Method for screening live sperms from inactive sperms based on magnetic suspension system

Abstract

The invention relates to a method for screening live sperms from inactive sperms based on a magnetic suspension system, and relates to the technical field of assisted reproduction. Comprising the following steps: taking inactive sperms as a to-be-screened sample, and carrying out magnetic suspension separation on the to-be-screened sample by adopting a magnetic suspension separation technology so as to separate live sperms from the to-be-screened sample. The invention develops a simple and safe sperm separation method based on a magnetic suspension technology, and realizes the separation of live sperms in inactive sperms; therefore, the method can provide a new method for selection of live sperms in clinical samples.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to a method for screening live sperms from inactive sperms based on a magnetic suspension system. Background Art

[0002] The development of assisted reproductive technology (ART), especially the application of intracytoplasmic sperm injection (ICSI), has provided many men with severe semen quality abnormalities with the opportunity to have children. ICSI is a technique that uses a microscope to manipulate a single sperm to inject it into an oocyte for fertilization. Because dead sperm lack biological activity, the chromatin inside them is usually degraded compared to live sperm [Nordhoff V. How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view. ANDROLOGY. 2014; 3(2): 156-62.]. Therefore, the selection of live sperm for ICSI is the basis for its success. Assisted reproductive technology laboratories regard sperm motility as a sign of live sperm, which means that patients with sperm without motility may face greater challenges when undergoing ICSI. These patients typically have conditions such as primary ciliary dyskinesia, a genetic disorder that affects the structure and function of the cilia in the sperm tail, resulting in impaired sperm motility [Sironen A, Shoemark A, Patel M, Loebinger MR, Mitchison HM. Sperm defects in primary ciliary dyskinesia and related causes of male infertility. CELL MOL LIFE SCI. 2020; 77(11).], or polymorphic abnormalities of sperm flagella, a disease in which various morphological abnormalities of the sperm flagella occur, affecting sperm motility [Wang J, Wang W, Shen L, Zheng A, Meng Q, Li H, et al. Clinical detection, diagnosis and treatment of morphological abnormalities of sperm flagella: A review of literature. FRONT GENET. 2022; 13: 1034951.]; or they require surgical sperm retrieval. In some cases, such as obstructive azoospermia, sperm must be surgically extracted directly from the testicles, and the extracted sperm are usually very weak or immotile [Godart ES, Turek PJ. The evolution of testicular sperm extraction and preservation techniques. Faculty Reviews. 2020; 9.].

[0003] Immobile sperm does not necessarily mean they are dead. In fact, ICSI using immotile sperm can still result in live birth [S.Geber, Lemgruber M, Taitson PF, Valle M, Sampaio M. laser-assisted selection of viable spermatozoa before intracytoplasmatic sperm injection in a couple with male primary cilia dyskinesia. FERTIL STERIL. 2008; 89(6). theejaculate.HUM REPROD.1996.], which showed that there are immobile but alive sperm carrying normal genetic material and retaining potential fertility. In addition, selecting live sperm from immobile sperm for ICSI can significantly improve treatment outcomes.A randomized controlled trial showed that selective injection of live sperm from an immotile sperm sample significantly increased the fertilization rate of ICSI compared with random sperm injection (43.6% vs. 28.2%) [Sallam HN, Ashraf F, Abdel-Fattah A, Yehia EG, Fathy E. The use of the modified hypo-osmotic swelling test for the selection of immotile testicularspermatozoa in patients treated with ICSI: arandomized controlled study. HUMREPROD 2005(12): 3435.]. Similar results were reported by Aktan et al. [T., M., Aktan, M., Montag, S., et al. Use of a laser to detect viable but immotilespermatozoa. ANDROLOGIA. 2004.], in which the fertilization rate (45.4% vs. 20.4%) and live birth rate (19.0% vs. 5.9%) of the selective injection of live sperm were significantly higher than those of the random injection group. These findings suggest that accurate selection of live sperm for ICSI is critical to ensure a successful outcome in patients with sperm motility issues.

