IMA

1. Sperm
Cryopreservation
Dr. Chandan
M.sc. PhD
Senior Embryologist
ARC

2. Aim of this lecture…………….
• Overview of the current technologies and approaches utilized in sperm
cryopreservation.
• Consider the factors affect to results of cryopreservation
• Sperm Cryodamage.
• How to optimize semen cryopreservation.
• Sperm preparation prior cryopreservation
• Is cryopreservation induce DNA damage?
• whether using fresh rather than cryopreserved sperm cells has the same effect
on reproductive outcome in ICSI

3. What is sperm
cryopreservation?
• A procedure to preserve sperm cells (commonly called sperm banking)
• Cryopreservation is the freezing of cells or tissues to sub zero temperatures, typically -196 º C
(boiling point of liquid nitrogen).
• The first successful cryopreservation of spermatozoa was initiated 1960s.
• For human sperm, the longest reported successful storage is 22 years.
• All biological activity is stopped or paused until it is thawed.
• The freezing of sperm needs cryopreservation agents that minimize damage to the cells during the
freezing and thawing process

4. Why sperm cryopreservation is
performed ?
1. Semen containing a very limited number of spermatozoa or abnormal semen parameters.
2. For cancer patients to preserve their fertility prior to gonadotoxic chemotherapy or radiation.
3. Patients undergoing certain types of pelvic or testicular surgeries
4. Patients who suffer from degenerative illnesses such as diabetes or multiple sclerosis; spinal
cord disease or injury.
5. persons in occupations where a significant risk of gonadotoxicity prevails.
6. men undergoing surgical sterilization such as vasectomy.
7. allow donor semen samples to be quarantined while appropriate screening is performed to
prevent the transmission of infectious pathogens during therapeutic donor insemination (TDI).
8. used in combination with ART techniques.

5. How sperm cryopreservation is
performed?
Cryoprotective agents (CPAs):
• low molecular weight chemicals that serve to protect spermatozoa from freezing damage or ice
crystallization by decreasing the freezing point of materials.
• CPAs can be toxic if used at high concentrations.
1. Permeating CPAs (penetrate the plasma membrane)
 such as dimethylacetaldehyde; dimethyl sulfoxide, glycerol, glycol, ethylene and methanol
 stabilize cell plasma membrane proteins and reduce concentrations of electrolytes.
2. Nonpermeating CPAs (unable to penetrate the plasma membrane)
 such as albumins, dextrans, egg yolk citrate, hydroxyethyl, polyethylene glycols, polyvinyl
pyrollidone and sucrose
 minimize intracellular crystallization by increasing viscosity of the sample.

6. How sperm cryopreservation is
performed?
Benefits of cryoprotectants:
1. Maintaining sperm viability.
2. Improvement in the recovery of motile sperm by the use of zwitterion
buffers has been attributed to their ability to bind free hydrogen and hydroxy
ions in the surrounding medium, aiding in the dehydration process.
3. Improve sperm membrane fluidity.
4. Increase sperm longevity and percent survival.
5. Increase ability of sperm to penetrate cervical mucus post-thaw.

7. Techniques for Cryopreservation
1- Slow Freezing:
Slow programmable freezing
- Liquid nitrogen is poured into the tank, and the machine, once programmed, uses the software
data logging to obtain cooling from 20°C to −80°C at rate of 1.5°C/min and then at 6°C/min;
at completion of the freezing the straws are removed and stored into liquid nitrogen at
−196°C. This takes about 40 min.
- Simple to use
- does not require continuous operator intervention
- increase the reproducibility of the freezing operations

8. Techniques for Cryopreservation
2- Rapid Freezing:
• requires direct contact between the straws and the nitrogen vapors for 8–10 min and immersion in
liquid nitrogen at −196°C.
• Inside nitrogen vapors there is a thermal gradient, as a function of the distance and the volume of
the liquid below.
• The sample is initially mixed in dropwise manner with equal volume of cold cryoprotectant; the
mixture is loaded into the straws and left to incubate at 4°C for 10 minutes.
• The straws are then placed at a distance of 15–20 cm (1 hr WHO) above the level of liquid
nitrogen (−80°C) for 15 min; after this stage, the straws are immersed in liquid nitrogen.
• place the straws in horizontal position to minimize the heat difference between the two ends.
• Low reproducibility
• temperature drop curve cannot be controlled
• and the freezing temperatures may vary from −70, −80, and −99°C.

9. Techniques for Cryopreservation
3. Ultra-rapid freezing (Vitrification)
• Until only recently, vitrification of spermatozoa was unsuccessful, possibly due to high
concentrations of permeable CPAs (30-50% compared to 5-7% with slow freezing) and low
tolerance of spermatozoa to permeable agents.
• Even brief exposure to a high concentration of CPAs can lead to toxic and osmotic shock and
would be lethal for spermatozoa.
• One possible strategy to lower the concentration of CPAs could be to increase the speed of cooling
and warming temperatures as higher rates of cooling and warming, require lower concentrations of
CPAs; these conditions can help eliminate intracellular ice crystallization, and facilitate the
formation of a glassy state .
• Another option is to add non-permeable CPAs--such as carbohydrates--to permeable CPAs to
minimize osmotic shock by decreasing osmotic pressure and stabilizing the nuclear membrane.

