Cryopreservation of human sperm with H.S.A.  is a standard method for preserving human sperm with various indications.  We will discuss a exceptionally straightforward procedure in “Novel Technique to Cryopreserve  Human Sperm without H.S.A.  Which  do not need  any modern / expensive equipment.  By The technique “Novel Technique  to  Cryopreserve  Human Sperm without  H.S.A.” our aim is to give opportunity even for a basic  IUI / IVF center to preserve human sperm with all  safety and security.  Moreover,  this technique (Novel Technique  to  Cryopreserve  Human Sperm without  H.S.A.)  will even help the cancer patient. Who is hoping  to preserve their fertility  later on in the wake of completing their treatment.   This cryopreservative  media does not contain Human Serum Albumin (H.S.A.) or any crude protein from animal or avian source. Accordingly even there is no possibility to infection transmission through any far off source.

H.S.A.  == Human serum albumin

Sperm preservation  –  an introduction

Cryopreservation of human sperm began decades & decades back.  Many type of  cryopreservative media and technique has been used with subsequent improvement in technique and result.  But one thing remain persistent that some form of crude protein is being used either as nutrition or upgrade factor for human sperm.  We will discuss all basic phenomena  to make cryopreservation of human sperm easy and affordable without using any crude protein.

In “Novel Technique  to  Cryopreserve  Human Sperm without  H.S.A.”, we will discuss how to preserve  human sperm with 100% chemically defined  cryopreservative  compound.  Here  a cocktail of amino acid has been used to provide nutrition and as enhancement factor  to human sperm.

Side by side our emphasis will be to describe an easy and cost effective method to freeze human sperm

Conventional technique of Human sperm cryopreservation

In conventional technique of human sperm cryopreservation  we use permeable and non-permeable cryo-protectant  along with Human serum albumin. At the same time different salts are being added to make cryopreservative media sperm friendly.

Material and method

Below mentioned chemicals was purchased from  SRL , Himedia and  Sigma-Aldrich. All chemicals was of AR  grade. Only L-isomer of all  amino acids (Essential and non-essential)  selected for media preparation.

(L- Isolucine,  L-leucine, L-lycine, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine,     L-valine, L-proline, L-aspartate, L-asparginine, L-glutamate, L-serine, L-cystine, L-histidine, L-threonine,  L-arginine, Alanyl glutamine, NaCl, KCl, Lactic acid, MgSO4, Pot. Phosphate, Sodium bicab,  Sucrose, glycine, Hepes, Gentamicin, Taurine, EDTA, KH2PO4,  CaCl2, Sodium pyruvate, Glycerol in double distilled water)

Glycerol concentration was kept at 6 % (vol / vol). An invented  cocktail of amino acid was used instead of human serum albumin. Different salts was added in a predefined  ratio.  We set the pH of solution at 7.35 approx.

Six donors were selected for sperm preservation with  Novel Technique  to  Cryopreserve  Human Sperm without  H.S.A.. All of them belong to B+ blood group.  The donor’s ejaculate was with 50-60 million sperm per ml,   more than 50% progressive motility and  40% or more morphological typical sperm. Donors  were informed and consent taken.  Semen was collected after 48 to 72 hour of abstinence in the morning hour of the by masturbation.  Each ejaculate divided in two part.  Part-A – only 1 /3   of semen was taken,  Part-B  – rest of the semen. 

Both parts was prepared by swim down technique.  In short, Part-A  and Part-B of each ejaculate was washed twice , first time by density gradient 75% at 380 G for 15 minutes. Prepared pellet was  mixed  with 2 ml oh washing media and centrifuged again at 380 G for 4 minutes.  Used  swim down media(discontinuous gradient & washing media)  was Den-1  of Frozen Cell.

After second centrifugation in Part -A appropriate quantity of our newly invented Novel  cryopreservative without  H.S.A.  was added  to make sperm concentration 30 to 50 million of each ml. 

In Part-B  we used conventional cryopreservative with H.S.A..   Samples was filled in cryo-vials and  kept at room temperature for 10 to 20 minutes.  Cryovials of Part-A  and Part-B (appropriately labeled) was freezed  together according as per  our standard  freezing protocol.

