- Email: [email protected]
Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential
Middle East Fertility Society Journal xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Middle East Fertility Society Journal journal homepage: www.sciencedirect.com
Original Article
Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential Sahabeh Etebary a, Nahid Yari a, Mohammad Ali Khalili a,⇑, Seyed Mehdi Kalantar b, Morteza Anvari a a b
Research and Clinical Center for Infertility, ShahidSadoughi University of Medical Sciences, Bouali Ave, Safaeyeh, Yazd, Iran Abortion Research Center, Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
a r t i c l e
i n f o
Article history: Received 30 April 2018 Revised 20 June 2018 Accepted 3 July 2018 Available online xxxx Keywords: Spermatozoa Azoospermia MSOME Cryopreservation Vacuoles DNA fragmentation
a b s t r a c t Background: There are difficulties associated with testicular sperm freezing. Different methods of sperm cryopreservation developed. Not enough detailed studies about the real efficacy of these techniques exist. For sperm morphologic assessment, motile sperm organelle morphology examination (MSOME) is able to identify not only conventional morphological sperm alterations but also sperm head vacuoles. Objective: To assess the effect of cryopreservation on testicular spermatozoa vacuoles by MSOME and its correlation to DNA fragmentation and mitochondrial membrane potential. Materials and methods: After preparation, testicular sperm extraction samples of 15 azoospermic men, aged 20–40 years old were divided into three groups. Group 1 was assessed freshly. Group 2 was cryopreserved with vitrification method and Group 3 with cooling in liquid nitrogen vapor using droplet. Pre and post warming assessment in terms of spermatozoa head vacuoles by MSOME, DNA fragmentation, and mitochondrial membrane potential were performed. Results: The number of spermatozoa with no vacuoles significantly decreased after two cryopreservation techniques (P < 0.001). There were no significant differences between groups regarding small and large vacuoles (p > 0.05). DNA fragmentation and mitochondrial membrane potential increased after cryopreservation (P < 0.001). There was a significant positive correlation between spermatozoa with large vacuoles in group 2 and DNA fragmentation and a significant positive correlation between spermatozoa with no vacuole and mitochondrial membrane potential in groups 2 and 3. Conclusion: Cryopreservation affects spermatozoa vacuolization, DNA structure, and mitochondrial membrane potential. Using MSOME in the selection of post-thaw morphologically normal testicular spermatozoa for ICSI procedure will be of particular value. Ó 2018 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Cryopreservation of human spermatozoa was first described many years ago [1]. Despite the success of normal sperm cryopreservation, there are difficulties associated with testicular sperm freezing. What makes testicular sperm cryopreservation and using frozen-thawed sperm in ICSI cycles, beneficial and even necessary are difficulties associated with testicular sperm extraction (TESE) in azoospermic patients such as the need for repetition of biopsy
Peer review under responsibility of Middle East Fertility Society. ⇑ Corresponding author at: Bouali Ave., Safaeyeh Street, Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. E-mail addresses: [email protected] (M.A. Khalili), [email protected] (S.M. Kalantar).
in each ICSI cycle and probable risk of damage to the testis [2].Testicular sperm cryopreservation permits the couple to perform surgical procedures in different days and prevents the need to do testicular biopsy and oocyte retrieval on the same day. On the other hand, it avoids difficulties associated with expenses and repeating surgery [3,4]. There are different methods of sperm cryopreservation, such as slow freezing [5], freezing in liquid nitrogen vapor [6] and vitrification [7]. Rapid freezing is routinely used for the cryopreservation and it results in better post-thaw sperm motility and recovery in comparison to slow freezing [8]. Other data showed that vitrification may be more applicable in comparison to rapid freezing technique [9]. However, there are not enough detailed studies about the real efficacy of these techniques in large series [10]. On the other hand, intracytoplasmic sperm injection (ICSI) is usually performed under a magnification of x400, which only
https://doi.org/10.1016/j.mefs.2018.07.001 1110-5690/Ó 2018 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
2
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
enables the observation of major morphological defects. So in this way, the selection of the normal morphology spermatozoa don’t guarantee the selection of spermatozoa free of morphological abnormalities [11]. Many methods have been proposed to assess sperm morphology for improving diagnosis and treatment of male infertility. One of them is motile sperm organelle morphology examination (MSOME), which is based on a morphological analysis of isolated motile spermatozoa in real-time at high magnification (up to 6600), and was introduced by Bartoov et al. [12]. MSOME is able to identify not only conventional morphological sperm alterations but also more specifically sperm head vacuoles, which are considered as nuclear defects [12]. Even after several studies about the origin and creation of sperm head vacuoles, it is not clear if sperm vacuoles are normal features of the sperm head or degenerative structures with no physiological effect [11]. It has been proven that vacuole is a concavity extending from the surface of the sperm head to the nucleus through the acrosome that only a high magnification enables its visualization [13]. Some studies showed that sperm vacuoles are a physiological feature of the sperm head [13], while others suggested that presence of vacuoles is related to sperm chromatin packaging/DNA abnormalities [14,15] and lower mitochondrial membrane potential [16]. Also, studies proposed that selection of morphologically normal sperm with no vacuoles is related to better assisted reproductive technology (ART) outcomes [17]. The aim of present study was to assess the effect of vitrification and cooling in LN vapor on testicular spermatozoa vacuoles by MSOME and to evaluate the correlation between spermatozoa vacuoles, DNA fragmentation, and mitochondrial membrane potential.
