J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/10.47277/JIRB/8(3)/43  
Sperm Cryopreservation: Principles and Biology  
Yashaswi Sharma, Mona Sharma*  
Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi, India  
Received: 28/07/2020  
Accepted: 13/09/2020  
Published: 20/09/2020  
Abstract  
Cryopreservation is a widely used method to preserve sperm prior to any cytotoxic therapy or testicular surgical intervention to be  
used later for assisted conception. Despite of multiple modifications in cryopreservation protocols, the yield of post-thaw good quality  
sperm has not been improved much. There is little data regarding factors affecting cryopreservation techniques and outcomes. Present  
review focuses on the basic biology of cryopreservation, its current protocols and effects on sperm proteomic and epigenomic  
modifications.  
Keywords: Cryopreservation, Spermatozoa, Fertility preservation, Cryoprotective agents, Cryobiology  
this since at such low temperatures no detectable biochemical  
1
Introduction1  
activity is possible due to the lack of sufficient thermal energy.  
Moreover, the absence of liquid water limits all metabolic  
processes [5]. Thus, the storage in LN2 at -196°C has become  
the standard approach for sperm freezing.  
Human semen cryopreservation history stretches back to  
over 200 years. The first attempt to cryopreserve semen was  
made by Lazaro Spallanzani in 1776 by cooling it in snow [1].  
He is also known to have performed the first successful  
artificial insemination owing to the property of sperm  
recovering motility after freeze thaw. Further, in 1886,  
Mantegazza from Italy reported that human sperm survived  
cooling to −17°C for more than 4 days [1]. This led to the idea  
of semen banking for the military personnel to safeguard the  
ability to procreate in the future. Thereafter, in an attempt to  
increase the time duration for which sperm could be frozen,  
various studies were conducted which came up with their own  
individualized techniques and protocols. However, only after  
about sixty years of time, significant scientific progress was  
made with the discovery of glycerol as an effective  
cryoprotectant for sperm by Polge [2].  
Since then, glycerol has been regularly employed in animal  
sperm freezing, including that of humans and this is considered  
as a cornerstone in the field of fertility preservation. With the  
discovery of glycerol as sperm cryoprotective agent, there was  
a quick development in cryopreservation techniques that  
maintained sperm motility as well as its fertilizing ability post  
thaw. The earliest human offspring produced from  
cryopreserved spermatozoa were reported by Bunge and  
Sherman in 1953 [3]. Three pregnancies were reported on using  
the sperm which had been frozen in dry ice using glycerol [4].  
After a decade to this, Sherman further showed how sperm  
Sperm has small size with a large surface area. It is made  
up of considerable amount of water and has a high membrane  
fluidity as a result of the high sterol content of its membrane.  
The high amounts of polyunsaturated fatty acids (PUFA)  
present enables the sperm to survive rapid drops in temperature  
and makes it less susceptible to cold shock, possibly, since high  
cholesterol levels stabilize membranes during cooling. The  
lipid composition of the sperm plasma membrane is thus  
considered to be a major factor affecting the cryotolerance as  
well as the cold sensitivity of sperm [6].  
The small size and large surface area of sperm makes them  
less susceptible to potential damage of cryopreservation [6].  
However, the cellular survival is largely dependent on the  
cooling and thawing rate imposed during the cryopreservation  
process. The cooling rate must be such that it is slow enough to  
bypass intracellular ice crystal formation while fast enough not  
to cause immoderate cell dehydration followed by a rapid  
thawing rate. Solid crystalline structures called ice is formed on  
cooling water below its freezing point which is lighter and  
occupy a larger volume than liquid water. The expansion of  
these ice crystals during the solidification of water is the main  
factor that causes pressure and has a shearing effect on  
intracellular organelles resulting in appreciable damage [4].  
In addition to this, as water solidifies, the solutes that were  
present in the water are excluded from the so formed ice, as a  
result the freezing point of the rest of the solution is lowered.  
The solution gets highly concentrated as the temperature drops  
leading to osmotic shock. There may be further damage to the  
cell as a result of disparity in osmolality gradient, causing  
release of reactive oxygen species which is highly detrimental  
to sperms [4].  
o
could be stored for a longer duration if kept at -196 C in liquid  
nitrogen (LN2). Cryopreservation of sperm has become a  
common practice today for artificial insemination of animals  
and human assisted reproductive technology. But it was an  
outcome of 20 years of continuous research before the first  
commercial sperm banks were created in the USA [4].  
