Seminal plasma-like diluent for detecting high-concentration sperm quality of human by CASA and application of seminal plasma-like diluent

Abstract

The invention discloses a seminal plasma diluent for detecting the quality of human high-concentration sperms through CASA, and relates to the technical field of male fertility detection, and the seminal plasma diluent comprises an osmotic pressure regulator and an anti-sperm agglutination agent. The seminal plasma osmotic pressure after liquefaction is simulated by analyzing the osmotic pressure change rule of in-vitro liquefaction of human seminal fluid, and meanwhile, the sperm agglutinant is added, so that sperm energy supply substances and stimulants are not additionally added on the premise of ensuring the accuracy of sperm detection of a patient, and the detection accuracy is improved. The sperm motility is prevented from being excessively optimized while it is guaranteed that the dilution effect of the seminal plasma-like diluent is consistent with that of the seminal plasma, and a real basis is provided for sperm quality detection of a patient. Meanwhile, few substances are added, the cost is low, the operation is simple, and clinical popularization and application are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of male fertility testing, and in particular to a seminal plasma-like diluent for CASA testing of high-concentration human sperm quality and its application. Background Art

[0002] In the field of male reproductive health and infertility diagnosis, routine semen analysis is a basic and mandatory diagnostic test. Currently, the CASA system is the preferred method for routine semen examination. The CASA system can quickly and objectively detect important indicators such as sperm motility, sperm viability, the percentage of rapidly moving sperm, and sperm concentration. However, when processing high-concentration semen samples, phenomena such as sperm agglutination, aggregation, and clustering can greatly affect the accuracy of the CASA system's detection. On the one hand, this clustering makes it difficult for the CASA system to identify some sperm, resulting in the test value being inconsistent with the actual quality of the semen. On the other hand, because sperm has agglutinative and tactic properties, if the sperm concentration is too high, a large number of sperm will agglutinate and adhere, resulting in an increased misjudgment rate for the CASA system.

[0003] The latest World Health Organization (WHO) "Human Semen Examination and Processing Laboratory Manual" (6th edition) stipulates that if there is no excessive debris or other impurities in the undiluted semen and the sperm concentration is within 2×10 6 -50×10 6 / mL range, the CASA system can be used for routine semen evaluation. 6 / mL, the CASA test will have a large evaluation error.

[0004] Therefore, high-concentration sperm samples should be diluted before testing. The WHO (sixth edition) recommends using the patient's own seminal plasma for semen dilution. However, preparing seminal plasma requires the use of a high-speed centrifuge. Taking a portion of the patient's own semen sample and centrifuging it at high speed (16,000g) for 6 minutes increases the workload and places high demands on the centrifuge equipment in the andrology sperm testing laboratory. Currently, more than 90% of andrology laboratories are not equipped with high-speed centrifuges.

[0005] The study found that more than half of the men who came to the hospital had sperm concentrations greater than 50×10 6 / mL, meaning that more than half of male patients require semen dilution. Using their own semen for dilution would significantly increase the clinical workload. Furthermore, most patients undergo other sperm testing in addition to routine semen examinations. Furthermore, some patients have low ejaculation volumes. Clinically, approximately 20% of patients have ejaculation volumes less than 1.5mL, making it difficult to separate sufficient semen for dilution.

[0006] The 2023 edition of the “Chinese Expert Consensus on Routine Analysis of Semen” points out that if the semen volume is too small to separate the seminal plasma for dilution, physiological saline can be used instead of seminal plasma in a ratio of 1:3 for dilution. However, studies have found that sperm motility decreases after dilution with physiological saline. This is because the osmotic pressure of semen increases after liquefaction (about 340-350mOsm / kg), which is higher than the osmotic pressure of physiological saline. This causes a hypotonic shock to sperm during dilution, resulting in decreased sperm motility and a decrease in the accuracy of the CASA system for detecting routine semen indicators. Therefore, the development of a seminal plasma-like diluent for CASA detection of high-concentration human sperm quality is particularly critical for clinical examinations of men's health.

