An integrated rare sperm evaluation positioning and cryocontainer and method of use

Abstract

The application discloses an integrated rare sperm evaluation positioning and freezing carrier and a use method, wherein the carrier comprises a carrier main body, an observation scanning cavity, a coordinate positioning mark component, a special interface, a sealing section and a data recording component; the carrier main body is a microfluidic structure, and the observation scanning cavity is arranged in the middle of the carrier main body and is a transparent structure; the coordinate positioning mark component is arranged on the periphery or inside of the observation scanning cavity and is used for establishing a carrier coordinate system; the special interface comprises a sample injection port and a sample recovery interface; the sealing section is arranged on one side or both sides of the carrier main body and is used for sealing a stage of confirming that there are sperms; and the data recording component is used for recording corresponding data. The application realizes the integration of evaluation, positioning, positioning counting, freezing, rewarming and recovery, reduces the loss of rare sperm transfer, improves the retrieval efficiency and traceability, and is suitable for outpatient rare sperm analysis and testicular microdissecting sperm extraction and other rare sperm scenes.

Description

Technical Field

[0001] This invention relates to the field of assisted reproduction and fertility preservation technology, and in particular to an integrated cryopreservation carrier for assessing and locating rare sperm and its usage method. Background Technology

[0002] In outpatient cases of oligospermia, asthenospermia, and non-obstructive azoospermia (NOA), sperm often exists in a "very small, scattered, and morphologically atypical" manner in the suspension following microsurgical sperm retrieval or mechanical dispersion. Traditional procedures typically involve repeated scanning in wet slides / culture dishes to search for sperm, followed by micromanipulation to aspirate and transfer to cryopreservation media (such as straws, cryopreservation drops, or microdrop systems). This process presents the following challenges:

[0003] 1) Multiple transfers carry a high risk of losing single sperm or a small number of sperm;

[0004] 2) The scanning-recording-relocation link is broken, making re-location difficult;

[0005] 3) The cryogenic carrier and the microscopic scanning carrier are inconsistent, resulting in more operational steps and a higher risk of contamination;

[0006] 4) The search time is long, it relies heavily on the experience of embryologists, and its efficiency and consistency are insufficient.

[0007] Therefore, there is an urgent need for an integrated solution that can complete sample carrying, microscopic scanning evaluation, AI positioning and recording, sealing and freezing, and rewarming recovery within the same carrier. Summary of the Invention

[0008] In view of the aforementioned deficiencies in the prior art, the technical problem to be solved by the present invention is that existing methods for detecting, locating, losing, retrieving, and cryopreserving rare sperm are difficult. The present invention provides an integrated rare sperm assessment, location, and cryopreservation carrier and its usage method, achieving integrated "assessment—location—location counting—cryopreservation—thawing and recovery," reducing rare sperm transfer and loss, improving retrieval efficiency and traceability, and is suitable for rare sperm analysis in outpatient settings and for rare sperm retrieval via testicular microsurgery.

[0009] To achieve the above objectives, this invention provides an integrated cryopreservation carrier for rare sperm assessment and localization, comprising a carrier body, an observation scanning cavity, a coordinate positioning marker component, a dedicated interface, a sealing section, and a data recording component. The carrier body has a microfluidic structure, and the observation scanning cavity is located in the middle of the carrier body and is transparent. The coordinate positioning marker component is located on the periphery or inside the observation scanning cavity and is used to establish a carrier coordinate system. The dedicated interface includes a sample injection port and a sample retrieval port, and the dedicated interface is threadedly connected to the end of the carrier. The sealing section is located on one or both sides of the carrier body and is used to seal the stage confirming the presence of sperm.

[0010] The data recording component communicates with the AI ​​recognition system to record relevant data.

[0011] Furthermore, the carrier body is configured as a tubular, flat tubular, or laminated microfluidic structure.

[0012] Furthermore, the carrier body is made of a low-temperature resistant transparent polymer or composite film material, including one or more of COP / COC, PET composite film, and PTFE composite material.

[0013] Furthermore, the observation scanning cavity is configured to include a spacer structure to define the liquid layer thickness.

[0014] Furthermore, the coordinate positioning and marking component is disposed around and / or inside the observation scanning cavity, and includes one or more of two-dimensional grid lines, reference points, and identifiable micro-marker patterns; the identifiable micro-marker patterns include micro-marker arrays and / or alignment marks. The aforementioned two-dimensional grid lines, reference points, and identifiable micro-marker patterns together constitute the carrier coordinate reference, and are identified, matched, and registered by external positioning and recognition software after image acquisition, thereby establishing a mapping relationship between the image coordinate system and the carrier coordinate system.