2
Basic biology of sperm cryopreservation  
Cryopreservation refers to the maintenance of cellular life  
The freeze-thaw process involves cooling and warming  
processes that may be lethal to the cells especially in the critical  
temperature range of -10°C to -60°C [7]. The sperm must  
undergo this temperature change two times during cooling and  
warming process. On warming, water re-enter the sperm and  
thus intracellular volume is restored, however, this invites the  
at subzero temperatures for a definite period of time [4]. Long  
term storage of cells including reproductive cells can be  
achieved if their metabolism is arrested and their cellular  
reactions retarded. The temperature of LN2 (-196°C) allows  
*
Corresponding authors: Mona Sharma, Associate Professor, Department of Reproductive Biology, All India Institute of Medical Sciences, New  
Delhi-110029, India. Contact No. 011-26594166; 9968147821. Email address: dr.mona18sharma@gmail.com  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/10.47277/JIRB/8(3)/43  
risk of intracellular ice crystal formation and subsequent cell  
it has been argued that conventional slow freezing leads to cell  
damage due to ice crystal formation, cell shrinkage, or osmotic  
changes [6].  
injury. Hence, any cryopreservation protocol ensuring good  
sperm viability post thaw should target on controlled cell  
volume and avoid membrane damage by inhibiting ice crystal  
formation [4].  
In an attempt to overcome these drawbacks of slow  
conventional cooling,  
a remarkable fast approach to  
cryopreservation called vitrification has been proposed. The  
technique has already been widely explored and found its uses  
on embryos and oocytes and lately also for spermatozoa. [16-  
3
Clinical applications of sperm  
cryopreservation  
2
1]. Different studies have described individual protocols using  
Sperm cryopreservation is extensively used today as a  
routine technique in the clinical settings for fertility  
preservation, infertility treatment and establishment of donor  
banks. Men can choose to preserve their semen prior to any  
circumstances that may impair their fertility such as cancer  
patients before commencing treatment such as chemotherapy  
or radiotherapy, before undergoing surgery that may affect  
testicular function or lead to any form of sexual dysfunction,  
before onset of certain diseases and conditions such as an  
autoimmune disease or AIDS known to compromise fertility  
and in adult men who are seeking sterilization or sex  
reassignment surgery in case they want to have children in the  
future [7-11].  
Moreover, sperm cryopreservation is also an important  
cornerstone in assisted reproduction technologies which  
primarily address infertility issues [12-14]. Patients with severe  
male infertility conditions like oligozoospermia or presence of  
intermittent motile spermatozoa are advised to freeze their  
sperms. Cryopreservation is also commonly used for surgical  
sperm recovery like percutaneous epididymal sperm aspiration  
different carriers and methods for standardization of the  
vitrification procedure. But regardless of the protocol  
proposed, the principle behind the process is based on freezing  
of sperm by direct immersing it into LN2 which is at a freezing  
o
temperature of -196 C. [4]. This bypass the formation of  
intracellular ice crystals and thus the ensuing damage to the  
cell. In vitrification, water solidifies as an amorphous glass-like  
structure, and not as ice [7]. Vitrifying requires extremely high  
cooling rates (>100,000 °C/min) which can be obtained with  
different specifically designed packaging systems such as the  
open pulled straws, Flexipet denuding pipette, Cryotop,  
Cryoleaf, Cryotip and other carrier devices [4]. An alternative  
protocol involves the direct dropping of spermatozoa  
suspension in liquid nitrogen [7]. Rapid cooling rates have  
prevented cell shrinkage and osmotic cell damage. Since  
cryopreservation of sperm by vitrification avoids ice formation,  
requires minimal equipment, is time efficient and cost  
effective, it stands as a promising alternative to conventional  
methods of freezing [21].  
(PESA), testicular sperm aspiration (TESA), or testicular  
sperm extraction (TESE) [15]. It also finds its applications in  
procedures like IUI, IVF and ICSI wherein sperm can be frozen  
from patients requiring assisted reproduction but face problems  
in sperm collection. Cryopreservation is also opted for patients  
with spinal cord injury coming for assisted ejaculation [4].  
