To our knowledge, this study invent First-Ever: Successful Vitrification of Human Sperm in an optimum quantity. Multiple attempts taken by many researchers to vitrify sperm of different living organism. But resulted either in partial success with controversy or failure. Moreover in our present work we got success in vitrifying human sperm (not semen) in an optimum quantity of 0.6 ml. Most importantly we performed it in a closed cryovial.  Which is effective enough to prevent cross contamination through liquid nitrogen.

We used only three culture media for whole process. All three media and plastic disposable prepared & developed by Frozen Cell itself.

  1. VITRI MEDIA (Post Wash)  ———- developed & named by Frozen Cell.

  2. Den-1 (75% density gradient) ——– for sperm separation.

  3. Enhance ———————————– sperm washing media.

VITRI MEDIA (Vitrification Medium)

The newly developed VITRI MEDIA (Post Wash)  does not have any macro-molecule like human serum albumin. Molecular weight of all chemicals are within limit (less than 200 millimoles) + Glycerol 6% (V/V). Out of 200 millimoles sucrose contributes only 20 millimols. Therefore osmotic pressure of VITRI MEDIA (Post wash) is not high to affect  human sperm adversely.

We also tested VITRI MEDIA (Post wash) as sperm preparation (extender) medium & sperm culture medium. We noted, sperm can be separated effectively  using VITRI MEDIA (Post wash)  either by swim up or swim down method. Following washing we used VITRI MEDIA (Post Wash)  as nutritional media. We got an acceptable survival of sperm in VITRI MEDIA (Post wash) even after 72 hour and more in some case. It suggests that VITRI MEDIA (Post wash)provide optimum environment to sperm. Additionally I mention again that VITRI MEDIA (Post Wash) does not contain human serum albumin or any substance derived from human, animal, bird, aquatic or plant origin. In other words it is 100% chemically defined.

Failure of previous attempt of sperm vitrification.

Many researchers tried to vitrify human / animal sperm by different method. But resulted in either partial success or complete failure.

Main reason behind failure seems ———

  1. Possibility of cross contamination because vitrification performed on open system.

  2. Use of very high concentration of compound (mainly sucrose).

  3. Vitrification in inadequate quantity (10 to 20 micro liter).

  4.  Inadequate trial, etc.

MATERIAL AND METHOD

Chemicals and Reagents —  Chemicals / reagents purchased from either Sisco Research Lab. Pvt. Ltd (SRL), India or Himedia laboratories, India. All culture media & reagents prepared in-house only.

A laboratory with basic equipment is only requirement for sperm vitrification. The equipment required are laminar hood, incubator, microscope, centrifuge machine, liquid nitrogen. For thawing additional requirement is a timer, apparatus to boil water & forceps.

Presently our study is limited with good quality human sperm. We noted better outcome in term of progressive motility. Maintenance of motility also seems good enough.

We compared the result with control sample. Semen sample after collection divided in two parts. Part one freezed by conventional slow freeze- thaw method. While part two preserved by vitrification-thaw method. Motility of the samples checked up to after 24 hour for this particular study.

Vitrification Process

We opted very simple process to vitrify human sperm. All media and plastic disposable kept in incubator at 32 – 34 degree C. approx one hour before starting the process. Donors ware asked to maintain abstinence for 3 to 5 days. We perform semen collection in morning only.

  • Just after semen collection – added 2 ml of Enhance & mixed.

  • Kept in incubator for 40 minutes to liquefy completely and mixed well.

  • Semen & Enhance mixture slowly layered over Den – 1 in a conical centrifuge test tube.

  • Centrifuged for 18 minutes at 380 G.

  • Supernatant was discarded.

  • Added required quantity of VITRI MEDIA.

  • 0.6 ml of content filled in cryovials

  • Kept at room temp for 15 minutes.

  • Cryovial directly plunged in liquid nitrogen.

Thawing of vitrified sperm.

  • Cryovial taken out from liquid nitrogen.

  • Waved in air for 30 seconds.

  • Dipped in Boiling water for 50 seconds.

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

  • Mixed the content of cryovial gently with pasture pipette.

  • Sample is ready for post thaw motility assessment.

Post Thaw Motility after Vitrification

Post thaw motility after conventional freezing

Difference in progressive motility can be clearly noted. Sperms preserved by vitrification is showing much better progressive motility. Here it is worth to mention that better progressive motility gives higher success rate. Moreover vitrification technique is in primitive stage. Therefore there is lot of scope of further development.

Please note — Regarding Vitrification of human sperm

  1. Recovery and motility varies according to quality of semen.

  2. Addition of Hyaluronic acid does not improve post thaw motility.

  3. Used VITRI MEDIA does not have human serum albumin. Once inseminated, sperm get natural / favorable environment.

  4. Addition of human serum albumin may improve the outcome of VITRI MEDIA.

  5. VITRI MEDIA contain only one permeable cryo-preservative — glycerol. It does not have any toxic cryoprotectant like dimethyl sulfoxide (DMSO).

  6. Right now we have not used any ice blockers in VITRI MEDIA.

Similar articles

Article & post of similar type has been published in either our website (www.frozencell.org) or on our Linkedin site (www.linkedin.com/in/frozencell). Hence we authenticate originality of this post.

Conclusion

According to  findings of present study we say with all confidence that human sperm can be vitrified successfully. Most importantly post thaw recovery of vitrified sperm is better in comparison to conventional freezing. And above all we noted much better progressive motility following vitrification.

Conflict of Interest

As of now we did not get any conflict of interest. But we invite conflict and critics on this issue. First ever: Vitrification of Human Sperm is a new concept in human sperm freezing. Therefore we are always open to any discussion and live demonstration of this technique.

Funding

The whole study and technique conducted by our own resources. No other organization or any system is involved in financial assistance for this study.

Acknowledgement

We are thankful to our whole team of Frozen Cell.

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.

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

  4. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. https://doi.org/10.1155/2012/854837

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