Scholarly article on topic 'Clinician-induced (iatrogenic) damage incurred during human infertility treatment: Detrimental effects of sperm selection methods and cryopreservation upon the viability, DNA integrity, and function of human sperm'

Clinician-induced (iatrogenic) damage incurred during human infertility treatment: Detrimental effects of sperm selection methods and cryopreservation upon the viability, DNA integrity, and function of human sperm Academic research paper on "Basic medicine"

Share paper
OECD Field of science
{"Assisted reproductive technology" / "Iatrogenic damage" / Sperm / Gametes / Cryopreservation / Freeze / Thaw}

Abstract of research paper on Basic medicine, author of scientific article — Suseela Yelumalai, Junaid Kashir, Celine Jones, Hani Bagheri, Su Lin Oo, et al.

Abstract Human infertility is becoming increasingly prevalent and is now estimated to affect 10% of couples worldwide. Although phenomenal development in assisted reproductive technology (ART) has yielded a powerful and sophisticated discipline with which to combat many types of human infertility, success rates vary markedly and rarely exceed 40%. Mounting evidence suggests that laboratory techniques used routinely in ART may inadvertently impart detrimental effects upon gamete and embryo viability and competence. In this mini-review, we discuss how routine cryopreservation methods, commonly used in ART, may exert iatrogenic (clinician-induced) damage upon sperm structure, DNA integrity, and function. It is recommended that future research programmes aim to refine or replace current cryobiology protocols in order that the efficacy of ART can be optimised accordingly.

Academic research paper on topic "Clinician-induced (iatrogenic) damage incurred during human infertility treatment: Detrimental effects of sperm selection methods and cryopreservation upon the viability, DNA integrity, and function of human sperm"

F V't I

Document heading 10.1016/S2305-0500(13)60052-4

Clinician-induced (iatrogenic) damage incurred during human infertility treatment: Detrimental effects of sperm selection methods and cryopreservation upon the viability, DNA integrity, and function of human sperm

Suseela Yelumalai*, Junaid Kashir*, Celine Jones, Hani Bagheri, Su Lin Oo, Laura McLaren, Kevin Coward*

Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU. UK


Human infertility is becoming increasingly prevalent and is now estimated to affect 10% of couples worldwide. Although phenomenal development in assisted reproductive technology (ART) has yielded a powerful and sophisticated discipline with which to combat many types of human infertility, success rates vary markedly and rarely exceed 40%. Mounting evidence suggests that laboratory techniques used routinely in ART may inadvertently impart detrimental effects upon gamete and embryo viability and competence. In this mini-review, we discuss how routine cryopreservation methods, commonly used in ART, may exert iatrogenic (clinician-induced) damage upon sperm structure, DNA integrity, and function. It is recommended that future research programmes aim to refine or replace current cryobiology protocols in order that the efficacy of ART can be optimised accordingly.

Contents lists available at ScienceDirect

Asian Pacific Journal of Reproduction

Journal homepage:


Article history: Received 26 December 2011 Received in revised form 5 January 2012 Accepted 4 March 2012 Available online 20 March 2012


Assisted reproductive technology

Iatrogenic damage




1. Assisted reproductive technology (ART)

Infertility affects approximately 10% of couples globally and has led to phenomenal growth in a series of clinical laboratory techniques designed to combat such conditions, collectively termed assisted reproductive technology (ART)[1,2], resulting in the birth of more than 4.6 million babies worldwide. Infertility may arise as a result of male or female factors, a combination of both factors, or may arise for unknown reasons (idiopathic infertility[2]. Female infertility often occurs as a result of mechanical factors such as endometriosis, uterine lining defects,

Corresponding author: Kevin Coward, Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU. UK. Tel: 01865 782878

E-mail: #These authors contributed equally to this work.

anovulation, or cervical polyps and cysts[3], while male factor infertility is commonly associated with significant reductions in seminal sperm (azoospermia), reductions in sperm concentration (oligozoospermia), poor sperm motility (asthenozoospermia), or abnormal sperm morphology (teratozoospermia). Other major causative male factors include varicocele (anomalous enlargement of veins in the scrotum resulting in compromised blood drainage in the testicles), cryptorchidism (absence of one or both testes from the scrotum), or idiopathic factors[4].