transferred to a conical tube. 2 ml of the sperm-washing medium was added to the tissue fragments in the petri dish. With a 5-cc syringe attached to a 21-gauge needle, the tissue suspension was aspirated repeatedly. The suspension then was transferred to the same conical tube and let to settle for 5 min. The supernatant was transferred to a new conical tube. 1–2 ml of the spermwashing medium was added to the tissue pellet and mixed with a pipette. After 5 min the tissue fragments were settled and the supernatant was transferred to the conical tube and centrifuged at 400g for 10 min. 3–4 ml of RBC lysis buffer was added to the pellet and centrifuged again. The pellet was re-suspended in 3 ml of the sperm-washing medium and centrifuged at 400g for 10 min to wash off the RBC lysis buffer [18]. The supernatant was discarded and the pellet was suspended in 0.5 ml of the spermwashing medium. 2.3. Vitrification Vitrification was done according to Isachenko [7]. The sperm suspension was mixed gently with sperm freezing solution (Vitrolife, Sweden) briefly at a ratio of 1:1 and left at room temperature for 10 min. By using a micropipette in 45° and 10 cm above from liquid nitrogen level, 30 µl drops of sperm suspension were dropped into a basket was plunging into a container filled with liquid nitrogen. The spherically shaped droplets in contact with LN were collected and put into cryovials and stored in liquid nitrogen for at least 1 week until warming. 2.4. Cooling in liquid nitrogen vapor
2. Materials and methods 2.1. Study design The patients were selected between February and July 2016. TESE samples were obtained from 15 men aged 20–40 (32.07 ± 5. 06) years old with obstructive azoospermia. Informed written consent was obtained from all participants. The study protocol was approved by the ethics committee of our institute (code: 197059). Also, the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed written consent was obtained from all participants. Demographic data and medical history of patients were documented from their medical records. For each patient, the inclusion criteria were as follows: the presence of at least 1 million spermatozoa in TESE samples, no history of varicocele or diabetes and tobacco addiction. Additionally, cases with no motile spermatozoa in TESE samples were excluded. The prepared TESE samples were divided into three aliquots. The fresh part (group 1), was assessed regarding the morphometric analysis of the vacuoles, DNA fragmentation, and mitochondrial membrane potential. The second part (Group 2) was cryopreserved with vitrification method, and the third part (Group 3) was cryopreserved with cooling in LN vapor. After a week, warming was done for the last two groups and all the aforementioned parameters were examined. 2.2. Preparation of TESE samples The collected testicular tissue was rinsed in sperm washing medium (Quinn’s Sperm Washing Medium, SAGE IVF Inc., CT, USA) to remove the blood and transferred to a petri dish. The tissue was then minced and 1 ml of the sperm washing medium was added to it. The medium was aspirated, and the suspension was
Cooling in LN vapor using droplet was performed as described by Isachenko [19]. Sperm suspension was mixed gently with sperm freezing solution in a ratio of 1:1 and was kept at room temperature for 10 min. Then 30 ml aliquots of sperm suspension were located onto pre-cooled sterile aluminum foil at distance of 30 cm of LN vapor. After 5 min of cooling, the solidified droplets were placed into cryovials, pre-cooled in liquid nitrogen. Then, the vials were plunged into liquid nitrogen for subsequent storage until warming. 2.5. Warming five spheres, one by one, were plunged into 5 ml pre-warmed Ham’s F10 medium (37 °C) supplemented with 5% HSA and vortexes for 5–10 s. Finally; the spermatozoa were centrifuged at 400g for 5 min. The post-warming sperm suspension was maintained at 37° C under 5% CO2 for 1 hr and sperm analysis assays were done as described above. 2.6. Spermatozoa evaluation for vacuoles under high magnification 5 µl drops of MOPS medium (Vitrolife, Sweden) overlaid with mineral oil (Ovoil 100, Vitrolife, Goteborg, Sweden) placed in glass bottom dish (WillCo-Dish, Amsterdam, Netherlands) were incubated for 20 min. Then, 1 µl of sperm suspension was added to the microdroplets and assessed under the inverted microscope (TE300; Nikon, Tokyo, Japan) equipped with high power differential interference contrast optics (Hoffman) which was used for MSOME (6600). The image capturing for further analysis was performed with software (OCTAX Eyewear; Octax). The morphological assessment was done on at least 100 spermatozoa on the monitor and the captured pictures, by one operator who was blinded to the sample groups. The area of vacuoles was recorded. A vacuole was considered large when its area was >4% of the total head area and small if the area was 4% [20].
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
3
2.7. DNA fragmentation assessment The TUNEL assay was carried out according to the manufacturer’s (Roche Diagnostics GmbH, Manheim, Germany) protocol. PBS/1% BSA was added to the prepared testicular pellet to reach a final concentration of 1 106 cells/100 ml of sample. Then on a shaker, a 100 µl aliquot of 1% paraformaldehyde was added for 1 h at 15–25 °C. The cells were washed once with 1 ml of PBS/1% BSA after fixation. Then, the cells were re-suspended in 100 ml of permeabilization solution (consisting of 0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. After being washed once with PBS/1% BSA, the labeling reaction was done by incubation with 50 ml of labeling solution (supplied within situ Cell Death Detection Kit, Fluorescing, Roche Diagnostics GmbH, Mannheim, Germany) which contains terminal deoxynucleotidyl transferase (TdT) for 1 h at 37 °C in the dark. A negative control was prepared by omitting TdT from the reaction mixture for each sample. After labeling, two subsequent washes in PBS/1% BSA were performed and the sample was resuspended in PBS/0.1% BSA to a volume of 500–700 ml and counterstained with propidium iodide (0.5 ml/ml). Preparation of positive controls for each sample was done as mentioned above, but with further treatment with 2 IU of DNase (Roche Diagnostics GmbH) for 15 min at 37 °C before doing the labeling reaction. Green fluorescence (TUNEL-positive cells) and red fluorescence (propidium iodidelabeled cells) were measured using a 560 nm dichroic filter. 2.8. Mitochondrial membrane potential evaluation Mitochondrial membrane potential was assessed by fluorescent dye JC-1. 5,50 ,6,60 -tetra-chloro-1,10 ,3,30 -tetraethyl ben-imidazolylcarbocyanine iodide (JC-1, CS0390, Staining Kit, SIGMA ALDRICH, USA). The stock solution was prepared at 1 mg/mL in dimethylsulfoxide (DMSO). 1 ml of sperm suspension was mixed with 1 ml of Staining Solution (25 ml of the 200´ JC-1 Stock Solution in 4 ml of ultrapure water) and incubated for 20 min at 37 °C in 5% CO2. The cell suspension was centrifuged at 600g for 3–4 min at 2–8 °C. The cell pellet was placed on ice. The cells were washed with 5 ml of the ice-cold 1´ JC-1 Staining Buffer (10 ml of the 1´ JC-1 staining buffer by diluting 2 ml of the JC-1 staining buffer 5´ with 8 ml of water), and then the cells were re-suspended in 5 ml of the ice-cold 1´ JC-1 staining buffer. The sample was kept on the ice and analyzed within 30 mins after staining. Under fluorescent microscope, the monomeric dye structure emits at 527 nm, while J-aggregates in non-damaged (healthy) mitochondria emit at 590 nm. When mitochondria were intact, the JC-1 reagent aggregate inside the healthy mitochondria and fluoresce red color. If the mitochondria are damaged, the mitochondrial membrane potential was breaking down and the JC-1 reagent was seen dispersed through the entire cells and fluoresce green color [7]. 2.9. Statistical analysis Data were analyzed using SPSS version 20 (SPSS, Inc., Chicago, IL, USA). The data are means ± standard deviation (SD). Comparison of means between groups was evaluated by ANOVA test. Spearman’s correlations between parametric and Pearson correlations between non-parametric variables of groups were obtained. P-value <0.05 was considered as statistically significant.
