WO2021121077A1 - 一种在人工授精时筛选精子的方法 - Google Patents

一种在人工授精时筛选精子的方法 Download PDF

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WO2021121077A1
WO2021121077A1 PCT/CN2020/134481 CN2020134481W WO2021121077A1 WO 2021121077 A1 WO2021121077 A1 WO 2021121077A1 CN 2020134481 W CN2020134481 W CN 2020134481W WO 2021121077 A1 WO2021121077 A1 WO 2021121077A1
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transparent material
sperm
artificial insemination
microscope
arranging
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于毅欣
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于毅欣
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present disclosure relates to the field of human assisted reproduction technology, and in particular, to a method for screening sperm during artificial insemination.
  • Test-tube baby is currently one of the main methods for the treatment of infertility.
  • Female patients with ovulation disorders or male patients with oligospermia can both give birth through test-tube baby technology to solve the infertility caused by the disease Infertility is troubled, but the test tube baby technology is limited in the screening of sperm.
  • This method can at least increase the screening of sperm to a certain extent, and can at least avoid the phenomenon of "polyspermy" (polyspermy).
  • a feature of the present invention is to change the difficulty of insemination by extending the distance between the egg and the sperm and adjusting the viscosity of the culture solution.
  • a method for screening sperm during artificial insemination which is characterized in that it comprises:
  • Step 1 Place the culture solution on the transparent material.
  • Step 2 Arrange the eggs at the designated position and place the transparent material under the microscope.
  • Step 3 Arrange the sperm on the other end of the transparent material, and observe that the artificial insemination is satisfied.
  • the transparent material is a supporting material of sufficient length that can be placed under a microscope for observation, such as a glass sheet or a transparent sheet (or a transparent pick) of other materials.
  • the area used for arranging the culture solution in the transparent material can be a straight line or a curve, a U-shaped, L-shaped, and other more complicated shapes.
  • the culture medium is a liquid used to connect sperm and eggs.
  • the main components of the culture solution may be water, fructose, and propylene glycol, and an appropriate amount of lysozyme and lactic acid may be added to be closer to the vaginal fluid components.
  • the proportion of the main components of the culture solution by weight fructose (about 2%), propylene glycol (about 0.8%), and the rest is mainly water.
  • the proportion can be modified according to actual needs.
  • the content of propylene glycol can be modified according to requirements to modify the viscosity of the culture solution.
  • the main components of the culture solution can be water, fructose, propylene glycol (or similar substances), and the pH value of the culture solution can be between 3.5 and 6.5 by modifying the ratio of lactic acid. And can add lysozyme in a small amount according to sperm quality or demand.
  • the ratio of the main components of the culture solution by weight fructose (about 2%), propylene glycol (about 0.8%), and the rest is mainly water, and this ratio can be modified according to actual needs.
  • the egg is usually arranged at one end of the transparent material, and then the transparent material is placed under a microscope to observe the egg.
  • the objective lens and the eyepiece are adjusted to a proper position, and it should be considered here that the objective lens should avoid touching the sample.
  • a sufficient amount of sperm is arranged at the other end of the transparent material connected by the culture medium.
  • the microscope is observed to determine that the artificial insemination is basically completed.
  • an optional configuration method is to select a 20x objective lens, combined with a 30x eyepiece (or a camera with a similar magnification and a display), adjust the objective lens and When the eyepiece magnification is combined, you can use a microscope ruler to check whether the 0.01mm scale can be observed as expected.
  • a scale may be added at the edge of the sperm placement area to indicate the distance from the egg placement area.
  • a computer can be used to obtain the image frames of the camera connected to the microscope to monitor the image. If the image change of a continuous image frame meets greater than a certain threshold, a sound or ⁇ And flashing the screen to reduce the number of initial observations by personnel.
  • the image change of the continuous picture frame refers to the pixel change of the continuous image frame, or the change ratio of the pixel-by-pixel comparison of two image frames separated by several frames satisfies a value greater than a certain value.
  • Fig. 1 schematically shows a schematic diagram of a layout placed on the transparent material in the present invention, a top view.
  • Fig. 2 schematically shows a schematic diagram of a layout placed on the transparent material in the present invention, a top view.
  • Step 1 Add culture solution to the transparent sheet, the addition method can refer to FIG. 1.
  • Step 2 Add eggs and place them under the microscope.
  • Step 3 Adjust the microscope to meet the insemination situation of the human eye to observe the egg.
  • an optional configuration method is to choose a 20x objective lens combined with a 30x eyepiece (or a camera with a similar magnification and a display).
  • it can also be based on It is necessary to adjust the objective lens and eyepiece magnification with imaging.
  • you can use a microscope ruler to check whether the scale of 0.01mm can be observed as expected.
  • Step 4 Continue to observe (or observe multiple times) until the insemination is found to be completed.
  • Step 1 Add culture solution to the transparent sheet, the addition method can refer to FIG. 1.
  • Step 2 Add eggs, place them under the microscope, and use the camera instead of the eyepiece to transmit the image frame information to the computer.
  • Step 3 Adjust the focal length of the microscope and the objective lens used to meet the insemination condition of the observation egg in the image obtained by the computer.
  • an optional configuration method is to select a 20x objective lens combined with a 30x camera (camera or microscope camera)
  • Step 4 Initially, you can wait for the computer to send out a prompt stating that the sperm appears around the egg.
  • Step 5 Continue to observe (or observe multiple times) until the insemination is found to be completed.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Reproductive Health (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Gynecology & Obstetrics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种在人工授精时筛选精子的方法，其特征在于，包括：步骤一：向透明材质上布置培养液。步骤二：在指定位置布置卵子，并将透明材质布置到显微镜下。步骤三：在透明材质的另一端布置精子，观察满足人工授精后完成。所述方法能够至少一定程度上增加对精子的筛选，并且能够至少一定程度上避免"多精入卵"(polyspermy)的现象。所述方法的一个特征是通过延长卵子和精子之间的距离和调整培养液的粘稠程度来改变授精难度。本方法也可以用于代孕时替代其他人工授精方案。

