Kit for detecting spermatogenic ability of azoospermia patient and application thereof

Abstract

The invention provides a kit for detecting spermatogenic ability of azoospermia patients and application thereof, and belongs to the technical field of reproductive medicines.The kit comprises the following reagents: testis tissue digestive juice and testis single cell flow staining fluid; the testis single cell flow type staining solution comprises a DNA (Deoxyribose Nucleic Acid) staining solution Hoechst 33342 and an RNA (Ribonucleic Acid) staining solution # imgabs0 # RNASelectTM green fluorescent cell staining agent; according to the kit, double staining of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) is carried out on testis single cells, and a fluorescence activated cell sorting technique is matched, so that refining of testis single cell subgroups of azoospermia patients is realized, and the spermatogenic ability of the azoospermia patients is determined according to a testis single cell subgroup subdivision result. The kit disclosed by the invention is used for pre-operation evaluation of a microscopic sperm extraction operation, is used for judging whether a patient has spermatogenic ability and is suitable for microscopic sperm extraction or not, can also be used for guiding the patient to further clarify the cause of disease, and has important clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of reproductive medicine technology, and in particular relates to a reagent kit for detecting the spermatogenic capacity of patients with azoospermia and its application. Background Technology

[0002] Azoospermia is defined as the absence of sperm in multiple semen analyses (generally more than three times). It is one of the causes of male infertility. Testicular biopsy, vasography, and radioimmunoassay of endocrine hormones may be necessary to help differentiate between obstructive and congenital azoospermia. These methods play an important role in differentiating between non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) and in assessing spermatogenesis, but they have significant limitations. Hormone testing and ultrasound can only provide general information about testicular function but cannot precisely locate the spermatogenesis disorder. Although testicular biopsy is considered the gold standard, it is an invasive procedure with potential risks. Molecular diagnostic tools, such as gene testing and metabolomics analysis, are still insufficient in identifying the exact developmental stage of spermatogenesis arrest.

[0003] When infertile patients seek medical attention, they first undergo hormone testing and semen analysis. If the semen analysis report shows no sperm, the patient undergoes ultrasound and other tests to determine if the obstruction is the cause. Next, the patient undergoes fine-needle aspiration to check for sperm in the testicular tissue and determine if they meet the criteria for assisted reproductive technology. After the aspiration examination, the patient undergoes microsurgical sperm retrieval. However, among patients with non-obstructive azoospermia, only about 35% can obtain sperm through microsurgical sperm retrieval, meaning that 65% of patients have to endure the trauma of surgery without receiving any substantial treatment. Therefore, preoperative assessment of whether the patient still has spermatogenic capacity and is suitable for microsurgical sperm retrieval is of significant clinical importance.

[0004] However, current preoperative assessment methods are limited to needle biopsy. The biopsy methods can be divided into fine-needle aspiration and core-needle aspiration. If the patient undergoes core-needle aspiration, although the obtained tissue is larger and can be used for pathological examination, it causes further damage to the already fragile testicles and causes greater pain. If the patient undergoes fine-needle aspiration, due to the small amount of tissue, only microscopic observation of sperm is possible. Because there is no staining, it is impossible to distinguish other cells besides sperm. Therefore, it is impossible to assess the patient's spermatogenesis ability, and thus, it cannot be guaranteed that sperm will be retrieved during microsurgical sperm retrieval. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a kit for detecting spermatogenesis in patients with azoospermia and its application. The kit of this invention uses dual fluorescent staining of testicular cells for RNA and DNA, combined with flow cytometry, to clearly separate and classify human testicular cells based on RNA and DNA content, determining the abnormal distribution of spermatogenic cells at each stage. Only a small amount of testicular tissue is needed to directly and effectively locate abnormalities in the spermatogenic stage in a single step. The kit provided by this invention significantly improves the accuracy and resolution of NOA diagnosis, providing a novel tool for clinical pathological analysis and personalized treatment strategies.

[0006] This invention provides a kit for detecting the spermatogenic capacity of patients with azoospermia, comprising the following reagents: testicular tissue digestion fluid and testicular single-cell flow cytometry staining solution; wherein the testicular single-cell flow cytometry staining solution includes DNA staining solution and RNA staining solution.

[0007] Preferably, the DNA staining solution is Hoechst 33342, and the RNA staining solution is... RNASelect TM Green fluorescent cell staining agent.

[0008] Preferably, the testicular tissue digestion solution includes a first digestion solution and a second digestion solution, wherein the first digestion solution is a Durbeco phosphate buffer containing type I collagenase and DNase I, and the second digestion solution is a trypsin solution.