Fig. 1. Comparison of Presence of vacuoles in 15 individuals before freezing, after vitrification and freezing in liquid nitrogen vapor. Note: Different superscript letters on columns indicate significant differences (ANOVA test).
number of spermatozoa with no vacuoles significantly decreased after cryopreservation (P < 0.001). There was no significant difference regarding no vacuoles between groups 2 and 3 (P > 0.05). Large vacuoles, in groups 2 and 3, and small vacuoles in group 3 also increased after cryopreservation, but this increase was not significant between the three groups (P > 0.05). 3.2. DNA fragmentation and mitochondrial membrane potential assessment in three groups DNA fragmentation significantly increased after two cryopreservation techniques (P < 0.001). Mitochondrial membrane potential decreased significantly after freezing in group 2 and 3. Table 1 shows a comparison of DNA fragmentation and mitochondrial membrane potential in three groups. 3.3. Correlation between presence of vacuoles with DNA fragmentation and mitochondrial membrane potential There was a significant positive correlation between spermatozoa with large vacuoles in group 2 and DNA fragmentation. In all samples together, a significant negative correlation between spermatozoa with no vacuoles and DNA fragmentation also a significant positive correlation was shown between spermatozoa with large vacuoles and DNA fragmentation. There was a significant positive correlation between spermatozoa with no vacuole and mitochondrial membrane potential in groups 2 and 3. In all samples together a significant positive correlation was shown between spermatozoa with no vacuole and a significant negative correlation between spermatozoa with large vacuoles and mitochondrial membrane potential (Table 2). 4. Discussion
3. Results 3.1. Descriptive assessment of samples regarding the presence of vacuoles Fig. 1 showed the comparison of 3 groups regarding vacuoles. In the fresh sample, 15.73% of spermatozoa had no vacuoles and the
There is not enough information about detailed post-thaw morphologic assessment of spermatozoa even after using MSOME [21]. According to this study, mitochondrial membrane potential decreased to almost 31% after vitrification and freezing in liquid nitrogen vapor. Sperm is consisting of several parts enclosed within the plasma and mitochondrial membranes. These
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
4
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
Table 1 Comparison of DNA fragmentation and mitochondrial membrane potential (by JC1) in three groups. Variable (mean ± SD)
Groups
DNA fragmentation (%) Mitochondrial membrane potential (%)
P-value
Fresh sample (Group 1)
Vitrification (Group 2)
Freezing in LN vapor (Group 3)
11.653 ± 3.10a 54.755 ± 4.05a
29.487 ± 6.69b 31.814 ± 2.74b
29.727 ± 6.28b 31.981 ± 2.92b
<0.001 <0.001
*Values are means ± SD; LN: Liquid Nitrogen. Different superscript letters within a row indicate significant differences (ANOVA test).
Table 2 Correlation between presence of vacuoles with DNA fragmentation and mitochondrial membrane potential. Variables
Groups
No vacuole
Small vacuole
Large vacuole
DNA fragmentation (%)
1
r = 0.130 P = 0.644 r = 0.34 P = 0.204 r = 0.15 P = 0.58 r =0.58 P < 0.001
r = 0.169 P = 0.548 r = 0.45 P = 0.090 r = 0.08 P = 0.762 r = 0.06 P = 0.684
r = 0.173 P = 0.537 r = 0.53 P = 0.041 r = 0.03 P = 0.90 r = 0.36 P = 0.015
r = 0.22 P = 0.426 r = 0.51 P = 0.04 r = 0.53 P = 0.038 r = 0.64 P < 0.001
r = 0.11 P = 0.69 r = 0.23 P = 0.403 r = 0.19 P = 0.488 r = 0.01 P = 0.92
r = 0.058 P = 0.839 r = 0.01 P = 0.964 r = 0.33 P = 0.21 r = 0.34 P = 0.02
2 3 All JC1 (%)
1 2 3 All
Significant values of the Spearman rank correlation coefficient are presented in the table. P-value < 0.05 was statistically significant.
membranes should remain intact and functionally normal to keep cell competence. Mitochondria, by producing ATP, are the cell’s main source of oxidative energy [22,23]. It has been shown that cryopreservation alters spermatozoa plasma, acrosomal and mitochondrial membranes composition thus affects the acrosomal reaction, gamete interaction and mitochondrial function [24,25]. Sperm membrane disruption induced by cryopreservation may be the result of liquid phase transition changes and increased lipid peroxidation [26]. In a study by O’Connell et al. the effects of cryoinjury were determined on the function of mitochondria, motility, morphology, and viability of ejaculated human spermatozoa. They showed that these parameters are equally susceptible to cryopreservation-induced damage [24]. As previously suggested [27], we also found that cryopreservation increased spermatozoa DNA integrity. It has been demonstrated that sperm DNA damage is strongly correlated with mutagenic events [28]. Fertilization is still possible for spermatozoa with the damaged genetic material, and the defects could be apparent when the embryo has divided or the fetus developed [29]. Hazout et al. (2006), assessed sperm DNA integrity in 72 infertile patients. Improved implantation and birth rates with intracytoplasmic morphologically selected sperm injection (IMSI) were observed in patients with an elevated degree of sperm DNA fragmentation. So choosing a technique which selects the healthy spermatozoa as much as possible is of particular value. The reason for the occurrence of vacuoles in the sperm head is unclear and it needs more studies. Tanaka et al. presented that vacuoles are physiological structures with no negative effect, while other researchers reported vacuoles as pathological structures [30,31]. Increasing studies showed the association between the presence of vacuoles and decrease in fertilization rate [32], blastocyst formation [33], embryo development [34] and pregnancy outcomes [32]. Only high-magnification analysis by MSOME enables
more detailed assessment of spermatozoa head vacuoles. In this study, the percentage of spermatozoa with no vacuole significantly decreased to 10.12% and 8.83%, after vitrification and rapid freezing, respectively. Also, the spermatozoa with small and large vacuoles increased after two cryopreservation techniques; although it wasn’t significant. Although it is debatable if these newly shaped vacuoles are of the same origin of in vivo shaped ones. These results were different with those of a study that showed no difference in position or size of vacuoles pre and post- cryopreservation [21]. The reason for this disagreement was thatGatimel et al. assessed fertile normospermia samples but our samples were TESE samples of infertile men. It has been shown that spermatozoa of infertile men are more susceptible to cryopreservation in comparison to fertile men regarding for example chromatin condensation [35]. Compatible with our results, Biotrelle et al. demonstrated that cryopreservation induced sperm nuclear vacuolization and increased spermatozoa with non-condensed chromatin [36]. The present observations showed a significant positive correlation between spermatozoa with large vacuoles and DNA fragmentation which was more pronounced in vitrification group. In line with our findings, Franco et al. demonstrated that DNA fragmentation in spermatozoa with large nuclear vacuole was significantly higher in spermatozoa with normal nucleus [37]. Although, MSOME is necessary for detailed evaluation of vacuoles which is not possible in 400 magnification, its drawbacks is that the process of finding normal morphology spermatozoa under high magnification takes too much time and requires expert laboratory personnel [11].The limitation of our study is that IMSI wasn’t performed after MSOME to follow up the clinical outcome of using MSOME for post-thaw selection of spermatozoa, because in our center MSOME and IMSI are not used in a clinical setting yet. Also, our study TESE samples were from diagnostic cases, not clinical ones. So Further studies with a view to investigating the clinical outcome (fertilization, embryo development, chemical and clinical pregnancy and live birth rate) of using testicular sperm selected by MSOME and cryopreserved testicular sperm will be useful.
