In-Vitro Application of Pentoxifylline Preserved Ultrastructure of Spermatozoa After Vitrification in Asthenozoospermic Patients Purpose: To evaluate the effect of in vitro application of pentoxifylline (PX) on sperm parameters and ultrastruc- ture after vitrification in asthenozoospermic patients. Materials and methods: A total of 30 asthenozoospermic semen samples (aged 25-45 years) were divided into four groups before vitrification, after vitrification, control (without PX) and experimental (with PX). In experi- mental group, each sample was exposed for 30 min to 3.6mmol/l PX and the control group without any treatment apposing in 370C for 30 min. After incubation, the samples were washed and analyzed again. Vitrification was done according to straw method. Eosin–nigrosin and Papanicolaou staining were applied for assessment of sperm viability and morphology, respectively. The samples without PX and post treatment with PX were assessed by transmission electron microscopy (TEM). Results: A significant decrease in sperm motility (P ≤ .001), morphology (11.47 ± 2.9 versus 6.73 ± 2.01) and viability (73.37 ± 6.26 versus 54.67 ± 6.73) was observed post vitrification, but sperm motility (19.85 ± 4.75 versus 32.07 ± 5.58, P ≤ .001) was increased significantly following application of PX. This drug had no significant (P >.05) detrimental neither negative effect on ultrastructure acrosome, plasma membrane and coiled tail statues of spermatozoa. Conclusion: Vitrification had detrimental effects on sperm parameters, but PX reversed detrimental effects on sperm motility. However, PX had no alteration on ultrastructure morphology of human spermatozoa after vitrifi- cation. Keywords: asthenozoospermia; pentoxifylline; vitrification; ultrastructure. INTRODUCTION Cryopreservation of human spermatozoa is widely used in many assisted conception units to preserve male fertility, for example before cytotoxic chemother- apy, radiotherapy or certain surgical treatments that may lead to testicular failure or ejaculatory dysfunc- tion(1). Vitrification is a method that can be useful to achieve the same purpose and does not use the special extenders. It is based on the ultrarapid freezing of the cell by quick immersion in liquid nitrogen. Moreover, it is a simple procedure that requires less time and prob- ably will become safer and more cost-effective than conventional freezing(2,3). A normal motility pattern in ejaculated spermatozoa is a basic condition for male fertility. Because adenosine triphosphate (ATP) sup- ports the chemical–mechanical coupling catalyzed by dyneins (specific ATPases of the flagellum), it is usu- ally accepted that both glycolysis and oxidative phos- phorylation are required for optimal sperm function(4). Thus, it is supposed that impaired motility might follow a bioenergetic shortage, although other mechanisms are still possible(5). Mohamed et al. showed the significant affection of the progressive motility, viability and mitochondrial membrane potential of human sper- matozoa by cryopreservation. Although, both con- 1Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Rome, Italy. *Correspondence: Research and Clinical Center for Infertility, Safayeh, Bou-Ali Ave., BOX: 89195-999, Yazd, Iran. Tel.: +983518247085. Fax: +983518247087. E-mail: Khalili59@hotmail.com. Received August 2016 & Accepted June 2017 ventional slow and the vitrification techniques had similar results, vitrification is faster, easier with less toxicity and costs(6). Kuznyetsov showed that vit- rification was an alternative method for freezing human spermatozoa without toxic permeable cryoprotectants (CPAs). It can be used to optimize the vitrification and post thaw recovery of a small number of spermatozoa in normozoospermic and severe oligozoospermic sam- ples(7). Pentoxifylline (PX) is a non-specific phosphodiesterase (PDE) inhibitor of the methylxantine group, inhibiting the breakdown of cyclic adenosine monophosphate (cAMP). It is known that intracellular cAMP concentra- tion plays a central role in sperm motility. It is consid- ered to be a sperm movement enhancer, hyperactivation agent, inhibitor of reactive oxygen species formation and acrosome reaction-improving agent(8). Electron microscope has been used for examining the ultrastructure of spermatozoa. Studies by means of TEM showed that cryopreservation has deleterious effects on spermatozoa, especially on plasmalemma, acrosomes and tails(9,10). After thawing, the rate of spermatozoa that were considered normal by means of TEM evaluations decline. Among defined abnormalities, the defects of acrosomal change and subacrosomal swelling increase. SEXUAL DYSFUNCTION AND INFERTILITY Ali Nabi1, Mohammad Ali Khalili1*, Ali Reza Talebi., Esmat Mangoli1, Nahid Yari1, Stefania