Our standard  freezing protocol  —

  • Take approx  2 inch of liquid nitrogen in a thermocol  insulating box.
  • Set a plastic mesh approx 3 inch above  liquid nitrogen.
  • Place the cryovials  over plastic mesh.
  • Place the censor of temperature meter  above plastic mesh along with cryovials .
  • Reduce the temperature of plastic mesh at the rate of 3 to 5  degree per minute ( it can be done adding or removing the liquid nitrogen.  Also slow blowing of air is very effective in maintaining the temperature reduction.
  • Once temperature is less than (-) 130 degree C. Plunge  all cryovial in  liquid nitrogen.
  • Store in liquid nitrogen for 24 hours to check post thaw recovery at different parameter.
Frozen Cell Cryovial – Double walled , highly secured

Thawing process takes 45 to 50 minutes. First temperature of  incubator was set at 32 to 340C.  Cryovials  taken out from liquid nitrogen  and kept in incubator for 45 minutes. Content of cryovial was mixed properly after thawing.

We checked following parameters  after complete thawing of each cryovials  independently.

  1. Post thaw sperm motility.
  2. Progressive motility.
  3. Motility after 24 hour.
  4. Sperm morphology.

POST THAW SPERM MOTILITY

In general we noticed post thaw motility  was somewhere less than conventional cryopreservative  with  H.S.A.  As an average post thaw recovery was 10 to 20% inferior to conventional cryopreservative  with  H.S.A.

PROGRESSIVE MOTILITY

Surprisingly sperm preserved with cryopreservative without H.S.A. had  a very good progressive motility in comparison to conventional cryopreservative  with  H.S.A. .  Most of the motile sperm was moving in a  straight line.

One negative point  – speed of sperm even in forward direction was less in compare to conventional cryopreservative with H.S.A..

MOTILITY  AFTER  24 HOUR

To check the motility after 24 hours, we kept the firmly closed cryovials at room temperature (15 to 25 degree C).  Before checking the motility  all cryovials was kept in incubator for 40 minutes at 33 degree C.

            Motility after 24 hour was distinctly less with Part-A (Novel  cryopreservative without  H.S.A.)

In comparison to  Part-B (conventional cryopreservative with H.S.A.), The difference  was 20% to 50%.

SPERM MORPHOLOGY

Sperm morphology following freezing and thawing  was noted better in case of Part-A (Novel  cryopreservative without  H.S.A.).  That might be the reason of better post thaw progressive motility when preserved  with Part-A (Novel  cryopreservative without  H.S.A.).

DISCUSSION

Till date the majority of the cryobiological  work on sperm were based on  study of various type of conventional cryopreservative with H.S.A.  or different type of animal or avian serum.   In the meantime extraordinary sort of modern freezer (programmable and non-programmable) has been upheld to utilize. Which might be hard to manage.

            Besides, this novel cryopreservative without H.S.A., as name recommend is without H.S.A.. Human serum albumin is a potential source of disease . Consequently it must be kept away from at whatever point conceivable.

CONCLUSION

We finished up following focuses with the theme “Novel Technique to Cryopreserve Human Sperm without H.S.A.”.

  1. Human sperm can be effectively  preserved  without Human Serum Albumin (H.S.A.)
  2. Costly freezer (programmable or non-programmable ) isn’t compulsory to freeze  human sperm.
  3. We  assume that carbohydrate and amino acid can give nourishment to sperm.
  4. More investigation is required to build up an ideal media for human sperm preservation.

Conflict of interest  —-  Till now we did not notice any  conflict on the topic “Novel Technique  to  Cryopreserve  Human Sperm without  H.S.A.”.

Acknowledgements  —  Frozen Cell Team is in a nonstop procedure to find out an ideal media to preserve  human sperm.  We are in an undertaking to find a better strategy to preserve human sperm  by various procedure.

Funding  —  Frozen Cell do not accept any fund from any outside source.  100%  funded by Frozen Cell

Limitations —   We work with besic equipments. Therefore not able to give figures regarding osmolarity,  DNA fragmentation, Acrosomal status,  Sperm viability etc.

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