5. Conclusion This study showed that cryopreservation affects spermatozoa vacuolization, DNA structure and mitochondrial membrane potential in azoospermic patients. We consider that using MSOME for post-thaw morphologic assessment of spermatozoa will be of particular value, especially in the selection of normal morphologic testicular spermatozoa of infertile men for ICSI procedure. Assessment of clinical outcome of using testicular spermatozoa selected by MSOME will be valuable in future studies.
6. Conflict of interest The authors declared that there is no conflict of interest.
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
Acknowledgements This article extracted from the Ph.D. thesis of Sahabeh Etebary that was financially supported by Research and Clinical Center for Infertility, Yazd, Iran. The authors would like to thank the colleagues who helped with experiments and data collection. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] J.K. Sherman, Freezing, and freeze-drying of human spermatozoa, Fertil. Steril. 5 (4) (1954) 357. [2] G.S. Prins et al., Quality of cryopreserved testicular sperm in patients with obstructive and nonobstructive azoospermia, J. Urol. 161 (5) (1999) 1504– 1508. [3] F. Abdel-Hafez et al., Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review, Hum. Reprod. Update 15 (2) (2009) 153–164. [4] B.K. Gangrade, Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception, Clinics 68 (2013) 131–140. [5] M. Hammadeh et al., Sperm morphology and chromatin condensation before and after semen processing, Arch. Androl. 44 (3) (2000) 221–226. [6] N. Fukunaga et al., Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections, J. Assist. Reprod. Genetics 18 (12) (2001) 634–637. [7] E. Isachenko et al., Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose, Reproduction 136 (2) (2008) 167–173. [8] T. Vutyavanich, W. Piromlertamorn, S. Nunta, Rapid freezing versus slow programmable freezing of human spermatozoa, Fertil. Steril. 93 (6) (2010) 1921–1928. [9] M. Slabbert, S. Du Plessis, C. Huyser, Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa, Andrologia. 47 (5) (2015) 594–599. [10] M. Gil-Salom et al., Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa, Mol. Cell. Endocrinol. 169 (1) (2000) 15–19. [11] A.S. Setti et al., Twelve years of MSOME and IMSI: a review, Reprod. Biomed. Online 27 (4) (2013) 338–352. [12] B. Bartoov, A. Berkovitz, F. Ellis, Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection, N. Engl. J. Med. 345 (14) (2001) 1067–1068. [13] A. Tanaka et al., Human sperm head vacuoles are physiological structures formed during the sperm development and maturation process, Fertil. Steril. 98 (2) (2012) 315–320. [14] N.G. Cassuto et al., Correlation between DNA defect and sperm-head morphology, Reprod. Biomed. Online 24 (2) (2012) 211–218. [15] S. Watanabe et al., An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay, Hum. Reprod. 26 (5) (2011) 978–986. [16] A. Garolla et al., High-power microscopy for selecting spermatozoa for ICSI by physiological status, Reprod. Biomed. Online 17 (5) (2008) 610–616. [17] K. Knez et al., The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: A comparative prospective randomized study, Reprod. Biol. Endocrinol. 9 (1) (2011) 1.
5
[18] Z.P. Nagy et al., An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series, Fertil. Steril. 68 (2) (1997) 376–379. [19] V. Isachenko et al., a Clean technique for cryoprotectant-free vitrification of human spermatozoa, Reprod. Biomed. Online 10 (3) (2005) 350–354. [20] B. Bartoov et al., Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome, J. Androl. 23 (1) (2002) 1–8. [21] N. Gatimel, R. Leandro, J. Parinaud, Sperm vacuoles are not modified by freezing-thawing procedures, Reprod. Biomed. Online 26 (3) (2013) 240–246. [22] L. Zamboni, The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality, Fertil. Steril. 48 (5) (1987) 711–734. [23] D.L. Garner et al., Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa, Biol. Reprod. 57 (6) (1997) 1401–1406. [24] M. O’Connell, N. McClure, S. Lewis, The effects of cryopreservation on sperm morphology, motility and mitochondrial function, Hum Reprod. 17 (3) (2002) 704–709. [25] M. Bell et al., Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm, J. Androl. 14 (6) (1993) 472–478. [26] J.G. Alvarez, B.T. Storey, Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as a sole cryoprotectant, J Androl. 14 (3) (1993) 199–209. [27] E.T. Donnelly et al., Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation, Hum Reprod. 16 (6) (2001) 1191–1199. [28] C.G. Fraga et al., Ascorbic acid protects against endogenous oxidative DNA damage in human sperm, Proc Natl Acad Sci U S A. 88 (24) (1991) 11003– 11006. [29] J. Twigg, D. Irvine, R. Aitken, Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection, Hum. Reprod. 13 (7) (1998) 1864–1871. [30] Boitrelle, F., et al., Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to the failure of chromatin condensation, Human Reproduction. (2011) der129 [31] I. Hammoud et al., Selection of normal spermatozoa with a vacuole-free head (x6300) improves the selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates, Andrologia. 45 (3) (2013) 163–170. [32] A. Berkovitz et al., The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm, Hum. Reprod. 20 (1) (2005) 185–190. [33] P. Vanderzwalmen et al., Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles, Reproductive biomedicine online. 17 (5) (2008) 617–627. [34] N.G. Cassuto et al., A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality, Fertility and sterility. 92 (5) (2009) 1616–1625. [35] M. Hammadeh et al., Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men, Int J Androl. 22 (3) (1999) 155–162. [36] F. Boitrelle et al., Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation, J. Androl. 33 (6) (2012) 1371–1378. [37] J. Franco et al., Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI, Reprod. Biomed. Online 17 (1) (2008) 42–45.