[0009] Preferably, the kit performs dual staining of testicular single cells for DNA and RNA, combined with fluorescence-activated cell sorting, to achieve the following refined grouping of human testicular single cells with normal spermatogenesis: differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids, and Sertoli cells.

[0010] After refining and clustering testicular single cells from patients with azoospermia, the results were compared with those from patients with normal spermatogenesis.

[0011] If, after the testicular single-cell subgrouping of an azoospermic patient, only Sertoli cells are found, the diagnosis is Sertoli-only syndrome.

[0012] Blocking any of the nine groups except Sertoli supporting cells will result in weaker spermatogenesis as the blockade progresses further forward.

[0013] If the arrest occurs at the Diplotene spermatocyte, which includes differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, and Pachytene spermatocytes, but no subsequent MI spermatocytes, then the diagnosis is spermatocyte first meiotic arrest.

[0014] The present invention also provides the application of the kit in the preparation of reagents for evaluating the feasibility of microsurgical sperm retrieval.

[0015] The present invention also provides the application of the kit in the preparation of reagents for the accurate diagnosis of azoospermia.

[0016] Compared with existing technologies, the present invention has the following beneficial effects: The kit for detecting spermatogenesis in azoospermic patients provided by the present invention includes testicular tissue digestion fluid and testicular single-cell flow cytometry staining solution; the testicular single-cell flow cytometry staining solution includes DNA staining solution and RNA staining solution; by performing dual fluorescent staining of testicular cells for RNA and DNA, combined with flow cytometry, human testicular cells are clearly separated and classified according to RNA and DNA content, determining the abnormal distribution of spermatogenic cells at each stage. Only a small amount of testicular tissue is needed to directly and effectively locate abnormalities in the spermatogenesis stage in a single operation. This kit is of significant clinical importance for the accurate diagnosis of azoospermia patients and preoperative evaluation for microsurgical sperm retrieval surgery, determining whether the patient still has spermatogenic capacity and is suitable for microsurgical sperm retrieval. Attached Figure Description

[0017] Figure 1 The correlation analysis of different cell subpopulations in single testicular cells is shown in Figure A, where mRNA expression profile is shown in Figure B, the correlation of different cell subpopulations is shown in Principal Component Analysis (PCA) and Pearson correlation analysis, and the expression of cell-specific marker genes in different cell subpopulations is shown in Figure C.

[0018] Figure 2 The Hoechst Blue / FITC fluorescence map generated by double fluorescence staining showed nine distinct subgroups, which were merged into the Hoechst Blue / Hoechst Red map. Blue markers indicated the corresponding positions of these subgroups on the Hoechst Blue / Hoechst Red map.

[0019] Figure 3 This outlines the workflow for low-input RNA-seq, describing how to extract RNA from sorted subpopulations for sequencing.

[0020] Figure 4The analysis of different cell subpopulations in single testicular cells is presented in section A, which is a heatmap of selected spermatogenic cell marker genes, showing the different expression characteristics in nine subpopulations; section B is a Pearson correlation analysis of RNA expression profiles among the nine subpopulations, illustrating the relationships between these subpopulations; and section C is a heatmap showing the correlation between bulk RNA-seq data obtained from sorted testicular cells and pseudo-bulk RNA-seq gene expression matrices obtained from single-cell transcriptomics.

[0021] Figure 5 To verify the spermatogenic subsets and their RNA expression profiles using capillary electrophoresis; A) Chromosome spreading of P1 and P2 cells co-stained with γ-H2AX (green, marking double-strand breaks [DSBs]) and SYCP3 (red, marking axial elements), P1 mainly consists of ditene spermatocytes, while P2 consists of pachytene spermatocytes, scale bar = 5 μm; B) DAPI staining of P6 and P9 subsets, showing chromosome distribution, scale bar = 10 μm;