[0007] Application No. 202410856314.5 discloses a composition, a semen diluent and a method for detecting semen. It provides energy to the diluted sperm by simulating glucose, fructose, etc. in seminal plasma, thereby maintaining the true motility of sperm. Its defect is that it only focuses on simulating seminal plasma components and ignores the core problem of increased osmotic pressure after semen liquefaction. When high-concentration semen is diluted and tested on the machine within 1 minute, the direct impact on sperm motility is the change in osmotic pressure. Other nutrients have limited protective effects on sperm because the time is too short and the sperm has not been fully metabolized. In addition, the invention adds more substances, which increases economic costs, and the addition of multiple protective and energy-supplying substances may lead to higher sperm motility. The most critical thing is that it simulates seminal plasma, but the relevant results compared with seminal plasma are not disclosed, which is a major defect.

[0008] Existing diluents are difficult to use for clinical testing of high-concentration human sperm. Therefore, the inventors have developed a CASA system-based seminal plasma diluent that can replace the patient's own seminal plasma for high-concentration semen dilution testing. This low-cost, easy-to-use alternative to seminal plasma improves the efficiency and accuracy of clinical sperm laboratory testing in men's health. Summary of the Invention

[0009] Purpose of the invention: The purpose of the present invention is to provide a seminal plasma diluent for CASA detection of high-concentration human sperm quality.

[0010] Technical solution:

[0011] The main purpose of the present invention is to propose a CASA system-type semen diluent that can replace seminal plasma for high-concentration semen dilution testing, so as to solve the clinical problems of difficulty in preparing the patient's own semen in andrology laboratories and the lack of relevant alternative diluents, and to improve the efficiency and accuracy of sperm testing in andrology laboratories.

[0012] Another object of the present invention is to provide the above-mentioned seminal plasma-like diluent that replaces seminal plasma, which includes an osmotic pressure regulator and an anti-sperm agglutination agent, but does not contain sperm energy-supplying substances and stimulants, so as to ensure that sperm motility is not over-activated, thereby ensuring that the test results after using the seminal plasma-like diluent are equivalent to those after diluting the semen with the patient's own seminal plasma.

[0013] The present invention measures and analyzes the osmotic pressure of the patient's semen before and after liquefaction, analyzes the osmotic pressure change pattern of its in vitro liquefaction, and thus simulates the osmotic pressure of the seminal plasma after liquefaction. At the same time, an anti-sperm agglutination agent is added. Under the premise of ensuring the accuracy of the patient's sperm quality detection, the addition of protective substances to the seminal plasma-like diluent is minimized to prevent excessive optimization of sperm motility, while reducing costs and improving its accuracy and economy.

[0014] Among them, the osmotic pressure regulator is sodium chloride, which is cheaper than glucose and fructose and does not artificially increase sperm motility. The sperm anticoagulant is bovine serum albumin (BSA), which has multiple functions such as anti-aggregation, antioxidant, and osmotic pressure buffering, which can maintain sperm motility and morphological stability.

[0015] Furthermore, the seminal plasma-like diluent is specifically:

[0016] A sperm seminal plasma diluent is provided, which comprises the following components per 100 mL by weight: 0.9-1.5 parts by weight of sodium chloride and 0.1-0.5 parts by weight of BSA.

[0017] The present invention also provides a method for using a special seminal plasma diluent in semen testing, comprising:

[0018] The CASA system detects and obtains sperm concentration data of the patient's semen sample to be tested

[0019] If the sperm concentration of the patient's semen sample is X, when X>50×10 6 / mL, take 1 volume of semen sample to be tested and add N-1 volumes of special diluent. in To round up to an integer, for example If there is a decimal place, round up to the nearest integer, so N=3.

[0020] The diluted semen sample to be tested (sperm concentration of 2×10 6 / mL-50×10 6 / mL) and perform sperm quality testing using the CASA system, with the testing temperature maintained at 37°C. Specifically, the following procedures were performed:

[0021] In one embodiment, 5 μL of semen sample is added to a disposable counting cell with a depth of 20 μm, and the cell is placed on a 37°C constant temperature testing table. After standing for 30 seconds, the CASA system test is performed. The motility of at least 200 sperm should be evaluated in each counting cell.

[0022] In one embodiment, the diluted semen sample should not be tested more than 5 minutes after the sample is diluted.