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
Contents lists available at ScienceDirect
Middle East Fertility Society Journal journal homepage: www.sciencedirect.com
Original Article
Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential Sahabeh Etebary a, Nahid Yari a, Mohammad Ali Khalili a,⇑, Seyed Mehdi Kalantar b, Morteza Anvari a a b
Research and Clinical Center for Infertility, ShahidSadoughi University of Medical Sciences, Bouali Ave, Safaeyeh, Yazd, Iran Abortion Research Center, Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
a r t i c l e
i n f o
Article history: Received 30 April 2018 Revised 20 June 2018 Accepted 3 July 2018 Available online xxxx Keywords: Spermatozoa Azoospermia MSOME Cryopreservation Vacuoles DNA fragmentation
a b s t r a c t Background: There are difficulties associated with testicular sperm freezing. Different methods of sperm cryopreservation developed. Not enough detailed studies about the real efficacy of these techniques exist. For sperm morphologic assessment, motile sperm organelle morphology examination (MSOME) is able to identify not only conventional morphological sperm alterations but also sperm head vacuoles. Objective: To assess the effect of cryopreservation on testicular spermatozoa vacuoles by MSOME and its correlation to DNA fragmentation and mitochondrial membrane potential. Materials and methods: After preparation, testicular sperm extraction samples of 15 azoospermic men, aged 20–40 years old were divided into three groups. Group 1 was assessed freshly. Group 2 was cryopreserved with vitrification method and Group 3 with cooling in liquid nitrogen vapor using droplet. Pre and post warming assessment in terms of spermatozoa head vacuoles by MSOME, DNA fragmentation, and mitochondrial membrane potential were performed. Results: The number of spermatozoa with no vacuoles significantly decreased after two cryopreservation techniques (P < 0.001). There were no significant differences between groups regarding small and large vacuoles (p > 0.05). DNA fragmentation and mitochondrial membrane potential increased after cryopreservation (P < 0.001). There was a significant positive correlation between spermatozoa with large vacuoles in group 2 and DNA fragmentation and a significant positive correlation between spermatozoa with no vacuole and mitochondrial membrane potential in groups 2 and 3. Conclusion: Cryopreservation affects spermatozoa vacuolization, DNA structure, and mitochondrial membrane potential. Using MSOME in the selection of post-thaw morphologically normal testicular spermatozoa for ICSI procedure will be of particular value. Ó 2018 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Cryopreservation of human spermatozoa was first described many years ago [1]. Despite the success of normal sperm cryopreservation, there are difficulties associated with testicular sperm freezing. What makes testicular sperm cryopreservation and using frozen-thawed sperm in ICSI cycles, beneficial and even necessary are difficulties associated with testicular sperm extraction (TESE) in azoospermic patients such as the need for repetition of biopsy
Peer review under responsibility of Middle East Fertility Society. ⇑ Corresponding author at: Bouali Ave., Safaeyeh Street, Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. E-mail addresses: [email protected] (M.A. Khalili), [email protected] (S.M. Kalantar).
in each ICSI cycle and probable risk of damage to the testis [2].Testicular sperm cryopreservation permits the couple to perform surgical procedures in different days and prevents the need to do testicular biopsy and oocyte retrieval on the same day. On the other hand, it avoids difficulties associated with expenses and repeating surgery [3,4]. There are different methods of sperm cryopreservation, such as slow freezing [5], freezing in liquid nitrogen vapor [6] and vitrification [7]. Rapid freezing is routinely used for the cryopreservation and it results in better post-thaw sperm motility and recovery in comparison to slow freezing [8]. Other data showed that vitrification may be more applicable in comparison to rapid freezing technique [9]. However, there are not enough detailed studies about the real efficacy of these techniques in large series [10]. On the other hand, intracytoplasmic sperm injection (ICSI) is usually performed under a magnification of x400, which only
https://doi.org/10.1016/j.mefs.2018.07.001 1110-5690/Ó 2018 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
2
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
enables the observation of major morphological defects. So in this way, the selection of the normal morphology spermatozoa don’t guarantee the selection of spermatozoa free of morphological abnormalities [11]. Many methods have been proposed to assess sperm morphology for improving diagnosis and treatment of male infertility. One of them is motile sperm organelle morphology examination (MSOME), which is based on a morphological analysis of isolated motile spermatozoa in real-time at high magnification (up to 6600), and was introduced by Bartoov et al. [12]. MSOME is able to identify not only conventional morphological sperm alterations but also more specifically sperm head vacuoles, which are considered as nuclear defects [12]. Even after several studies about the origin and creation of sperm head vacuoles, it is not clear if sperm vacuoles are normal features of the sperm head or degenerative structures with no physiological effect [11]. It has been proven that vacuole is a concavity extending from the surface of the sperm head to the nucleus through the acrosome that only a high magnification enables its visualization [13]. Some studies showed that sperm vacuoles are a physiological feature of the sperm head [13], while others suggested that presence of vacuoles is related to sperm chromatin packaging/DNA abnormalities [14,15] and lower mitochondrial membrane potential [16]. Also, studies proposed that selection of morphologically normal sperm with no vacuoles is related to better assisted reproductive technology (ART) outcomes [17]. The aim of present study was to assess the effect of vitrification and cooling in LN vapor on testicular spermatozoa vacuoles by MSOME and to evaluate the correlation between spermatozoa vacuoles, DNA fragmentation, and mitochondrial membrane potential.
