Successful vitrification of human sperm tried many times by researchers. But resulted only with partial success and with controversy or failure. This is first-ever successful vitrification of human sperm performed. Moreover quantity also remained in an optimum volume of 0.6 ml (600 micro liter). Which is appropriate for IUI, IVF or any infertility management.
We used only three culture media for whole process. All three medium and disposable prepared & developed by Frozen Cell itself.
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Den -1 (75% density gradient) — for sperm separation.
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VITRI MEDIA — developed & named by Frozen Cell.
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Enhance — sperm washing media.
VITRI MEDIA (Vitrification Medium)
The newly developed VITRI MEDIA does not have any macro-molecule like human serum albumin. Molecular weight of all chemicals are within limit (less than 200 millimoles) + Glycerol 6% (V/V). Out of 200 millimoles sucrose contribute 20 millimols only. Therefore osmotic pressure of VITRI MEDIA is not high enough to harm sperm.
We also tested VITRI MEDIA as sperm preparation medium & sperm culture medium. We noted, sperm can be separated effectively using VITRI MEDIA either by swim up or swim down method. Following washing we used VITRI MEDIA as nutritional media. We got an acceptable survival of sperm in VITRI MEDIA even after 72 hour. It simply confirm that VITRI MEDIA provide optimum environment to sperms. Additionally I mention here that VITRI MEDIA does not contain human serum albumin or any substance derived from human, animal, bird, aquatic or plant origin. Rather it is 100% chemically defined.
Presently our study is limited only with good quality sperm. We noted better outcome in term of progressive motility and maintenance of motility. We compared the result with control sample.
Many researchers tried to vitrify human / animal sperm by different method. But resulted in either partial success or complete failure.
Main reason behind seems ———
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Vitrification in inadequate quantity (10 to 20 micro liter).
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Possibility of cross contamination because vitrification performed on open copper loop.
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Use of very high concentration of compound (mainly sucrose).
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Inadequate trial, etc.
MATERIAL AND METHOD
Chemicals and Reagents — Chemicals / reagents purchased from either Sisco Research Lab. Pvt. Ltd (SRL), India or Himedia laboratories, India. All culture media / reagents prepared in-house only.
As mentioned earlier, culture medium/ compound used for successful vitrification of human sperm was — Vitri Media , Den – 1 (75% density gradient) and Enhance (sperm washing media). A basic laboratory is only requirement for sperm vitrification / thawing. The required equipment are laminar flow, simple incubator, microscope, centrifuge machine, apparatus to boil water, liquid nitrogen and a simple timer, etc.
Vitrification Process
We adopted very simple process for successful vitrification of human sperm.
All media and plastic disposable kept in incubator at 32 – 34 degree C. approx one hour before starting the process. Donor was asked to keep abstinence for 3 to 5 days.. We perform semen collection in morning only.
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Just after semen collection – added 2 ml of Enhance.
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Kept in Incubator for 40 minutes to liquefy completely and mixed well.
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Semen & Enhance mixture slowly layered over Den – 1 in a conical bottom centrifuge test tube.
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Centrifuged for 15 minutes at 380 G.
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Supernatant discarded.
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Added required quantity of Vitri Media.
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0.6 ml of content filled in cryovials.
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Kept at room temp for 15 minutes.
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Cryovial directly plunged in liquid nitrogen.
Thawing of vitrified sperm
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Cryovial taken out from liquid nitrogen.
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Waved in air for 30 seconds.
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Dipped in Boiling water for 50 seconds.
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Kept in Incubator at 34 Degree C for 40 minutes to liquefy completely.
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Mixed the content of cryovial gently with pasture pipette.
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Watch the motility under microscope.
Maintenance of motility in Vitri Media.
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Post thaw — 25 to 50% motility.
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After four hours — Like post thaw motility
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Eight hours after thawing – 50% of post thaw motility.
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24 hours after thawing — Approx 10-20% motility noted in comparison to immediate post thaw motility.
Washing of vitrified sample by Swim Up method
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Taken whole content of cryovial ( liquefied) in round bottom centrifuge test tube.
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Added 1.5 ml of Enhance (washing media) — mixed well
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Centrifuged at 350 G for 15 minutes
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Supernatant discarded.
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Very slowly added 2 ml of Enhance over pellet.
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Kept round bottom test tube inclined at 30 degree in Incubator for 50 minutes
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Supernatant taken in another conical bottom centrifuge test tube.
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Centrifuged for 5 minutes at 350 G.
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Supernatant discarded.
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0.5 ml of Enhance (washing media) added with pellet – mixed well
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Very good progressive motility motility noted. Please watch video.
https://youtu.be/7DY_M0GqxqY
Maintenance of motility after vitrification
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Keep the content in tightly closed centrifuged test tube at room temperature (approx 30 degree centigrade).
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After 24 hour warm the tube in incubator at 34 degree C.
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Mix the content.
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Watch the motility under microscope.
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We noted 10% to 30% motility after 24 hour of thawing and detectable motility after 48 hour.
Note
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Recovery and motility varies according to quality of semen.
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Addition of Hyaluronic acid does not improve the outcome in term post thaw motility.
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The prepared Vitri media does not have any toxic cryoprotectant like dimethyl sulfoxide (DMSO).
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Vitri Media does not have Human serum albumin.
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Addition of Human serum albumin may improve the result
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Presently we have not used ice blocker. Although we are in plan to use it in next step.
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