Description

一种在人工授精时筛选精子的方法 技术领域
本公开涉及人类辅助生殖技术领域，具体而言，涉及一种在人工授精时筛选精子的方法。
背景技术
试管婴儿是目前治疗不孕不育的主要方法之一，患有排卵障碍的女性患者或患有少精弱精的男性患者，都能够通过试管婴儿技术进行生育，从而解决疾病带来的不孕不育困扰，但试管婴儿技术对精子的筛选有限，本方法能够至少一定程度上增加对精子的筛选，并且能够至少一定程度上避免“多精入卵”(polyspermy)的现象。本发明的一个特征是通过延长卵子和精子之间的距离和调整培养液的粘稠程度来改变授精难度。
发明内容
根据本公开的一个方面，提供一种在人工授精时筛选精子的方法，其特征在于，包括：
步骤一：向透明材质上布置培养液。
步骤二：在指定位置布置卵子，并将透明材质布置到显微镜下。
步骤三：在透明材质的另一端布置精子，观察满足人工授精后完成。
优选地，所述透明材质是拥有足够长度的，能够放置在显微镜下观察的承托物质，例如玻璃片或其他材质的透明薄片(或者说透明拨片)。
优选地，所述透明材质中用来布置培养液的区域可以是直线也可以是曲线，U型，L型等较复杂形状。
优选地，所述培养液是用来连接精子和卵子的液体。
优选地，作为一种可选的实施方式，培养液的主要成分可以是水，果糖，丙二醇，可以增加适量溶菌酶和乳酸来更接近阴道液成分。
进一步地，培养液的主要成分的按重量配比情况：果糖(约2％左右)、丙二醇(约0.8％左右)，其余主要是水，可根据实际需要修改配比。
优选地，可以根据需求来修改丙二醇的含量来修改培养液的粘稠程度。
优选地，作为一种可选的实施方式，培养液的主要成分可以是水、果糖、丙二醇(或类似物质)，可以通过修改乳酸的比率来使培养液的ph值在3.5到6.5 之间，并可根据精子质量或需求少量添加溶菌酶。
进一步地，培养液的主要成分的按重量配比情况：果糖(约2％左右)、丙二醇(约0.8％左右)，其余主要是水，并可根据实际需求修改此配比。
优选地，通常是在所述透明材质的一端布置卵子，之后将所述透明材质放置在显微镜下观察卵子。
优选地，调整物镜和目镜到合适的位置，此处要考虑到物镜应避免碰到样本。
优选地，在通过培养液连接的所述透明材质的另一端布置足够量的精子。
优选地，观察显微镜来判定基本完成了人工授精。
可选地，调整显微镜来满足人眼观察卵子的授精情况，例如一种可选的配置方法是选择20倍的物镜，结合30倍的目镜(或者类似放大倍数的摄像头连显示器)，调整物镜和目镜倍数组合的时候，可以使用显微镜尺来查看0.01mm的刻度是否能够按照预期的情况观察到。
可选地，为了较明确的标识与卵子布置点的距离，可以在精子布置区域的边缘处增加刻度来标明与卵子布置区的距离。
可选地，作为一种可选的实施方式，可以使用计算机来获取显微镜相连的摄像机的图像帧以监测画面，如果某个连续画面帧的图像变化满足大于某一阈值的时候，提示声音或\和闪烁屏幕，来减少人员对初期的观察次数。
进一步地，所述连续画面帧的图像变化是指连续的图像帧的像素变化，或者间隔几帧的两个图像帧信息的逐像素比较的变化比率满足大于某一值。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1示意性示出一种本发明中所述透明材质上放置的布局示意图，顶视图。