transferred to a conical tube. 2 ml of the sperm-washing medium was added to the tissue fragments in the petri dish. With a 5-cc syringe attached to a 21-gauge needle, the tissue suspension was aspirated repeatedly. The suspension then was transferred to the same conical tube and let to settle for 5 min. The supernatant was transferred to a new conical tube. 1–2 ml of the spermwashing medium was added to the tissue pellet and mixed with a pipette. After 5 min the tissue fragments were settled and the supernatant was transferred to the conical tube and centrifuged at 400g for 10 min. 3–4 ml of RBC lysis buffer was added to the pellet and centrifuged again. The pellet was re-suspended in 3 ml of the sperm-washing medium and centrifuged at 400g for 10 min to wash off the RBC lysis buffer [18]. The supernatant was discarded and the pellet was suspended in 0.5 ml of the spermwashing medium. 2.3. Vitrification Vitrification was done according to Isachenko [7]. The sperm suspension was mixed gently with sperm freezing solution (Vitrolife, Sweden) briefly at a ratio of 1:1 and left at room temperature for 10 min. By using a micropipette in 45° and 10 cm above from liquid nitrogen level, 30 µl drops of sperm suspension were dropped into a basket was plunging into a container filled with liquid nitrogen. The spherically shaped droplets in contact with LN were collected and put into cryovials and stored in liquid nitrogen for at least 1 week until warming. 2.4. Cooling in liquid nitrogen vapor
2. Materials and methods 2.1. Study design The patients were selected between February and July 2016. TESE samples were obtained from 15 men aged 20–40 (32.07 ± 5. 06) years old with obstructive azoospermia. Informed written consent was obtained from all participants. The study protocol was approved by the ethics committee of our institute (code: 197059). Also, the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed written consent was obtained from all participants. Demographic data and medical history of patients were documented from their medical records. For each patient, the inclusion criteria were as follows: the presence of at least 1 million spermatozoa in TESE samples, no history of varicocele or diabetes and tobacco addiction. Additionally, cases with no motile spermatozoa in TESE samples were excluded. The prepared TESE samples were divided into three aliquots. The fresh part (group 1), was assessed regarding the morphometric analysis of the vacuoles, DNA fragmentation, and mitochondrial membrane potential. The second part (Group 2) was cryopreserved with vitrification method, and the third part (Group 3) was cryopreserved with cooling in LN vapor. After a week, warming was done for the last two groups and all the aforementioned parameters were examined. 2.2. Preparation of TESE samples The collected testicular tissue was rinsed in sperm washing medium (Quinn’s Sperm Washing Medium, SAGE IVF Inc., CT, USA) to remove the blood and transferred to a petri dish. The tissue was then minced and 1 ml of the sperm washing medium was added to it. The medium was aspirated, and the suspension was
Cooling in LN vapor using droplet was performed as described by Isachenko [19]. Sperm suspension was mixed gently with sperm freezing solution in a ratio of 1:1 and was kept at room temperature for 10 min. Then 30 ml aliquots of sperm suspension were located onto pre-cooled sterile aluminum foil at distance of 30 cm of LN vapor. After 5 min of cooling, the solidified droplets were placed into cryovials, pre-cooled in liquid nitrogen. Then, the vials were plunged into liquid nitrogen for subsequent storage until warming. 2.5. Warming five spheres, one by one, were plunged into 5 ml pre-warmed Ham’s F10 medium (37 °C) supplemented with 5% HSA and vortexes for 5–10 s. Finally; the spermatozoa were centrifuged at 400g for 5 min. The post-warming sperm suspension was maintained at 37° C under 5% CO2 for 1 hr and sperm analysis assays were done as described above. 2.6. Spermatozoa evaluation for vacuoles under high magnification 5 µl drops of MOPS medium (Vitrolife, Sweden) overlaid with mineral oil (Ovoil 100, Vitrolife, Goteborg, Sweden) placed in glass bottom dish (WillCo-Dish, Amsterdam, Netherlands) were incubated for 20 min. Then, 1 µl of sperm suspension was added to the microdroplets and assessed under the inverted microscope (TE300; Nikon, Tokyo, Japan) equipped with high power differential interference contrast optics (Hoffman) which was used for MSOME (6600). The image capturing for further analysis was performed with software (OCTAX Eyewear; Octax). The morphological assessment was done on at least 100 spermatozoa on the monitor and the captured pictures, by one operator who was blinded to the sample groups. The area of vacuoles was recorded. A vacuole was considered large when its area was >4% of the total head area and small if the area was 4% [20].
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
3
2.7. DNA fragmentation assessment The TUNEL assay was carried out according to the manufacturer’s (Roche Diagnostics GmbH, Manheim, Germany) protocol. PBS/1% BSA was added to the prepared testicular pellet to reach a final concentration of 1 106 cells/100 ml of sample. Then on a shaker, a 100 µl aliquot of 1% paraformaldehyde was added for 1 h at 15–25 °C. The cells were washed once with 1 ml of PBS/1% BSA after fixation. Then, the cells were re-suspended in 100 ml of permeabilization solution (consisting of 0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. After being washed once with PBS/1% BSA, the labeling reaction was done by incubation with 50 ml of labeling solution (supplied within situ Cell Death Detection Kit, Fluorescing, Roche Diagnostics GmbH, Mannheim, Germany) which contains terminal deoxynucleotidyl transferase (TdT) for 1 h at 37 °C in the dark. A negative control was prepared by omitting TdT from the reaction mixture for each sample. After labeling, two subsequent washes in PBS/1% BSA were performed and the sample was resuspended in PBS/0.1% BSA to a volume of 500–700 ml and counterstained with propidium iodide (0.5 ml/ml). Preparation of positive controls for each sample was done as mentioned above, but with further treatment with 2 IU of DNase (Roche Diagnostics GmbH) for 15 min at 37 °C before doing the labeling reaction. Green fluorescence (TUNEL-positive cells) and red fluorescence (propidium iodidelabeled cells) were measured using a 560 nm dichroic filter. 2.8. Mitochondrial membrane potential evaluation Mitochondrial membrane potential was assessed by fluorescent dye JC-1. 5,50 ,6,60 -tetra-chloro-1,10 ,3,30 -tetraethyl ben-imidazolylcarbocyanine iodide (JC-1, CS0390, Staining Kit, SIGMA ALDRICH, USA). The stock solution was prepared at 1 mg/mL in dimethylsulfoxide (DMSO). 1 ml of sperm suspension was mixed with 1 ml of Staining Solution (25 ml of the 200´ JC-1 Stock Solution in 4 ml of ultrapure water) and incubated for 20 min at 37 °C in 5% CO2. The cell suspension was centrifuged at 600g for 3–4 min at 2–8 °C. The cell pellet was placed on ice. The cells were washed with 5 ml of the ice-cold 1´ JC-1 Staining Buffer (10 ml of the 1´ JC-1 staining buffer by diluting 2 ml of the JC-1 staining buffer 5´ with 8 ml of water), and then the cells were re-suspended in 5 ml of the ice-cold 1´ JC-1 staining buffer. The sample was kept on the ice and analyzed within 30 mins after staining. Under fluorescent microscope, the monomeric dye structure emits at 527 nm, while J-aggregates in non-damaged (healthy) mitochondria emit at 590 nm. When mitochondria were intact, the JC-1 reagent aggregate inside the healthy mitochondria and fluoresce red color. If the mitochondria are damaged, the mitochondrial membrane potential was breaking down and the JC-1 reagent was seen dispersed through the entire cells and fluoresce green color [7]. 2.9. Statistical analysis Data were analyzed using SPSS version 20 (SPSS, Inc., Chicago, IL, USA). The data are means ± standard deviation (SD). Comparison of means between groups was evaluated by ANOVA test. Spearman’s correlations between parametric and Pearson correlations between non-parametric variables of groups were obtained. P-value <0.05 was considered as statistically significant.
