Human Sperm Preservation with or without H.S.A. – A comparative study

Study on “Human Sperm preservation, with or without H.S.A. – A comparative study” is performed to know the role and necessity of H.S.A.  for human sperm preservation. We conducted this study ( Human Sperm preservation, with or without H.S.A. – A comparative study ) by  evaluating the outcome in term of post thaw motility, type of sperm motility after thawing and maintenance of sperm motility after thawing.  

Objective   —  The objective of this study ( Human Sperm preservation, with or without H.S.A. – A comparative study) is to evaluate the necessity of human serum albumin (H.S.A.) in the process of human sperm preservation. As well as we tried for an safe & practical option.

Design  — A prospective study.

Place   — Frozen Cell cryo-preservation center.

Donors   — Ten number of  healthy donors selected with good sperm parameter for this comparative study.

Material and Method

All selected donor for this study was from B +ve blood group.  We opted B +ve donors , because we have enough number of B +ve donors. Secondly we noticed that semen quality of B +ve donors was relatively better. As well as their post thaw recovery was also better.

Two type of Cryo-preservative medium used :–

1. Conventional sperm cryo-preservative medium  — Post Wash CP
2. Sperm preservative medium without H.S.A.  and with a fixed ratio  of amino acids

Semen sample collected in the morning by masturbation in sterile sample container. Taken care that all donors must have an abstinence of not less than three days and not more than five days.  On first collection day semen sample of five donor was taken for study. Remaining five donor”s semen was taken on next collection day.  Every sample was analyzed individually for – volume, total sperm count, total motile sperm and motility %, and type of motility. Every donor allotted a specific number (1) to (10).

All consumable including –  Den – 1, Enhance, Post Wash CP and newly prepared sperm preservative medium  without H.S.A.  along with required plastic disposable was kept in incubator at 33 degree C. one hour before starting the collection.

Immediately after collection 2 ml of Enhance (sperm washing media) was added with semen sample. We kept  semen sample + Enhance  in incubator at 33 degree C for 30 to 40 minutes.  Mixed well semen with Enhance.  Semen + Enhance mixture divided in two part  of each collection – Part –A   & Part – B.
We planned to preserve Part-A of each semen with Conventional sperm cryopreservative medium  — Post Wash CP.
And Part – B, we preserved with Sperm preservative medium without H.S.A. Which contain amino acid in a specific concentration.

Steps of preservation.

1. Sperm of Part –A & Part — B of each collection  separated by single layer density gradient method (Den-1).
2. Each pellet washed with Sperm Washing Media (Enhance)  at 250 G for 4 minutes.
3. In Part-A of each collection we added  Conventional sperm cryopreservative medium  — Post Wash CP.
4. In Part –B of each collection we added sperm cryopreservative medium without H.S.A. .
5. We tried to maintain the concentration of sperm approx 60 million per ml.
6. Both part (Part — A & Part –B) filled in cryovials (0.6 ml).
7. Kept cryovials at room temperature for 15-30 minutes.
8. All vials kept over vapor of liquid nitrogen.
9. We reduced temperature at the rate of 4-6 degree C per minute.
10. Once we achieved the temperature of (-) 130 degree C, we dipped all cryovials liquid nitrogen.
11. We took the Cryovials out of liquid nitrogen only after a minimum period of 24 hour for further evaluation.
12. Thawing of the cryovials performed at temperature of 33 degree C for 45 minutes.

RESULT

In real sense we got encouraging and surprising result of the conducted study “ Human Sperm preservation, with or without H.S.A. – A comparative study ..

A/  POST THAW MOTILITY  — Post thaw motility was almost equal whether human sperm was preserved  with H.S.A. & without H.S.A..

2/  TYPE OF SPERM MOTILITY AFTER THAWING  — Most of the sperm preserved with amino acid (without H.S.A.)  demonstrated better progressive motility with straight line movement.

3/  We observed that maintenance of motility was poor when preserved with cryo-preservative media without H.S.A. (with amino acid)

Conclusion :–

1. Post thaw motility are almost same whether preserved by cryo-preservative media with H.S.A. or without H.S.A..
2. We observed much better progressive motility with Sperm preservative media without H.S.A..
3. Survival of sperm after 24 hour of thawing was poor when preserved with cryo-preservative medium without H.S.A..

Discussion :–  

We assume that once sperm is inside the uterus, they will get most favorable environment and nutrition. Therefore sperm will get  proper survival (maintenance of sperm motility) inside the uterus. But by using this sperm cryo-preservative medium, we can 100% prevent disease transmission by H.S.A.. By replacing H.S.A. by amino acids we also eliminate risk of transmission of un-identified & emerging virus .  Furthermore I don’t have any hesitation in saying that still this study is in initial stage. Therefore there is lot of scope of further improvement.

Conflict of interest  :–

Till now we did not observed any  conflict on the topic “
Human Sperm preservation, with or without H.S.A. – A comparative study”.

Acknowledgements  :–

Frozen Cell Team is in a nonstop endevour to find out an ideal technique to process and preserve human sperm.

Reference : —

1. Asian Pacific Journal of Reproduction 2015; 4(3): 222–227
2. Kundu CN , Chakraborty J, Dutta P, Bhattacharyya D, Ghosh A, Majumder GC. — Development of a simple sperm cryopreservation model.
3. Gerhard Gstraunthaler — Alternative to the use of Fetal Bovine serum.
4.  

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