Methods, uses, devices, computer equipment and storage media for diagnosing routine in vitro fertilization results of sperm

Abstract

The application relates to a method, device, computer equipment and storage medium for diagnosing the result of routine in vitro fertilization of sperm. The method for diagnosing and predicting the result of routine in vitro fertilization comprises the following steps: calculating the PLCzeta positive rate of a sperm sample, the PLCzeta positive rate being the proportion of the number of PLCzeta positive sperm in the sperm sample in the total number of sperm, the PLCzeta positive sperm being sperm in which PLCzeta can be detected in at least one of the following head regions of the sperm: acrosome, equator and post-acrosomal region; and diagnosing the fertilization result of routine in vitro fertilization according to the PLCzeta positive rate. The method can efficiently judge the fertilization capacity of sperm that has undergone or is to undergo routine in vitro fertilization, find sperm that may have low fertilization or fertilization failure in routine in vitro fertilization due to PLCzeta defects, provide a basis for the development of a fertilization scheme, and prevent the occurrence of fertilization failure or low fertilization.

Description

Technical Field

[0001] This application relates to the field of assisted reproduction, and in particular to a method, application, apparatus, computer device, and storage medium for diagnosing the results of routine in vitro fertilization of sperm. Background Technology

[0002] In mammals, fertilization refers to the fusion of an oocyte and a sperm to initiate the development of a new individual. Successful fertilization is also a crucial component of assisted reproductive technology (ART). With the use of advanced equipment and modern techniques, conventional in vitro fertilization (cIVF) laboratories have achieved fertilization rates approaching 70-80%. However, low fertilization rates, abnormalities, or failures still occur, significantly reducing oocyte utilization and the number of usable embryos, posing challenges for clinicians and embryologists.

[0003] The fertilization process mainly includes the following steps: (1) Sperm penetration of the cumulus oophorus: Complex glycoproteins in the cumulus oophorus cells help stimulate the potential of the sperm acrosome reaction. At the same time, the sperm also releases a series of enzymes such as hyaluronidase to help the sperm penetrate the cumulus granulosa cells; (2) Sperm penetration of the zona pellucida (ZP) and activation of the oocyte: The sperm binds to the ZP2-N terminus of the sperm receptor protein molecule on the oocyte's ZP. The sperm releases acrosomal enzymes, which can dissolve the zona pellucida into the pores, allowing the sperm to penetrate and activate the calcium release system in the oocyte, causing a sudden increase in the calcium concentration in the cytoplasm, thereby activating the oocyte. The most important function after the sperm and oocyte fuse is to activate the oocyte, including a series of early biochemical events within the oocyte: cortical granule exocytosis, which prevents multisperm penetration; the resumption and completion of meiosis; and the subsequent formation of the pronucleus (PN).

[0004] In conventional in-vitro fertilization (IVF) cycles, fertilization rates below 30% occur in 10% to 25% of cases. Male factors and related issues account for at least 50% of infertility cases in couples. Currently, clinical practice primarily relies on routine semen analysis, which measures sperm density, motility, and morphology, to evaluate male fertility. However, these indicators cannot effectively and comprehensively reflect sperm function; therefore, relying solely on routine semen analysis is far from sufficient to explain male infertility and assess male fertility. Consequently, an effective method for assessing sperm fertilization capacity remains lacking. Summary of the Invention

[0005] Based on this, this application provides a method for diagnosing cIVF results. This method can be used to analyze the causes of low fertilization in cIVF, evaluate sperm fertilization capacity, diagnose the results of in vitro fertilization of sperm, identify men who may have low fertilization in cIVF in advance, provide a basis for the formulation of fertilization plans, and prevent fertilization failure or low fertilization.

[0006] The technical solution to the above-mentioned technical problems in this application is as follows:

[0007] This application provides a method for diagnosing sperm cIVF results, comprising the following steps:

[0008] The PLCζ positivity rate of a sperm sample is detected and calculated, wherein the PLCζ positivity rate is the proportion of PLCζ-positive sperm in the sperm sample to the total number of sperm, and the PLCζ-positive sperm are sperm in which PLCζ can be detected in at least one of the following sperm head regions: acrosome, equator, and post-acrosome region; and

[0009] The cIVF result is diagnosed based on the PLCζ positive rate.