[0015] Furthermore, the dedicated interface is a Luer connector, snap-fit ​​micro-connector, or threaded sealing connector, and is equipped with a sealing cap and / or a one-way valve structure.

[0016] Furthermore, the sealing section is configured to include a heat-sealing area, a clamping area, or a fusible sealing membrane, which is used to form a closed cavity after sperm is detected.

[0017] Furthermore, the data recorded by the data recording component includes at least two of the following: carrier number, field of view number, sperm coordinates, confidence level or grade marker, and scan timestamp.

[0018] In a preferred embodiment of the present invention, an integrated method for assessing, locating, and cryopreserving rare sperm is provided, comprising the following steps:

[0019] A) A suspension containing rare sperm is injected into the observation and scanning chamber through a dedicated interface;

[0020] B) Acquire multi-field images under a microscope / scanning platform and perform AI recognition to obtain the location of rare sperm pixels;

[0021] C) The pixel position is mapped to carrier coordinates (X, Y) based on the coordinate positioning and identification component and recorded;

[0022] D) Seal and preserve the sperm-containing segments;

[0023] E) Pre-cool the carrier in liquid nitrogen vapor phase and then transfer it to liquid nitrogen for cryopreservation.

[0024] In another preferred embodiment of the present invention, a method for rewarming and recovering rare sperm using the above-mentioned integrated rare sperm assessment and positioning cryopreservation carrier is provided, comprising the following steps:

[0025] a) Remove the carrier from liquid nitrogen and reheat it in a 37°C water bath or temperature-controlled platform;

[0026] b) Open the recovery port and connect the injection device to blow the suspension in the observation scanning chamber into a culture dish or ICSI operating dish;

[0027] c) Based on the recorded carrier coordinate information, perform a second rapid confirmation or directly carry out assisted reproductive procedures.

[0028] The integrated rare sperm assessment and localization cryopreservation carrier and its usage method of the present invention have the following technical effects:

[0029] 1) The same carrier completes the "evaluation-location-cryopreservation-rewarming recovery" process, significantly reducing the number of transfers and the loss of rare sperm;

[0030] 2) The coordinate system, combined with AI recognition, enables sperm location to be recorded, traceable, and retrievable;

[0031] 3) Limiting the liquid layer thickness improves scanning focusing speed and imaging consistency, and shortens retrieval time;

[0032] 4) The cryopreservation sections can be segmented and managed according to "sperm-containing / non-sperm-containing" to improve the utilization of cryopreserved resources;

[0033] 5) Interface-based injection and recycling reduce the risk of contamination and adapt to clinical embryo laboratory procedures.

[0034] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of a preferred embodiment of the sperm evaluation and AI identification and localization process of the present invention;

[0036] Figure 2 This is a schematic diagram of a preferred embodiment of the sperm cryopreservation process of the present invention;

[0037] Figure 3 This is a schematic diagram of a preferred embodiment of the sperm rewarming and recovery process of the present invention;

[0038] Figure 4 This is a schematic diagram of the main body of an integrated rare sperm assessment and positioning cryopreservation carrier according to a preferred embodiment of the present invention. Detailed Implementation

[0039] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0040] In the following description, specific details, such as particular internal procedures and techniques, are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will appreciate that the invention may be practiced in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods have been omitted so as not to obscure the description of the invention with unnecessary detail.

[0041] like Figure 1-4 As shown, this invention provides an integrated cryopreservation carrier for rare sperm assessment and localization, comprising a carrier body, an observation scanning cavity, a coordinate positioning marker component, a dedicated interface, a sealing section, and a data recording component. The carrier body has a microfluidic structure, and the observation scanning cavity is located in the middle of the carrier body and is a transparent structure. The coordinate positioning marker component is located on the periphery or inside of the observation scanning cavity and is used to establish the carrier coordinate system. The dedicated interface includes a sample injection port and a sample retrieval port, and the dedicated interface is connected to the end of the carrier via a thread. The sealing section is located on one or both sides of the carrier body and is used to seal the stage confirming the presence of sperm.

[0042] The data recording component communicates with the AI ​​recognition system to record relevant data.

[0043] The carrier body is configured as a tubular, flat tubular, or laminated microfluidic structure. The carrier body is made of a low-temperature resistant transparent polymer or composite membrane material, including one or more of COP / COC, PET composite film, and PTFE composite material.

