 4 It is a schematic diagram of the discrimination efficiency of 4 miRNAs for 87 samples, where the abscissa TPR is the true positive rate, and the ordinate FPR is the false positive rate. Detailed implementation manners

[0020] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention rather than to limit the scope of the present invention.

[0021] In order to facilitate the study of sperm miRNAs as sperm-specific molecular markers, normal sperm samples from patients undergoing IVF treatment were selected in this example. According to their corresponding good-quality embryo rate (GQE rate), they were divided into a high GQE (≥75%; high rate of GQE, H-GQE) group and a low GQE group (≤25%; low rate of GQE, L-GQE). The relationship between miRNA changes and the good-quality embryo rate was analyzed to find miRNAs that can be used as detection indicators for male infertility.

[0022] Example 1

[0023] The operation flowchart of this example is as Figure 1 shown.

[0024] 1. Sample enrollment situation

[0025] In this example, patients who underwent IVF treatment for the first time were included. The female patients were aged between 23 and 40 years old and did not have polycystic ovary syndrome, ectopic pregnancy, premature ovarian failure and other diseases; the male patients were aged between 24 and 50 years old and did not have reproductive system diseases. Both parties were of Han ethnicity and did not have other diseases. Among them, there were no significant differences in the age, number of retrieved oocytes, and the number of oocytes at the MⅡ (metaphase Ⅱ stage) of female patients. There were no significant differences in the age, sperm density, sperm survival rate, and sperm (forward) motility rate (a + b grade sperm) of the corresponding male patients, but there were slight differences in sperm morphology (P < 0.05).

[0026] 2. Grouping of semen samples

[0027] A total of 87 normal male semen samples from IVF patients were selected. According to their corresponding GQE conditions, the semen samples were divided into the H-GQE group (≥75%) and the L-GQE group (≤25%). Among them, there were 23 cases in the high-quality embryo rate group (H-GQE) and 64 cases in the low-quality embryo rate group (L-GQE).

[0028] Among them, compared with the L-GQE group, there were significant differences in the number of corresponding 2PN (zygote developed to the 2-cell stage, two pronuclei stage) (P<0.05), the number of transferable embryos (P<0.001), the fertilization rate (P<0.05), the effective embryo rate (P<0.001), and the high-quality embryo rate (P<0.001) in the H-GQE group.

[0029] Therefore, the embryos in the H-GQE group developed better, and their corresponding sperm fertility was better. The embryo development quality in the L-GQE group was poorer, and the corresponding sperm fertility was poorer.

[0030] 3. Collection of semen samples

[0031] The research subjects abstained from sexual intercourse for 3 - 7 days, and the whole semen was obtained by masturbation method, placed in a 37°C water bath for incubation for 30 minutes, and used after liquefaction.

[0032] 4. Optimization of semen samples

[0033] According to the WHO Laboratory Manual for the Examination of Human Semen (5th Edition), the semen samples were optimized by the sperm swim-up method.

[0034] 5. Routine semen detection

[0035] According to the WHO Laboratory Manual for the Examination of Human Semen (5th Edition), the routine semen parameters of the optimized semen samples were detected by the CASA system, and normal sperm samples meeting the standards were selected for the study group.

[0036] 6. RNA extraction from semen samples

[0037] The optimized sperm (about 2×10 6 ) was resuspended in 1 ml of TRIzol (purchased from Invitrogen), and RNA extraction was performed (the method was referred to the TRIzol product instruction manual).

[0038] 7. High-throughput detection of sperm miRNAs by small RNAs

[0039] For each sample, 100 ng of total RNA was selected for small RNA high-throughput sequencing (for the method, see reference: Yang Q, Lin J, Liu M, et al. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Sci Adv. 2016, 10; 2(6): e1501482.). A total of 389 miRNAs were detected and divided into the H-GQE and L-GQE groups, and then software was used for experiments and analysis. Among them, the expression profiles of the top 100 miRNAs with relatively high expression levels can be seen in Figure 2 , such as hsa-miR-99a-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsa-miR-16-5p, etc., all had high expression levels, but the expression differences between the two groups were not significant.

[0040] The results of differential expression analysis are as shown in Figure 3 , hsa-miR-191-5p, hsa-miR-499a-5p, and hsa-miR-132-3p were specifically low-expressed in the L-GQE group, while hsa-miR-29a-3p was specifically highly expressed in the L-GQE group.

[0041] Conclusion: hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p can be used to predict sperm fertility. Therefore, it is necessary for male patients corresponding to these samples to receive personalized diagnosis and treatment for male infertility in order to improve the fertility outcome of IVF.

[0042] Example 2

[0043] Data analysis was further carried out by the method of support vector machines (SVMs). The discrimination efficiency of these 4 miRNAs (hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p) for the 87 samples in Example 1 could reach 71.54%. The results are as shown in Figure 4 .

[0044] Conclusion: By using the method of the present invention, the possibility for patients to obtain a high-quality embryo rate is 71.54%, which can effectively improve the success rate of IVF for patients.

[0045] In summary, the four miRNAs (hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p) of the present invention can be used as sperm-specific molecular markers for predicting sperm fertility, thereby providing a reliable scientific basis and ideas for personalized treatment for male infertility treatment and clinical diagnosis in IVF. Moreover, hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p can be further made into diagnostic reagents or diagnostic kits by methods known in the art. The diagnostic kit includes reagents for detecting the specific low expression or specific high expression of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and / or hsa-miR-29a-3p in a low high-quality embryo rate, and may also include sperm optimization-related reagents (BWW, HTF, etc.), RNA extraction reagent TRIzol, etc., which will be obvious to those skilled in the art.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a reagent for detecting the expression levels of sperm-specific miRNAs in the preparation of a diagnostic reagent for in vitro fertilization in assisted reproduction, wherein the sperm-specific miRNAs are a combination of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p; and the diagnostic reagent is for detecting the specific expression of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p in a biological sample in a low high-quality embryo rate group; wherein, hsa-miR-191-5p, hsa-miR-499a-5p, and hsa-miR-132-3p were specifically downregulated in the low high-quality embryo rate group, while hsa-miR-29a-3p was specifically upregulated in the low high-quality embryo rate group.

2. Use of a reagent for detecting the expression levels of sperm-specific miRNAs in the preparation of a diagnostic kit for in vitro fertilization in assisted reproduction, wherein the sperm-specific miRNAs are a combination of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p.

3. Use of the reagent for detecting the expression levels of sperm-specific miRNAs as described in claim 2 in the preparation of a diagnostic kit for in vitro fertilization in assisted reproduction, characterized in that, The diagnostic kit contains reagents for detecting the specific expression of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p in the low high-quality embryo rate group in biological samples.

4. Use of a reagent for detecting the expression levels of sperm-specific miRNAs in the preparation of a diagnostic reagent for predicting or evaluating sperm fertility during in vitro fertilization in assisted reproduction, wherein the sperm-specific miRNAs are a combination of hsa-miR-191-5p, hsa-miR-499a-5p, hsa-miR-132-3p, and hsa-miR-29a-3p.

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