A rapid staining reagent for sperm morphology and a staining method thereof

Abstract

The present application relates to the technical field of in vitro detection, in particular to a rapid sperm morphology staining reagent and a staining method thereof; the rapid sperm morphology staining reagent of the present application can make semen spread rapidly and uniformly after 5 minutes by using bromelain and L-cysteine in combination. The staining reagent of the present application greatly shortens the pretreatment time of the sample and improves the detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a rapid staining reagent for sperm morphology and its staining method. Background Technology

[0002] Sperm morphology examination is the most basic indicator of semen analysis. The World Health Organization's Laboratory Manual for Human Semen Examination and Processing (6th Edition) recommends the use of Papanicolaou staining, Diff-Quik staining, or Shorr staining. This is because proteins with different isoelectric points in sperm and cells carry different charges under the same acidity, and can selectively bind to the corresponding dyes for staining. All three staining methods can be used to observe sperm morphology under a bright field of an optical microscope.

[0003] Upon ejaculation into the collection container, semen quickly presents as a typical semi-solid gel or gel-like clump. Typically at room temperature, semen begins to liquefy (become thinner) within minutes, at which point a heterogeneous mixture of semi-solid clumps is visible. As liquefaction continues, the semen becomes more homogeneous and quite watery, but still has a higher viscosity than water. Only a small amount of clumps remain in the final stage of liquefaction. At room temperature, semen usually liquefies completely within 15–30 minutes.

[0004] The Diff-Quick method and Papanicolaou staining method are widely used in clinical practice, but these methods are complex to operate and require 15 to 30 minutes for sample liquefaction, resulting in a long overall testing time.

[0005] CN115266285A discloses a sperm morphology Diff-Quik staining solution and its application method, while CN118032464A discloses a rapid sperm morphology staining method. Both disclose that if the sample liquefies slowly or has excessive viscosity, it is necessary to slowly blow the semen sample with a pipette (avoiding the introduction of air) to promote complete liquefaction. In such cases, additional treatments such as mechanical mixing can promote semen liquefaction for further sample evaluation; however, these treatments may affect seminal plasma biochemistry, sperm motility, and sperm morphology.

[0006] Therefore, it is necessary to explore a pretreatment reagent that can shorten the sperm morphology examination, shorten the sperm liquefaction time, and shorten the detection time. Summary of the Invention

[0007] To address the issues of long staining operation time and high sample requirements, this invention provides a simple and rapid sperm morphology staining reagent and its application method.

[0008] Specifically, the first aspect of the present invention provides a rapid sperm morphology staining reagent, comprising reagent 1 and reagent 2, wherein,

[0009] Reagent 1 contains 2-4 mg / mL bromelain, 2-4 mmol / L L-cysteine, and phosphate buffer;

[0010] Reagent 2 contains 0.05-0.5 wt% azure B, 0.05-0.5 wt% methylene blue and phosphate buffer.

[0011] The staining method of this invention is quick and easy to operate, and the entire staining process can be completed within 20 seconds. This invention is suitable for immediate sample detection, and it provides good differentiation between sperm head, neck, midpiece, and tail, with uniform and clear staining, making it easy to apply in clinical settings. The use of preservatives improves reagent stability, extends shelf life, and extends the shelf life to two years.

[0012] The inventors of this invention discovered that using bromelain and L-cysteine ​​for short-term pre-liquefaction can promote semen liquefaction, while L-cysteine ​​can reduce the sulfhydryl groups in the active site of bromelain, significantly increasing the rate of enzyme catalysis. Surprisingly, the combination of the two can help liquefy routine semen samples in about 5 minutes without affecting sperm morphology.

[0013] In some embodiments, the content of the bromelain is 3 mg / mL.

[0014] In some embodiments, the L-cysteine ​​content is 4 mmol / L.

[0015] In some embodiments, the L-cysteine ​​content is 2 mmol / L.