[0004] At present, methods for selecting live sperm from immobile sperm include hypotonic swelling test, physical stimulation and chemical inducers. Although these methods are effective in clinical practice, there are still some shortcomings. The hypotonic swelling test is based on the principle that the tail of live sperm expands in a hypotonic solution, but long-term culture may increase the proportion of dead sperm [Bassiri F, Tavalaee M, Shiravi AH, Mansouri S, Nasr-Esfahani MH. Is there an association between HOST grades and sperm quality? HUM REPROD. 2012; 27 (8): 2277-84. Bloch ARE, Nicolas C. Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes. J ASSIST REPROD GEN. 2021: 1-9.]. In addition, the bending or swelling of the tail caused by hypotonic media makes it difficult to collect sperm with an ICSI needle [Esteves S, Varghese A. Laboratory handling of epididymal and testicular spermatozoa: What can be done to improve sperm injection outcome. J Hum Reprod Sci. 2012; 5 (3).]. In addition, it may not be applicable in some cases, such as cryopreserved sperm [Esteves SC, Sharma RK, Thomas AJ, Agarwal A. Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm. FERTIL STERIL. 1996; 66 (5): 798-804.]. Physical stimulation methods, such as mechanical contact of the sperm tail, require a high level of skill on the part of the operator and may result in abnormally high levels of ROS in sperm [Rossato M, Virgilio FD, Foresta C. Involvement of osmo-sensitive calcium influx in human sperm activation. MOL HUM REPROD. 1996; 2(12):903.].Chemical inducers can enhance sperm motility but may have a negative impact on early embryonic development of the fertilized egg and have long-term effects on offspring [Sandi-Monroy NL, Musanovic S, Zhu D, Szabó Z, Vogl A, Reeka N, et al. Use of dimethylxanthine theophylline. does not affect clinical, obstetric or perinatal outcomes. ARCH GYNECOL OBSTET. 2019; 300(5): 1435-43.]. Therefore, a safer, simpler and more universal method is needed to select live sperm from different sperm samples. In view of this, the present invention provides a method for selecting live sperm from inactive sperm based on a magnetic suspension system. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for screening live sperm from immobile sperm based on a magnetic suspension system. The purpose is to achieve automated, non-invasive separation and collection of live sperm from immobile sperm samples, providing a new method for ICSI sperm selection for patients with immobile sperm in clinical practice.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] A method for screening live sperm from inactive sperm based on a magnetic levitation system comprises the following steps: using inactive sperm as a sample to be screened, and performing magnetic levitation separation on the sample to be screened by using a magnetic levitation separation technique to separate live sperm from the sample to be screened.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Further, the following specific steps are included:

[0010] (1) suspending the sample to be screened in a suspending agent to obtain a suspended sample to be screened;

[0011] (2) loading the suspended sample to be screened into a magnetic suspension separation device, setting the operating parameters of the magnetic suspension separation device, and performing magnetic suspension separation until the separation band of the suspended sample to be screened is stable; setting a dividing line for the suspended sample to be screened, and obtaining a suspension enriched with live sperm above the dividing line.

[0012] Furthermore, the suspension in step (1) includes gadobutrol; the concentration of gadobutrol in the suspension is 50 mM to 200 mM.

[0013] Furthermore, the concentration of gadobutrol in the suspension is 150 mM.

[0014] Furthermore, the concentration of sperm in the suspended sample to be screened in step (1) is below 1 million / ml.

[0015] Furthermore, the concentration of sperm in the suspended sample to be screened in step (1) is below 500,000 / ml.

[0016] Furthermore, the position of the dividing line in step (2) is 0-5.

[0017] Furthermore, the position of the dividing line in step (2) is 5.

[0018] Furthermore, the separation and enrichment of live sperm in the sample to be screened is carried out using LeviCell TM 1.0 Label-free magnetic levitation high-throughput cell separation and enrichment platform.

[0019] The present invention adopts magnetic suspension separation technology to perform magnetic suspension separation on the sample to be screened, and optimizes the conditions such as the concentration of the suspending agent, the position of the stratification line, and the sperm concentration, so as to successfully separate the dead sperm and the live sperm from the inactive sperm; the survival rate of the upper layer sperm after separation is significantly higher than that of the lower layer sperm and the untreated control group sperm, and the survival rate of the upper layer sperm is 89.83±2.44%. There is no significant difference in sperm survival rate, DNA fragmentation index (DFI) and ROS level before and after treatment.

[0020] The beneficial effects of the present invention are: the present invention develops a simple and safe sperm separation method based on magnetic levitation technology, which realizes the separation of live sperm from inactive sperm; therefore, this method may provide a new method for selecting live sperm in clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A flow chart of the method for selecting live sperm from inactive sperm based on a magnetic suspension system of the present invention;

[0022] Figure 2 The diagram is the condition optimization diagram of the present invention; wherein, A: suspending agent concentration optimization; B: dividing line position optimization; C: sperm concentration optimization; D is the intuitive image of separation under the optimal conditions; unstained sperm is live sperm, and green sperm is dead sperm;

[0023] Figure 3 This is a comparison chart of the results of screening live sperm from samples of different patients in the present invention; **p<0.01; ***p<0.001;

[0024] Figure 4This is a diagram showing the effects of the experimental treatment of the present invention on sperm survival rate, DFI and ROS level; wherein, A: survival rate; B: DFI; C: ROS. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0026] Example

[0027] 1. Experimental methods

[0028] 1.1 Preparation of immotile sperm samples

[0029] This invention has been approved by the Research Ethics Committee of Shenzhen Zhongshan Gynecology and Obstetrics Hospital (Approval number: SZZSECHU-2023004).