[0044] The observation scanning cavity is configured with a spacer structure to limit the thickness of the liquid layer. This spacer structure is designed to limit the liquid layer thickness to 45-46 μm, allowing the suspension to form a stable thin layer in this area, facilitating microscopic imaging. The external dimensions of the observation scanning cavity are compatible with the scanning path of commonly used wet slide coverslips, preferably with a major axis of 9 cm, a minor axis of 0.9 cm, and a total volume of 28.6 μm. The two ends of the observation scanning cavity are sealing sections, approximately 2 cm in length and approximately 1.5 mm in inner diameter. In this embodiment, the observation scanning cavity height is limited by a shim, micropillar, height-limiting frame, or laminated spacer film, ensuring the liquid layer thickness is within the imaging range (45-46 μm, which can be selected based on the microscope depth of field and sample concentration), thereby shortening focusing time and improving AI recognition stability.

[0045] A coordinate positioning and identification component is disposed around and / or inside the observation scanning cavity, including one or more of two-dimensional grid lines, reference points, and identifiable micromarker patterns; wherein, the identifiable micromarker pattern is an array of micromarkers and / or alignment marks disposed on the carrier. The aforementioned two-dimensional grid lines, reference points, and identifiable micromarker patterns together form the carrier coordinate reference; after image acquisition, external positioning and identification software identifies, matches, and registers the two-dimensional grid lines, reference points, and / or identifiable micromarker patterns to establish a mapping relationship between the image coordinate system and the carrier coordinate system, and is used to subsequently convert the pixel coordinates of the sperm target into carrier coordinates.

[0046] The dedicated interface is a Luer connector, snap-fit ​​micro-connector, or threaded sealing connector, equipped with a sealing cap and / or a one-way valve structure. A sealing cap is also included.

[0047] The sealing section is configured with a heat-sealing area, a clamping area, or a fusible sealing membrane to form a sealed cavity after sperm is detected. Sealing areas located on either side or one side of the carrier body are equipped with heat-sealing areas / clamping areas / fusible sealing membranes for sealing segments where sperm is confirmed.

[0048] The data recording component records data including visual encoding and / or electronic storage units, and the data recorded by the data recording component includes at least two of the following: carrier number, field of view number, sperm coordinates, confidence level or grade marker, and scan timestamp.

[0049] In this embodiment of the invention, a protective sleeve or support is also included to protect the observation scanning cavity from compression and frost contamination during freezing, transportation and rewarming.

[0050] In a preferred embodiment of the present invention, an integrated method for assessing, locating, and cryopreserving rare sperm is provided, comprising the following steps:

[0051] A) Inject the suspension containing rare sperm into the observation scanning chamber through a special interface; specifically, take 15ul and drop it into the observation scanning chamber (on a 1.8cm*1.8cm film, stretch it into a strip of about 15-20ul), with a thickness of 45um.

[0052] B) Acquire multi-field images under a microscope / scanning platform and perform AI recognition to obtain the location of rare sperm pixels;

[0053] C) The pixel position is mapped to carrier coordinates (X, Y) based on the coordinate positioning and identification component and recorded;

[0054] D) Seal and preserve the sperm-containing segments;

[0055] E) Pre-cool the carrier in liquid nitrogen vapor phase and then transfer it to liquid nitrogen for cryopreservation.

[0056] Specifically, the following steps are included:

[0057] S1. A suspension containing rare sperm is injected into the observation scanning chamber via a dedicated interface, and the carrier carrying the rare sperm sample is then subjected to microscopic scanning. At least one identifiable reference feature (such as a boundary, scale line, or corner marker) is set on the carrier to establish its coordinate system. The system defines the lower left corner of the carrier as the origin O, the horizontal axis as the X-axis, and the vertical axis as the Y-axis. Before starting the identification process, the suspension containing rare sperm is loaded onto the carrier and then placed in the microscopic imaging module.

[0058] S2 utilizes a microscopic imaging module to perform field-by-field imaging scanning of the carrier and outputs sperm target detection results; multi-field images are acquired under a microscope / scanning platform and AI recognition is performed to obtain the location of sparse sperm pixels. Specifically, the microscopic imaging module performs serpentine or grid scanning with preset step distances Δx and Δy to form a sequence of fields of view. Each field of view record includes a field of view number. Platform position ,image ;

[0059] After scanning and imaging, each image is first processed using an AI recognition module. Preprocessing is performed, including: normalizing the brightness of the original image to reduce the impact of illumination differences under different fields of view on the recognition results; performing denoising processing on the image to suppress random noise in the microscopic imaging process; scaling or cropping the image according to the preset input size, and converting the image data into an input tensor that can be accepted by the target detection model.

