Sperm Cryopreservation

Dr. ChandanM.sc. PhD

Senior Embryologist ARC

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

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

Why sperm cryopreservation isperformed ? 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 surgeries4. 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.

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.

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.

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

Techniques for Cryopreservation2- 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.

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.

Straw holder

Straw or vails Forceps

Syringe

Vistube

Requirments for Cryopreservation

Tanks

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.

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

Guidelines

How to avoid Negative Impacts

Maintenance of media.Standard SOPTrained staffEquipped lab

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.

How to optimize semen cryopreservation ? Factors affecting the optimization of human sperm preservation:

Technical factors Biological factorsCryopreservation medium

Genetic factors

Addition/removal of cryoprotectant

Sexual abstinence

Cooling rate Seminal fluid quality packing Seminal quality Storage Thawing storage temperature

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).

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.

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.

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.

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.

Thank You!