Artificial insemination is a way forward for an infertile man  
with no live spermatozoa or a single mother with desire to  
conceive. Stored semen from healthy and fertile donors may  
result in successful pregnancy in cases of recurrent  
miscarriages. Also, simple long-term storage of known quality  
donor semen is advised in many cases [11].  
5
Use of Cryoprotectant  
Cryoprotectants are substances with high solubility used to  
protect biological tissue from cold shock during a freezing  
thawing process. They work by changing the solute  
concentration in the liquid phase by displacing water from  
intracellular to extracellular environment, lowering the  
freezing point of the solution. This limits water crystallization  
and thus cellular damage post-thaw/warm [4]. Depending upon  
their ability to cross the cell membrane, CPAs maybe of two  
types: permeating and non-permeating [7].  
Permeating CPAs are compounds with relatively low  
molecular weight (<100 g/mol) which freely cross membranes  
following osmolality gradient. Given to its ability to form  
hydrogen bonds with water, it prevents ice crystallization [4].  
Dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol  
4
Techniques with modified protocols  
Freezing techniques used in sperm cryopreservation  
involves two principal approaches- conventional freezing and  
vitrification. The conventional methods include slow freezing  
and fast freezing.  
(PROH) and glycerol are the most commonly used permeating  
cryoprotectants in sperm cryopreservation [22]. Almost all  
permeating CPAs for human sperm cryopreservation contain  
glycerol [22]. Glycerol, like other CPAs work by lowering the  
freezing point but also modify the lipid packing structure of cell  
membrane. Such induced changes bring about the reduction in  
the concentration of electrolyte in the unfrozen fraction [22].  
Non- permeating cryoprotective agents on the other hand, are  
large molecules that remain extracellular. They work by  
creating osmolality gradient by drawing water from within the  
cell and are mostly used in combination with permeating CPAs  
to decrease cytotoxicity. Sucrose, raffinose and trehalose are  
the common non-permeating cryoprotectants used for sperm  
Slow freezing can be manual or automatic and as the name  
implies is done in phases. In a number of steps, the sperm is  
subjected to progressive cooling over a 2-3-hour period [11].  
Manual method comprises of mixing the semen sample with an  
equal volume of cryoprotective agent (CPA) in a drop wise  
manner and then loading in straws or cryovials. It is further  
incubated at 40℃ for 10 minutes and placed 15-20 cm above  
the level of LN2 for 15 minutes and completed by immersing  
o
in LN2 at -196 C [4]. In the automated system, programmable  
o
freezers are used wherein software obtain cooling from 20 C to  
o
o
o
-
80 C at a rate of 1.5 C/min and then at 6 C/min. Once the  
cryopreservation [23]. Usually,  
a
combination of  
freezing is complete, the straws are plunged into LN2 at -  
cryoprotectants are used in routine cryobiology. In an attempt  
to find the optimal additive that significantly improves  
cryosurvival rates, the use of glycine, citrate, egg yolk and  
zwitterions have also been explored [4]. Almost all the  
commonly used cryoprotective agents in sperm freezing  
contain glycerol as a thermal shock protecting agent, sugars for  
energy and optimization of pH and osmolality, egg yolk for  
o
96 C. In vapor phase cooling, vapors of nitrogen that are  
1
constantly present around its tank is utilized for the cooling  
process. The vials are placed on cryocanes at certain specified  
heights above the liquid phase such that all vials are exposed to  
vapors equally. After a predetermined period of exposure, a  
cooling curve is attained [4]. Regardless of the method chosen  
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plasma membrane fluidity and integrity and antibiotics for  
safeguarding against microorganisms [22].  
clearly defined [30]. There is partial or complete disintegration  
of the acrosomal membrane with depletion of acrosomal  
content, swelling and thinning of the area between the cell  
membrane and the acrosome [7]. Changes in the middle piece  
and axial filament complex are also observed. Scanning  
electron microscopy studies report a significant increase of  
head and tail defects in post freeze sample where coiled and  
looped tails of sperms is the most common finding [32].  