While the clinical and laboratory aspects of ART have evolved enormously since their first successful implementation in 1978, global pregnancy and live birth rates remain relatively low and rarely exceed 40%[5]. Pregnancy and delivery via routine conventional ART procedures such as in vitro fertilisation (IVF; whereby sperm and oocytes are co-incubated in culture media) or intracytoplasmic sperm injection (ICSI; whereby a single

sperm is microinjected into the ooplasm) remain as low as 22.4% and 23.3% respectively!5]. As the number of couples seeking infertility treatment increases, it is essential to further develop ART success rates to improve chances of successful conception^.

Over recent years, the unpredictable success rate of ART has drawn much attention, particularly in terms of the potential for ART to promote clinician-induced (iatrogenic) damage to gametes and embryos. Characterising the extent of such damage and identifying potential sources would provide an opportunity to upgrade, modify, develop and improve routine protocols[7]. ART procedures such as IVF and ICSI are performed on pre-treated/manipulated gametes outside of their natural environment, mainly to enhance and facilitate the fertilisation process. For example, oocytes are normally exposed to the enzyme hyaluronidase prior to ICSI to remove cumulus cells, followed by mechanical corona removal to expose the oocyte for maturity identification and injection. Semen is also extensively processed in order to pre-select good quality sperm exhibiting high motility, normal morphology and concentration. It is a distinct possibility that such manipulative techniques may inadvertently compromise important features relating to gamete integrity, structure, and function.

2. Oxidative stress and DNA fragmentation

Reactive oxygen species (ROS) like hydrogen peroxide are oxygen-derived molecules that have the capacity to act as powerful oxidants and are normally formed in low concentration in the both the male and female genital tracts. Oxidative stress, an elevation in the steady-state levels of ROS that exceeds the body's antioxidant defences[8,9], has been implicated in a number of different reproductive scenarios ranging from endometriosis to oocyte maturation, as well as being indicated in the etiology of defective embryo development, and a number of sperm conditions such as asthenozoospermia, and sperm DNA damage (Figure 1)[9-11]. While it is generally accepted that it is difficult for an in vitro system to mimic the exact physiological conditions of an in vivo system, multiple factors inherent in an ART setting may lead to elevated levels of oxidative stress and a suboptimal ART outcome[9,i2].

Oxidative stress can induce damage to sperm membranes and DNA, ultimately leading to non-viable sperm. The use of damaged sperm in ART has been implicated in alterations in oocyte and/or embryo development. Critically, however, sperm selected for use in ART most likely originate from an environment experiencing oxidative stress. It follows, therefore, that DNA damage may have already occurred in a large percentage of such sperm prior to semen processing[9,i4]. Similarly, oocytes and embryos may contribute to increased levels of ROS due to inadequate protective antioxidant mechanisms present in vivo. Indeed, cells cultured in vitro may be exposed to a relative "hyperoxic" environment compared to conditions in


Figure 1. Representative micrographs of human sperm following an electrophoresis comet assay for DNA fragmentation. The majority of DNA from normal sperm migrates as a single cohesive mass, while in sperm with DNA fragmentation, a comet-like structure is created with the fragmented DNA forming the "tail". The further the migration, the more profound the damage produced. A, B and C represent undamaged DNA, intermediate DNA damage, and extensive DNA damage in human sperm, repectively. Figure was reproduced from Enciso et al.[13] with permission.

3. Effects of ART treatment upon sperm

The perceived role of sperm has tended to be generalised to the mere transportation of paternal DNA into the oocyte. However, sperm play a crucial role which extends beyond the early stages of fertilisation, including embryogenesis, and the events leading to implantation[16]. In an ART treatment, semen samples are subjected to a variety of processing methods and chemicals (e.g., sperm washing, retardation of motility by polyvinylpyrrolidone, cryopreservation), in order to enhance sperm parameters prior to use, to facilitate the fertilisation procedure, and to maximise the chances of successful fertilisation. However, it follows that each of these treatments may impart detrimental effect upon the sperm being processed.

