CN104403993A - Low-dosage high/low density gradient centrifugation method for washing human PESA sperms - Google Patents

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

The method of low dosage high/low density gradient centrifugation washing mankind PESA sperm

Technical field

The present invention relates to a kind of method reclaiming high quality sperm from people's epididymal fluid, be specifically related to the method for low dosage high/low density gradient centrifugation washing mankind PESA sperm.

Background technology

ICSI (ICSI) technology is unique effective ways [1] for the treatment of Obstructive azoospermia at present, and this technology makes countless Obstructive azoospermia patient have the offspring of oneself.The sperm obtained by mankind's Percutaneous epididymal sperm aspiration (PESA) is one of main source of ICSI sperm, but through the sperm concentration that PESA obtains, motility rate and propulsion rate very poor, traditional high/low density gradient centre of force method is only suitable for washing external injection, density and the higher seminal fluid of motility rate, if with this method washing PESA sperm, can not efficient recovery high quality sperm (traditional gradient centrifugation density culture medium add-on is 2.5-3.0 ml), the washing PESA sperm method generally used at present directly washs the centrifugal epididymal fluid of substratum with HET, this method effectively can not remove the red corpuscle in epididymal fluid, the impurity such as white corpuscle and the hydrocele of tunica vaginalis, when carrying out ICSI operation, these impurity often block and adhesion puncture needle, need even in operation to change puncture needle, add running cost and operating time, ovum was extended in incubator outer open-assembly time, the probability of ovum ultrastructure damage increases, the rate of fertilization of ovum of impact and the plantation potential [2] of embryo. (reference: [1] Dong Ruina, Guo Yihong, Sun Yingpu etc., 3106 different sources sperm ICSI cycle Clinical Outcomes analyze. reproduction and contraception, 2013, 4(33): 233-238 [2] Jin Lei, Zhu Guijin, Zhang Hanwang, Deng. PESA treats the retrospective study of its offspring's security of azoospermia in conjunction with ICSI. Chinese current clinical medicine magazine, 2006, 4 (11): 1025-1027).

Summary of the invention

The object of the invention is to overcome deficiency of the prior art, thus provide a kind of low dosage high/low density gradient centrifugation to wash the method for mankind PESA sperm.Principle is few by: low dosage density gradient centrifugation is added gradient media amount, for the 30%-40% of traditional method, liquid level is lower, motile sperm moves along the direction of precipitation gradient more on one's own initiative than unvital sperm when centrifugal, thus pass the feature at different densities interface, the sperm of more activity can be reclaimed, the rate of recovery is better than the HET washing substratum centrifuging generally applied at present, the present invention adopts low dosage high/low density gradient centrifugation to decrease the consumption of density gradient media, make effectively removing in epididymal fluid the motile sperm collected to greatest extent while impurity in epididymal fluid.

Solve the problems of the technologies described above adopted technical scheme by following step and condition:

Step one: add in tapered tube by 80% density gradient media 0.5ml, is then carefully added on the page by 40% density gradient media 0.5ml, makes it liquid level layering clear;

Step 2: fully mixing epididymal fluid sample, is added in 40% density gradient media slowly by 0.5ml epididymal fluid, makes it liquid level layering clear;

Step 3: centrifugal 400 × g15min, high-quality sperm will at tapered tube bottom settlings;

Step 4: gentle aspiration goes out precipitation, and be suspended in 5mlHET substratum, centrifugal 300 × g8min is to remove residual density gradient media;

Step 5: sucking-off precipitates, and be suspended in 3.0ml capacitation substratum, centrifugal 300 × g5min is to remove residual HTF substratum;

Step 6: remove supernatant liquor slowly, the capacitation liquid leaving and taking about 0.5ml fully mixes with precipitation, is placed in 37 DEG C, the substitute of 5%CO2 incubator.

The beneficial effect of employing technique scheme is:

Low dosage high/low density gradient centrifugation provided by the invention, a kind of simple to operate, practical liquefacient duration method, be applicable to washing mankind Percutaneous epididymal sperm aspiration (PESA, also known as doing epididymis paracentesis) sperm that extracts, effectively can remove the red corpuscle in epididymal fluid, white corpuscle, the impurity such as the hydrocele of tunica vaginalis and bacterium, be conducive to sperm microcytotoxicity, effectively prevent the generation of impurity blocking and adhesion puncture needle situation in injection (ICSI) operation in monosperm ooecium slurry, reduce the ovum manipulation in vitro time, reduce temperature to greatest extent, the change of humidity and CO2 concentration is to ovum spindle body, the Ultrastructural damage such as microfilament, thus ovum fertilization potential and fetal development potential can be improved, to improve ovum fertilization rate and Embryo Implantation Rate in.

Embodiment

The method of low dosage high/low density gradient centrifugation washing mankind PESA sperm, embodiment repeat under, it is made up of following step:

Step one: high/low density gradient centrifugation substratum: produced by Coopersurgical company of the U.S., article No.: ART-2024.80% density gradient media 0.5ml is added in tapered tube, then careful 40% density gradient media 0.5ml is added on the page, make it liquid level layering clear;

Step 2: fully mixing epididymal fluid sample, is added in 40% density gradient media slowly by 0.5ml epididymal fluid, makes it liquid level layering clear, as epididymal fluid volume is comparatively large, can use multiple tapered tube simultaneously;

Step 3: centrifugal 400 × g15min, high-quality sperm will at tapered tube bottom settlings;

Step 4: HTF Sperm washing substratum is produced by Coopersurgical company of the U.S., article No.: ART-1006.Gentle aspiration goes out precipitation, and be suspended in 5mlHET substratum, centrifugal 300 × g8min is to remove residual density gradient media;

Step 5: capacitation washing substratum is produced by Coopersurgical company of the U.S., article No.: ART-1020.Sucking-off precipitates, and be suspended in 3.0ml capacitation substratum, centrifugal 300 × g5min is to remove residual HTF substratum;

Step 6: remove supernatant liquor slowly, the capacitation liquid leaving and taking about 0.5ml fully mixes with precipitation, is placed in 37 DEG C, the substitute of 5%CO2 incubator.

Claims (1)

1. the method for low dosage high/low density gradient centrifugation washing mankind PESA sperm, is characterized in that: it is made up of following step:

Step one: add in tapered tube by 80% density gradient media 0.5ml, is then carefully added on the page by 40% density gradient media 0.5ml, makes it liquid level layering clear;

Step 2: fully mixing epididymal fluid sample, is added in 40% density gradient media slowly by 0.5ml epididymal fluid, makes it liquid level layering clear;

Step 3: centrifugal 400 × g15min, high-quality sperm will at tapered tube bottom settlings;

Step 4: gentle aspiration goes out precipitation, and be suspended in 5mlHET substratum, centrifugal 300 × g8min is to remove residual density gradient media;

Step 5: sucking-off precipitates, and be suspended in 3.0ml capacitation substratum, centrifugal 300 × g5min is to remove residual HTF substratum;

Step 6: remove supernatant liquor slowly, the capacitation liquid leaving and taking about 0.5ml fully mixes with precipitation, is placed in 37 DEG C, the substitute of 5%CO2 incubator.