Although the detrimental effects of freeze-thaw cycles on  
various sperm parameters and fertilization capacity is well  
elucidated, there are two opposite thoughts regarding the DNA  
damage imposed by such procedures. A set of studies suggest  
that cryopreservation of sperm exert a negative impact on DNA  
integrity and causes its fragmentation due to oxidative stress  
caused by ROS [33-36]. The second line of thought believes  
that sperm DNA integrity is not affected by freezing and  
thawing process [37-39].  
Studies carried out to assess the damage on DNA post  
cryopreservation utilize a battery of tests such as TUNEL,  
SCSA, SCD, Comet neutral or Comet alkaline which might  
help answer the discrepancies in findings of these studies. Also,  
disparity in the cryo-resistance of each different sample,  
protocol followed during the cryopreservation process, and the  
method of choice to assess DNA integrity may account for the  
non-consensus between studies.  
Although the use of cryoprotectants is prescribed with the  
aim to maximize cell survival, it has also been shown that  
freezing methods which use permeable cryoprotectants may  
lead to cell injuries [7, 21-23]. The CPAs are osmotically  
active and thus their addition as well as removal during the  
freeze thaw process can cause lethal mechanical stress to the  
cells [22]. The most well studied cause for this is by the  
formation of intracellular and extracellular ice crystals.  
Moreover, chemical toxicity of the cryoprotectants on  
membrane and cellular components is also a concern. [21].  
Permeable cryoprotectants are further hypothesized to have a  
negative influence on the genetic apparatus of spermatozoa  
[23]. Hence, some recent studies have been conducted with the  
aim to find the possibility of sperm cell freezing without the  
use of cryoprotectants [24, 25].  
6
Changes in sperm parameters during  
cryopreservation  
Although it has been well established that sperm are less  
susceptible to freezing damages than other cell types,  
significant structural and functional damage to sperm have  
been reported post cryopreservation. Thermal shock due to  
drastic changes in temperature, osmotic shock, dehydration of  
cells and formation of ice crystals are the most commonly  
reported mechanisms by which cryodamage to the cell is  
incurred [4]. The inevitable change involves the significant  
reduction in motility mostly due to membrane changes and  
acrosome degeneration among the others. A decrease in  
spermatozoa kinetics after thawing has been reported  
consistently in many studies [26-29].  
7 Other modifications in post-thaw sperm  
(
proteomic, transcriptomic/ epigenomic)  
The freeze thaw process is quite elaborative as it not only  
alters the sperm parameters but also the genes, mRNA stability,  
protein expression and the epigenetic content of the  
spermatozoa. In post thaw sperm samples, protein degradation  
and phosphorylation have been reported [6]. Protein  
phosphorylation has also been linked to capacitation like  
changes in sperm which is believed to shorten its lifespan.  
Protein expression differences have been reported in multiple  
proteins of boar sperm post thaw compared to its pre freeze  
samples [40]. Some proteins have been shown to increase in  
frozen thawed sperm such as AKAP3, superoxide dismutase 1  
Damage is incurred due to the peroxidation of the fatty  
acids present in the sperm plasma membrane. The lipid  
oxidation results in the inhibition of oxidative phosphorylation  
by loss of intracellular enzymes. The ultrastructure of the sperm  
mitochondria and plasma membrane is particularly susceptible  
to freezing damage as shown by studies employing electron  
microscopes [4]. The widespread damage to the cells is mostly  
either due to alteration in mitochondrial functions due to  
significant destruction of mitochondrial membrane or an  
alteration in the fluidity of the membrane leading to the  
liberation of reactive oxygen species (ROS) [30]. ROS have  
been well recognized as cell destructive agents most commonly  
causing single- or double-strand DNA breakage. They are also  
responsible for inducing apoptotic pathways in cell under low  
activity of antioxidant enzymes which subsequently leads to  
decreased sperm viability [31].  
The integrity of cell membrane is crucial for sustained  
functioning of the cell post freeze thaw. However, low  
temperatures have been suggested to alter the membrane  
proteins and carbohydrate structures either due to osmolality  
changes or intracellular ice crystal formation [7]. This may in  
turn impair ion transport and metabolism leading to cell  
disruption and consequently in reduction of sperm viability and  
fertilization capability [4].  