[0016] In some embodiments, reagent 1 comprises:

[0017] 3 mg / mL bromelain;

[0018] 2-4 mmol / L L-cysteine; and

[0019] Phosphate buffer.

[0020] In some embodiments, reagent 1 comprises:

[0021] 3 mg / mL bromelain;

[0022] 4 mmol / L L-cysteine; and

[0023] Phosphate buffer.

[0024] In some embodiments, reagent 1 comprises the following components:

[0025] 3 mg / mL bromelain;

[0026] 4 mmol / L L-cysteine; and

[0027] Phosphate buffer.

[0028] In some embodiments, reagent 2 further comprises a preservative, wherein the preservative is ProClin 300.

[0029] In some embodiments, the content of the preservative is 0.01 to 0.1 wt%.

[0030] In some embodiments, the preservative content is 0.05 wt%.

[0031] In some embodiments, reagent 2 comprises:

[0032] 0.05~0.5 wt% Azure B;

[0033] 0.05~0.5 wt% methylene blue;

[0034] 0.01~0.1wt% of ProClin 300;

[0035] 10-30% (v / v) methanol; and

[0036] Phosphate buffer.

[0037] In some embodiments, reagent 2 consists of the following components:

[0038] 0.05~0.5 wt% Azure B;

[0039] 0.05~0.5 wt% methylene blue;

[0040] 0.01~0.1wt% of ProClin 300;

[0041] 10-30% (v / v) methanol; and

[0042] Phosphate buffer.

[0043] In some implementations, the content of azurite B is 0.1 wt%.

[0044] In some embodiments, the methylene blue content is 0.1 wt%.

[0045] In some embodiments, the content of azure B is 0.1 wt%; the content of methylene blue is 0.1 wt%.

[0046] In some embodiments, the methanol content is 20% (v / v).

[0047] In some embodiments, the pH of the phosphate buffer solution is 7.0 to 8.0.

[0048] In some embodiments, the pH of the phosphate buffer solution is 7.2 to 7.4.

[0049] In some embodiments, the pH of the phosphate buffer solution is 7.3.

[0050] In some embodiments, the concentration of the phosphate buffer solution is 10 mmol / L.

[0051] A second aspect of the present invention provides an application of the rapid sperm morphology staining agent described in the first aspect above, for sperm morphology detection for non-diagnostic purposes, such as in vitro detection.

[0052] A third aspect of the present invention provides a rapid staining method for sperm morphology, which employs staining with the staining reagent described in the first aspect above, the staining method comprising:

[0053] Step S1: Treat the unliquefied semen sample to be treated with reagent 1 for 4 to 6 minutes to obtain a pretreated sample; take the pretreated sample and smear it on a glass slide;

[0054] Step S2: Immerse the smear obtained in step S1 into reagent 2.

[0055] In some implementations, in step S1, the time for treating the unliquefied semen sample with reagent 1 does not exceed 6 minutes.

[0056] In some implementations, in step S1, the time for treating the unliquefied semen sample with reagent 1 is 5 minutes.

[0057] In some implementations, the specific steps of the rapid staining method are as follows:

[0058] S1: Collect fresh, unliquefied semen samples, add reagent 1 in an equal volume to the sample, mix thoroughly, and let stand for 5 minutes to obtain a pretreated sample;

[0059] Apply the pretreated sample to a clean glass slide and smear it; place the smeared slide horizontally and allow it to air dry in a natural environment.

[0060] S2: Immerse the smear obtained in step S1 in reagent 2 for a period of time (preferably 15 seconds), remove the slide and place it perpendicular to the absorbent paper to remove excess staining solution.

[0061] Further, the smear after washing step S2 was observed.

[0062] In some embodiments, reagent 1 is prepared by adding bromelain and L-cysteine ​​to a 10 mmol / L phosphate buffer solution, wherein the concentration of bromelain is 2-4 mg / mL and the concentration of L-cysteine ​​is 2-4 mmol / L.