[0022] Figure 6 The following are the results of combined testicular FNAC and R&D-FACS examinations performed on patients in Example 2. Figure A shows the R&D-FACS results of two patients with obstructive azoospermia. These two patients were diagnosed with congenital absence of the vas deferens by palpation and ultrasound, but their testicular spermatogenesis was normal. R&D-FACS can be used to subdivide human testicular cells into three groups: differentiated spermatogonia, spermatocytes 1, 2, and 3 in prophase I of meiosis, MI in metaphase I, and M in metaphase II. These are combined into a Hoechst Blue / Hoechst Red chart, with blue markers indicating the corresponding positions of these subgroups on the Hoechst Blue / Hoechst Red chart, showing that the cell populations conform to developmental trajectories. Figure B shows the R&D-FACS results of a patient with severe oligoasthenospermia. Abnormalities were observed in the spermatocyte group 3 in prophase I of meiosis, and Figure C shows a decreased proportion of round sperm cells, indicating that the spermatogenesis abnormality occurred in prophase I of meiosis. Figure D shows the R&D-FACS results of a patient with non-obstructive azoospermia, revealing only Sertoli cells and no spermatogenic cells, suggesting Sertoli-only cell syndrome. Figure E shows the R&D-FACS results of a patient with oligoasthenospermia. This patient has a TTC29 gene mutation, which, according to literature, leads to abnormal sperm flagellar formation and changes in sperm volume. The R&D-FACS results show that the patient's elongated sperm morphology is indeed abnormal, with size and granule density approaching that of round sperm cells. Detailed Implementation

[0023] This invention provides a kit for detecting the spermatogenic capacity of patients with azoospermia, comprising the following reagents: testicular tissue digestion fluid and testicular single-cell flow cytometry staining solution; wherein the testicular single-cell flow cytometry staining solution includes DNA staining solution and RNA staining solution.

[0024] In this invention, the DNA staining solution is preferably Hoechst 33342, and the RNA staining solution is preferably... RNASelect TM Green fluorescent cell staining agent.

[0025] In this invention, the testicular tissue digestion solution includes a first digestion solution and a second digestion solution. The first digestion solution is preferably a Durbeco phosphate buffer containing type I collagenase and DNase I. In the first digestion solution, the concentration of type I collagenase is preferably 150-250 U / mL, more preferably 200 U / mL, and the concentration of DNase I is preferably 4-6 mg / mL, more preferably 5 mg / mL. The second digestion solution is preferably a trypsin solution, and the concentration of the trypsin solution is preferably 0.04-0.06%, more preferably 0.05%.

[0026] In this invention, the kit performs dual staining of DNA and RNA on single testicular cells, combined with fluorescence-activated cell sorting, to achieve the following refined grouping of single testicular cells from normally spermatogenic individuals: differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids, and Sertoli cells.

[0027] After refining and clustering testicular single cells from patients with azoospermia, the results were compared with those from patients with normal spermatogenesis.

[0028] If, after the testicular single-cell subgrouping of an azoospermic patient, only Sertoli cells are found, the diagnosis is Sertoli-only syndrome.

[0029] The spermatogenic capacity is weaker if any of the nine groups (excluding Sertoli supporting cells) are blocked.

[0030] If the arrest occurs at the Diplotene spermatocyte, which includes differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, and Pachytene spermatocytes, but no subsequent MI spermatocytes, then the diagnosis is spermatocyte first meiotic arrest.

[0031] The method of using the kit described in this invention includes the following steps: 1) digesting testicular tissue with the testicular tissue digestion solution in the kit; 2) performing double staining of DNA and RNA on the digested testicular cells; 3) performing flow cytometry analysis and fluorescence-activated cell sorting on the stained testicular cells.

[0032] In this invention, tissue is preferably obtained via testicular puncture. The tissue is placed in a first digestion solution and incubated at 36–38°C for 10–20 minutes, during which time it is inverted and mixed 8–12 times every 5 minutes until the seminiferous tubules are completely dissociated and settle. After removing the supernatant, the precipitated seminiferous tubules are resuspended in a second digestion solution and incubated at 36–38°C for another 10–20 minutes, during which time the tubules are pipetted 25–35 times every 5 minutes. The digestion time can be appropriately extended depending on the tissue lysis. Preferably, digestion is terminated by adding DMEM medium containing 10% fetal bovine serum.

[0033] In this invention, the double staining preferably includes the following steps:

[0034] 1) Mix DNA staining solution Hoechst 33342 and RNA staining solution. RNASelect TM 1) Mix the green fluorescent cell staining agent with the cell suspension and incubate; 2) After incubation, centrifuge and discard the supernatant, then resuspend the cells in PBS to obtain a cell suspension.

[0035] In this invention, the amount of the DNA staining solution Hoechst 33342 is preferably 3-8 μg / 10. 6 10 cells, more preferably 5 μg / 10 6 Cells; the RNA staining solution RNASelect TM The preferred amount of green fluorescent cell staining agent is 450–550 nmol / 10⁻¹. 6 Cells, preferably 500 nmol / 10 6 Cells. In this invention, the incubation temperature is preferably 36-38°C, more preferably 37°C, and the incubation time is preferably 25-35 min, more preferably 28-32 min. In this invention, the centrifugal force is preferably 450-550 × g, more preferably 500 × g, the centrifugation time is preferably 4-6 min, more preferably 5 min; and the centrifugation temperature is preferably 3-5°C, more preferably 4°C.