(SOD1), TPI1 and ODF2 proteins [41]. The expression levels  
of heat shock protein 90 (HSP90) having its direct role in  
motility of sperm is found to be significantly decreased after  
cryopreservation [42]. Significant changes in proteins related  
to motility, viability and acrosomal integrity of spermatozoa,  
such as mitochondrial aconitase hydratase (ACO2), alpha-  
enolase (ENO1), OXCT1, tektin1 (TEKT1), acrosome  
membrane-associated protein 3 (SPACA3), vimentin, etc. has  
been shown to change after freezing [43, 44].  
Cryopreservation has also been suggested to negatively  
influence the development of early embryo by increasing the  
risk of loss of important transcripts and mRNAprotein  
interactions in sperms [44, 45]. The correlation of sperm  
mRNA and early embryo development is shown for both  
humans and animals like pig. A number of transcripts having  
definite roles in human sperm fertility such as PRM1, PRM2,  
PEG1/MEST, eif2S1 and ADD1 have been reported to have  
decreased expression post cryopreservation [44]. Since damage  
to the sperm genetic material do not always restrict fertilization  
capability, mutations and defects may be evident only after  
sufficient growth of the embryo or on the development of fetus  
Another parameter affected by freezing is sperm  
morphology which mostly results due to the deformation in  
membrane structure due to dysregulated liquid influx [2, 7, 27,  
[46].  
2
9]. It has also been shown that freeze preservation of sperm is  
Some studies explain the possible cryogenic epigenetic  
followed by ultrastructural changes [11]. The most conspicuous  
of changes are reported in the acrosomal region wherein  
limiting membranes are found to be more wrinkled and less  
modifications in the sperm. Although most of these studies  
have been conducted on animal sperm samples, they report  
alterations in histone H1-DNA binding proteins, proteinDNA  
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disulphide bonds and high methylation in the vasa and cxcr4b  
involve stripping sperm from the protective seminal plasma and  
demonstrating the adverse effect of ROS on cell organelles  
including cell DNA [57]. Further, such damage has been shown  
to be successfully avoided by supplementing media with  
antioxidants.  
promoters in boar and zebrafish respectively [47]. A particular  
study on DNA methylation pattern of human sperm genes,  
however, report no changes pre and post freezing [48].  
However, there are studies suggesting that seminal  
leukocytes and damaged sperm in semen produce ROS which  
damage ejaculated sperm upon prolonged exposure leading to  
reduction in motility and viability of sperm [61, 62]. In  
accordance to this belief, it has been suggested that ejaculated  
sperm should be separated from the seminal environment as  
soon as possible.  
8
Solution to Cryoinjuries  
Cryoinjuries to sperm can be limited during the  
cryopreservation process by using certain substances that are  
known to have cryoprotective properties. Cryoprotectants, are  
one such substance which depending upon their size and  
permeability exert their cryoprotective effect by decreasing the  
freezing point of intracellular and extracellular water [22].  
They protect both cytoplasmic components as well as sperm  
membranes by forming a protective layer around them. Other  
additives that are reported to protect sperm against damage  
include antioxidants which primarily work to neutralize ROS  
generated during the cryopreservation process and thus  
improve post-thaw sperm function. The commonly employed  
antioxidants include vitamin E, vitamin C, catalase, l-carnitine,  
biotin, butylated hydroxytoluene, taurine, hyaluronic acid,  
resveratrol, honey, and nerve growth factor [49-53]. Antifreeze  
proteins and glycoproteins are another group of cryoprotective  
supplements that can be employed in sperm freezing that help  
to maintain the plasma membrane integrity by stabilizing the  
phospholipids and unsaturated fatty acids [50]. The use of such  
proteins is also reported to act by decreasing the freezing point  
and inhibiting ice crystal formation [22]. Although the  
beneficial role of these antifreeze compounds has not been well  
established for human sperm cryopreservation, promising  
results have been shown in animals. Other additives that help  
to reduce cryoinjuries include fatty acids, animal serum,  
nanoparticles or plant essential oils which are added to the  
freezing media to protect sperm against damage [6].  