[0023] Beneficial effects:

[0024] 1. Based on previous research on the changes in the in vitro osmotic pressure of human semen, the present invention has developed a seminal plasma-like sperm diluent that can replace the patient's own seminal plasma for semen sample dilution, thereby solving the problem of difficulty in preparing one's own seminal plasma and improving the efficiency and accuracy of clinical laboratory testing.

[0025] 2. The sperm motility (PR) of semen samples diluted with the diluent of the present invention and tested by the CASA system is comparable to the test results of semen samples diluted with the patient's own seminal plasma, and is significantly better than the results of semen samples diluted with physiological saline and tested by the CASA system.

[0026] 3. The present invention contains only sodium chloride as an osmotic pressure regulator and BSA as a sperm anticoagulant, without adding any additional sperm energizing substances or stimulants. This ensures that the dilution effect of the seminal plasma diluent is consistent with that of the seminal plasma itself, while avoiding excessive optimization of sperm motility. Furthermore, the present invention utilizes only a few additional substances, is low-cost, and is simple to operate, facilitating clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the change of osmotic pressure before and after liquefaction of semen of the present invention;

[0028] Figure 2 is the sperm acrosome integrity rate after dilution with different dilution media of the present invention;

[0029] Figure 3 It is the sperm DNA integrity rate after dilution with different dilution media of the present invention. DETAILED DESCRIPTION

[0030] The following is a further detailed description of the embodiments of the present invention in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, but the embodiments of the present invention are not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] Example 1

[0032] Study on the in vitro changes of male semen osmotic pressure and the preparation method of patients' own seminal plasma.

[0033] 20 normal semen samples (WHO, 6th edition)

[0034] (1) In this example, 20 semen samples were subjected to in vitro osmotic pressure change detection. Specifically, the semen samples were first tested for osmotic pressure within 5 minutes after removal (before liquefaction), and then again at 30 minutes (after liquefaction). 75 μL of semen sample was collected for osmotic pressure detection and analysis of its osmotic pressure change pattern.

[0035] (2) After liquefaction, 1 mL of semen samples from 20 patients were taken and centrifuged at 16,000 g for 6 min using a high-speed centrifuge. The supernatant seminal plasma was collected and 75 μL of seminal plasma was taken for osmotic pressure detection and analysis.

[0036] Final result ( Figure 1 ): This example found that the osmotic pressure of semen before liquefaction was 301.90±7.79mOsm / kg, which was significantly lower than the osmotic pressure of semen after liquefaction (30min) of 338.10±13.69mOsm / kg and the osmotic pressure of the patient's own seminal plasma after high-speed centrifugation of 340.45±13.95mOsm / kg.

[0037] Example 2

[0038] According to the average osmotic pressure of the patient's own seminal plasma measured in Example 1, a seminal plasma-like diluent for detecting human sperm quality based on CASA was prepared.

[0039] Preparation method:

[0040] (1) Accurately weigh 1.1 g of sodium chloride and 0.4 g of BSA.

[0041] (2) Place all the reagents weighed in step (1) in a clean, sterile glass beaker, add 100 mL of double-distilled water, and stir magnetically at 37°C for 10 min. After sufficient dissolution, take 75 μL of the diluent for osmotic pressure detection and analysis to make its osmotic pressure 340 mOsm / kg, filter with a 0.22 μm filter, seal, and store at 2-8°C.

[0042] (3) When using, preheat the semen diluent in a 37°C water bath in advance, and determine the dilution multiple according to the sperm concentration of the original semen, that is, dilute it at 1:(N-1). X is the original semen concentration.

[0043] Specifically, 100 μL of original semen was taken, diluted and mixed with (N-1)*100 μL of diluent, and then the sperm motility test was performed in the next step.

[0044] Example 3

[0045] Sample source: 20 sperm samples with a concentration > 50×10 6 / mL normal semen sample (WHO 6th edition).

[0046] In this example, physiological saline, the patient's own seminal plasma, and seminal plasma-like diluent were used to detect and analyze the sperm motility (PR, progressive motility) of 20 high-concentration semen samples after dilution using the CASA system.

[0047] Take 5 μL of diluted semen sample, add it into a disposable counting cell with a depth of 20 μm, place it on a 37°C constant temperature testing table, let it stand for 30 seconds, and then perform CASA system testing. The motility of at least 200 sperm should be evaluated in each counting cell.