图2示意性示出一种本发明中所述透明材质上放置的布局示意图，顶视图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
步骤1：向所述透明薄片添加培养液，添加方式可以参照图1。
步骤2：添加卵子，放置在显微镜下。
步骤3：调整显微镜来满足人眼观察卵子的授精情况，例如一种可选的配置方法是选择20倍的物镜，结合30倍的目镜(或者类似放大倍数的摄像头连显示器)，当然也可以根据需要和成像调整物镜和目镜倍数，调整物镜和目镜倍数组合的时候，可以使用显微镜尺来查看0.01mm的刻度是否能够按照预期的情况观察到刻度。
步骤4：持续观察(或多次观察)直到发现授精完成。
实施例2
步骤1：向所述透明薄片添加培养液，添加方式可以参照图1。
步骤2：添加卵子，放置在显微镜下，使用摄像机替代目镜将图像帧信息传送到电脑中。
步骤3：调整显微镜焦距和使用的物镜来满足电脑获取的图像中满足观察卵子的授精情况，例如一种可选的配置方法是选择20倍的物镜，结合30倍的摄像机(摄像头或显微镜摄像头)，当然也可以根据需要和成像调整物镜的倍数，调整物镜倍数时，可以使用显微镜尺来查看0.01mm的刻度是否能够按照预期的情况观察到刻度。
步骤4：初期可以等待电脑发出提示说明精子出现在卵子周围了。
步骤5：持续观察(或多次观察)直到发现授精完成。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (4)

  1. 一种在人工授精时筛选精子的方法，其特征在于，包括：
    步骤一：向透明材质上布置培养液；
    步骤二：在指定位置布置卵子，并将透明材质布置到显微镜下；
    步骤三：在透明材质的另一端布置精子，观察满足人工授精后完成。
  2. 根据权利要求1所述的“步骤一”，其特征在于，向透明材质上布置培养液，包括：
    所述透明材质是拥有足够长度的，能够放置在显微镜下观察的物质；
    所述培养液是用来连接精子和卵子的液体；
    作为一种可选的实施方式，培养液的主要成分可以是水，果糖，丙二醇，可以增加适量溶菌酶和乳酸来更接近阴道液成分。
  3. 根据权利要求1所述的“步骤二”，其特征在于，在指定位置布置卵子，并将透明材质布置到显微镜下，包括：
    通常是在所述透明材质的一端布置卵子，之后将所述透明材质放置在显微镜下观察卵子；
    调整物镜和目镜到合适的位置，此处要考虑到物镜应避免碰到样本。
  4. 根据权利要求1所述的“步骤三”，其特征在于，在透明材质的另一端布置精子，观察满足人工授精后完成，包括：
    在通过培养液连接的另一端布置足够量的精子；
    观察来判定基本完成了人工授精。
PCT/CN2020/134481 2019-12-18 2020-12-08 一种在人工授精时筛选精子的方法 WO2021121077A1 (zh)

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