[0060] After preprocessing, the processed image is input into a pre-trained target detection model. The target detection model is used to detect suspected sperm regions in the image and outputs a set of candidate targets.

[0061]

[0062] in, Let $r$ represent the center pixel coordinates of the $i$-th candidate bounding box in the $i$-th view. This represents the width and height of the candidate box in pixels. This indicates the confidence level that the corresponding candidate target is sperm.

[0063] Furthermore, for the candidate target set Perform threshold filtering and retain those that meet the criteria. The candidate targets, among which, A pre-set confidence threshold is used to filter out low-confidence false targets.

[0064] For candidate targets that have passed the threshold filtering, calculate the overlap (IoU) between any two candidate boxes, which is expressed as:

[0065]

[0066] in, and These represent two candidate bounding box regions. If the overlap between the two candidate bounding boxes exceeds a preset overlap threshold... If the candidate boxes have higher confidence, they will be retained, while those with lower confidence will be suppressed, in order to eliminate redundant boxes caused by repeated detection of the same sperm target.

[0067] After thresholding and overlap suppression, the sperm target set in the i-th field of view is obtained. The sperm target set is used for subsequent pixel coordinate extraction, carrier coordinate conversion, and scan map generation.

[0068] S3, based on the coordinate positioning and identification component, maps the pixel position to the carrier coordinates (X, Y) and records them, records the X / Y coordinates of the sperm on the carrier, and generates a scan map after structuring the X / Y coordinates.

[0069] Specifically, for the set of sperm targets in the i-th field of view obtained in step S2 The center pixel coordinates (u, v) of each sperm target are extracted. To convert the pixel coordinates to their physical location in the carrier coordinate system, the pixel-to-physical length conversion factor under the current imaging conditions is first determined based on the calibration results of the microscopic imaging module. and ,in This represents the physical length corresponding to a unit pixel in the X direction. This represents the physical length corresponding to a unit pixel in the Y direction; it also determines the coordinates of the current view center pixel. The physical displacement of the target relative to the current center of the field of view. for:

[0070]

[0071] Where, 𝑢 and 𝑣 represent the center pixel coordinates of the sperm target in the current field of view image, respectively. and These represent the pixel coordinates of the current center point of the field of view. and It was obtained in advance by microscope calibration.

[0072] Furthermore, considering the current position of the platform corresponding to the current field of view.

[0073]

[0074] By mapping the relative physical displacement to the carrier coordinate system, the absolute coordinates (X, Y) of the sperm target in the carrier coordinate system are obtained:

[0075]

[0076] in, and Let represent the positions of the stage in the carrier coordinate system during the acquisition of the i-th field of view. Thus, the unique spatial position of each sperm target on the carrier can be obtained.

[0077] The system records each sperm target as a structured data entry:

[0078]

[0079] in, This represents a temporary identifier for the target. X and Y represent the absolute coordinates of the target in the carrier coordinate system, p represents the target recognition confidence level, i represents the field of view number to which the target belongs, and t represents the timestamp when the target was identified. Multiple sperm targets correspond to form a set of target entries. .

[0080] Furthermore, the target item set The scan map is structured and generated. A carrier coordinate system is established based on the reference features on the carrier, and the carrier boundary range and coordinate orientation are determined. A two-dimensional scan area model corresponding to the carrier is constructed based on the actual size of the carrier, the starting position of the microscopic scan, the preset step distances Δx and Δy, and the scan sequence. The absolute coordinates (X, Y) of each sperm target are mapped to the corresponding position in the two-dimensional scan area model, and its corresponding map unit is determined. Map units are then associated with their corresponding temporary target identifiers. The confidence level p, field of view number i, and timestamp t are correlated to form a searchable structured coordinate record. Based on all the structured coordinate records, the target points of each sperm are marked in the carrier-level two-dimensional region model to generate a scanned carrier map that represents the spatial distribution relationship of sperm on the carrier.

[0081] The scanned carrier map supports retrieval, sorting, and display by confidence level, field of view number, and / or timestamp, and is used for rapid target location in subsequent carrier identification binding, cryopreservation tracking, and rewarming retrieval processes. An RFID tagging module binds coordinate data to carrier identity; the RFID tagging module assigns a unique ID (e.g., CID) to the carrier and establishes a carrier data package corresponding to the CID in a database or server.

[0082] S4. Seal and preserve the sperm-containing segments; Identify the sperm-containing segments according to the aforementioned method, then seal and preserve them in segments and label them.