3.1. Sperm washing as a means of selecting sperm for ART

Sperm preparation techniques such as density gradient wash (DGW) and swim-up (SUP) are routinely used in ART to produce a processed sample that is enriched with mature, motile, and morphologically normal sperm[17]. SUP depends on a migratory approach following the formation of a sperm pellet by brief centrifugation, whereby sperm with the greatest motility "swim-up" into culture medium, while DGW depends on sperm density and sedimentation[i8]. DGW and SUP sperm isolation techniques may play a role in selecting sperm without DNA damage, and they are able to select non-fragmented sperm following cryopreservation[i8,i9]. Sperm quality has been shown to be superior following density gradient compared to SUP processing[2°,21].

However, DGW and SUP require centrifugation, which may increase sperm ROS production, thereby damaging not only the sperm membrane which affects motility and fertilising capability but also sperm DNA[22]. Furthermore, time-dependant incubation of prepared sperm at RT or 37

°C reduces sperm motility and also significantly increases sperm DNA fragmentation and chromatin decondensation^.

3.2. Alternative methods for sperm selection

Several newer selection methods have been developed with the objective of improving sperm preparation protocols for ART, aiming to isolate mature, structurally intact, and non-apoptotic spermatozoa with high DNA integrity[17]. One group of new selection techniques separate sperm by size and electronegative charge[17,24], do not involve centrifugation and may avoid the generation of ROS[17,25]. Another sperm preparation technique, based on the externalisation of phosphatidylserine to the outer surface of the sperm membrane, a feature of early apoptosis, has been proposed as a basis for selection of non-apoptotic spermatozoa[17]. While not as efficient as established techniques such as DGW or SUP in selecting a large proportion of viable sperm, a combination of DGW and phosphatidylserine techniques resulted in the selection of sperm with a 50% reduction in DNA fragmentation rates[26], and significantly higher rates of survival following cryopreservation-thawing compared to samples prepared by DGW only[27].

Morphological selection techniques have also been proposed based largely upon sperm ultra-morphology. Assessing the sperm acrosome, post-acrosomal lamina, neck, tail, mitochondria, nuclear shape, and chromatin content, motile sperm organelle morphology evaluation has been utilised prior to a modified version of ICSI, termed intracytoplasmic morphologically-selected sperm injection[28]. While such an approach particularly benefits situations where identification of specific sperm organelles is required, e.g., the acrosomal component in cases of globozoospermia[29,30], there is a significant debate over the clinical viability of motile sperm organelle morphology evaluation followed by intracytoplasmic morphologically-selected sperm injection, considering that it is an elaborate and highly skilled procedure that involves prolonged sperm manipulation, adding significantly to routine ICSI processing times.

3.3. Cryopreservation

Cryopreservation is an essential tool for infertility treatment, and it is used not only in sperm donation programmes or in cases of poor semen quality but also following cases involving surgical sperm retrieval, or fertility preservation for cancer patients prior to treatment. Cryopreservation involves cooling of the sperm to subzero temperatures (-196 °C) by means of controlled-rate slow freezing or vitrification ('glass-like' solidification of cells), resulting in the temporary suspension of all biological activity (Figure 2). Samples treated in such a manner can be stored for long periods of time[31]. A combination of cryoprotectant solutions (glucose, sucrose, ethylene glycol, propylene glycol, dimethyl sulfoxide, and 2-methyl-2,4-pentanediol) is added to sperm samples in order to

avoid damage as a result of cryopreservation or thawing procedures. However, high cryoprotectant concentrations may result in cellular damage, cause biochemical changes, and induce lethal osmotic injury[32,33].

Cryopreservation techniques are under constant scrutiny to reduce potential risks to gametes and embryos. Newer cryoprotectants can replace water in cells during freezing[34], while vitrification has been utilised to replace slow-freezing technologies[35], allowing higher cryoprotectant concentrations within cells and faster freezing to preserve cells in a 'glass-like' state, which avoids the formation of damaging ice crystals[35,36]. Simplified or adapted vitrification protocols have reported even better survival and fertilisation rates compared to original protocols, and there is thus a significant hope for considerable further improvements in the near future[34,37-39].

- I: V & M

Figure 2. Students learning the intricacies of cryopreservation (A) and vitrification (B) as part of the MSc in Clinical Embryology at the University of Oxford, U.K.