[0010] The aforementioned method for diagnosing cIVF results diagnoses sperm fertilization capacity by calculating the PLCζ positivity rate of sperm samples. This helps in analyzing the causes of low fertilization rates in IVF, provides an efficient prediction of the fertilization capacity of sperm intended for cIVF, and can quickly identify sperm that may experience low fertilization rates in cIVF, thus providing a basis for developing fertilization protocols.

[0011] In some embodiments, the PLCζ positive rate is positively correlated with the total fertilization rate and normal fertilization rate of conventional in vitro fertilization.

[0012] In some embodiments, when the PLCζ positivity rate is less than 56.7%, the sperm sample is a low-fertilization sample from conventional in vitro fertilization.

[0013] Among them, a total fertilization rate of less than 30% in conventional in vitro fertilization is considered low fertilization.

[0014] In some embodiments, the step of detecting and calculating the PLCζ positivity rate of the sperm sample includes:

[0015] The expression and distribution of PLCζ in the sperm samples were detected by PLCζ antibody immunofluorescence staining.

[0016] Statistical analysis was performed on more than 200 sperm cells in the sperm sample to obtain the total sperm count; the number of PLCζ-positive sperm cells was also counted.

[0017] The PLCζ positivity rate is calculated based on the proportion of PLCζ positive sperm to the total number of sperm.

[0018] In some embodiments, the step of detecting the expression and distribution of PLCζ in the sperm sample by PLCζ antibody immunofluorescence staining includes:

[0019] The sperm sample was fixed using a fixative.

[0020] The fixed sperm sample is coated to obtain a sperm smear;

[0021] The sperm smear was incubated with 1% Triton X-100 to obtain an incubated sperm sample;

[0022] A blocking solution was added to the incubated sperm sample to seal it, resulting in a sealed sperm sample.

[0023] The sealed sperm sample was incubated with PLCζ antibody to obtain a sperm sample bound with PLCζ antibody.

[0024] The sperm sample containing the PLCζ antibody was combined with Alexa Fluor TM Incubation with secondary antibody containing 594 markers yielded sperm samples conjugated with the secondary antibody; and

[0025] The sperm samples labeled with the secondary antibody were counterstained with 4',6'-diamino-2-phenylindole to obtain sperm samples stained with immunofluorescence.

[0026] This application also provides an application of the above-described method in diagnosing the results of routine in vitro fertilization of sperm.

[0027] A device for diagnosing the results of routine in vitro fertilization of sperm, comprising:

[0028] A calculation module is used to detect and calculate the PLCζ positivity rate of a sperm sample, wherein the PLCζ positivity rate is the proportion of PLCζ-positive sperm in the sperm sample to the total number of sperm, and the PLCζ-positive sperm are sperm in which PLCζ can be detected in at least one of the following sperm head regions: acrosome, equator, and post-acrosomal region; and

[0029] The data module is used to provide the calculation module with the total number of sperm in the sperm sample and the number of PLCζ positive sperm.

[0030] In some embodiments, the device further includes a detection module that is communicatively connected to the data module, the data module being able to obtain from the detection module the total number of sperm and the number of PLCζ positive sperm in the sperm sample;

[0031] The detection module includes a sperm count unit and a PLCζ positive unit. The sperm count unit is used to detect and count sperm in the sperm sample, and the PLCζ positive unit is used to detect the distribution of PLCζ for each sperm in the sperm sample and count the number of PLCζ positive sperm.

[0032] A computer device includes a memory and a processor, the memory storing a computer program, the processor executing the computer program to implement the steps of the method described above.

[0033] A computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the method described above. Attached Figure Description

[0034] Figure 1 The results of sperm PLCζ immunofluorescence staining in Example 1;

[0035] Figure 2 The graph shows a comparison of sperm PLCζ positivity rate and different positioning patterns in men in the normal fertilization group and low fertilization group in Example 1, as well as an ROC curve analysis graph of sperm PLCζ positivity rate predicting low fertilization in cIVF. Detailed Implementation

[0036] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below, and preferred embodiments are given. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. Reagents or instruments used herein without a specified manufacturer are all conventionally available products.

