Micro-sperm cryopreservation method

Abstract

The invention discloses a micro-sperm cryopreservation method and aims to provide a micro-sperm (including single sperm like testis puncture sperm, epididymis puncture sperm and sperms of patients suffering serious oligozoospermia and asthenozoospermia) cryopreservation carrier, which has the advantages of relatively small volume, closed space, easiness in operation and high sperm activity, and acryopreservation method. The cryopreservation method comprises the following steps: when the sperm thaws, taking out a cryovial from a liquid nitrogen tank; taking out the cryopreservation carrier, byusing a pipet, from the cryovial, and quickly adding the cryopreservation carrier into a sperm culture dish (37 DEG C); carefully searching for the sperm in the cryopreservation carrier under an inverted microscope (100-200-time magnification); grabbing the sperm with a single sperm injection needle in ovarian follicle, and transferring into a sperm culture medium of the sperm culture dish, for follow-up sperm immobilization and ICSI of ripe ovum.

Description

technical field [0001] The present invention relates to the field of assisted reproduction in medicine, in particular to cryopreservation techniques for trace sperm (single or dozens of sperm), including testicular puncture sperm, epididymal puncture sperm, and sperm from patients with severe oligospermia and asthenospermia. Background technique [0002] Infertility is a major disease that seriously endangers human reproductive health. Its incidence accounts for about 1 / 6 of couples of childbearing age in the world, and about 50% of them are caused by male factors. Sperm or azoospermia, wherein azoospermia accounts for about 15% of male infertility patients. There is no sperm in the semen of patients with azoospermia, including obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA patients are mainly caused by spermatogenesis or maturation disorders. Currently, there is no treatment to restore spermatogenesis in patients, accounting for about 60% of azoosp...

AI Technical Summary

Problems solved by technology

However, this method requires a special wet box under saturated humidity conditions to ensure that the droplets do not evaporate during the freezing process. The operation process is cumbersome and time-consuming, which greatly limits the wide application of this method in clinical practice.

In 2011, Lu Hui and Peng Qiuping developed a moisturizing device for freezing rare sperm. This moisturizing device provides constant humidity conditions. During the sperm transfer process, it can ensure that the humidity of the transfer environment remains unchanged and prevent sperm from being removed from the petri dish. In the process of transferring to a frozen carrier (such as a frozen sheet), the osmotic pressure in the liquid increases due to the evaporation of the droplets, causing sperm damage or even death, and reducing sperm activity. However, this device is only a moisturizing device and not a frozen carrier loaded with sperm. sperm cryopreservation

However, this method also has some problems. Firstly, when freezing and thawing, the liquid droplets are in direct contact with mineral oil and need to be cleaned and removed. If the removal is not clean, it may have an impact on sperm activity and ICSI treatment results. Secondly, the droplet volume is relatively large (0.1 -10.0μL), it takes a lot of time to find and grab the cryopreserved sperm when recovering the sperm by micromanipulation after thawing, thus limiting its clinical application

[0004] In summary, the current micro-sperm cryopreservation methods have certain defects, which limit their clinical application. Therefore, it is urgent to develop a sperm cryopreservation carrier with a smaller volume and a closed space.

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