Successful vitrification of human sperm  tried many times by  researchers. But resulted  only  with  partial success and with controversy.   This is  first time  – we performed  successful vitrification of human sperm in an optimum volume of 0.6 ml (600 micro liter). Which is enough & sufficient for IUI, IVF or any infertility management.

We used only three culture  media for whole process of successful vitrification of human sperm.

  1. Den -1 — 75%  density gradient  — for sperm separation.

  2. Vitri Media — developed & named by Frozen Cell.

  3. Enhance — sperm washing media.

ABSTRACT                                                                                                                                                      

We performed Successful vitrification of human sperm with an specially developed Vitri  Media.  This  compound (Vitri Media) does not have any macro-molecule like Human Serum Albumin. Glycerol is in concentration of 6% only V/V.  And sucrose concentration has been fixed to 20 milli  Mol only. Other chemicals are within limit, therefore hardly affect the osmolarilty.

Presently our study is limited  only with person with good quality sperm(normozoospermic sample) .  Even the result in term of progressive motility and maintenance of motility is certainly better than conventional cryo-preservation.

Previous trials to vitrify human sperm failed .  Main reason of failure seems  —-

  • Inadequate quantity (2 to 20 micro liter) of sperm preservation

  • Possibility of cross contamination because of  preservation on open copper loop  in liquid nitrogen.

  • Use of inadequate cryo-preservative.

  • Inadequate trial,  etc.

Lastly to mention, this technique, we developed, does not require any costly equipment like programmable freezer. Even this technique do not require any technically expert person.

MATERIAL AND METHOD

Chemicals and Reagents  —  Chemicals / salts  purchased from either  Sisco Research Lab. Pvt. Ltd (SRL), India or  Himedia laboratories, India. All culture media / reagents prepared in-house only.  We didn’t use any ready to use or prepared culture medium or compound.

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 with laminar flow, simple incubator, microscope, centrifuge machine,  boiling water apparatus, liquid nitrogen storage and a simple timer, etc are  only requirement.  Even the same equipment is enough for  thawing and  washing   of  vitrified sperm.

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 performed semen collection in morning only.

  • Just after  semen collection – added 2 ml of  Enhance.

  • Kept in Incubator – for 40 minutes – to get complete liquefaction – mixed well.

  • Semen sample + Enhance mixture  slowly layered over Den – 1 (single layer density gradient) in a conical bottom centrifuge test tube.

  • Centrifuged for 15 minutes at 350 G.

  • Supernatant discarded.

  • Added required quantity of Vitri Media.

  • 0.6 ml of content filled in double walled cryovials

  • Kept at room temp for 15 minutes.

  • Cryovial directly plunged in  liquid nitrogen.

Cryovial
Vial to keep semen sample in liquid nitrogen

Thawing of vitrified sperm

  • Water was boiled in an apparatus.

  • Cryovial taken out from liquid nitrogen.

  • Kept at room temperature  for 20 second and waved in air.

  • Dipped in Boiling water for 50 second.

  • Kept in Incubator at  34 Degree C for 40 minutes to liquefy completely. And also to  regain the motility of sperm.

  • Watch the motility under microscope.

Maintenance of motility in Vitri Media itself

  • Post thaw  — Very good motility 

  • After eight  hour of thawing   – 25% of post thaw motility.

  • After eighteen hour of thawing  — All sperm  immotile.

Washing of vitrified  sample by Swim Up method

 

Maintenance of motility after vitrification

  • Keep the content in tightly closed centrifuged test tube at room temperature (approx 30 degree centigrade).

  • After 24 hour warm the tube in incubator at 34 degree C.

  • Mix the content.

  • Watch the motility under microscope.

  • We noted 10% to 30% motility after 24 hour of thawing and detectable motility after 48 hour.

Note

  • Recovery and motility varies according to post-collection semen parameter.

  • Addition of Hyaluronic acid does not improve the outcome in term post thaw motility.

  • The prepared Vitri media does not have any toxic cryoprotectant like dimethyl sulfoxide (DMSO).

  • Vitri Media does not have Human serum albumin.

  • Addition of Human serum albumin may improve the result

  • Presently we have not used ice blocker. Although we are in plan to use it in next step.

Limitations

We are working with constrained resource, especially in term of expensive equipment.  We don’t have osmometer, in this manner not ready to say about osmolarity of our recently developed “Vitri Media”. Furthermore we need ample of time to obtain some basic chemicals. Which makes our investigation delay.

Conclusion

After this comprehensive study we can say that human sperm can be vitrified successfully.  Our aim is to achieve  greatest number of pregnancy with this technique.   Ultimate goal of our organization “FROZEN CELL” is to make human sperm preservation effective, simple and productive as well. Vitrification of human sperm is a yardstick in the field of ART. I understand that lot of research and study  is required to continue ahead.  But successful start is constantly significant.

Conflict of Interest

 The topic “Successful vitrification of human sperm” did not have any conflict at present. But we welcome conflict & critics on this study.  Rather we invite all researchers who are working in same field to come forward.  And  perform it and evaluate as well.  Always we are  open for live demo of this method.

Acknowledgement

We are thankful to Contipro a. s,  561 02 DolniDobroc 401, Czech Republic. They provided us Sodium Hyaluronate  – pharmaceutical for  experiment purpose.

Funding 

This method & vitri media for “Successful vitrification of human sperm ” has been exclusively developed  by Frozen Cell.  No other organization or any system is not at all involved.  We did not take any  financially  help in any way  for  this study.

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