[0038] In human and mouse sperm, PLCζ is primarily located in the acrosome, equatorial, and post-acrosomal regions of the perinuclear sheath, a specialized structure beneath the acrosome surrounding the sperm head nucleus. This protein possesses four main domains: the EF-hand region, X and Y catalytic domains, the XY connector, and the C2 domain. It is released after sperm entry into the oocyte and activates the oocyte to resume meiosis. cIVF is an insemination method that co-incubates oocytes with hundreds of thousands of sperm in an in vitro fertilization system. Unlike intracytoplasmic sperm injection (ICSI), cIVF involves several unique key events, such as sperm binding and zona pellucida crossing. Whether and how PLCζ defects (reduced expression levels, deletion, or abnormal localization of the PLCζ protein) affect human cIVF outcomes remains unclear. Based on this, the technical personnel of this application have broken through the constraints of the prior art and creatively combined sperm PLCζ with the fertilization outcome of cIVF, making it a biological indicator for evaluating the fertilization capacity of sperm, thereby obtaining a diagnostic biomarker for cIVF outcome.

[0039] One embodiment of this application provides a method for diagnosing cIVF results, the method comprising steps S100 and S200. Specifically:

[0040] Step S100: Detect and calculate the PLCζ positivity rate of the sperm sample.

[0041] The PLCζ positivity rate is the proportion of PLCζ-positive sperm in a sperm sample to the total number of sperm. PLCζ-positive sperm are sperm whose head regions can detect PLCζ in at least one of the following: acrosome region, equatorial region, and post-acrosomal region.

[0042] Specifically, the steps for calculating the positive rate of PLCζ include steps S110, S120, and S130.

[0043] Step S110: Count the number of sperm in the sperm sample to obtain the total number of sperm.

[0044] Specifically, the number of sperm in the male sperm sample is multiple. In some embodiments, the total number of male sperm samples analyzed is more than 200. For example, the number of male sperm samples is 200, 500, or 1000. It is understood that the larger the number of sperm analyzed in the male sperm sample, the more accurate the PLCζ positivity rate of the tested semen sample calculated by the above method. That is, the PLCζ positivity rate can be continuously corrected by increasing the sample size, thereby making the accuracy of the PLCζ positivity rate in diagnosing cIVF results higher. In this document, "multiple" means a number greater than 1; "more than" used with numbers to indicate a range of numbers includes the number itself, for example, 200 or more includes 200.

[0045] Step S120: Detect the distribution of PLCζ in the sperm sample by PLCζ antibody immunofluorescence staining and count the number of PLCζ positive sperm.

[0046] In some embodiments, the above detection includes the step of detecting the distribution of PLCζ in a sperm sample using PLCζ antibody immunofluorescence staining.

[0047] Optionally, the steps for detecting the distribution of PLCζ in a sperm sample using immunofluorescence staining include steps a, b, c, d, e, and f.

[0048] Step a: Spread the fixed sperm sample to obtain a sperm smear.

[0049] Step b: Add 1% Triton X-100 for incubation to obtain incubated sperm samples.

[0050] Step c: Add blocking solution to the incubated sperm sample to seal it, and obtain the sealed sperm sample.

[0051] Step d: Incubate the sealed sperm sample with PLCζ antibody to obtain a sperm sample bound with PLCζ antibody.

[0052] Step e: Combine the sperm sample conjugated with the PLCζ antibody with Alexa Fluor TM Sperm samples containing the 594-labeled secondary antibody were obtained by incubation.

[0053] Step f: Counterstain the sperm sample labeled with the secondary antibody with 4',6'-diamino-2-phenylindole to obtain the sperm sample stained with immunofluorescence.

[0054] In some embodiments, the fixative is 4% paraformaldehyde.

[0055] In some embodiments, a PBST washing step is further included after the PLCζ antibody incubation step, the secondary antibody incubation step, and the counterstaining step.