Comprehensive training, coupled with the continued modification of such techniques as a result of investigative research, is highly likely to lead to improved success rates. For more information on the Oxford MSc in Clinical Embryology, see

4. Vulnerability of sperm DNA integrity to freeze-thaw cycles

Sperm cryopreservation can detrimentally affect sperm integrity and the dynamics of DNA fragmentation[40,41]. Increased levels of DNA fragmentation and DNA oxidative damage are often observed in thawed sperm[42]. Further incubation of post-thaw semen also results in a significant increase in DNA fragmentation^]. The act of cooling, freezing, and thawing also damages sperm membrane structures, leading to a significant reduction in sperm viability and motility. Cryopreserved sperm are prone to oxidative stress damage, leading to an increase in the number of ROS present, but with a coincident reduction in the level of important antioxidants such as glutathione[43]. Cellular lipids and proteins can be damaged by changes in the concentration of oxidants/antioxidants[43]. Furthermore,

oxidative stress is known to reduce conception rate, compromise embryonic development, and correlate to an increased risk of miscarriage and childhood mortality!44-47!].

However, while several studies indicate that sperm chromosomes and sperm viability were not affected by cryopreservation[48], other studies suggest that DNA fragmentation measurements are often under-estimated due to skewing of the TUNEL assay by DNA compaction and cell vitality[49]. Furthermore, fertilisation rates following ICSI were higher for fresh sperm (73.8%) compared with frozen sperm (68.7%) in a study carried out by Borges et al[50]. Cryopreserved asthenozoospermic (sperm with reduced motility) and oligoasthenozoospermic (low sperm count with reduced motility) were prone to further severe damage and consequently reduced fertilisation rates. Studies suggest that DNA fragmentation in sperm is mostly induced during sperm transport through the seminiferous tubules and the epididymis[9,51,52], mediated by ROS production by immature sperm and by the nitric oxide-producing epithelial cells that line the epididymis, where sperm are highly packed, facilitating transfer of free radicals from immature to mature sperm. This is particularly a reason for concern regarding ART protocols, where a similar mechanism may occur within the pellet of centrifuged semen where sperm would also be highly packed[9].

Interestingly, the eggs of marine invertebrates are known to undergo a 'respiratory burst' at fertilisation, involving an increase in levels of ROS within the zygote, suggesting that mitochondrial activity is stimulated by calcium release in the ooplasm following gamete fusion[53-55]. Intriguingly, Lopes et al. demonstrated that levels of oxygen consumption and ROS production were higher during fertilisation and cell cleavage stages within bovine zygotes[55], while Vandaele et al. indicated that the short term exposure of bovine cumulus-oocyte-complexes to levels of hydrogen peroxide led to improved embryo development[56]. While these studies indicate a possible role for ROS within embryo development, it is also possible that an abundance of ROS, introduced either within the oocyte or sperm as a result of ART, may account for deficient/abnormal embryogenesis. Indeed, cumulus cells are routinely removed for clinical procedures like ICSI, which may result in a deficiency of ROS within the zygote when it is required. It is therefore important that routine ART techniques associated with the potential induction of ROS production, such as overnight incubation and SUP, are investigated further in order to improve success rates.

5. Sperm function may be compromised by the use of cryopreservation

Besides DNA damage, cryopreservation may exert detrimental effects upon key sperm proteins associated with fertility[57]. However, specific effects of iatrogenic damage incurred by ART upon critical sperm proteins have not yet been extensively determined, mostly pertaining

to difficulties in determining specific functions for the numerous proteins involved in sperm viability. The human sperm proteome, characterised in great detail by Johnston et al.[58], is thought to contain more than 1 760 proteins, 1 350 being identified in the soluble fraction, 719 in the insoluble fraction, and 309 identified in both fractions.

A number of studies have begun to explore the human sperm proteome in order to identify immune-dominant sperm surface antigens[59], and to identify new biomarkers for male infertility[60]. Furthermore, Pixton et al. mapped the sperm proteome of a human male who had experienced failed IVF and discovered at least 20 proteins with abnormal expression patterns compared to fertile controls[6i]. Indeed, knock-out mouse models have shed light on numerous sperm proteins which may play crucial roles in spermatogenesis or sperm function during fertilisation (Table 1)[62]. More recently, the human sperm proteome has been used to compile a protein profile of capacitated versus freshly ejaculated human sperm[63]. However, while the effects of cryopreservation upon sperm DNA integrity has been exhaustively investigated, the effects of cryopreservation upon the vast array of sperm proteins thought to be involved in determining fertility remain largely un-investigated. As such, it is crucial that new studies aim to investigate related changes in vital sperm proteins. One key point to consider is how clinical activity/processing may exert influence over the quality of sperm DNA and protein, and ultimately, the 'fitness' of sperm to carry out their required reproductive function.