[0036] Following the double staining, this invention performs flow cytometry analysis and fluorescence-activated cell sorting (FACS) on the stained testicular cells. Preferably, the cell suspension after double staining is filtered through a 70 μm cell sieve before flow cytometry analysis and FACS. In this invention, a BD Biosciences FACS AriaII flow cytometer and a Beckman Coulter MoFLOXDP high-speed flow cytometry sorting system are used for flow cytometry and cell sorting analysis. Different cell populations are distinguished based on fluorescence intensity. The laser setup includes a 488 nm laser for forward scattering (FSC-A) and side scattering (SSC-A) detection, a 355 nm UV laser for Hoechst 33342 excitation, a 450 / 40 nm bandpass filter for detecting the "Hoechst blue" signal, and a 670 nm longpass filter for detecting the "Hoechst red" signal. RNASelect TM The green fluorescent cell staining agent was detected via the FITC channel at absorption / emission wavelengths of 490 / 530 nm. Cell sorting was performed using a 70 μm nozzle at a sorting rate of 4000–5000 events per second.

[0037] The kit described in this invention uses dual staining of DNA and RNA in single testicular cells, combined with fluorescence-activated cell sorting, to achieve the following refined grouping of single testicular cells from normally spermatogenic individuals: differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids, and Sertoli cells.

[0038] After refining and clustering testicular single cells from patients with azoospermia, the results were compared with those from patients with normal spermatogenesis.

[0039] If, after the testicular single-cell subgrouping of an azoospermic patient, only Sertoli cells are found, the diagnosis is Sertoli-only syndrome.

[0040] The spermatogenic capacity is weaker if any of the nine groups (excluding Sertoli supporting cells) are blocked.

[0041] If the arrest occurs at the Diplotene spermatocyte, which includes differentiated spermatogonia, pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, and Pachytene spermatocytes, but no subsequent MI spermatocytes, then the diagnosis is spermatocyte first meiotic arrest.

[0042] The present invention also provides the application of the kit in the preparation of reagents for evaluating the feasibility of microsurgical sperm retrieval.

[0043] The present invention also provides the application of the kit in the preparation of reagents for the accurate diagnosis of azoospermia.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Mouse testicular cell subset subgroups

[0047] Mice were euthanized by cervical dislocation, and the testes were dissected and removed. After removing the tunica albuginea, the testes were placed in 5 mL of Durbecoxib phosphate-buffered saline (DPBS) containing 200 U / mL L-type collagenase (Thermo Fisher Scientific, 17100017) and 5 mg / mL DNase I (Sigma-Aldrich, DN25), and incubated at 37°C for 15 minutes until the seminiferous tubules were completely dispersed and precipitated. The supernatant was removed, and the precipitated tubules were resuspended in 20 mL of 0.05% trypsin (Gibco, 25200072), and digestion was continued at 37°C for 15 minutes. Digestion was terminated by adding 30 mL of Durbecoxib modified Eagle medium (DMEM) (Gibco, C11995500BT) containing 10% fetal bovine serum (FBS). After digestion was terminated, no washing was required before proceeding with subsequent staining procedures.

[0048] Add Hoechst 33342 (5 μg / 10) to the cell suspension 6 (cells) (purchased from Thermo Fisher Scientific) and 500 nmol / 10 6 individual cells RNASelect TM Green fluorescent cell staining agent (ThermoFisher Scientific, S32703) was gently mixed and incubated at 37°C for 30 minutes. Subsequently, the cells were centrifuged at 500×g at 4°C for 5 minutes, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) and filtered through a 70 μm cell sieve (Corning, 352340) for flow cytometry analysis and fluorescence-activated cell sorting (FACS).

[0049] Flow cytometry and cell sorting analysis were performed using a BD Biosciences FACS Aria II flow cytometer and a Beckman Coulter MoFLOXDP high-speed flow cytometry sorting system. Staining protocols included the use of Hoechst 33342 or Hoechst 33342 / SYTO. TM Different combinations of green fluorescent nucleic acid dyes were used to distinguish different cell populations based on fluorescence intensity. The laser setup included a 488 nm laser for forward scattering (FSC-A) and side scattering (SSC-A) detection, a 355 nm UV laser for Hoechst 33342 excitation, a 450 / 40 nm bandpass filter for detecting the "Hoechst blue" signal, and a 670 nm longpass filter for detecting the "Hoechst red" signal. SYTO dye was detected via the FITC channel at absorption / emission wavelengths of 490 / 530 nm. Cell sorting was performed using a 70 μm nozzle, with a sorting rate maintained at 4000–5000 events per second.