10. Straw holder
Straw or vails
Forceps
Syringe
Vistube
Requirments for Cryopreservation
Tanks

11. Thawing Procedure
• The thawing procedure is an equally important
step: the cell must be allowed to recover its normal
biological activities trying to avoid abrupt thermal
changes as far as possible. Generally speaking, the
cryopreservation protocols use a thawing
temperature of 37°C; even if higher thawing
temperatures allow for more rapid heating, they
are not used because of the risks associated to cell
damage.

12. At the present time, several thawing techniques
are used, among which are
• (i) thawing at room temperature for 10 min and
subsequent thermostat pass at 37°C for another
10 min,
• (ii) thawing in a thermostat and water-bath at 37°C
for 10 min,
• (iii) thawing at room temperature for 15 min.
• Once the semen is thawed, it is separated from the
cryopreservation medium by washing in culture
medium and centrifuging

13. Guidelines

14. How to avoid Negative Impacts
Maintenance of media.
Standard SOP
Trained staff
Equipped lab

15. Semen preparation pre-freeze and
post-thaw
• The quality of ejaculated semen is also related to the outcome of cryopreservation.
• For example, dead spermatozoa or leukocytes in pre-freezing semen detrimentally affect the
sperm survival rate and the fertility potential after thawing through the ROS generation
process
• Sperm preparation before cryopreservation should be considered in routine sperm
cryopreservation.
• Optimizing the concentration of progressive motile sperm cells before the freezing process is
recommended to ameliorate the fertilizing capacity of the frozen spermatozoa.
• Semen preparation before freezing resulted in better sperm quality and fewer apoptotic sperm.

17. How to optimize semen
cryopreservation ?
Factors affecting the optimization of human sperm preservation:
Technical factorsBiological factors
Cryopreservation mediumGenetic factors
Addition/removal of cryoprotectantSexual abstinence
Cooling rateSeminal fluid quality
packingSeminal quality
Storage
Thawing
storage temperature

18. Cryopreservation and DNA Damage
• There is no agreement in the literature neither on whether cryopreservation induces DNA
damage nor on the amount of damage.
Because:
(1) different freezing procedures
(2) different tests to evaluate the DNA integrity
(3) different semen preparation techniques before cryopreservation (i.e., swimup or density
gradient centrifugation).

19. Cryopreservation and Reproductive
Outcome
Testicular Spermatozoa:
• No statistically significant differences in all parameters examined (fertilization rate,
cleavage rate, embryo quality, implantation rate, clinical pregnancy rate, and ongoing
pregnancy rate) between ICSI cycles with fresh or cryopreserved testicular spermatozoa.
• Only, De Croo and colleagues stated that fertilization, implantation, and live-birth rates per
embryo transfer are significantly lower after ICSI.
Epididymal Spermatozoa:
• Tournaye and colleagues reported that the clinical pregnancy rate in ICSI cycles was
comparable between fresh and frozen-thawed epididymal spermatozoa.
• Sukcharoen and colleagues came to the same conclusion; also Cayan and colleagues [64]
supported the same opinion.

20. Cryopreservation and Reproductive
Outcome
Ejaculated Spermatozoa:
• Kucznynski and colleagues did not report of any statistically significant differences in
fertilization rate between fresh and frozen semen patients.
• ongoing pregnancies are significantly higher in ICSI patients when human sperm samples are
cryopreserved.
this suggests that properly performed cryopreservation selectively affects defective rather
than normal spermatozoa
The freezing-thawing procedure causes more damage in patients with alterations in semen
quality than that in patients with normal semen. However, once the oocyte is fertilized,
implantation and pregnancy rates are similar in patients with or without sperm anomalies.

21. Future issues
• Cryopreservation of human semen is extremely important to the field of male infertility.
However, there is dissension regarding the best cryopreservation protocol for human semen.
• To date there is no agreement in the literature on whether or not cryopreservation affects
sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-
thawing procedure.
• Further investigations are needed to fully understand the real influence of cryopreservation on
sperm DNA integrity and the impact of the use of cryopreserved spermatozoa on the
reproductive outcome, technical measures should be applied to provide maximum protection
to the male gametes: appropriate use of cryoprotectants before and sperm selection
technologies after cryopreservation seems to have the greatest impact on preventing DNA
fragmentation, thus improving sperm cryosurvival rates.

22. References
[1] Marlea Di Santo, Nicoletta Tarozzi, Marco Nadalini, and Andrea Borini. Human Sperm
Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART.
Advances in Urology. Volume 2012 (2012), Article ID 854837, 12 pages.
[2] 2014 Andrology and Embryology Review Course Manual of the American Board of
Bioanalysis (ABB).
[3] World Health Organisation: Department of Reproductive Health and Research WHO
laboratory manual for the examination and processing of human semen. 5th edition. 2010.
[4] Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted Reproductive
Techniques. 3rd ed. Vol. 1. In: Agarwal A, Erenpreiss J, Sharma R. Sperm chromatin assessment.
United Kingdom: Informa healthcare, 2009:67-84.

23. Thank You!