Kashir et al. reported that cryopreservation of human sperm significantly reduced levels of the sperm-specific oocyte activation factor, phospholipase C (PLC) zeta compared to fresh human sperm (by 20%-56%), possibly leading to a compromised fertilisation capacity of such sperm[6]. Indeed, specific levels of PLC zeta correspond to successful fertilisation and embryonic development, with an absence or reduction of this protein associated with male factor infertility[4,64]. Cao et al. further observed that levels of heat shock protein 90 were significantly reduced in thawed human sperm following cryopreservation[65]. Similarly, sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis on freeze-thawed boar sperm further revealed significantly reduced levels of heat shock protein 90[66]. Surface proteins that act as mediators in the fertilisation pathway[67], such as P25b, are reduced in bull sperm following cryopreservation with consequential reduction in fertilisation rates[68]. Moreover, 2-dimensional poly acrylamide gel electrophoresis (2D-PAGE) analysis of cryopreserved sea-bass sperm revealed degradation in a total of 21 proteins[69].

Prior to fertilisation, sperm endure several membrane and biochemical processes in the female tract, collectively known as capacitation, in order to facilitate penetration into the oocyte[70]. Among the many intracellular modifications occurring during sperm capacitation, are elevations in protein tryrosine phosphorylation (P-Tyr)[71] and cAMP[72]. In a recent study, cryopreservation was not found to affect the regulatory pathways of capacitation and the acrosome

Table 1

Key genes involved in fertilisation (modified from Matsuk & Lamb[62]).

Gene Effect of mutation upon functional outcome in knock-out mice Fertility status

Ace Compromised ability of sperm to fertilise ova Sub-fertile

Acr Delayed fertilisation in vitro Fertile

Adam2 Altered sperm protein expression and adhesion defects during fertilisation Infertile

Adam3 Altered sperm protein expression and adhesion defects during fertilisation Infertile

B4galtl Defects in sperm-egg interaction Infertile

Cadml Oligoasthenoteratozoospermia, block at elongating spermatid stage Infertile

Camk4 Impaired chromatin packaging during spermiogenesis Infertile

Cplxl Zona pellucida penetration defect in vivo secondary to abnormal acrosomal exocytosis Sub-fertile

Crem Defective spermiogenesis with aberrant post-meiotic gene expression Infertile

Crisp Decreased sperm-egg fusion in vitro Fertile

Fndc3a Spermatid defects with multinucleated syncytia resulting in azoospermia Infertile

HexbB Reduced in vitro fertilisation rates at older ages Progressive infertility

Mfge8 Defective sperm-egg binding Sub-fertile

Mmell Decreased fertilisation and early embryo loss Sub-fertile

pgapl Sperm-egg interaction defects, defective sperm entry into oviduct Infertile

Piwill Block in spermatogenesis beginning at the round spermatid stage Infertile

Plcbl Apparent decrease in fertilisation rates Sub-fertile

Plcd4 Defective zona-pellucida induced acrosome reaction Infertile

Prnd Dysmorphic spermatids with loss of motility of spermatozoa, defective acrosome reaction Infertile

Pvrl2 Sperm morphological defects, impaired egg binding and penetration Infertile

Rasipl Defective fusion of sperm with zona-free egg Infertile

Rbmxl2 Abnormal sperm incapable of fertilisation Infertile

Spaml Sperm defects in hyaluronic-acid binding Sub-fertile

Tyst2 Defective sperm-egg binding and motility in viscous media Infertile

Wipf3 Sperm head defects and decreased fertilisation Infertile

Zpbp Abnormal acrosome and globozoospermia Infertile

reaction; however, levels of P-Tyr and cAMP were significantly reduced in cryopreserved sperm[73].

6. Conclusions

Sperm selection and cryopreservation are commonly-used techniques in ART. However, current selection protocols may underlie an increased incidence of DNA fragmentation in sperm, thus compromising viability. Growing evidence suggests that cryopreservation may also exert a number of detrimental effects upon key sperm proteins involved in capacitation, acrosome reaction and fertilisation. However, our understanding of such effects is hindered by our limited understanding of the exact functional role of the multitude of proteins making up the sperm proteome.