[0050] Sorting was performed using R&D-FACS, followed by low-input RNA sequencing to analyze the specific gene expression profiles of each group of spermatogenic cells (the workflow of low-input RNA sequencing is as follows). Figure 1 (As shown in B in the original study). Furthermore, the SMART-seq2 single-cell transcriptome gene expression matrix provided by Chen et al., which covers all stages of spermatogenesis in mouse testes (Chen, Y., Zheng, Y., Gao, Y., Lin, Z., Yang, S., Wang, T., Wang, Q., Xie, N., Hua, R., Liu, M., et al. (2018). Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis. Cell Res 28, 879-896). Based on these data, a pseudo-batch RNA-seq dataset was generated. t-SNE analysis was performed following the data processing methods described in the original study (…). Figure 1 (A) and applied the DotPlot function to visualize the expression of cell-specific marker genes. Figure 1(C in the original text). Finally, the low-input RNA-seq results of these nine cell populations were cross-referenced with pseudo-batch RNA-seq datasets. Combining the positions of these populations in the classic Hoechst fluorescence signature map (Gaysinskaya, V., Soh, IY, vander Heijden, GW, and Bortvin, A. (2014). Optimized flow cytometry isolation of murinespermatocytes. Cytometry A 85, 556-565) and identification by chromosome spreading and immunofluorescence staining, the types of these nine cell populations were finally determined.

[0051] In the R&D-FACS feature map, P1 shows diploid DNA content and is located in the position corresponding to spermatogonia in the classic fluorescence map. Figure 2 Low-input RNA-seq analysis revealed that P1 highly expressed markers of differentiated spermatogonia, including Stra8, Uchl1, Crabp1, and Rhox13. Furthermore, Pearson correlation analysis with pseudo-batch RNA-seq datasets showed that the transcriptomic profile of P1 was highly similar to that of differentiated spermatogonia (A1, In, S phase B-type spermatogonia and G2 / M phase B-type spermatogonia) and prolaminar spermatocytes. Therefore, the P1 population was identified as representative of differentiated spermatogonia (…). Figure 4 ).

[0052] P2 exhibits tetraploid DNA content and is located at the L / Z position in the Hoechst fluorescence signature map. Figure 2 Low-input RNA-seq analysis revealed that P2 cells highly express markers of leptotene and syncytium spermatocytes, such as Gm960, Meiob, and Dmc1. Pearson correlation analysis with pseudo-batch RNA-seq datasets showed that the transcriptional profile of P2 cells was highly similar to that of leptotene spermatocytes (sc-L), syncytium spermatocytes (sc-Z), differentiated spermatogonia, and pre-leptotene spermatocytes. Therefore, the P2 population is primarily composed of leptotene and syncytium spermatocytes.

[0053] Both P3 and P4 are tetraploid cells and are located at the P / D position in the Hoechst fluorescence signature map. Figure 2 Low-input RNA-seq results showed that both P3 and P4 highly expressed Psma8, Piwil1, and Pou5f2, indicating that they correspond to pachytene / diplotene spermatocytes. Figure 4The expression level of Ccna1 in P4 was significantly higher than that in P3, suggesting that P4 may represent the diplotene stage, while P3 corresponds to the pachytene stage. Principal component analysis (PCA) and Pearson correlation analysis revealed a strong correlation between P3 and P4. Figure 4 B in Figure 1 (B in the original text). By comparing low-input RNA-seq results with pseudo-batch RNA-seq data, P3 and P4 were found to be closely associated with the P and D populations. Chromosome spreading and immunofluorescence staining of P3 and P4 sorted using R&D-FACS confirmed that P3 was mainly composed of pachytene spermatocytes, while P4 was mainly composed of diplotene spermatocytes, as indicated by SYCP3 and H2AX staining. Figure 5 (A) Therefore, population P3 corresponds to pachytene spermatocytes, while P4 represents diplotene spermatocytes.

