It is postulated that Ultra Rapid Freezing (Vitrification – ?) of Human Sperm require extremely high concentration of salt. Specially sucrose up to 1 mole or even more. My work on Ultra Rapid Freezing (Vitrification-?) of Human Sperm and Salt Concentration isn’t favoring this idea. Rather my work shows that even at adequate salt concentration it is possible to freeze human sperm by ultra rapid freezing (vitrification -?). Obviously we have to choose proper salt/chemical in proper quantity. Since we work just on human sperm, therefore our this discussion will focus on ultra rapid freezing (Vitrification -?) of human sperm only. Still we need to decide it is ultra rapid freezing or vitrification.  We assume this low salt concentration concept of ultra rapid freezing (vitrification-?) may also be applicable on other cell or a thin slice of tissue. Moreover disadvantage of high concentration of Cryo- protective agent does not work here. Additionally there is no requirement of step wise addition and removal of Crypprotective agent or Vitrification media.

I emphasize again that newly developed Vitri Media work successfully with human sperm. It is effective to vitrify sperm in a closed container and an optimum quantity of 0.6 ml.

INTRODUCTION

My work on vitrification started with a trial of finding freezing media without H.S.A.(Human serum albumin). And as a result of lateral approach I proceeded toward ultra rapid freezing (vitrification-2) of sperm.

Most of the author of this field claim that sperm (particularly Human Sperm) can not be freezed by ultra rapid freezing (vitrification-?).  Specially in optimum quantity and in a closed system. The major drawback with previously developed / used vitrification media was very high concentration of salt (particularly sucrose). But according to our study it is possible to freeze human sperm in a closed container of 0.6 ml by ultra rapid freezing (vitrification-?).

MATERIAL & METHOD –

The basic material for this study is our newly developed Vitri Media. We are in continuous process to reduce the salt concentration in said Vitri Media. As well as working hard to improve its quality and outcome. We have adjusted its salt concentration to less than 200 millimoles + 6 % Glycerol  (V/V).   Out of 200 millimoles, sucrose contributes only 20 millimoles.

Presently we are following very simple & basic model to separate and freeze the sperm by ultra rapid freezing (Vitrification -?). Once we got a desire result, we will work to improve and refine method of sperm separation and vitrification. Next step is certainly thawing and checking the sperm motility at predetermined  period of time.

Sperm separation

    • Semen collected after proper abstinence & in morning hour.

    • Immediately after  collection semen mixed with 2 ml Enhance (Sperm washing media).

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

    • Layered over 75% density gradient  in centrifuged test tube.

    • Centrifuged at 370 G for 18 minutes.

    • Supernatant discarded.

    • Sperm rich pellet mixed with Vitri Media (quantity of Vitri Media decided according to sperm concentration – motile sperm / ml).

Ultra rapid freezing (Vitrification -?)

  • Sperm mixed Vitri Media filled in cryovial.

  • Kept at room temperature for 15 minutes.

  • Cryovial plunged in liquid nitrogen.

We checked post thaw motility  and  maintenance of motility.  As well as we also check maintenance of  motility of sperms in Vitri media without freezing.  To check the maintenance of motility in  cryovial or Centrifuge test tube, we kept it at room temperature. Before checking motility we keep  sample in incubator for 30 minutes at 34 degree C.

  1. Post thaw motility.        

      •  We noticed 25 to 50 % post thaw motility  depending on quality of semen.

      • We noted better progressive motility in comparison to conventional freezing.

      2.  Post thaw maintenance of motility.

      • Thawed sperm was kept in same cryovial. Quality checked at predetermined  intervals.

      • Motility was like post thaw even after 4 hours.

      • Again checked after 8 hours – it was very good.

      • Finally checked after 24 hours.  Approx 10 – 20 % motility was noted in comparison to post thaw motility.

        3.  Sperm motility in vitri media without freezing.

      • First step was to separate the sperm by density gradient in a centrifuged test tube.

      • Sperm rich pellet mixed with vitri media and kept at room temperature.

      • After 4 hours motility was as good as normal.

      • Again checked after 8 hours we noted some reduction in motility.  But still very good.

      • Next day (after 24 hours) motility was  approx 40%.

      • On second day (after 48 hours)   – 10 to 25 %  of motility.

      • After 72 hours – there was detectable motility or some time no motility.

Discussion.

Our motive is not to say that we are getting better result than conventional sperm freezing. Rather we claim that it is possible to freeze human sperm by ultra rapid freezing (Vitrification – ?). Moreover there is  lot of further scope of  research this technique.

Water content of sperm.   — Human sperm is a tightly packed cell. Therefore water content of human sperm is somewhere 25% to 50%. Rest of the space inside the sperm is occupied with genetic material. Therefore osmotically human sperm cell has to perform less trans-membrane  activity.  Since less solution need to transfer  to attain stability before vitrification.

We assume that by reducing the salt & glycerol concentration we reduce toxicity of cryopratactent. According to our concept it can be achieved by using simple and minimum quantity of salts. Moreover we prefer to use only those salts which makes a part of  human body.   And using simple cryoprotactent  in minimum quantity. Also our preference is to use only one permeable cryoprotactent (glycerol).

Additionally it is worth to mention that this Vitri Media do not have Human serum albumin or any natural protein. Therefore extracellular protein denaturation  by cryosystem or cold is not applicable here. Presently we do not comment anything on intra-cellular  protein of sperm.

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.

Limitation

Our study is with basic equipment only. Therefore we use only basic data and outcome like post thaw motility, maintenance of motility, progressive motility, etc only.

Presently we do not have access to perform ——

  • Osmotic pressure of 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

Conflict of Interest

Presently the  study “Ultra Rapid freezing (Vitrification-?) of Human Sperm  and Salt Concentration”   did not have any conflict. Rather we welcome conflict & critics on this topic.  Always we are open for any discussion and  live demonstration related to ultra rapid freezing (Vitrification – ?) of human sperm  and salt concentration.

Acknowledgement

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

References

  1. Principles of Cryopreservation by Vitrification, Gregory M. Fahy and Brian Wowk, https://www.researchgate.net/publication/268875004
  2. 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.
  3. 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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