[0056] In some embodiments, the PLCζ distribution type is divided into four modes: acrosome region, equatorial region, post-acrosomal region, and no-signal region. PLCζ-positive sperm are sperm in which PLCζ can be detected in at least one of the acrosome region, equatorial region, and post-acrosomal region of the sperm head.

[0057] Step S130: Calculate the PLCζ positivity rate based on the proportion of PLCζ positive sperm to the total number of sperm.

[0058] Step S200: Diagnose cIVF results based on PLCζ positive rate.

[0059] The aforementioned method for diagnosing cIVF results diagnoses sperm fertilization capacity by calculating the PLCζ positivity rate of sperm samples. This helps in analyzing the causes of low fertilization rates in IVF, provides an efficient prediction of the fertilization capacity of sperm intended for cIVF, and can quickly identify sperm that may experience low fertilization rates in cIVF, thus providing a basis for developing fertilization protocols.

[0060] In some embodiments, the PLCζ positive rate is positively correlated with the total fertilization rate and normal fertilization rate of conventional in vitro fertilization.

[0061] In some embodiments, the cIVF result of a sperm sample is diagnosed based on a threshold for the PLCζ positivity rate.

[0062] It is understandable that the threshold was obtained based on the PLCζ positivity rate analysis of multiple normal sperm samples and multiple low-fertilization samples, and the analysis methods included correlation analysis, regression analysis and ROC curve analysis.

[0063] In some embodiments, the threshold is 56.7%. That is, when the PLCζ positivity rate of a sperm sample is less than 56.7%, the sperm sample can be determined to be a low-fertilization sample of cIVF, wherein the total fertilization rate of cIVF is less than 30% and is considered low-fertilization.

[0064] It is understandable that the total fertilization rate refers to the percentage of eggs that form pronuclei after cIVF out of the total number of cIVF eggs. A total fertilization rate of less than 30% in cIVF is considered low fertilization, and when this sperm sample is used for cIVF, the risk of a fertilization rate of less than 30% is high.

[0065] The aforementioned method for determining the PLCζ positivity rate threshold involves dynamically collecting male semen, continuously refining the detection of the PLCζ positivity rate, detecting the distribution of PLCζ in each sperm sample using PLCζ antibody immunofluorescence staining, and counting the number of PLCζ-positive sperm. Based on the number of PLCζ-positive sperm and the total sperm count, the PLCζ positivity rate is calculated. This allows for an efficient assessment of the fertilization capacity of sperm in patients who have undergone or are planning cIVF. It can quickly identify the causes of low fertilization or fertilization failure in patients, or identify sperm that may be at risk of low fertilization or fertilization failure in cIVF, providing a basis for developing fertilization protocols and preventing fertilization failure or low fertilization.

[0066] One embodiment of this application also provides an application of the above-described method in diagnosing sperm cIVF results.

[0067] Furthermore, one embodiment of this application also provides an apparatus for diagnosing sperm cIVF results, the apparatus including a calculation module and a data module.

[0068] Specifically, the calculation module is used to calculate the PLCζ positivity rate of the sperm sample. The PLCζ positivity rate is the proportion of PLCζ positive sperm in the sperm sample to the total number of sperm. PLCζ positive sperm are sperm that can detect PLCζ in at least one of the following sperm head regions: acrosome, equator, and post-acrosomal region.

[0069] Specifically, the calculation module is used to provide the total number of sperm in the sperm sample and the number of PLCζ positive sperm to the calculation module.

[0070] In some embodiments, the above-described apparatus further includes a detection module, which is communicatively connected to a data module. The data module is capable of obtaining the total number of sperm and the number of PLCζ-positive sperm from the sperm sample via the detection module.

[0071] Specifically, the detection module includes a sperm count unit and a PLCζ positive unit. The sperm count unit is used to detect and count sperm in the sperm sample, and the PLCζ positive unit is used to detect the distribution of PLCζ for each sperm in the sperm sample and count the number of PLCζ positive sperm.

[0072] The various modules in the aforementioned device for diagnosing cIVF results of sperm can be implemented entirely or partially through software, hardware, or a combination thereof. These modules can be embedded in or independent of the processor in a computer device, or stored in the memory of a computer device as software, so that the processor can call and execute the corresponding operations of each module.