[0054] Comprehensive analysis of the R&D-FACS fluorescence signatures showed that the DNA content of P5 was between 2C and 4C. Figure 2 This population highly expressed the diplotene spermatocyte markers Psma8 and Piwil1, as well as elevated classical meiotic second division markers Ccna1 and Tbp1. Figure 4 (A in the text). PCA and Pearson correlation analyses of low-input RNA data showed a strong correlation among P5, P6, and P7. Figure 4 B in Figure 1 (B in the original text). Furthermore, the mRNA expression profile of P5 is highly similar to that of the sc-MII and sc-D populations in the pseudo-batch RNA-seq data (…). Figure 4 Based on these findings, the P5 population may correspond to cells in the late stage of the first meiotic division (MI).

[0055] Both P6 and P7 showed diploid DNA content and were located at the MII position in the Hoechst fluorescence signature map. Figure 2 Low-input RNA-seq analysis showed that the mRNA expression profiles of the two populations were highly similar. Figure 1 In the A group, both subpopulations highly expressed the meiotic second division (MII) marker genes Ccna1 and Tbp1. Figure 4 (A) Correlation analysis with pseudo-batch RNA-seq datasets confirmed that the mRNA profiles of these two populations were highly similar to those of spermatocyte remnants (sc-RS) and meiotic second-phase spermatocytes (sc-MII) populations. Figure 4 (C in the text). Therefore, the P6 and P7 populations represent two subpopulations of secondary sperm cells (MII).

[0056] Both P8 and P9 are haploid cells, indicating that they have undergone the second meiotic division and developed into gametes. Figure 2 Furthermore, low-input RNA-seq results showed that P8 highly expressed round spermatocyte markers such as Sun5, Tex29, and Spaca3, while P9 was characterized by high expression of elongated spermatocyte markers Prm1, Prm2, Ttc24, Reep6, and Cstl1. Figure 4 (A) DAPI staining of the P8 and P9 populations sorted using R&D-FACS confirmed that P8 was mainly composed of round spermatocytes, while P9 was mainly composed of elongated spermatocytes. Figure 5 (B in the middle).

[0057] As can be seen, the present invention, employing dual fluorescent staining of DNA and RNA combined with flow cytometry R&D-FACS, not only expands the capabilities of traditional Hoechst-based FACS, enabling the separation of known cell populations, but also accurately classifies previously difficult-to-distinguish cell types, such as pachytene spermatocytes, diplotene spermatocytes, round spermatocytes, and elongating spermatocytes. The R&D-FACS method of this invention also successfully distinguished two MII cell subtypes and identified cells in the M phase of meiosis I (MI). These results demonstrate the superior accuracy and versatility of R&D-FACS in characterizing spermatogenic cell populations.

[0058] Example 2

[0059] reagent kit composition

[0060] Testicular tissue digestion solution: Durbeco phosphate-buffered saline (DPBS) containing 200 U / mL type I collagenase and 5 mg / mL DNase I.

[0061] DNA staining solution: Hoechst 33342

[0062] RNA staining solution: RNASelect TM Green fluorescent cell staining agent

[0063] Although non-obstructive azoospermia (NOA) patients may lack sperm in their ejaculate, localized spermatogenesis may still exist in their testes. Therefore, it is possible to obtain sperm through microsurgical testicular sperm extraction (micro-TESE) and subsequently achieve biological offspring through in vitro fertilization. However, the uncertainty of sperm retrieval makes prognostic methods particularly important. Therefore, before attempting micro-TESE, a more comprehensive assessment of testicular spermatogenesis should be conducted by combining fine-needle aspiration cytology (FNAC) and R&D-FACS.

[0064] In this embodiment, five patients were selected for combined testicular FNAC and R&D-FACS examinations. Patients #1 and #2 were both diagnosed with obstructive azoospermia (OA) due to congenital absence of the vas deferens. Patients #3 and #5 were diagnosed with extremely severe oligoasthenospermia, while patient #4 was diagnosed with NOA.

[0065] The specific method is as follows:

[0066] 1) Digestion of testicular tissue

[0067] The tissue was obtained via testicular puncture and had been fragmented with a needle. If the tissue pieces were large, they could be fragmented with forceps first. The tissue was placed in 5 mL of PBS containing 200 U / mL L-type collagenase and 5 mg / mL DNase I and incubated at 37°C for 15 minutes, inverting and mixing approximately 10 times every 5 minutes until the seminiferous tubules were completely dissociated and settled. After removing the supernatant, the precipitated seminiferous tubules were resuspended in 20 mL of 0.05% trypsin and digested at 37°C for 15 minutes, pipetting approximately 30 times every 5 minutes. The digestion time could be extended appropriately depending on the tissue lysis. The digestion reaction was terminated by adding 30 mL of DMEM medium containing 10% fetal bovine serum. No washing was required afterward; the dye was added directly and incubated at 37°C for 30 minutes. After staining, the tissue was centrifuged (500g, 5 min), the supernatant was discarded, and the tissue was resuspended in PBS, sieved through a 200-mesh sieve, and then processed.

