Abstract  —  We designed an experiment to evaluate  the effect  and utility of H.S.A.  in Human Sperm Vitrification.  We compared post thaw recovery of sperm after vitrification  with and without using H.S.A..  Also we had a control sample which was preserved by slow freezing method. Motility and progressive motility was main yardstick in our this evaluation. We noticed that human sperms can be well preserved by vitrification even without H.S.A..  Certainly addition of H.S.A. gives better result in term of  post thaw recovery. I realize  that our this  study is in primitive stage. In any case , this concept of vitrification is going to reveal an extraordinary improvement in human sperm freezing. However, further extensive studies is still in demand to find out best possible result of Human Sperm Vitrification with or without H.S.A..

H.S.A.  — Human Serum Albumin

Introduction —  According to topic  “Human sperm vitrification & H.S.A.” it appears  that H.S.A.  is mandatory for human sperm freezing.  In current practice of human sperm preservation by conventional slow freezing method, H.S.A.  is an integral part of sperm freezing medium. Therefore  conventionally in almost all case of human sperm freezing H.S.A. is used. Somewhere use of animal or avian protein (BSA, FSA,  Egg albumin, milk powder, etc)  has been also proposed. But invariably preferred one is H.S.A..

In our this discussion “Human sperm vitrification & H.S.A.” we will  focus on role of H.S.A. in our recently developed Vitri Media. Initially we started our experiment of sperm vitrification with Vitri Media, which was without H.S.A.. Even it did not have  any protein from natural source. Rather we used amino acids. We used 26 different concentration of salts to prepare Vitri Media. And still we are struggling with different composition of salts to get better result.

Since our newly designed Vitri Media is without H.S.A..  Even it did not have any protein of human, animal or avian origin. In the same Vitri Media we added  H.S.A. to conduct further experiments. 

Material & Method — 

Chemicals and reagents    Chemicals / salts  obtained either from Sisco  Research Lab. Pvt. Ltd (SRL), India or  Himedia laboratories, India. In case of unavailability of product with these manufacturer, we opted for Sigma aldrich (Merck).   All culture media / reagents prepared in-house only.  We didn’t use any ready to use or prepared culture medium or compound.

We prepared Vitri Media with H.S.A. by adding  5mg of H.S.A.  per ml of Vitri Media.  After addition of H.S.A.  we checked the pH and filter with 0.2 micron syringe filter.

Work place & consent  —  The whole study related to article “Human Sperm Vitrification & H.S.A.”  conducted in cryo-preservation center of Frozen Cell  itself. We informed and took verbal  consent from sperm donors with all possible explanation. We discarded all semen sample and processed sperm  after completion of experiments.

Our team selected total eight donors for this study. All donors was healthy and had good quality of sperm. We requested all the donors to maintain abstinence for 3 to 5 days.  Semen was collected in morning hour by masturbation in a sterile sample container specially designed for semen collection.

Sample Container

After collection immediately we added 2 ml  Enhance (sperm washing media) with each semen sample.  Semen sample was kept in in Incubator (at 34 degree C) for 30  to 40 minutes  to  liquefy completely. We checked every semen sample  macroscopically for liquefaction and volume. In the same way also we evaluated every semen sample microscopically for total sperm count, motility %, total motile sperm. We started processing only after complete liquefaction.

For a comparative study we divided each semen sample in three parts.   —-   Part – A, Part – B & Part –C.

Every part of semen sample prepared separately by single layer density gradient (Den -1). We added different freezing media with different part (A, B, & C) in below mentioned sequence —

Cryovials with level
      Ideal cryovials for sperm vitrification

We prepared one cryovials with each part  and leveled accordingly.                                                                   —  Part – A was freezed in usual way of slow freezing method.                                                                               —  Part – B  &  Part – C  was  vitrified according to vitrification protocol we used .

Vitrification Protocol  —

After adding one ml of  Vitri Media with pellet we mixed pellet and vitri media properly.  Viewed a drop under microscope to check motility.  Filled the cryovials up 0.5 ml to with content.  Every cryovials was leveled appropriately.  Incubated at room temperature for 15 minutes.  After 15 minutes we plunged cryovials directly  in liquid nitrogen.

We checked & evaluated preserved cryovials after 24 hours of preservation in liquid nitrogen. Thawing of Part – A performed in usual way.

Thawing of vitrified sample —

Cryovials of  Part – B & Part – C  was first waved  in air for 25 sec after taking out from liquid nitrogen.  Then immediately dipped in boiling water for 55 second. After taking out from boiling water cryovials was again dipped in water at 34 degree C for approx 10 second. Finally cryovials kept in incubator at 34 degree C for 30 minutes.

