Vitrification of Human Semen

Human Semen Vitrification.

Human Semen Vitrification, we first worked to vitrify semen (without washing) in November 2019. No doubt its was continuation of our previous work on sperm vitrification.  As usual we had obstacles in beginning. Gradually we adjusted the concentration of components. As well we continuously reviewed  whole protocol.  Anyway now we are getting an acceptable and almost consistent result of Human Semen Vitrification.

Our vitrification history –     Our journey of new concept of sperm freezing started in 2018, perhaps June 2018.  I commenced my work with the apprehension that biological product can  transmit disease. Therefore why not to preserve sperm with 100 % chemically defined ingredients. 

During initial few months I explored, discussed and got the references only.  In the mean time also I discussed, convinced and selected semen donors. My initial work was to freeze sperm (washed sperm) without H.S.A. or any biological product.  I worked continuously with different composition and got some success with time. I became able to preserved sperm without use of any biological  product even H.S.A.. However I observed that post thaw recovery of sperm was not good like freezing with H.S.A.. Secondly I failed to preserve sperm of poor quality. 

One day unexpectedly and coincidentally  I got a lead to vitrify washed sperm somewhere in the month of  Jan. 2019 .  Initially the result was not consistent. After a continuous trial and frequent adjustment  I successfully vitrify sperm (washed sperm) in July 2019. Off course result was not better than conventional freezing  but justifiable.

From November 2019 I started to work on semen  vitrification.

Why I preferred to vitrify whole semen ?

  1. In most part of the world whole semen is preserved. Semen is washed after thawing and used. Only in few places people use ready to use post thaw sperm.

  2. Whole semen has antioxidants, which protect the sperm during vitrification-thawing process.

  3. Whole semen has adequate nutrient and other protective elements for sperm.

  4. After vitrification, thawed sperms can be separated by any method.

Everyone is aware that semen freezing by conventional slow freezing is an established technique.  This technique invented in 1960s. Thereafter lot of improvement and analysis remained in progress.  Now it is well accepted that conventional slow freezing of human semen  have many issue: like  ——–

  1. Low pregnancy rate.

  2. Poor post thaw recovery.

  3. Decreased sperm motility and viability after thawing.

  4. Change in morphology of sperm.

  5. Costly equipment.

  6. Training , etc

With work of “Human Semen Vitrification” we are trying to explore an option to freeze human semen with fewer demerits.  Simultaneously we also focused to search a simple technique. Moreover vitrification of whole semen is less expensive and easy to perform. It does not require any additional equipment.  We are able to vitrify 1 ml of whole semen in one cryovial (1.6 ml.). We can use more cryovials to vitrify more volume of semen.  Moreover the medium used for semen vitrification does not have any biological product. In other words it is 100% chemically defined.

Material & Method

The basic material required  for this study is Pre Wash Vitri Media. We developed it with our  experience of Vitri Media for washed sperm. It has same ingredients as we used in VITRI MEDIA.  We have only adjusted the concentration of components to make it suitable with whole semen.

Pre Wash Vitri Media

Pre Wash Vitri Media is medium used for vitrification of whole/unwashed human semen. After getting successful result with VITRI MEDIA (post wash)  we adjusted quantity of ingredient. The fine tune in ingredient adjustment of Vitri Media enabled us to prepare Pre Wash Vitri Media.

Donor Consent  — We took verbal consent for this experimental procedure from each concerned donor. We explained them whole process.

Additionally we  require a basic laboratory, liquid nitrogen, long forceps, a timer, boiling water apparatus and incubator.

Vitrification of Semen

We used a very simple protocol to vitrify human semen.

  1. Start to keep Pre Wash Vitri Media in incubator at 34 degree C.

  2. Collect semen after proper abstinence in a sterile container.

  3. Incubate at 34 degree C for 40 minutes or till liquefaction.

  4. Mix semen gently.

  5. Take one ml of semen in a sterile tube.

  6. Add  drop by drop 0.6 ml of Pre Wash Vitri Media & mix regularly.

    Cryovial
    Double walled cryovial – suitable for vitrification

       7. Fill in cryovial and keep at room temperature for 15 minutes.

      8. Plunge directly in liquid nitrogen.

Thawing

As we are at experiment stage, we thaw  after 24 or 48 hours.

  1. Take cryovial out of liquid nitrogen with help of a long forceps.

  2. Wave in air for 30 seconds.

  3. Dip in boiling water for 60 seconds.

  4. Put in water at room temperature for a while.

  5. Place in incubator for 50 minutes.

Sample is ready to view and evaluate under microscope. Please mix properly before taking a drop on slide.

What we observed

  1. Human semen can be vitrified successfully.

  2. Post thaw motility is good enough.

  3. Progressive motility of sperm is better than conventional freezing.

  4. Un-liquefied or viscid sample give poor result.

  5. Sometime we are getting better result than conventional freezing and sometime poor even.  But  it confirms without doubt that human semen vitrification is possible.

Please note

  • After completion of whole procedure we discard the semen according to norm.

  • It is worth to mention that the technique is still under trial. We are not using vitrified semen  for any fertility management.

  • Lot of further study, improvement, discussion and even critics required to establish human semen vitrification.

Conflict of interest

We did not receive any conflict on this study. Rather we are simply waiting for conflict & critics.  In our opinion conflict & critics are strongest tool to improve ant innovation. Therefore we are always open to any discussion or live demonstration.

Constraints

We work with basic facility and basic equipment. Therefore we are not able to provide more elaborate findings like:

  • Osmotic pressure of Pre Wash Vitri Media.

  • Rate of reduction of temperature during plunging in liquid nitrogen

  • Rate of increase of temperature during de-vitrification

  • DNA fragmentation test

  • Viability test.

  • HOS Test

Ethical Issue

Not applicable

Funding

The whole study conducted by our own resources.  We did not receive any grant or assistance from any organization.

Acknowledgement

We are thankful to our whole team of Frozen Cell for their supportive approach and co-operation. Additionally we ate thankful to semen donors for their continuous co-operation.

References

  1. Principles of Cryopreservation by Vitrification, By Gregory M. Fahy and Brian Wowk. https://www.researchgate.net/publication/268875004.

  2. The Process of Sperm Cryopreservation,  Thawing and Washing Techniques. Sajal Gupta,

  3. https://en.wikipedia.org/wiki/Vitrification

  4. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis.  Li YX1Zhou L2Lv MQ1Ge P1Liu YC3Zhou DX4.

                                                                                      —  End of Blog —

 

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