[0083] S5. The carrier containing sperm segments was pre-cooled in liquid nitrogen vapor and then transferred to liquid nitrogen for cryopreservation.

[0084] In another preferred embodiment of the present invention, a method for rewarming and recovering rare sperm using the above-mentioned integrated rare sperm assessment and positioning cryopreservation carrier is provided, comprising the following steps:

[0085] a) Remove the carrier from liquid nitrogen and reheat it in a 37°C water bath or temperature-controlled platform;

[0086] b) Open the recovery interface and connect the injection device to blow the suspension in the observation scanning chamber into a culture dish or ICSI operating dish; wherein, the injection device is one of a micro-syringe, a gas injection device or a peristaltic pump.

[0087] c) Based on the recorded carrier coordinate information, perform a second rapid confirmation or directly carry out assisted reproductive procedures.

[0088] The carrier in this invention has similar specifications to commercially available carriers and can be recycled using existing equipment.

[0089] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An integrated cryopreservation medium for assessing and locating rare sperm, characterized in that, The device includes a carrier body, an observation scanning cavity, a coordinate positioning marker component, a dedicated interface, a sealing section, and a data recording component. The carrier body is a microfluidic structure. The observation scanning cavity is located in the middle of the carrier body and is a transparent structure. The coordinate positioning marker component is located on the periphery and / or inside the observation scanning cavity. The identifiable micro-marker pattern includes a micro-marker array and / or alignment marks, used to form a carrier coordinate reference that can be recognized by external positioning and recognition software to support the mapping from image coordinates to carrier coordinates. The dedicated interface includes a sample injection port and a sample retrieval port, which are threadedly connected to the end of the carrier. The sealing section is located on one or both sides of the carrier body and is used to seal the stage confirming the presence of sperm. The data recording component is connected to the AI ​​recognition system and is used to record relevant data.

2. The integrated rare sperm assessment and cryopreservation medium as described in claim 1, characterized in that, The carrier body is configured as a tubular, flat tubular, or laminated microfluidic structure.

3. The integrated rare sperm assessment and cryopreservation carrier as described in claim 2, characterized in that, The carrier body is made of a low-temperature resistant transparent polymer or composite film material, including one or more of COP / COC, PET composite film, and PTFE composite material.

4. The integrated cryopreservation medium for rare sperm assessment and localization as described in claim 1, characterized in that, The observation scanning cavity is configured to include a spacer structure to define the thickness of the liquid layer.

5. The integrated cryopreservation medium for rare sperm assessment and localization as described in claim 1, characterized in that, The coordinate positioning and marking component includes one or more of the following: two-dimensional grid lines, reference points, micro-marker arrays, and alignment marks, to establish a carrier coordinate system and support the mapping from image coordinates to carrier coordinates.

6. The integrated cryopreservation medium for rare sperm assessment and localization as described in claim 1, characterized in that, The dedicated interface is a Luer connector, snap-fit ​​micro connector, or threaded sealing connector, and is equipped with a sealing cap and / or a one-way valve structure.

7. The integrated cryopreservation medium for rare sperm assessment and localization as described in claim 1, characterized in that, The sealing section is configured to include a heat-sealing area, a clamping area, or a fusible sealing membrane, which is used to form a closed cavity after sperm is detected.

8. The integrated cryopreservation medium for rare sperm assessment and localization as described in claim 1, characterized in that, The data recorded by the data recording component includes at least two of the following: carrier number, field of view number, sperm coordinates, confidence level or grade marker, and scan timestamp.

9. A method for integrated assessment, localization, and cryopreservation of rare sperm, characterized in that, Includes the following steps: A) A suspension containing rare sperm is injected into the observation and scanning chamber through a dedicated interface; B) Acquire multi-field images under a microscope / scanning platform and perform AI recognition to obtain the location of rare sperm pixels; C) The pixel position is mapped to carrier coordinates (X, Y) based on the coordinate positioning and identification component and recorded; D) Seal and preserve the sperm-containing segments; E) Pre-cool the carrier in liquid nitrogen vapor phase and then transfer it to liquid nitrogen for cryopreservation.

10. A method for rewarming and recovering rare sperm using an integrated rare sperm assessment and positioning cryopreservation carrier as described in any one of claims 1-8, characterized in that, Includes the following steps: a) Remove the carrier from liquid nitrogen and reheat it in a 37°C water bath or temperature-controlled platform; b) Open the recovery port and connect the injection device to blow the suspension in the observation scanning chamber into a culture dish or ICSI operating dish; c) Based on the recorded carrier coordinate information, perform a second rapid confirmation or directly carry out assisted reproductive procedures.

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