Annarita Nottola2, Selenia Miglietta2, Fatemeh Taheri1 Vol 14 No 04 July-August 2017 4038 Acrosomal change defect, which is described by natu- ral equatorial acrosomal content but an altered apical acrosomal region. Apical head alterations in acrosomal change defect are lack of stability, loss of acrosomal content and appearance of vesiculations. Another ab- normality was subacrosomal swelling, which is charac- terised by detachment of the inner acrosomal membrane from the nuclear envelope and filamentous widening of the subacrosomal space(10,11). So far, some studies eval- uated the effects of PX on cryopreserved spermatozoa and were focused mainly on post thaw sperm motility and characteristics like velocity, head movements, and linearity(1,8,12) rather than ultrastructure of it. In this study, we attempt to evaluate the effect of In-vitro ap- plication of PX on sperm parameters and ultrastructure after vitrification in asthenozoospermic patients. MATERIAL AND METHODS Study Population A total of 30 asthenozoospermic semen samples were selected according to WHO criteria for sperm total motility of <32%. Sperm specimens were obtained by masturbation into sterile cups. All subjects were asked to abstain from ejaculation from 2 to 3 days before col- lection. After liquefaction at 37ºC, a small aliquot was removed from each specimen and the sperm parameters were determined(1). Inclusion and exclusion criteria In this study, smokers, patient with varicocele and pa- tients with history of varicocelectomy were excluded. The patients agreed to participate in this project via fill- ing out the consent forms. The ethics committee of the institution approved this study. Evaluation of sperm parameters The seminal samples were prepared using the density gradient technique. Sperm parameters of motility, vi- ability and normal morphology (%) were evaluated for 100 spermatozoa in each sample. Sperm motility was assessed using Makler chamber and light microscopy (Olympus, Tokyo, Japan). Motility was expressed as % of progressive and non-progressive. Sperm viability and morphology were evaluated by Eosin and Papan- icolaou staining tests, respectively(13). Assessments of sperm parameters were carried out before vitrification (group I), after warming (group II), after the warming and without PTX treatment (group III), and PTX treat- ment (group IV). Final analysis of sperm parameters and ultrastructure were undertaken (Figure 1). Vitrification and Warming Vitrification was done by straw to straw method accord- ing to Isachenko et al(14). In this process, the suspension was mixed with Ham’s F10 medium supplemented with 5% HSA/0.5 mol/l sucrose (1:1) at room temperature, 100 μl of the sperm suspension was transferred into a 0.25 ml plastic straw maintained horizontal. Then, it was placed into 0.5ml straws and sealed at the end, and were immersed directly into liquid nitrogen. At least after one week for warming, after cutting one end side of the straw, the samples were vacated in pre-warmed Ham’s F10 with 5% HSA. The simple-wash method was applied and sperm parameters were evaluated ac- cording to WHO criteria(14). Incubation with pentoxifylline A stock solution of PX (Sigma, USA) in distilled water was prepared each week and kept at 4°C until used. The samples were divided into two groups of control and Vol 14 No 02 March-April 2017 3029 PX and ultrastructure of human spermatozoa-Nabi et al. Variables* Before vitrification (group III) Mean ± SD After warming (group IV) Mean ± SD P value Progressive motility (%) 40.57 ± 6.49 21.03 ± 4.57 <.001 Non progressive motility (%) 14.57 ± 3.23 15.63 ± 3.22 .097 Immotile (%) 43.47 ± 9.33 63 ± 5.32 <.001 Normal morphology (%) 11.47 ± 2.9 6.73 ± 2.01 <.001 Viability (%) 73.37 ± 6.26 54.67 ± 6.73 <.001 Table 1. The results of sperm analysis between before vitrification and after warming Abbreviations: PX, pentoxifylline; SD, Standard Deviation. *Values are mean ± SEM. Variables* Without PX treatment (group III) Mean ± SD PX treatment (group IV) Mean ± SD P value Progressive motility (%) 19.87 ± 4.75 32.07 ± 5.58 <.001 Non progressive motility (%) 15.63 ± 3.76 14.97 ± 3.7 .219 Immotile (%) 64.3 ± 5.37 53.23 ± 6.16 <.001 Normal morphology (%) 6.63 ± 1.97 6.53 ± 1.97 .083 Viability (%) 53.07 ± 5.71 53.2 ± 5.85 .161 Abbreviations: PX, pentoxifylline; SD, Standard Deviation. *Values are mean ± SEM. Table 2. The results of sperm analysis between Without PX treatment and PX treatment Sexual Dysfunction and Infertility 4039 experimental. In experimental group, each sample was exposed for 20 minutes to 3.6mmol/l PX at 370C and the control group in 370C for 30 minutes. After incu- bation, the samples were washed and analyzed again(1). Transmission Electron Microscopy (TEM) The samples without