Fig. 1. Comparison of Presence of vacuoles in 15 individuals before freezing, after vitrification and freezing in liquid nitrogen vapor. Note: Different superscript letters on columns indicate significant differences (ANOVA test).
number of spermatozoa with no vacuoles significantly decreased after cryopreservation (P < 0.001). There was no significant difference regarding no vacuoles between groups 2 and 3 (P > 0.05). Large vacuoles, in groups 2 and 3, and small vacuoles in group 3 also increased after cryopreservation, but this increase was not significant between the three groups (P > 0.05). 3.2. DNA fragmentation and mitochondrial membrane potential assessment in three groups DNA fragmentation significantly increased after two cryopreservation techniques (P < 0.001). Mitochondrial membrane potential decreased significantly after freezing in group 2 and 3. Table 1 shows a comparison of DNA fragmentation and mitochondrial membrane potential in three groups. 3.3. Correlation between presence of vacuoles with DNA fragmentation and mitochondrial membrane potential There was a significant positive correlation between spermatozoa with large vacuoles in group 2 and DNA fragmentation. In all samples together, a significant negative correlation between spermatozoa with no vacuoles and DNA fragmentation also a significant positive correlation was shown between spermatozoa with large vacuoles and DNA fragmentation. There was a significant positive correlation between spermatozoa with no vacuole and mitochondrial membrane potential in groups 2 and 3. In all samples together a significant positive correlation was shown between spermatozoa with no vacuole and a significant negative correlation between spermatozoa with large vacuoles and mitochondrial membrane potential (Table 2). 4. Discussion
3. Results 3.1. Descriptive assessment of samples regarding the presence of vacuoles Fig. 1 showed the comparison of 3 groups regarding vacuoles. In the fresh sample, 15.73% of spermatozoa had no vacuoles and the
There is not enough information about detailed post-thaw morphologic assessment of spermatozoa even after using MSOME [21]. According to this study, mitochondrial membrane potential decreased to almost 31% after vitrification and freezing in liquid nitrogen vapor. Sperm is consisting of several parts enclosed within the plasma and mitochondrial membranes. These
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
4
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
Table 1 Comparison of DNA fragmentation and mitochondrial membrane potential (by JC1) in three groups. Variable (mean ± SD)
Groups
DNA fragmentation (%) Mitochondrial membrane potential (%)
P-value
Fresh sample (Group 1)
Vitrification (Group 2)
Freezing in LN vapor (Group 3)
11.653 ± 3.10a 54.755 ± 4.05a
29.487 ± 6.69b 31.814 ± 2.74b
29.727 ± 6.28b 31.981 ± 2.92b
<0.001 <0.001
*Values are means ± SD; LN: Liquid Nitrogen. Different superscript letters within a row indicate significant differences (ANOVA test).
Table 2 Correlation between presence of vacuoles with DNA fragmentation and mitochondrial membrane potential. Variables
Groups
No vacuole
Small vacuole
Large vacuole
DNA fragmentation (%)
1
r = 0.130 P = 0.644 r = 0.34 P = 0.204 r = 0.15 P = 0.58 r =0.58 P < 0.001
r = 0.169 P = 0.548 r = 0.45 P = 0.090 r = 0.08 P = 0.762 r = 0.06 P = 0.684
r = 0.173 P = 0.537 r = 0.53 P = 0.041 r = 0.03 P = 0.90 r = 0.36 P = 0.015
r = 0.22 P = 0.426 r = 0.51 P = 0.04 r = 0.53 P = 0.038 r = 0.64 P < 0.001
r = 0.11 P = 0.69 r = 0.23 P = 0.403 r = 0.19 P = 0.488 r = 0.01 P = 0.92
r = 0.058 P = 0.839 r = 0.01 P = 0.964 r = 0.33 P = 0.21 r = 0.34 P = 0.02
2 3 All JC1 (%)
1 2 3 All
Significant values of the Spearman rank correlation coefficient are presented in the table. P-value < 0.05 was statistically significant.
membranes should remain intact and functionally normal to keep cell competence. Mitochondria, by producing ATP, are the cell’s main source of oxidative energy [22,23]. It has been shown that cryopreservation alters spermatozoa plasma, acrosomal and mitochondrial membranes composition thus affects the acrosomal reaction, gamete interaction and mitochondrial function [24,25]. Sperm membrane disruption induced by cryopreservation may be the result of liquid phase transition changes and increased lipid peroxidation [26]. In a study by O’Connell et al. the effects of cryoinjury were determined on the function of mitochondria, motility, morphology, and viability of ejaculated human spermatozoa. They showed that these parameters are equally susceptible to cryopreservation-induced damage [24]. As previously suggested [27], we also found that cryopreservation increased spermatozoa DNA integrity. It has been demonstrated that sperm DNA damage is strongly correlated with mutagenic events [28]. Fertilization is still possible for spermatozoa with the damaged genetic material, and the defects could be apparent when the embryo has divided or the fetus developed [29]. Hazout et al. (2006), assessed sperm DNA integrity in 72 infertile patients. Improved implantation and birth rates with intracytoplasmic morphologically selected sperm injection (IMSI) were observed in patients with an elevated degree of sperm DNA fragmentation. So choosing a technique which selects the healthy spermatozoa as much as possible is of particular value. The reason for the occurrence of vacuoles in the sperm head is unclear and it needs more studies. Tanaka et al. presented that vacuoles are physiological structures with no negative effect, while other researchers reported vacuoles as pathological structures [30,31]. Increasing studies showed the association between the presence of vacuoles and decrease in fertilization rate [32], blastocyst formation [33], embryo development [34] and pregnancy outcomes [32]. Only high-magnification analysis by MSOME enables
more detailed assessment of spermatozoa head vacuoles. In this study, the percentage of spermatozoa with no vacuole significantly decreased to 10.12% and 8.83%, after vitrification and rapid freezing, respectively. Also, the spermatozoa with small and large vacuoles increased after two cryopreservation techniques; although it wasn’t significant. Although it is debatable if these newly shaped vacuoles are of the same origin of in vivo shaped ones. These results were different with those of a study that showed no difference in position or size of vacuoles pre and post- cryopreservation [21]. The reason for this disagreement was thatGatimel et al. assessed fertile normospermia samples but our samples were TESE samples of infertile men. It has been shown that spermatozoa of infertile men are more susceptible to cryopreservation in comparison to fertile men regarding for example chromatin condensation [35]. Compatible with our results, Biotrelle et al. demonstrated that cryopreservation induced sperm nuclear vacuolization and increased spermatozoa with non-condensed chromatin [36]. The present observations showed a significant positive correlation between spermatozoa with large vacuoles and DNA fragmentation which was more pronounced in vitrification group. In line with our findings, Franco et al. demonstrated that DNA fragmentation in spermatozoa with large nuclear vacuole was significantly higher in spermatozoa with normal nucleus [37]. Although, MSOME is necessary for detailed evaluation of vacuoles which is not possible in 400 magnification, its drawbacks is that the process of finding normal morphology spermatozoa under high magnification takes too much time and requires expert laboratory personnel [11].The limitation of our study is that IMSI wasn’t performed after MSOME to follow up the clinical outcome of using MSOME for post-thaw selection of spermatozoa, because in our center MSOME and IMSI are not used in a clinical setting yet. Also, our study TESE samples were from diagnostic cases, not clinical ones. So Further studies with a view to investigating the clinical outcome (fertilization, embryo development, chemical and clinical pregnancy and live birth rate) of using testicular sperm selected by MSOME and cryopreserved testicular sperm will be useful.