The extensive development of ART has not only revolutionised clinical treatment of infertility but also facilitated significant advances in our understanding of human reproduction. However, pregnancy and birth rates resulting from such technology remain frustratingly low. Modifications of current ART protocols and the development of novel techniques associated with advances in science and technology are essential in order to provide improved clinical and diagnostic tools for patients. Recognising the potential for iatrogenic damage upon gametes and embryos

during ART will facilitate the improvement, development, and introduction of modified ART protocols which are highly likely to improve success.

Conflict of interest statement

We declare that we have no conflict of interest.


[1] Gnoth C, Godehardt D, Godehardt E, Frank-Herrmann P, Freundl G. Time to pregnancy: results of the German prospective study and impact on the management of infertility. Hum Reprod

2003; 18(9): 1959-1966.

[2] Nygren KG, Sullivan E, Zegers-Hochschild F, Mansour R, Ishihara O, Adamson GD, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) world report: assisted reproductive technology 2003. Fertil Steril 2011; 95(7): 2209-2222, 2222. e1-17.

[3] HFEA: Latest UK IVF figures-2009 and 2010. [Online]. Avaible from: Accessed on 17 June 2012.

[4] Healy DL, Trounson AO, Andersen AN. Female infertility: causes and treatment. Lancet 1994; 343(8912): 1539-1544.

[5] Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J,

Coward K. Oocyte activation, phospholipase C zeta and human infertility. Hum Reprod Update 2010; 16(6): 690-703.

[6] Kashir J, Heynen A, Jones C, Durrans C, Craig J, Gadea J, et al. Effects of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa.

Reprod Biomed Online 2011; 23(2): 263-267.

[7] Rienzi L, Vajta G, Ubaldi F. New culture devices in ART. Placenta 2011; 32(Suppl 3): S248-251.

[8] Agarwal A, Allamaneni SS. Role of free radicals in female reproductive diseases and assisted reproduction. Reprod Biomed

Online 2004; 9(3): 338-347.

[9] Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. Oxidative stress in an assisted reproductive techniques setting.

Fertil Steril 2006; 86(3): 503-512.

[10] Goto Y, Noda Y, Mori T, Nakano M. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic

Biol Med 1993; 15(1): 69-75.

[11] Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 2001; 7(2): 175-189.

[12] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010; 42(10): 1634-1650.

[13] Enciso M, Sarasa J, Agarwal A, Fernandez JL, Gosalvez J. A two-tailed Comet assay for assessing DNA damage in spermatozoa.

Reprod Biomed Online 2009; 18(5): 609-616.

[14] Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003; 79(Suppl 3): 1597-1605.

[15] Nasr-Esfahani MH, Johnson MH. How does transferrin overcome the in vitro block to development of the mouse preimplantation embryo? J Reprod Fertil 1992; 96(1): 41-48.

[16] Barroso G, Valdespin C, Vega E, Kershenovich R, Avila R, Avendano C, et al. Developmental sperm contributions: fertilization and beyond. Fertil Steril 2009; 92(3): 835-848.

[17] Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum

Reprod Update 2011; 17(6): 719-733.

[18] Enciso M, Iglesias M, Galan I, Sarasa J, Gosalvez A, Gosalvez J. The ability of sperm selection techniques to remove single-or double-strand DNA damage. Asian J Androl 2011; 13(5): 764-768.

[19] Gosalvez J, de la Torre J, Lopez-Fernandez C, Perez-Gutierrez L, Ortega L, Caballero P, et al. DNA fragmentation dynamics in fresh versus frozen thawed plus gradient-isolated human spermatozoa. Syst Biol Reprod Med 2010; 56(1): 27-36.

[20] Allamaneni SS, Agarwal A, Rama S, Ranganathan P, Sharma RK. Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa. Asian J

Androl 2005; 7(1): 86-92.

[21] Niu ZH, Shi HJ, Zhang HQ, Zhang AJ, Sun YJ, Feng Y. Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF. Asian J Androl 2011; 13(6): 862-866.

[22] Tremellen K. Oxidative stress and male infertility-- a clinical perspective. Hum Reprod Update 2008; 14(3): 243-258.

[23] Matsuura R, Takeuchi T, Yoshida A. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa. Asian J Androl 2010; 12(5): 753-759.

