Low-dosage high/low density gradient centrifugation method for washing human PESA sperms

Abstract

The invention discloses a low-dosage high / low density gradient centrifugation method for washing human PESA sperms. The method comprises the following steps: adding 0.5 mL of a culture medium with a concentration gradient of 80% into a conical pipe, then adding 0.5 mL of a culture medium with a concentration gradient of 40% on the liquid surface to form clear liquid layering; fully mixing an epididymis liquid sample, slowly adding 0.5 mL of epididymis liquid on the liquid surface of the culture medium with a density gradient of 40% to form clear liquid layering; carrying out centrifugation under 400*g for 15 minutes to precipitate high quality sperms in the bottom of the conical pipe; carefully absorbing the sperms out of the conical pipe, suspending the sperms on an HET culture medium with a volume of 5 mL, carrying out centrifugation under 300*g for 8 minutes to remove the residual density gradient culture medium; absorbing the sperms out, suspending the sperms on a sperm capacitation culture medium, carrying out centrifugation under 300*g for 5 minutes to remove the residual HTF culture medium; removing the supernate, fully mixing the capacitation liquid with a volume around 0.5 mL with the sperms, and finally storing the mixture in an incubator with a temperature of 37 DEG C and a CO2 concentration of 5% for later use.

Description

technical field [0001] The invention relates to a method for recovering high-quality sperm from human epididymis fluid, in particular to a method for washing human PESA sperm by low-dose high-low density gradient centrifugation. Background technique [0002] Intracytoplasmic sperm injection (ICSI) technology is currently the only effective method for the treatment of obstructive azoospermia [1], this technology allows countless patients with obstructive azoospermia to have their own offspring. Sperm obtained by human percutaneous epididymal sperm aspiration (PESA) is one of the main sources of ICSI sperm, but the sperm density, motility and forward movement rate obtained by PESA are very poor. The method is only suitable for washing semen with high density and activity rate ejaculated from the body. If this method is used to wash PESA sperm, high-quality sperm cannot be effectively recovered (the addition of traditional gradient centrifugal density medium is 2.5-3.0 ml), whi...

AI Technical Summary

Problems solved by technology

Sperm obtained by human percutaneous epididymal sperm aspiration (PESA) is one of the main sources of ICSI sperm, but the sperm density, motility and forward movement rate obtained by PESA are very poor. The method is only suitable for washing semen with high density and activity rate ejaculated from the body. If this method is used to wash PESA sperm, high-quality sperm cannot be effectively recovered (the addition of traditional gradient centrifugal density medium is 2.5-3.0 ml), which is currently widely used The current PESA sperm washing method is to directly centrifuge the epididymis fluid with HET washing medium. This method cannot effectively remove impurities such as red blood cells, white blood cells, and hydrocele in the epididymis fluid. These impurities often block and adhere to the puncture needle during ICSI operations. Even the puncture needle needs to be replaced during the operation, which increases the operation cost and operation time, prolongs the exposure time of eggs outside the incubator, increases the probability of egg ultrastructural damage, and affects the fertilization rate of eggs and the implantation potential of embryos[2 ].(References: [1] Dong Ruina, Guo Yihong, Sun Yingpu, etc. Analysis of clinical pregnancy outcomes in 3106 ICSI cycles of sperm from different sources. Reproduction and Contraception, 2013, 4 (33): 233-238 [2] Jin Lei, Zhu Guijin, Zhang Hanwang, et al. A retrospective study on the safety of PESA combined with ICSI in the treatment of offspring of azoospermia. Chinese Journal of Modern Clinical Medicine, 2006,4(11): 1025-1027)