10 Factors affecting outcome of freeze thaw  
cycle  
The outcome of a freeze thaw cycle of sperm is dependent  
on multiple factors. The type of cell undergoing freezing, its  
size and maturity, composition of the intracellular lipids,  
amount of water present, function and morphology all  
determine the cryosurvival potency of the frozen cell  
irrespective of the freezing method employed. Resistance to  
cryoinjuries is also dependent on sperm size, shape and lipid  
composition as it varies between different animals and between  
different species of the same animal [6]. The difference in lipid  
composition in terms of fatty acid profile and omega-3/ omega-  
6
ratio also decide their cryotolerance level [4].  
The pre-thaw properties of sperm are also likely to have an  
effect on the post-thaw quality of sperm. For instance, sperm  
with abnormal motility traits such as asthenozoospermia and  
oligoasthenozoospermia are found to be more susceptible to  
cryodamage. Studies have concluded that sub-normal semen  
samples do not withstand freezing and the yield of the sperm  
post-freezing bears a linear relationship with the pre-freeze  
quality [63, 64]. Also, the period of abstinence of sperm donors  
has been shown to affect the cryosurvival rate of post-thaw  
sperm. The subnormal samples are more prone to DNA damage  
induced during freeze-thawing and has a higher incidence of  
irregular chromatin organization and decreased resistance to  
thermal denaturation as compared to normal sperm samples  
Besides the use of supplements, other noble strategies to  
reduce cryodamage in sperm include the use of magnetized  
water as one of the components of freezing media which allow  
only small ice crystals to form on freezing, stress  
preconditioning of spermatozoa before cryopreservation to  
induce adaptation to stress and the use of low-level laser  
irradiation to affect the sperm's mitochondrial respiratory chain  
such that ATP production is increased while ROS decreased  
[65].  
Besides these factors, the method of cryopreservation  
employed (slow freezing or vitrification), the use or disuse of  
cryoprotectant, the type (permeable or non-permeable or mix  
of both), concentration of cryoprotectant or other additives  
used and the protocol followed significantly impacts the  
outcome of cryopreservation [4]. Among the other variation in  
protocols, the cooling and warming rate employed and the  
temperature at which the sample is plunged into LN2 and the  
subsequent sample storage temperature has a significant effect  
on the outcome of the freeze thaw cycle.  
[54, 55, 56].  
9
Use of sperm cryopreservation with or  
without semen  
The somatic cells contain antioxidants in cytoplasm which  
protect them from oxidative insult. During maturation  
however, sperm lose a large portion of their cytoplasm and as  
a result fall short on enzymes and repairing mechanism for  
oxidative damage leaving them susceptible to freeze-thaw  
damage by ROS [57]. However, it has been shown that the  
seminal plasma has an anti ROS activity against O2 and H2O2  
by virtue of its enzymes: superoxide dismutase, glutathione  
reductase/peroxidase and catalase [58]. It has also been  
proposed that the lipoproteins are responsible for stabilizing the  
plasma membrane by maintaining the optimal lipid  
composition. It has been observed that cryostability of the  
cellular and plasma membrane of sperm is increased during  
cryopreservation [57]. Studies on such antioxidant properties  
of human, rat and mouse seminal plasma have been described  
and proven [59, 60].  
11 Future of sperm cryopreservation  
The field of cryobiology is in a rapid state of flux and has  
found its applications in fertility medicine. Since it is a cutting-  
edge technology, it has  
a great potential for further  
development. It is also utmost important to assess the extent of  
sperm cryopreservation being used clinically for assisted  
conception. Although sperm cryopreservation has been used  
for preserving fertility in adult men, it further needs to find its  
application in fertility preservation in boys preceding puberty.  
The germ stem cells of the seminiferous epithelium in the testes  
called spermatogonial stem cells and even testicular tissue  
stand as promising alternatives to ejaculated spermatozoa  
which may be cryopreserved to be retrieved later [66,67,68].  