[0030] The inactive sperm samples used in the present invention were obtained from patients who needed in vitro fertilization or ICSI treatment at Shenzhen Zhongshan Obstetrics and Gynecology Hospital. These samples were the discarded portion of semen collected after density gradient centrifugation. Briefly, 30% and 80% sperm gradient separation solutions (SpermGrad, Vitrolife Sweden AB, V. Sweden) prepared two layers of gradient, each layer 1.5 ml. Semen was centrifuged for 15 minutes using a density gradient centrifuge at a speed of 300g. Sperm with motility remained at the bottom of the centrifuge tube and was collected for subsequent processing and fertilization. The supernatant containing inactive sperm was collected and used as a sample of the present invention.

[0031] 1.2 Screening of live sperm

[0032] The separation and enrichment of live sperm from the immobile sperm sample of the present invention was performed using the LeviCell 1.0 system (LevitasBio, CA, USA) according to the user manual. Briefly, the sample was centrifuged at 800 g for 5 minutes, and then the precipitate was resuspended in a suspension containing a suspending agent (gadobutrol solution, product number: 1003001) to obtain a suspended sample to be screened. TM 1.0 Label-free magnetic suspension high-throughput cell separation and enrichment platform, the suspension sample to be screened is loaded into the injection port of a dedicated chip (gadobutrol solution, product number: 1003001). After the separation band is stable, set the segmentation line and run the recovery program ( Figure 1 ).

[0033] In order to visually observe the separation of dead sperm from live sperm, PI (propidium iodide) was used to stain dead sperm, and the instrument's built-in fluorescence microscope was used to take pictures.

[0034] 1.3 Sperm parameter analysis

[0035] Sperm viability was assessed using eosin-Y staining (Huakang Biotechnology, Shenzhen, China). After staining, sperm that appeared white were considered alive, while those that appeared red or pink were dead. Sperm viability was calculated according to the WHO standard (fifth edition), i.e., the number of live sperm divided by the total number of sperm, counting 200 sperm. Sperm DNA fragmentation index (DFI) was analyzed using the chromatin structure analysis (SCSA) kit (Anke Biotechnology, Hefei, China) and was performed according to the operating instructions of the kit. Reactive oxygen species (ROS) were detected by DCFH-DA staining (BrayDe Biotech, Shenzhen, China) and detected using flow cytometry, and analyzed according to the operating instructions of the kit.

[0036] 1.4 Statistical analysis

[0037] Statistical analysis was performed using SPSS version 23 (IBM company, Chicago, IL, USA). The chi-square test was used to analyze the differences in ratios, and the paired t-test was used to analyze the effects of different treatments on sperm parameters. p < 0.05 was considered significant.

[0038] 2. Results and Discussion

[0039] 2.1 Condition Optimization

[0040] The suspension concentration, the position of the dividing line, and the sperm concentration were optimized. The optimal conditions were determined based on the survival rates of the sperm in the upper and lower layers. The upper layer showed the highest sperm survival rate at a suspension concentration of 150 mM ( Figure 2 A), while the survival rate of the lower layer is lower. Therefore, this concentration is selected as the optimal concentration of the suspension. Figure 2B), the instrument is equipped with a built-in reference line, which is located at 0. Values ​​above the reference line are considered positive, and values ​​below the reference line are negative, with a minimum moving distance of 1 unit. As the reference line moves upward, the survival rates of both the upper and lower sperm increase. The upper sperm showed a higher survival rate when the dividing line position was 5, while the survival rate of the lower sperm was relatively low, so it was selected as the optimal dividing line position. In addition, as the sperm loading concentration decreased, the survival rate of the upper sperm gradually increased and remained stable after reaching 1 million / ml ( Figure 2 C). The survival rate of the lower layer sperm is low at ≤ 1 million / mL. Therefore, a sperm loading concentration of ≤ 1 million / mL was selected.

[0041] 2.2 Effectiveness Analysis

[0042] In the visualization results, live sperm and dead sperm were observed to accumulate in the upper and lower layers of the channel, respectively. This indicates that under optimal conditions, live and dead sperm can be effectively separated ( Figure 2 D). Samples from 6 different patients were separated and analyzed ( Figure 3 ). The results showed that although there were differences in sperm survival rates among different samples, the survival rate of the upper layer sperm after separation was significantly higher than that of the lower layer sperm and the original sperm of the sample. Specifically, the average survival rate of the upper layer sperm reached 89.83±2.44%.

[0043] 2.3 Security Analysis

[0044] In order to evaluate the safety of this method, the present invention examined its effects on sperm survival rate, DFI and ROS level. The results showed that compared with the control group, there was no significant change in sperm survival rate (p = 0.448), DFI (p = 0.369) and ROS (p = 0.983) in the experimental group after receiving magnetic levitation treatment ( Figure 4 AC in ), indicating that this method is safe for sperm processing.