[0068] 2) Dual fluorescent staining of RNA and DNA

[0069] Add Hoechst 33342 (5 μg / 10) to the cell suspension 6 (1 cell) (Thermo Fisher Scientific) and 500 nM SYTO TM The cells were incubated with a green fluorescent nucleic acid dye (Thermo Fisher Scientific) at 37°C for 30 minutes after gentle mixing. Then, they were centrifuged at 500×g at 4°C for 5 minutes, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) and filtered through a 70 μm cell sieve for flow cytometry analysis and fluorescence-activated cell sorting (FACS).

[0070] 3) Flow cytometry analysis and fluorescence-activated cell sorting were performed on stained testicular cells.

[0071] Flow cytometry and cell sorting analysis were performed using a BD Biosciences FACS Aria II flow cytometer and a Beckman Coulter MoFLOXDP high-speed flow cytometry sorting system. Different cell populations were distinguished based on fluorescence intensity. The laser setup included a 488 nm laser for forward scattering (FSC-A) and side scattering (SSC-A) detection, a 355 nm UV laser for Hoechst 33342 excitation, a 450 / 40 nm bandpass filter for detecting the "Hoechst blue" signal, and a 670 nm longpass filter for detecting the "Hoechst red" signal. RNASelect TM The green fluorescent cell staining agent was detected via the FITC channel at absorption / emission wavelengths of 490 / 530 nm. Cell sorting was performed using a 70 μm nozzle at a sorting rate of 4000–5000 events per second.

[0072] Results analysis:

[0073] R&D-FACS analysis of testicular tissues from patients #1 and #2 after FNAC revealed that, despite differences in fluorescence characteristics between human and mouse testicular cells, human testicular cells could be clearly separated and classified based on RNA and DNA content. In the Hoechst blue channel, cells were divided into haploid, diploid, and tetraploid groups based on DNA content, consistent with the characteristics of testicular cells. Referring to cell population localization and corresponding Hoechst fluorescence spectra in mouse testicular R&D-FACS, and using the fluorescence characteristics of OA patients as a baseline, a classification scheme for human testicular cell populations was proposed, dividing them into nine cell subpopulations, such as... Figure 6 As shown in A, it is specifically divided into differentiated spermatogonia (Mi-SPG), pre-Leptotene spermatocytes (pre-L), leptotene / zygote spermatocytes (SPC-1), pachytene spermatocytes (SPC-2), diplotene spermatocytes (SPC-3), a subset of secondary spermatocytes MII1, a subset of secondary spermatocytes MII2, round spermatocytes (RS), and elongated spermatocytes (ES).

[0074] Hormone assessment of patient #3 showed a follicle-stimulating hormone (FSH) level of 7.62 mIU / mL, and FNAC analysis revealed a small number of sperm. Remaining tissue samples underwent R&D-FACS analysis. The R&D-FACS characteristic curve showed that subset 3 of pre-lepidocrocus spermatocytes (SPC3) in stage I was divided into two distinct subsets, indicating abnormal RNA levels in these cells. Furthermore, the proportion of RS was significantly lower than in the control group (…). Figure 6(as shown in B and C). This finding suggests that the spermatogenesis abnormalities in patient #3 may be closely related to developmental abnormalities occurring during the prospermatocyte stage I. Abnormal RNA metabolism may disrupt the normal process of meiosis, ultimately leading to reduced sperm count and dysfunction.

[0075] R&D-FACS analysis of patient #4 showed only a diploid cell population, indicating a complete absence of spermatogenesis, and the testes were primarily composed of Sertoli cells. Figure 6 (D in the text). Furthermore, patient #4's FSH level was 17.19 mIU / mL, and no sperm was detected by FNAC. Additionally, m-TESE surgery also failed to retrieve sperm from patient #4. Pathologically, the Johnsen score was 1, indicating a diagnosis of Sertoli-only syndrome (SCOS). Based on this patient's results, it is concluded that the combination of FNAC and R&D-FACS has the potential to diagnose SCOS. This diagnostic result would encourage patients to avoid m-TESE surgery, thus avoiding unnecessary suffering and ineffective treatment associated with this procedure.