[0063] In some embodiments, reagent 2 is prepared by adding methanol (20% v / v), azure B (0.1 wt%), methylene blue (0.1 wt%) and ProClin 300 (0.05 wt%) to a 10 mmol / L phosphate buffer solution and adjusting the pH to 7.3 (25 ± 0.5 °C).

[0064] The beneficial effects of this invention are:

[0065] This invention provides a rapid staining reagent combination, wherein bromelain and L-cysteine ​​are used for short-time pre-liquefaction. L-cysteine ​​can reduce the sulfhydryl groups in the active site of bromelain, significantly increasing the enzyme catalytic reaction rate, thereby reducing the sperm liquefaction time, so that the semen can be rapidly and evenly spread on the glass slide after short-time pre-liquefaction.

[0066] The staining reagent formulation of this invention is simple, using azure B and methylene blue to stain the acrosome, nucleus, midpiece, and tail of sperm heads. The raw materials are low in cost and readily available, and the operation is quick, making it suitable for rapid detection of laboratory samples. Attached Figure Description

[0067] Figure 1 The staining results of Example 1 are shown.

[0068] Figure 2 The staining results for Comparative Example 1 are shown. Detailed Implementation

[0069] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are all widely used terms and routine procedures in the respective fields. To better understand this invention, definitions and explanations of related terms are provided below.

[0070] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0071] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0072] liquefaction

[0073] Upon ejaculation into the collection container, semen quickly presents as a typical semi-solid gel or gel-like clump. Typically at room temperature, semen begins to liquefy (become thinner) within minutes, at which point an uneven mixture of semi-solid clumps is visible. As liquefaction continues, the semen becomes more homogeneous and quite watery, but still has a higher viscosity than water. Only a small amount of clumps remain in the final stage of liquefaction.

[0074] At room temperature, semen usually liquefies completely within 15 to 30 minutes.

[0075] Sometimes semen may not liquefy, making diagnostic semen analysis difficult. In such cases, additional treatments such as mechanical mixing or enzymatic digestion can promote semen liquefaction for further sample evaluation, but these treatments can affect seminal plasma biochemistry, sperm motility, and sperm morphology.

[0076] Repeatedly (6-10 times) slowly passing semen through an 18-gauge blunt needle (0.84 mm inner diameter) or a 19-gauge needle (0.69 mm inner diameter) attached to a syringe can reduce the heterogeneity of the semen. However, the shearing force may damage sperm, thus having a side effect on DNA integrity.

[0077] Digestion with bromelain, a broad-spectrum proteolytic enzyme, can help promote semen liquefaction. However, this treatment can alter seminal plasma biochemical markers and sperm motility.

[0078] Sperm morphology

[0079] The value of sperm morphology assessment lies not only in its predictive role in natural pregnancy and assisted reproductive technology (ART) outcomes, but also in providing more diagnostic information about the status of male reproductive organs, primarily the testes and epididymis. Determining only the percentage of "normal" sperm is insufficient for evaluating male reproductive organs. Assessing the specific morphology of the sperm head, neck / midpiece, and tail, as well as any abnormal residual cytoplasm, is particularly important. All human semen contains many sperm with diverse morphological appearances. Early definitions of sperm morphology were primarily based on veterinary experience and microscopic observation.

[0080] Under an optical microscope, a sperm cell can be considered to consist of a head and a tail. The thicker portion containing mitochondria at the junction of the tail and head is called the midpiece. The remaining tail consists of a main segment with a long columnar fibrous sheath (with dense peripheral fibers surrounding an axonal or ciliary structure) and a terminal segment. Because the terminal segment is difficult to observe under an optical microscope, a sperm cell can be considered to include both the head (and neck) and the tail (midpiece and mainpiece). For a sperm cell to be considered morphologically normal, the head, midpiece, mainpiece, and residual cytoplasm must all be normal. Any morphological borderline is considered abnormal.