[0045] 2.4 Discussion

[0046] For ICSI treatment, sperm selection is crucial, especially for patients with immotile sperm. Since it is impossible to judge the activity of sperm based on its motility, ICSI sperm screening for these patients has always been a problem that troubles embryologists. In the present invention, magnetic levitation separation technology is innovatively introduced, and a new method for separating live sperm from immotile sperm samples is established.

[0047] The principle of using magnetic levitation technology for cell separation is simply that in a magnetic field and paramagnetic solution environment, different types of cells will be suspended at different levels in the solution, and the suspension height is mainly related to the cell density. Studies have shown that there are density differences between dead and living cells of the same type (25,27). The results of the present invention show that dead sperm and live sperm are suspended at different positions in the channel ( Figure 2 D), indicating that there is a density difference between dead sperm and live sperm. This is the theoretical basis for separating live sperm from immobile sperm in the present invention.

[0048] In the results of the present invention, the proportion of live sperm in the recovered live sperm layer (upper layer) was as high as 89.83±2.44%, which was significantly higher than that of the dead sperm layer (lower layer) and the untreated control group, indicating that the method of the present invention can effectively separate live sperm from inactive sperm samples. However, the survival rate of sperm recovered by the present invention cannot reach 100%. The possible reason is that there are some dying sperm in the sample, whose membrane integrity and contents have not been completely destroyed (28), so their density is less different from that of live sperm. In addition, the instrument used in the present invention is a commercial instrument, and it is impossible to optimize parameters such as magnetic field strength and channel size. Therefore, we believe that with the optimization of technology, better separation effects can be achieved in the future.

[0049] Magnetic levitation separation is a gentle physical separation method that does not cause physical damage to cells. The main component of the suspending agent, gadobutrol, is a biosafe chemical that is widely used as a contrast agent in clinical practice. Therefore, magnetic levitation separation is a biosafe method. In addition, the safety of magnetic levitation separation has also been widely recognized in the separation of other cell types (30). The results of the present invention show that there is no significant change in sperm survival rate, DFI, and ROS levels after magnetic levitation treatment, which further proves the safety of this method.

[0050] Despite the encouraging results, this study has some limitations. First, the survival rate of recovered sperm did not reach 100%, and the possible reasons are described in the discussion section of this paper. Second, the present invention only evaluated the feasibility of separating live sperm from immobile sperm using magnetic levitation technology. Further animal experiments and prospective cohort studies are needed for clinical application.

[0051] 3. Conclusion

[0052] The present invention proposes for the first time to use magnetic levitation technology to separate live sperm from immobile sperm. Although there are certain limitations, the results show that this method has good separation efficiency and safety. Therefore, the present invention can provide a new method for ICSI sperm selection for patients with immobile sperm in clinical practice.

[0053] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for selecting live sperm from immobile sperm based on a magnetic suspension system, characterized in that: The method comprises the following steps: taking inactive sperm as a sample to be screened, and using magnetic suspension separation technology to perform magnetic suspension separation on the sample to be screened, so as to separate active sperm from the sample to be screened.

2. According to claim 1, a method for selecting live sperm from immobile sperm based on a magnetic suspension system, characterized in that: The specific steps include: (1) suspending the sample to be screened in a suspending agent to obtain a suspended sample to be screened; (2) loading the suspended sample to be screened into a magnetic suspension separation device, setting operating parameters of the magnetic suspension separation device, and performing magnetic suspension separation until the separation band of the suspended sample to be screened is stable; A dividing line is set for the suspension sample to be screened, and a suspension enriched with live sperm is obtained above the dividing line.

3. A method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 2, characterized in that: The suspending agent in step (1) includes gadobutrol; the concentration of gadobutrol in the suspending agent is 50 mM to 200 mM.

4. The method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 3, characterized in that: The concentration of gadobutrol in the suspension is 150 mM.

5. The method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 2, characterized in that: The concentration of sperm in the suspended sample to be screened in step (1) is below 1 million / ml.

6. The method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 2, characterized in that: The concentration of sperm in the suspended sample to be screened in step (1) is below 500,000 / ml.

7. The method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 2, characterized in that: The position of the dividing line in step (2) is 0 to 5.

8. The method for selecting live sperm from immobile sperm based on a magnetic suspension system according to claim 2, characterized in that: The position of the dividing line in step (2) is 5.

9. A method for selecting live sperm from immobile sperm based on a magnetic suspension system according to any one of claims 1 to 8, characterized in that: The separation and enrichment of live sperm in the sample to be screened was carried out using LeviCell TM 1.0 Label-free magnetic levitation high-throughput cell separation and enrichment platform.

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