Result  of slow freezing verses vitrification 

The result obtained by different part are summarized below.  In this comparative study, we are considering   Part – A  as control sample.

Part  – A  (Post Wash CP – Slow conventional freezing)  —  Control Sample 

  • Result was as usual, 30 to 60 post thaw recovery depend upon semen quality.

  • Variable progressive motility. 

  • Maintenance of motility —  10   to 15 %  sperm was motile after 24 hour of thawing of post thaw motility

Post thaw motility of conventional slow freezing

Part – B  (Vitri Media without H.S.A.)

  • Post thaw recovery –   inferior to Part – A.

  • Progressive motility — better progressive motility in comparison to Part – A

  • Maintenance of motility —  poor maintenance of motility. Hardly 0- 2 motile sperm per field was motile after 24 hour.

Post thaw motility following vitrification of sperm without H.S.A.

Part – C  (Vitri Media with H.S.A.)

  • Post thaw recovery —  Either inferior or equal to Part – A. But better in comparison to Part – B

  • Progressive motility — Better progressive motility in comparison to Part – A. As good as Part – B or even better.

  • Maintenance of motility — Maintenance of motility was as good as normal. In some semen sample we noticed even better maintenance of motility than part – A.

Post thaw motility following vitrification of Human sperm  – with H.S.A.

Additional information  —– 

We saw in other investigation that semen sample (sperm)  of some person was difficult to vitrified. Or on the other hand showing exceptionally poor post thaw motility. Additionally we observed that it was also hard to preserve semen sample (sperm) of those person  even by slow freezing technique.  Also we observed that after a period of time (four or five month) sperm of the same person was freezable  by both the method.

Discussion : —  Wright brothers did not invent Boeing or Concorde. They invented a very basic model of a  plane. But that was the pathfinder of today’s sky.

  1. Many paper has been published regarding human sperm vitrification. But all was either non-conclusive.  Or quantity was not sufficient, to say  (2 to 3 micro liter)  of sperm and cryoprotectant  mixture.

  2. Presently vitrification is a successful method to preserve egg & embryo in ART industry. They require very high concentration of cryoprotectant. That high concentration of cryoprotectant will damage or destroy the sperms permanently. Our newly developed Vitri Media does not have that much high concentration of any or total salt.

  3. With this method and our newly developed Vitri Media, we are successfully performing vitrification of human sperm in a volume of 0.5 ml. We have a plan to  proceed  ahead to  vitrify of sperm in a volume of 1.5 ml.

  4. Most important by this method we vitrified sperm in a high security closed cryovial.  Hence there is no chance of cross contamination between vitrified samples.

  5. Still we have to decide the use H.S.A. for  freezing of human sperm by  method of vitrification.

  6. H.S.A. certainly improve  post thaw maintenance of motility after vitrification. It may be because of nutrition provided by H.S.A. or some other unknown factor.  But a more natural environment  is available inside uterus. Which seems too provide best atmosphere for sperm and its function. Therefore in vitro maintenance of motility more than an hour does not seem very important.

Conclusion :—

  1. Human sperm can be vitrified even without use of H.S.A.

  2. Progressive motility was better after vitrification in comparison to slow freezing method.

  3. H.S.A. improve post thaw recovery of sperm after vitrification in most of the  case.

  4. H.S.A.  provides very good maintenance of motility.

  5. Progressive motility was equally good with or without H.S.A.

  6. We are presently not in condition to say that post thaw recovery of Vitri Media is better than conventional slow freezing media. But we are in ongoing endeavor to get a best possible Vitri Media with maximum or 100% post thaw recovery.

Conflict of interest —  Presently the  study related to “Human sperm vitrification & H.S.A. did not have any conflict. Rather we welcome conflict & critics on this topic.  Always we are open for any discussion or live demo related to Human sperm vitrification.

Acknowledgement : — We are thankful to persons , who donated their sperm for this study. Moreover they encouraged our whole team with their support and kindness.

Funding —  The study cost  and total other expenses managed by  Frozen Cell only.  Any other organization or system  did not have  involvement  in any funding.

References  –

  1. CRYOPRESERVATION OF HUMAN SPERMATOZOA BY VITRIFICATION:  IMPACTS ON SPERM PARAMETERS AND APOPTOSIS.  M. A. Khalili, M. Adib, M. Ramezani. Research and Clinical Center for Infertility,  Shahid Sadoughi University of Medical Sciences, Yazd, Islamic Republic  of Iran; Department of Biology, University of Payam-nour, Yazd,  Ireland.  https://www.fertstert.org/article/S0015-0282(10)01552-9/abstract.

  2. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for AR     Marlea Di Santo, Nicoletta Tarozzi, Marco Nadalini, and Andrea Borini

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