[0081] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0082] Example 1

[0083] (1) Preparation of reagent A: Add bromelain (3 mg / mL) and L-cysteine ​​(4 mmol / L) to a 10 mmol / L phosphate buffer solution and adjust the pH to 7.3 (25±0.5℃).

[0084] (2) Preparation of reagent B: Add methanol (20%, v / v), azure B (0.1wt%), methylene blue (0.1wt%) and ProClin 300 (0.05wt%) to a 10 mmol / L phosphate buffer solution and adjust the pH to 7.3 (25±0.5℃).

[0085] (3) Collect fresh, unliquefied semen samples, add reagent A in equal volume to the sample, mix thoroughly, and let stand for about 5 minutes to obtain a pretreated sample; observe that the semen rapidly changes from a gel-like state to a fluid state.

[0086] A 10 μL sample of pretreated sample was applied to a clean glass slide and smeared. A uniform thin layer was observed to form on the slide, with no areas of uneven thickness. The prepared slide was then placed horizontally and allowed to air dry naturally.

[0087] (4) Immerse the smear obtained in step (3) in reagent B for 15 seconds, remove the slide and place it perpendicular to the absorbent paper to remove excess staining solution.

[0088] (5) Til the smear obtained in step (4) at 60 degrees and gently immerse it in sterile water to wash 1-2 times, each time for 1 second, to remove excess staining solution. Place the slide vertically on absorbent paper to remove excess water and air dry in a natural environment. Observe the sperm morphology under a 100× objective lens of a regular optical microscope. Observation can be performed with or without mounting the slide.

[0089] Staining results as follows Figure 1 As shown, the top region of the sperm head is light blue, the nucleus is dark blue, and the midpiece and tail are light blue. The staining of different parts of the sperm is well distinguished, and there is no overlap or aggregation between sperm, making it easy to observe.

[0090] Example 2

[0091] (1) Preparation of reagent A: Add bromelain (3 mg / mL) and L-cysteine ​​(2 mmol / L) to a 10 mmol / L phosphate buffer solution and adjust the pH to 7.3 (25 ± 0.5℃).

[0092] (2) Preparation of reagent B: Add methanol (20%, v / v), azure B (0.1wt%), methylene blue (0.1wt%) and ProClin 300 (0.05wt%) to a 10 mmol / L phosphate buffer solution and adjust the pH to 7.3 (25±0.5℃).

[0093] (3) Collect fresh, unliquefied semen samples, add reagent A in equal volume to the sample, mix thoroughly, and let stand for about 5 minutes to obtain a pretreated sample; observe that the semen rapidly changes from a gel-like state to a fluid state.

[0094] A 10 μL sample of pretreated sample was applied to a clean glass slide and smeared. A uniform thin layer was observed to form on the slide, with no areas of uneven thickness. The prepared slide was then placed horizontally and allowed to air dry naturally.

[0095] Comparative Example 1 (Diff-Quik Method)

[0096] (1) Collect fresh, unliquefied semen samples and liquefy them at 37°C for about 15-30 minutes until completely liquefied. On a clean glass slide, drop 10 μL of the liquefied semen onto the slide, make a smear, and place the prepared slide horizontally to air dry naturally.

[0097] (2) Immerse the air-dried glass slide in the fixative (95% (v / v) methanol or 1.8 mg triarylmethane dissolved in 1000 mL 95% (v / v) methanol) for 15 s, and then remove it and blot the remaining stain on absorbent paper.

[0098] (3) Immerse the glass slide in staining solution I (the main component of which is the acid dye eosin) for 10-20 seconds, and then place the glass slide upright on absorbent paper to remove excess liquid.

[0099] (4) Immerse the slide in staining solution II (the main components of which are basic dyes methylene blue and azure B) for 5-10 seconds, and then place the slide upright on absorbent paper to remove excess liquid.