[0073] Furthermore, one embodiment of this application also provides a computer device, including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the steps of the method for diagnosing cIVF results of any of the above embodiments.

[0074] It is understood that the aforementioned computer device can be a terminal. This computer device includes a processor, memory, communication interface, display screen, and input device connected via a system bus. Specifically: the processor provides computing and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage media. The communication interface of the computer device is used for wired or wireless communication with external terminals. Wireless communication can be achieved through Wi-Fi, mobile cellular networks, NFC (Near Field Communication), or other technologies. When the computer program is executed by the processor, it implements a method for diagnosing cIVF results. The display screen of the computer device can be an LCD screen or an e-ink display screen. The input device can be a touch layer covering the display screen, buttons, a trackball, or a touchpad mounted on the computer device casing, or an external keyboard, touchpad, or mouse, etc.

[0075] Furthermore, one embodiment of this application also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the method for diagnosing cIVF results described in any of the above embodiments.

[0076] Furthermore, one embodiment of this application provides a computer program product or computer program including computer instructions stored in a computer-readable storage medium. A processor of a computer device reads the computer instructions from the computer-readable storage medium and executes the computer instructions, causing the computer device to perform the steps of the method for diagnosing cIVF results described in any of the above embodiments. Detailed Implementation

[0078] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0079] Example 1

[0080] 1. cIVF insemination

[0081] 1. Sperm preparation

[0082] Semen samples were collected from 49 patients undergoing cIVF (excluding patients with severe oligoasthenospermia, teratospermia, and malformed sperm and patients who underwent surgical sperm retrieval; all cIVF cycles yielded ≥3 oocytes). Patients ejaculated after abstinence for 2-7 days, liquefied the semen at 37°C for 30 minutes, and then underwent density gradient centrifugation. The total sperm density and the number of progressively motile sperm were counted before and after the treatment.

[0083] 2.Insemination

[0084] Sperm subjected to density gradient centrifugation were added to cIVF medium containing the ovum-corona-cumulus complex. The final density of progressively motile sperm was 1 × 10⁻⁶. 5 / mL, fertilized overnight in an incubator at 37℃ and 6% CO2.

[0085] 3. Fertilization assessment

[0086] 16-18 hours after cIVF fertilization, granulosa cells are removed, and the second polar body and the pronucleus (PN) in the cytoplasm of the egg are observed under an inverted microscope. The formation of two polar bodies and two male and female pronuclei indicates normal fertilization.

[0087] Normal fertilization rate = Number of normally fertilized (2PN) eggs / Number of cIVF eggs

[0088] Total fertilization rate = number of pronuclear eggs formed / number of cIVF eggs.

[0089] 4. Results

[0090] Based on fertilization outcomes, the participants were divided into two groups: (1) Normal fertilization group (NF) with 31 participants, normal fertilization rate ≥50%, 1PN + multiple PN <30%; (2) Low fertilization group (LF) with 18 participants, total fertilization rate ≤30%.

[0091] II. Immunofluorescence staining of human sperm

[0092] Each sperm sample underwent immunofluorescence staining, with a total of 200 or more sperm cells counted in each sample. The specific steps for immunofluorescence staining are as follows:

[0093] (1) Fixation: The sperm samples treated by the upstream method or density gradient centrifugation method were co-incubated with 4% paraformaldehyde (4% PFA) at room temperature for 30 min;

[0094] (2) After fixing, the sperm sample was smeared and dried at room temperature;

[0095] (3) Membrane permeation: Add 1% Triton X-100 and incubate at room temperature for 15 min;

[0096] (4) Remove the permeabilizing solution and wash twice with DPBS, 5 min each time;

[0097] (5) Blocking: Add blocking solution (PBS containing 5% donkey serum, 2% BSA, 0.1M glycine and 0.01% Triton X-100), and block at room temperature for 1 hour or at 4°C overnight;

[0098] (6) Primary antibody incubation: Rabbit anti-human PLCζ antibody (1:100; Covalab, pab0367-P) was diluted with blocking buffer and incubated overnight at 4°C;

[0099] (7) Carefully remove the primary antibody and wash three times with PBST (containing 0.05% Tween 20), 15 min each time;

[0100] (8) Secondary Antibody Incubation: Alexa Fluor TM 594 donkey anti-rabbit secondary antibody (1:800; A21207, Invitrogen) was diluted with PBST and incubated at room temperature in the dark for 1 h.