[24] Ainsworth C, Nixon B, Aitken RJ. Development of a novel

electrophoretic system for the isolation of human spermatozoa.

Hum Reprod 2005; 20(8): 2261-2270.

[25] Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988; 9(6): 367-376.

[26] Donnelly ET, O'Connell M, McClure N, Lewis SE. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod 2000; 15(7): 1552-1561.

[27] Said TM, Grunewald S, Paasch U, Glander HJ, Baumann T, Kriegel C, et al. Advantage of combining magnetic cell separation with sperm preparation techniques. Reprod Biomed Online 2005; 10(6): 740-746.

[28] Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, et al. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril 2003; 80(6): 1413-1419.

[29]Check JH, Levito MC, Summers-Chase D, Marmar J, Barci H. A comparison of the efficacy of intracytoplasmic sperm injection (ICSI) using ejaculated sperm selected by high magnification versus ICSI with testicular sperm both followed by oocyte activation with calcium ionophore. Clin Exp Obstet Gynecol 2007; 34(2): 111-112.

[30] Sermondade N, Hafhouf E, Dupont C, Bechoua S, Palacios C, Eustache F, et al. Successful childbirth after intracytoplasmic morphologically selected sperm injection without assisted oocyte activation in a patient with globozoospermia. Hum Reprod 2011; 26(11): 2944-2949.

[31] Jain JK, Paulson RJ. Oocyte cryopreservation. Fertil Steril 2006; 86(Suppl 4): 1037-1046.

[32] Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod

Genet 2008; 25(8): 403-411.

[33] Vutyavanich T, Piromlertamorn W, Nunta S. Rapid freezing versus slow programmable freezing of human spermatozoa. Fertil

Steril 2010; 93(6): 1921-1928.

[34] Kim BY, Yoon SY, Cha SK, Kwak KH, Fissore RA, Parys JB, et al. Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development. Pflugers Arch 2011; 461(5): 515-526.

[35] Herrero L, Martinez M, Garcia-Velasco JA. Current status of human oocyte and embryo cryopreservation. Curr Opin Obstet

Gynecol 2011; 23(4): 245-250.

[36] Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner DK. 1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology. Hum Reprod 2007; 22(1): 250-259.

[37] Lane M, Gardner DK. Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 2001; 58(3): 342-347.

[38] Kim SH, Ku SY, Sung KC, Kang MJ, Kim SA, Kim HS, et al. Simplified EM grid vitrification is a convenient and efficient method for mouse mature oocyte cryopreservation. Yonsei Med J

2006; 47(3): 399-404.

[39] Lee DR, Yang YH, Eum JH, Seo JS, Ko JJ, Chung HM, et al. Effect of using slush nitrogen (SN2) on development of microsurgically manipulated vitrified/warmed mouse embryos.

Hum Reprod 2007; 22(9): 2509-2514.

[40] Gosalvez J, Lopez-Fernandez C, Fernandez JL, Gouraud A, Holt WV. Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol Reprod Dev 2011; 78(12): 951-961.

[41] Johnston SD, Zee YP, Lopez-Fernandez C, Gosalvez J. The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas. J

Androl 2012; 33(5): 1007-1015.

[42] Zribi N, Feki Chakroun N, El Euch H, Gargouri J, Bahloul A, Ammar Keskes L. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril 2010; 93(1): 159-166.

[43] Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology

2011; 62(1): 40-46.

[44] Merlob P, Sapir O, Sulkes J, Fisch B. The prevalence of major congenital malformations during two periods of time, 1986-1994 and 1995-2002 in newborns conceived by assisted reproduction technology. Eur J Med Genet 2005; 48(1): 5-11.

[45] Farr SL, Schieve LA, Jamieson DJ. Pregnancy loss among pregnancies conceived through assisted reproductive technology,

United States, 1999-2002. Am J Epidemiol 2007; 165(12): 1380-1388.

[46] Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril

2008; 89(4): 823-831.

[47] Aitken RJ, De Iuliis GN, McLachlan RI. Biological and clinical significance of DNA damage in the male germ line. Int J Androl 2009; 32(1): 46-56.

[48] Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci U S A 2001; 98(24): 13501-13506.

[49] Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl 2011; 34(1): 2-13.