[0048] In an embodiment, the diluted semen sample should not be tested more than 5 minutes after the sample is diluted.

[0049] Semen sample information and test results are shown in Table 1

[0050]

[0051] Table 1 shows a comparison of sperm motility after dilution using the seminal plasma-like diluent prepared in an embodiment of the present invention, the patient's own seminal plasma, and physiological saline. As can be seen from Table 1, there was no significant difference in sperm motility after dilution using the seminal plasma-like diluent prepared in this embodiment and the patient's own seminal plasma, and sperm motility was significantly higher than that after dilution with physiological saline.

[0052] Example 4

[0053] In this example, physiological saline, the patient's own seminal plasma, and seminal plasma-like diluent were used to dilute 6 high-concentration semen samples, and the sperm acrosome integrity of the diluted semen samples was detected and analyzed.

[0054] Figure 2 The intact rate of sperm acrosome after dilution with different dilution media.

[0055] The seminal plasma diluent prepared in the embodiment of the present invention was compared with the patient's own seminal plasma and semen diluted with physiological saline. Figure 2 This is a comparison of the sperm acrosome integrity rate after dilution with different dilution media. Figure 2 It can be seen that there is no significant difference in the sperm acrosome integrity rate after dilution using the seminal plasma-like diluent in this example, physiological saline, or the patient's own seminal plasma. This shows that the seminal plasma-like diluent maintains a dilution effect similar to that of the patient's own seminal plasma and does not damage the sperm acrosome structure.

[0056] Example 5

[0057] In this example, physiological saline, the patient's own seminal plasma, and seminal plasma-like diluent were used to dilute 6 high-concentration semen samples, and the sperm DNA fragmentation rate (DFI) of the diluted semen samples was detected and analyzed.

[0058] Figure 3 is the sperm DNA integrity rate after dilution with different dilution media

[0059] The seminal plasma diluent prepared in the embodiment of the present invention was compared with the patient's own seminal plasma and semen diluted with physiological saline. Figure 3 It is a comparison of the sperm DNA integrity rate after dilution with different dilution media. Figure 3 A is a flow cytometric scatter plot of sperm DNA fragmentation rate detected by flow cytometry. Figure 3 B shows that there is no significant difference in sperm DNA fragmentation rate after dilution with the seminal plasma-like diluent in this example, normal saline, or the patient's own seminal plasma. This shows that the seminal plasma-like diluent is as effective as the patient's own seminal plasma and does not cause additional damage to sperm DNA.

[0060] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A seminal plasma diluent for CASA detection of high-concentration human sperm quality, characterized in that: The sperm diluent comprises an osmotic pressure regulator and an anti-sperm agglutinating agent, wherein the osmotic pressure regulator is sodium chloride and the anti-sperm agglutinating agent is bovine serum albumin.

2. The seminal plasma-like diluent for CASA detection of high-concentration human sperm quality according to claim 1, characterized in that: Each 100 mL is composed of the following components by mass: 0.9-1.5 parts by mass of sodium chloride, 0.1-0.5 parts by mass of BSA, and the remainder being double-distilled water.

3. An application of a seminal plasma diluent for detecting the quality of high-concentration human sperm by CASA according to any one of claims 1-2, characterized in that: The application of the high-concentration sperm detection seminal plasma diluent in human sperm quality detection.

4. The use of a seminal plasma-like diluent for CASA detection of high-concentration human sperm quality according to claim 3, characterized in that: The following steps are involved: S1, weigh sodium chloride and bovine serum albumin according to the recipe, add double distilled water, stir magnetically at 37°C for 10 min, filter after fully dissolving, seal, and store at 2-8°C; S2, when used, preheat the semen-like diluent in a 37℃ water bath in advance, and determine the dilution multiple according to the sperm concentration of the original semen, that is, dilute it at 1:(N-1). X is the original semen concentration.

5. The use of a seminal plasma diluent for CASA detection of high-concentration human sperm quality according to claim 4, characterized in that: The CASA system is used to test diluted semen, specifically including: testing within 5 minutes after dilution, and the motility of at least 200 sperm should be evaluated for each semen sample.

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