[0101] (9) Wash twice with PBST, 10 min each time;

[0102] (10) DAPI counterstaining for 10 min;

[0103] (11) Wash twice with PBST, 10 min each time;

[0104] (12) Mounting: Add 15 μL of anti-quenching mounting medium to the glass slide and gently cover it with a coverslip;

[0105] (13) Bake the slices in an oven at 37℃ for 15 minutes;

[0106] (14) Fluorescence signals were acquired using a Zeiss Cell Discoverer 7 fluorescence microscope and Adobe Photoshop software, and 200 or more sperm were counted.

[0107] Results and Analysis

[0108] Observe each sample, such as Figure 1 As shown, the sperm PLCζ localization area in each sample can be divided into four modes: (1) acrosome region (AcEq): acrosome region or simultaneously accompanied by equatorial / post-acrosome region; (2) equatorial region (Eq); (3) post-acrosome region (Pa): post-acrosome region or simultaneously accompanied by equatorial region; (4) no signal (None). Among them, acrosome, equatorial and post-acrosome are normal localization modes, while no signal is an abnormal mode.

[0109] III. Calculation of PLCζ Positive Rate

[0110] By counting the number of sperm in the sperm sample and counting the number of PLCζ-positive sperm, which are defined as sperm whose head contains at least one of the acrosome region, equatorial region, and post-acrosomal region, the PLCζ positivity rate was calculated based on the number of PLCζ-positive sperm and the proportion of the total sperm count, as shown in Table 1.

[0111] Table 1

[0112]

[0113] IV. Comparison of sperm PLCζ positivity rate and different positioning patterns in the normal fertilization group and the low fertilization group

[0114] To investigate the relationship between the positive rate of sperm PLCζ, different localization patterns, and cIVF fertilization outcomes, PLCζ staining analysis was performed on sperm from both groups of patients, as shown in Table 2. Figure 2 As shown in (a) of the table, median comparison revealed that the PLCζ positivity rate of sperm in men in the low-fertilization group of cIVF was significantly lower than that in the normal-fertilization group (64.1% (95% CI, 46.8–84.3) vs 82.2% (73.1–87.7), P = 0.0176). (See Table 2 and...) Figure 2 As shown in (b) to (d), the comparison of median values ​​revealed that, regardless of the PLCζ positioning mode, there was no significant difference in the sperm ratio between the low fertilization group and the normal fertilization group (P > 0.05).

[0115] Table 2

[0116]

[0117] a P = 0.0176 (low fertilization group vs. normal fertilization group).

[0118] V. Correlation analysis of sperm PLCζ positivity rate, different positioning patterns, and cIVF fertilization rate

[0119] To investigate the positive rate of sperm PLCζ and the influence of different positioning patterns on cIVF fertilization results, a correlation analysis was performed using the Pearson coefficient, as shown in Table 3. The positive rate of sperm PLCζ was significantly positively correlated with the 2PN rate (r = 0.459, P = 0.0005) and the total fertilization rate (r = 0.438, P = 0.0008). Although the post-apical positioning pattern of PLCζ was statistically significantly positively correlated with the total fertilization rate (r = 0.247, P = 0.044), the correlation coefficient was low.

[0120] Table 3

[0121]

[0122] a Based on data from the normal fertilization group and the low fertilization group; b P = 0.0005, c P = 0.0008, d P = 0.044.