[0076] Patient #5 underwent m-TESE surgery and intracytoplasmic sperm injection (ICSI) 12 years ago and successfully gave birth to a healthy 11-year-old child. She recently sought treatment at the hospital hoping to have a second child through assisted reproductive technology (ART). Whole-exome sequencing revealed a homozygous mutation in the TTC29 gene, with nucleotide and amino acid alterations of c.1107C>G (p.Y369*). R&D-FACS analysis was performed on testicular tissue obtained from patient #5 via FNAC. The R&D-FACS analysis showed that patient #5's cell proportions were similar to those of OA patients, but with a population of sperm cells exhibiting abnormal morphology. For further investigation, a detailed comparison of the sperm cell populations in patient #5 and OA patients was performed using side-scattered (SSC) and forward-scattered (FSC) parameters. In the OA group, haploid cells clearly divided into two populations along the FSC axis, representing smaller, elongated sperm cells and larger, round sperm cells, respectively. Conversely, in patient #5, the elongated spermatocyte group on the left shifted to the left from its original position, closer to the rounded spermatocyte group on the right, indicating an increase in the volume of the elongated spermatocytes in patient #5. Figure 6(E in the text). TTC29 mutations have been shown to cause sperm abnormalities, including flagellar loss, shortening, curling, and structural disorder. As a result, these cells exhibit a more spherical or irregular shape and are enlarged (Liu C, He X, Liu W, Yang S, Wang L, LiW, Wu H, Tang S, Ni X, Wang J, Gao Y, Tian S, Zhang L, Cong J, Zhang Z, Tan Q, Zhang J, Li H, Zhong Y, Lv M, Li J, Jin L, Cao Y, Zhang F. Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice. Am J HumGenet. 2019 Dec 5; 105(6):1168-1181.). This is consistent with observations detected by R&D-FACS. This indicates that the combination of FNAC and R&D-FACS can indicate whether there are abnormalities in the sperm morphology of patients, thereby guiding subsequent tests to focus more on the gene mutations that cause abnormal sperm morphology.

[0077] As demonstrated by the above embodiments, R&D-FACS exhibits unique advantages as a powerful diagnostic tool for analyzing spermatogenesis abnormalities. Unlike traditional methods, R&D-FACS requires only a very small amount of testicular tissue and maintains cell viability throughout the analysis process, avoiding cell loss due to repeated processing. This technology uses flow cytometry to generate clear images of cell populations and automatically calculates the proportion of each cell population, significantly reducing human error and subjective bias. These operational advantages enable R&D-FACS to efficiently and directly identify abnormalities in the spermatogenesis stage in a single step. By significantly improving diagnostic accuracy and resolution, particularly in the diagnosis of non-obstructive azoospermia (NOA), R&D-FACS highlights its significant potential in clinicopathological analysis and provides strong support for developing more accurate personalized treatment strategies.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting the spermatogenic ability of a patient with azoospermia, characterized in that: The method comprises the following reagents: testicular tissue digestion solution and testicular single cell flow cytometry staining solution; the testicular single cell flow cytometry staining solution comprises DNA staining solution and RNA staining solution.

2. The kit according to claim 1, characterized in that The DNA staining solution is Hoechst 33342, and the RNA staining solution is RNASelect TM Green fluorescent cell stain.

3. The kit according to claim 2, characterized in that The testicular tissue digestion solution comprises a first digestion solution and a second digestion solution, wherein the first digestion solution is a Dulbecco's phosphate buffer containing type I collagenase and DNase I, and the second digestion solution is a trypsin solution.

4. The kit according to claim 1, characterized in that The kit performs double staining of DNA and RNA on testicular single cells, and cooperates with fluorescence activated cell sorting to achieve the following subdivision and grouping of human testicular single cells with normal spermatogenesis: differentiated spermatogonia, Pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids and Sertoli supporting cells; After the refinement and grouping of testicular single cells of patients with azoospermia, they were compared with the refinement and grouping of testicular single cells of patients with normal spermatogenesis: If only Sertoli cells are found after single-cell analysis of the testicles of an azoospermic patient, the diagnosis is Sertoli cell-only syndrome. If the blockage is in any of the above 9 groups except Sertoli supporting cells, the more anterior the blockage is, the weaker the spermatogenesis capacity is; If the blockage occurs in Diplotene spermatocytes, that is, there are differentiated spermatogonia, Pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, and Pachytene spermatocytes, but no subsequent MI spermatocytes, it is diagnosed as the first meiotic arrest of spermatocytes.

5. Use of the kit according to any one of claims 1 to 4 in the preparation of reagents for evaluating the feasibility of microsperm retrieval surgery.

6. Use of the kit according to any one of claims 1 to 4 in the preparation of reagents for accurate diagnosis of azoospermia patients.

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