[0100] (5) Rinse with running water 10-15 times to remove excess dye, air dry, and observe under a microscope.

[0101] Staining results as follows Figure 2 As shown, the sperm head nucleus region is bluish-purple, the acrosome region is pale blue, and the sperm tail is light pink. Figure 2 .

[0102] Comparative Example 2

[0103] (1) Collect fresh, unliquefied semen samples, add an equal volume of reagent C (phosphate buffer solution containing 3 mg / mL bromelain), mix thoroughly, and let stand for about 10 minutes to obtain pretreated samples. It was observed that the semen slowly changed from a gel-like state to a liquid state with uneven viscosity.

[0104] (2) Take 10 μL of pretreated sample onto a clean glass slide and smear it. It was found that the sample was difficult to spread and accumulated in the form of droplets on the glass slide, resulting in uneven areas on the glass slide.

[0105] (3) The remaining pretreated samples were left to stand for about 10 minutes, and the semen was observed to slowly transform into a fluid with good flowability.

[0106] Comparative Example 3

[0107] (1) Collect fresh, unliquefied semen samples, add an equal volume of reagent D (phosphate buffer solution containing 4 mmol / L L-cysteine), mix thoroughly, and let stand for about 10 minutes to obtain a pretreated sample. Observe that the semen slowly changes from a gel-like state to a liquid state with uneven viscosity. After 15 minutes, it becomes a liquid with good fluidity.

[0108] (2) Take 10 μL of pretreated sample onto a clean glass slide and smear it. It was found that the sample was difficult to spread and accumulated in the form of droplets on the glass slide, resulting in uneven areas on the glass slide.

[0109] (3) The remaining pretreated samples were left to stand for about 20 minutes, and the semen was observed to slowly transform into a fluid with good flowability.

[0110] While certain features of the invention have been set forth and described herein, many modifications, substitutions, variations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations that fall within the true scope of the invention.

Claims

1. A rapid sperm morphology staining reagent, comprising reagent 1 and reagent 2, wherein, Reagent 1 contains 2-4 mg / mL bromelain, 2-4 mmol / L L-cysteine, and phosphate buffer; Reagent 2 contains 0.05-0.5 wt% azure B, 0.05-0.5 wt% methylene blue and phosphate buffer.

2. The rapid sperm morphology staining reagent according to claim 1, wherein, The content of bromelain is 3 mg / mL.

3. The rapid sperm morphology staining reagent according to claim 1, wherein, The L-cysteine ​​content is 4 mmol / L.

4. The rapid sperm morphology staining reagent according to claim 1, wherein, The reagent 2 also contains a preservative, namely ProClin 300.

5. The rapid sperm morphology staining reagent according to claim 4, wherein, The reagent 2 comprises: 0.05~0.5 wt% Azure B; 0.05~0.5 wt% methylene blue; 0.01~0.1 wt% of ProClin 300; 10-30% (v / v) methanol; and Phosphate buffer.

6. The rapid sperm morphology staining reagent according to claim 5, wherein, The content of azurite B is 0.1 wt%; The content of methylene blue is 0.1 wt%.

7. The rapid sperm morphology staining reagent according to any one of claims 1-6, wherein, The pH of the phosphate buffer solution is 7.0 to 8.

0.

8. The rapid sperm morphology staining reagent according to claim 7, wherein, The pH of the phosphate buffer solution is 7.

3.

9. A rapid sperm morphology staining method, comprising staining with the staining reagent according to any one of claims 1-7, wherein the rapid sperm morphology staining method includes: Step S1: Treat the unliquefied semen sample to be treated with reagent 1 for 4-6 minutes to obtain a pretreated sample; Take the pretreated sample and smear it onto a glass slide; Step S2: Immerse the smear obtained in step S1 into reagent 2.

10. The rapid sperm morphology staining method according to claim 9, wherein, In step S1, the time for treating the unliquefied semen sample with reagent 1 shall not exceed 5 minutes.

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