5. Conclusion This study showed that cryopreservation affects spermatozoa vacuolization, DNA structure and mitochondrial membrane potential in azoospermic patients. We consider that using MSOME for post-thaw morphologic assessment of spermatozoa will be of particular value, especially in the selection of normal morphologic testicular spermatozoa of infertile men for ICSI procedure. Assessment of clinical outcome of using testicular spermatozoa selected by MSOME will be valuable in future studies.
6. Conflict of interest The authors declared that there is no conflict of interest.
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001
S. Etebary et al. / Middle East Fertility Society Journal xxx (2018) xxx–xxx
Acknowledgements This article extracted from the Ph.D. thesis of Sahabeh Etebary that was financially supported by Research and Clinical Center for Infertility, Yazd, Iran. The authors would like to thank the colleagues who helped with experiments and data collection. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] J.K. Sherman, Freezing, and freeze-drying of human spermatozoa, Fertil. Steril. 5 (4) (1954) 357. [2] G.S. Prins et al., Quality of cryopreserved testicular sperm in patients with obstructive and nonobstructive azoospermia, J. Urol. 161 (5) (1999) 1504– 1508. [3] F. Abdel-Hafez et al., Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review, Hum. Reprod. Update 15 (2) (2009) 153–164. [4] B.K. Gangrade, Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception, Clinics 68 (2013) 131–140. [5] M. Hammadeh et al., Sperm morphology and chromatin condensation before and after semen processing, Arch. Androl. 44 (3) (2000) 221–226. [6] N. Fukunaga et al., Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections, J. Assist. Reprod. Genetics 18 (12) (2001) 634–637. [7] E. Isachenko et al., Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose, Reproduction 136 (2) (2008) 167–173. [8] T. Vutyavanich, W. Piromlertamorn, S. Nunta, Rapid freezing versus slow programmable freezing of human spermatozoa, Fertil. Steril. 93 (6) (2010) 1921–1928. [9] M. Slabbert, S. Du Plessis, C. Huyser, Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa, Andrologia. 47 (5) (2015) 594–599. [10] M. Gil-Salom et al., Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa, Mol. Cell. Endocrinol. 169 (1) (2000) 15–19. [11] A.S. Setti et al., Twelve years of MSOME and IMSI: a review, Reprod. Biomed. Online 27 (4) (2013) 338–352. [12] B. Bartoov, A. Berkovitz, F. Ellis, Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection, N. Engl. J. Med. 345 (14) (2001) 1067–1068. [13] A. Tanaka et al., Human sperm head vacuoles are physiological structures formed during the sperm development and maturation process, Fertil. Steril. 98 (2) (2012) 315–320. [14] N.G. Cassuto et al., Correlation between DNA defect and sperm-head morphology, Reprod. Biomed. Online 24 (2) (2012) 211–218. [15] S. Watanabe et al., An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay, Hum. Reprod. 26 (5) (2011) 978–986. [16] A. Garolla et al., High-power microscopy for selecting spermatozoa for ICSI by physiological status, Reprod. Biomed. Online 17 (5) (2008) 610–616. [17] K. Knez et al., The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: A comparative prospective randomized study, Reprod. Biol. Endocrinol. 9 (1) (2011) 1.
5
[18] Z.P. Nagy et al., An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series, Fertil. Steril. 68 (2) (1997) 376–379. [19] V. Isachenko et al., a Clean technique for cryoprotectant-free vitrification of human spermatozoa, Reprod. Biomed. Online 10 (3) (2005) 350–354. [20] B. Bartoov et al., Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome, J. Androl. 23 (1) (2002) 1–8. [21] N. Gatimel, R. Leandro, J. Parinaud, Sperm vacuoles are not modified by freezing-thawing procedures, Reprod. Biomed. Online 26 (3) (2013) 240–246. [22] L. Zamboni, The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality, Fertil. Steril. 48 (5) (1987) 711–734. [23] D.L. Garner et al., Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa, Biol. Reprod. 57 (6) (1997) 1401–1406. [24] M. O’Connell, N. McClure, S. Lewis, The effects of cryopreservation on sperm morphology, motility and mitochondrial function, Hum Reprod. 17 (3) (2002) 704–709. [25] M. Bell et al., Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm, J. Androl. 14 (6) (1993) 472–478. [26] J.G. Alvarez, B.T. Storey, Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as a sole cryoprotectant, J Androl. 14 (3) (1993) 199–209. [27] E.T. Donnelly et al., Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation, Hum Reprod. 16 (6) (2001) 1191–1199. [28] C.G. Fraga et al., Ascorbic acid protects against endogenous oxidative DNA damage in human sperm, Proc Natl Acad Sci U S A. 88 (24) (1991) 11003– 11006. [29] J. Twigg, D. Irvine, R. Aitken, Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection, Hum. Reprod. 13 (7) (1998) 1864–1871. [30] Boitrelle, F., et al., Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to the failure of chromatin condensation, Human Reproduction. (2011) der129 [31] I. Hammoud et al., Selection of normal spermatozoa with a vacuole-free head (x6300) improves the selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates, Andrologia. 45 (3) (2013) 163–170. [32] A. Berkovitz et al., The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm, Hum. Reprod. 20 (1) (2005) 185–190. [33] P. Vanderzwalmen et al., Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles, Reproductive biomedicine online. 17 (5) (2008) 617–627. [34] N.G. Cassuto et al., A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality, Fertility and sterility. 92 (5) (2009) 1616–1625. [35] M. Hammadeh et al., Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men, Int J Androl. 22 (3) (1999) 155–162. [36] F. Boitrelle et al., Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation, J. Androl. 33 (6) (2012) 1371–1378. [37] J. Franco et al., Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI, Reprod. Biomed. Online 17 (1) (2008) 42–45.
Please cite this article in press as: S. Etebary et al., Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.07.001