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.

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

  2. VITRI MEDIA — developed & named by Frozen Cell.

  3. 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  ———

  • Vitrification in inadequate quantity (10 to 20 micro liter).

  • Possibility of cross contamination because vitrification performed on open copper loop.

  • Use of very high concentration of compound (mainly sucrose).

  • 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.

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

  • Kept in Incubator for 40 minutes  to liquefy completely  and mixed well.

  • Semen  & Enhance mixture  slowly layered over Den – 1  in a conical bottom centrifuge test tube.

  • Centrifuged for 15 minutes at 380 G.

  • Supernatant discarded.

  • Added required quantity of Vitri Media.

  • 0.6 ml of content filled in 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

  • Cryovial taken out from liquid nitrogen.

  • Waved in air for 30 seconds.

  • Dipped in Boiling water for 50 seconds.

  • Kept in Incubator at  34 Degree C for 40 minutes to liquefy completely.

  • Mixed the content of cryovial gently with pasture pipette.

  • Watch the motility under microscope.

Maintenance of motility in Vitri Media.

  • Post thaw  — 25 to 50% motility.

  • After four hours  — Like post thaw motility 

  • Eight  hours after thawing   – 50% of post thaw motility.

  • 24 hours after thawing  —  Approx 10-20% motility noted in comparison to immediate post thaw motility.

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 quality of  semen.

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

Conclusion

After this comprehensive study we can say that human sperm can be vitrified successfully.   Ultimate goal of our organization “FROZEN CELL” is to make human sperm preservation effective, simple and productive. 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 this field to come forward & comment.  Always we are  open for live demo.

Acknowledgement

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

Funding 

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

14 thoughts on “Successful Vitrification of Human Sperm

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