PX (group III) and post treatment with PX (group IV) were assessed by TEM. For this goal, spermatozoa were fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) for 2 h at 4°C. The sperm samples were washed three times in PBS by cen- trifugation suspension at 1000×g for 10 minutes and post fixed in 1% osmium tetroxide for 1 h. The samples were then centrifuged at 1000×g for 10 minutes and the pellets were dehydrated in graded acetone. Finally, the pellets were embedded in Epon. Ultra-thin sections (50- 70 nm) were obtained with Leica EM UC7 ultra-mi- crotome and stained with uranyl acetate and lead citrate. The samples were observed with the TEM (Zeiss, EM 10) to evaluate intact (with acrosome), reacting (with ruptured and irregular outer acrosomal membrane), re- acted sperm (without acrosome), Nucleus , Vacuole and coil tail(10,15). Evaluations were accord- ing to Visual Atlas(16). Different parts of hundred sperm samples were studied and illustrated as the percentage of healthy sperm in Table 2. Statistical analysis Data were analyzed using SPSS version 20 software (SPSS, Inc., Chicago, IL, USA).The data were shown as mean ± SD. Paired-samples T test was used for compar- ison of sperm parameters between before vitrification and after warming, also this test was used for evaluated of sperm ultrastructure between without PTX treatment and PTX treatment. The term ‘statistically significant’ was used to signify a two-sided P value < .05 for sperm parameters and ultrastructure. RESULTS Table 1 shows sperm parameters between before vitri- fication and after warming samples. This table reveals that sperm motility, morphology and viability revealed significant differences (P < .001) between before vitri- fication and after warming. Table 2 shows the sperm parameters between without PX treatment and PX treat- ment. This table reveals that sperm motility had signif- icant differences (19.85 ± 4.75 vs. 32.07 ± 5.58, P ≤ .001) between without PX treatment and PX treatment. The data showed that vitrification impaired sperm pa- rameters and PX can reverse detrimental effects of vitrification on sperm parameters. Figure 2 shows the results of sperm analysis between after warming and PX treatment. The results of sperm ultrastructure evalu- ation between groups III and groups VI are presented in Table 3. There were insignificant differences (P >.05) between the groups with regards to acrosome, plasma membrane, vacuole, nucleus and coiled tail statues (Figure 3). DISCUSSION The findings of present study showed that almost all sperm parameters revealed significant decrease follow- ing vitrification. The decline in sperm parameters, es- pecially motility after cryopreservation, is a matter of current study, since this is a vital factor affecting post freezing–thawing success rate. PX indirectly increases intracellular cAMP concentration and plays a critical role in sperm motility improvement(17). The technique of vitrification is based on the ultra-rapid freezing of the cell by quick immersion in liquid nitrogen. Thereby, formation of intracellular ice crystals is avoided with- Figure 1. Schematic diagram for study design. Variables* Without PX treatment (group III) Mean ± SD PX treatment (group IV) Mean ± SD P value Acrosome (%) 38 ± 5.71 37.5 ± 5.56 .914 Plasma membrane(%) 32.5 ± 3.31 31 ± 3.46 .620 Nucleus (%) 46.75 ± 7.89 43.5 ± 8.06 .655 Vacuole (%) 48.5 ± 13.8 47.5 ± 15.1 .929 Coiled tail(%) 64.5 ± 9.39 67.25 ± 9.17 .621 Abbreviations: PX, pentoxifylline; SD, Standard Deviation *Values are mean ± SEM. Table 3. The results of sperm ultrastructure evaluation between without PX and with PX groups. PX and ultrastructure of human spermatozoa-Nabi et al. Vol 14 No 04 July-August 2017 4040 in sperm cells(2). Morris discussed that the intracellular compartment of the cell will vitrify during cooling and will not devitrify until approx-30ºC during warming. This caused the outer surface of the plasmalemma being exposed to the stresses of osmotic shock, therefore kills the sperm cell. Ultrastructure analysis of rapidly cooled samples during warming showed wide ice recrystalliza- tion occurred around the cell during the warming phase. At 40 ºC, the solution around the cell would be fluid, but the intracellular compartment would still be a glass, this occasion cause extensive membrane impairment. It is described that rapid rates of cooling has frequently been elated to the formation of intra cellular ice and cel- lular damage(18). Iasachenko et al. reported function of mitochondria decreased after vitrification(19). Assuming that oxida- tive phosphorylation and proton transport are mem- brane-bound phenomena, it is possible that ATP pro- Figure 2. The results of sperm analysis between after warming and PX treatment