Also, sperm vitrification, which stands as a promising  
Hence, cryopreserving sperm with semen or seminal  
plasma has been supported by a number of studies for the above  
reasons. These ideas are further endorsed by studies which  
4
6
J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/10.47277/JIRB/8(3)/43  
invaluable technology in the field of reproductive medicine  
10. Multidisciplinary Working Group convened by the British Fertility  
Society. A strategy for fertility services for survivors of childhood  
cancer. Hum Fertil (Camb). 2003;6 (2):A1-A39.  
needs further studying. As of now only protocols describing the  
vitrification of very small volume of semen sample are  
reported; upto about 10 μL with isolation from LN2 and upto  
1
1. Di Santo M, Tarozzi N, Nadalini M, and Borini A. Human Sperm  
Cryopreservation: Update on Techniques, Effect on DNA  
Integrity, and Implications for ART. Adv Urol. 2012; 2012:1-12.  
2. Poongothai J. Etiology, investigation and treatment of Human  
men’s infertility. J. Infertil. Reprod. Biol. 2013; 1(2): 31-36.  
3
0 μL with direct plunging into LN2 [24, 38]. Although use of  
vitrification has been encouraged, still some studies have  
shown detrimental effects on post-thaw quality of gametes [69,  
1
7
0]. Shortcomings need to be studied and may be reflected in  
13. Sheykhhasan M., Ghias M. Semen quality and age-dependent  
changes among male participants with normal sperm count in  
Qom, Iran. . J. Infertil. Reprod. Biol.2016; 4(2):35-39.  
non-uniformity of the vitrification regime.  
1
4. Eftekhar M., Pourmasumi S., and Razi MH. Efficacy of rescue  
ICSI after total fertilization failure in conventional IVF. J. Infertil.  
Reprod. Biol.2013; 1(2):58-62.  
5. Gangrade BK. Cryopreservation of testicular and epididymal  
sperm: techniques and clinical outcomes of assisted  
conception. Clinics (Sao Paulo). 2013;68 Suppl 1(Suppl 1):131-  
140.  
1
2 Conclusions  
A thorough understanding of current concepts of sperm  
cryopreservation influenced by epigenomic and proteomic  
modifications will enable efficient clinical application in  
assisted reproduction. There is a serious need for the  
vitrification process to be standardized owing to its promising  
advantages over the conventional methods.  
1
16. Konc J, Kanyó K, Kriston R, et al. Cryopreservation of embryos  
and oocytes in human assisted reproduction. Biomed Res Int.  
2
014; 2014:307268 (1-9).  
Ethical issue  
1
1
7. Wilmut I. The low temperature preservation of mammalian  
embryos. J ReprodFertil. 1972;31 (3):513-514.  
8. Le MT, Nguyen TTT, Nguyen TT, et al. Cryopreservation of  
human spermatozoa by vitrification versus conventional rapid  
freezing: Effects on motility, viability, morphology and cellular  
defects. Eur J ObstetGynecolReprod Biol. 2019;234:14-20.  
19. Khalili M, Adib M and Ramezani M. Cryopreservation of human  
spermatozoa by vitrification: impacts on sperm parameters and  
apoptosis. FertilSteril. 2010;94(4):S108.  
Authors are aware of, and comply with, best practice in  
publication ethics specifically with regard to authorship  
(avoidance of guest authorship), dual submission, manipulation  
of figures, competing interests and compliance with policies on  
research ethics. Authors adhere to publication requirements  
that submitted work is original and has not been published  
elsewhere in any language.  
2
0. Berkovitz A, Miller N, Silberman M, Belenky M, Itsykson P. A  
novel solution for freezing small numbers of spermatozoa using a  
sperm vitrification device. Hum Reprod. 2018;33(11):1975-1983.  
Competing interests  
The authors declare that there is no conflict of interest that  
would prejudice the impartiality of this scientific work.  
21. He X, Park EY, Fowler A, Yarmush ML, Toner M. Vitrification  
by ultra-fast cooling at a low concentration of cryoprotectants in a  
quartz micro-capillary: a study using murine embryonic stem  
cells. Cryobiology. 2008;56 (3):223-232.  
Authors’ contribution  
All authors of this study have a complete contribution for  
data collection, data analyses and manuscript writing.  
2
2. Sieme H, Oldenhof H, Wolkers WF. Mode of action of  
cryoprotectants for sperm preservation. AnimReprod Sci.  
2
016;169:2-5.  
References  
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Permeating andNonpermeating Cryoprotectants for Mouse Sperm  
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