[50] Borges E, Jr., Rossi LM, Locambo de Freitas CV, Guilherme P, Bonetti TC, Iaconelli A, et al. Fertilization and pregnancy outcome after intracytoplasmic injection with fresh or cryopreserved ejaculated spermatozoa. Fertil Steril 2007; 87(2): 316-320.

[51] Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod 2001; 16(9): 1912-1921.

[52] Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod 2005; 20(1): 226-230.

[53] Heinecke JW, Shapiro BM. The respiratory burst oxidase of fertilization. A physiological target for regulation by protein

kinase C. J Biol Chem 1992; 267(12): 7959-7962.

[54] Schomer B, Epel D. Redox changes during fertilization and maturation of marine invertebrate eggs. Dev Biol 1998; 203(1): 1-11.

[55] Lopes AS, Lane M, Thompson JG. Oxygen consumption and ROS production are increased at the time of fertilization and cell cleavage in bovine zygotes. Hum Reprod 2010; 25(11): 2762-2773.

[56] Vandaele L, Thys M, Bijttebier J, Van Langendonckt A, Donnay I, Maes D, et al. Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development.

Reproduction 2010; 139(3): 505-511.

[57] Lin C, Tsai S. The effect of cryopreservation on DNA damage, gene expression and protein abundance in vertebrate. Ital J Anim

Sci 2012; 11(1): 119-122.

[58] Johnston DS, Wooters J, Kopf GS, Qiu Y, Roberts KP. Analysis of the human sperm proteome. Ann N Y Acad Sci 2005; 1061: 190-202.

[59] Shetty J, Naaby-Hansen S, Shibahara H, Bronson R, Flickinger CJ, Herr JC. Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women. Biol Reprod 1999; 61(1): 61-69.

[60] Barratt CL. The human sperm proteome: the potential for new biomarkers of male fertility and a transformation in our understanding of the spermatozoon as a machine: commentary on the article Identification of proteomic differences in asthenozoospermic sperm samples by Martinez et al. Hum

Reprod 2008; 23(6): 1240-1241.

[61] Pixton KL, Deeks ED, Flesch FM, Moseley FL, Bjorndahl L, Ashton PR, et al. Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report.

Hum Reprod 2004; 19(6): 1438-1447.

[62] Matzuk MM, Lamb DJ. The biology of infertility: research advances and clinical challenges. Nat Med 2008; 14(11): 1197-1213.

[63] Secciani F, Bianchi L, Ermini L, Cianti R, Armini A, La Sala GB, et al. Protein profile of capacitated versus ejaculated human

sperm. J Proteome Res 2009; 8(7): 3377-3389.

[64] Ramadan WM, Kashir J, Jones C, Coward K. Oocyte activation and phospholipase C zeta (PLCzeta): diagnostic and therapeutic implications for assisted reproductive technology. Cell Commun

Signal 2012; 10(1): 12.

[65] Cao WL, Wang YX, Xiang ZQ, Li Z. Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism. Asian J Androl 2003; 5(1): 43-46.

[66] Huang SY, Kuo YH, Lee WC, Tsou HL, Lee YP, Chang HL, et al. Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa.

Theriogenology 1999; 51(5): 1007-1016.

[67] McLeskey SB, Dowds C, Carballada R, White RR, Saling PM. Molecules involved in mammalian sperm-egg interaction. Int Rev

Cytol 1998; 177: 57-113.

[68] Lessard C, Parent S, Leclerc P, Bailey JL, Sullivan R. Cryopreservation alters the levels of the bull sperm surface

protein P25b. J Androl 2000; 21(5): 700-707.

[69] Zilli L, Schiavone R, Zonno V, Rossano R, Storelli C, Vilella S. Effect of cryopreservation on sea bass sperm proteins. Biol Reprod

2005; 72(5): 1262-1267.

[70] Yanagimachi R. Fertility of mammalian spermatozoa: its development and relativity. Zygote 1994; 2(4): 371-372.

[71] Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, et al. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995; 121(4): 1139-1150.

[72] Parrish JJ, Susko-Parrish JL, Uguz C, First NL. Differences in the role of cyclic adenosine 3 ,5 -monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biol Reprod 1994; 51(6): 1099-1108.

[73] Pons-Rejraji H, Bailey JL, Leclerc P. Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis. Reprod Fertil Dev 2009; 21(4): 525-537.