Abbreviations: PX, pentoxifylline Figure 3. Ultra micrograph of human spermatozoa (A) Without PX, shows acrosome discharged completely and vesiculating, the remnant of plasma membrane was seen. The midpiece disrupted. Thin arrow shows the arrangement of tubulin in was section of tail disrupted and two sections of tail adherent together that means rolled tail. Thick arrow shows two sections of tail adherent together that means rolled tail. The sperm was seen morbid. (B) Without pentoxifylline (PX), Nucleolus show with large vacuoles, acrosome intact. Thick Arrow shows several sections of tail ad- herent together that means rolled tail. Thin arrow shows the arrangement of tubulin in was section of tail disrupted and several sections of tail adherent together that means rolled tail. (C) Addition with PX, Acrosome shows partly intact. Disrupted midpiece and tail and shows several sections of different area of tail meaning rolling tail. (D) Treatment with PX, acrosome discharged completely, the remnant of plasma membrane was seen. The plasma membrane midpiece was swollen. One vacuoles is seen in chromatin. Thick arrow shows a vesiculated acrosome. PX and ultrastructure of human spermatozoa-Nabi et al. Sexual Dysfunction and Infertility 4041 duction would be reduced. Also it is probable that change of the energy availability or damage to the axonemal components could contribute to the cause of motility failure after cryopreservation(20). In this study we observed some nuclear defect that were not statis- tically significant. After cryopreservation, the structure of chromatin is less condenses. The number of nuclear vacuole was increased and chromatin may have granular view. In some spermatozoa, a vacuole may expand and cause chromatin to marginalize. There are two factors may cause these expansions: ice crystal formation and osmotic shock. Ice crystal in nuclear was not formed, because this structure is highly viscose and intracellular ice was not occurred. But, osmotic shock is more proba- ble. The marginalized chromatin is the sign of apoptosis according to Baccettişs study(21). No significant differ- ences in the DNA integrity of prepared spermatozoa was reported related to the freezing method or presence of a cryoprotectant(19). Recently, reported vitrification did not show any adverse effect on DNA integrity of human spermatozoa with TUNEL test(2). Simultaneous- ly, more study should be done to confirm that the shape of chromatin causes DNA damage or not. Our results confirm the findings of Moein et al.(22) that patients with poor sperm motility can benefit from PX added straight to the washed semen specimens. Johan- son and colleagues studied the role of PX-treated cry- opreservation ejaculates on pregnancy rate. The higher pregnancy rate in the PX group proved that stimulated sperm motility is good predictor of fertility potential in an infertility treatment program(23). There are some studies that are in line with our results. Esteves et al. showed that the use of PX improves the capability of thawed spermatozoa to undergo the acrosome reaction in reaction to calcium ionophore. Also, the treatment of poor quality human sperm with PX may enhance post- thaw sperm fertilizing ability(1). Also, Rashidi et al. ob- served an increase of motile spermatozoa and fertiliza- tion rate after addition of 3 mmol/L PX to mouse sperm cells(24). PX has been used as a useful compound prior to oocytes injection for improving ICSI outcome in asthe- nozoospermic patients(17). Since, the motility of sperm cells is reduced after freezing, adding PX as a stimulant factor can improve it. To the best of our knowledge, this is the first investigation on the relationship between sperm ultrastructure and administration of the PX after vitrification in asthenozoospermic patients. Our results showed that treatment of spermatozoa with PX after thawing did not have significant detrimental effect on sperm ultrastructure (acrosome, plasma membrane and coiled tail statues). CONCLUSIONS PX had no alteration on ultrastructure morphology (acrosome, plasma membrane and coiled tail statues) of spermatozoa post vitrification of asthenospermia sam- ples. However, vitrification has detrimental effects on human sperm parameters of motility, morphology and viability, but PX reversed detrimental effects on sperm motility. ACKNOWLEDGEMENTS This study was supported by a grant from the Research and Clinical Center for Infertility, Shahid Sadougi Uni- versity of Medical Sciences, Yazd, Iran. The authors specially thank Dr. Iman Halvaei and Dr. Azam Agha- rahimi for their skillful assistance during the course of study. CONFLICT OF INTEREST None declared REFERENCES 1. Esteves SC, Spaine DM, Cedenho AP. 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