[0123] VI. Identification of individuals with low fertility

[0124] To explore the predictive value of PLCζ positivity rate for cIVF patients with low fertilization rate (total fertilization rate ≤30%), the PLCζ positivity rate, which showed significant differences between the two groups and was significantly correlated with fertilization rate, was selected as an indicator. Based on data from 49 men in the normal fertilization group and the low fertilization group, ROC curves were plotted, as shown below. Figure 2 As shown in (e), the optimal cutoff value for the positive rate of PLCζ associated with low fertilization in cIVF was 56.7% (sensitivity 44.4%, specificity 100%), with an area under the curve (AUC) of 0.705 (95% CI, 0.538–0.872). The results indicate that using a threshold of 56.7% resulted in a correct prediction in 79.6% of patients, suggesting that the positive rate of sperm PLCζ can serve as an indicator for diagnosing low fertilization in cIVF patients.

[0125] VII. Test Results of Clinical Samples

[0126] Table 4

[0127]

[0128] a A positive result means that the PLCζ positivity rate of the sperm sample is lower than the threshold of 56.7%; b Negative means that the PLCζ positivity rate of the sperm sample is higher than the threshold of 56.7%.

[0129] As shown in Table 4 above, the PLCζ positivity rate threshold of this application is used to diagnose sperm cIVF results and is suitable for detecting low fertilization samples in cIVF. It provides an efficient prediction of the fertilization capacity of sperm intended for cIVF and can quickly identify sperm that may have low fertilization due to PLCζ defects. Patients should be advised to switch to intracytoplasmic sperm injection (ICSI) and assisted oocyte activation (AOA) during assisted reproductive treatment to improve treatment outcomes.

[0130] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A device for diagnosing the results of routine in vitro fertilization of sperm, characterized in that, include: A calculation module is used to detect and calculate the PLCζ positivity rate of a sperm sample, wherein the PLCζ positivity rate is the proportion of PLCζ-positive sperm in the sperm sample to the total number of sperm, and the PLCζ-positive sperm are sperm in which PLCζ can be detected in at least one of the following sperm head regions: acrosome, equator, and post-acrosomal region; and A data module is used to provide the calculation module with the total sperm count and the number of PLCζ-positive sperm in the sperm sample; the PLCζ positivity rate is positively correlated with the total fertilization rate and normal fertilization rate of conventional in vitro fertilization; when the PLCζ positivity rate is lower than 56.7%, the sperm sample is considered a low-fertilization sample for conventional in vitro fertilization; wherein, a total fertilization rate of conventional in vitro fertilization lower than 30% is considered low-fertilization; the steps of detecting and calculating the PLCζ positivity rate of the sperm sample include: The expression and distribution of PLCζ in the sperm samples were detected by PLCζ antibody immunofluorescence staining. Statistical analysis was performed on more than 200 sperm cells in the sperm sample to obtain the total sperm count; the number of PLCζ-positive sperm cells was also counted; and The PLCζ positivity rate is calculated based on the proportion of PLCζ positive sperm to the total number of sperm.

2. The apparatus according to claim 1, characterized in that, The step of detecting the expression and distribution of PLCζ in the sperm sample by PLCζ antibody immunofluorescence staining includes: The sperm sample was fixed using a fixative. The fixed sperm sample is coated to obtain a sperm smear; The sperm smear was incubated with 1% Triton X-100 to obtain an incubated sperm sample; A blocking solution was added to the incubated sperm sample to seal it, resulting in a sealed sperm sample. The sealed sperm sample was incubated with PLCζ antibody to obtain a sperm sample bound with PLCζ antibody. Sperm samples conjugated with PLCζ antibody were incubated with a secondary antibody labeled with Alexa Fluor™ 594 to obtain sperm samples conjugated with the secondary antibody; and The sperm samples labeled with the secondary antibody were counterstained with 4',6'-diamino-2-phenylindole to obtain sperm samples stained with immunofluorescence.

3. The apparatus according to claim 1, characterized in that, The device also includes a detection module, which is communicatively connected to the data module. The data module is able to obtain the total number of sperm and the number of PLCζ positive sperm from the sperm sample. The detection module includes a sperm count unit and a PLCζ positive unit. The sperm count unit is used to detect and count sperm in the sperm sample, and the PLCζ positive unit is used to detect the distribution of PLCζ for each sperm in the sperm sample and count the number of PLCζ positive sperm.

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