Sperm Cryopreservation: Principles and Biology

J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/1 0.47277/JIRB/8(3)/43

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

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].

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].

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

J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/1 0.47277/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-

Clinical applications of sperm

cryopreservation

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

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].

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

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

in LN2 at -196 C [4]. In the automated system, programmable

freezers are used wherein software obtain cooling from 20 C to

80 C at a rate of 1.5 C/min and then at 6 C/min. Once the

cryopreservation [23]. Usually,

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

96 C. In vapor phase cooling, vapors of nitrogen that are

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

J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/1 0.47277/JIRB/8(3)/43

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].

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 mRNA–protein

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].

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, protein–DNA

J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/1 0.47277/JIRB/8(3)/43

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.

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-

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].

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

J Infertil Reprod Biol, 2020, Volume 8, Issue 3, Pages: 43-48. https://doi.org/1 0.47277/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. 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.

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,

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.

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.

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.

16. Konc J, Kanyó K, Kriston R, et al. Cryopreservation of embryos

and oocytes in human assisted reproduction. Biomed Res Int.

014; 2014:307268 (1-9).

Ethical issue

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.

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. Sieme H, Oldenhof H, Wolkers WF. Mode of action of

cryoprotectants for sperm preservation. AnimReprod Sci.

016;169:2-5.

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