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Leptin in sperm analysis can be a new indicator
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Leptin in sperm analysis can be a new indicator ⁎
Tuğçe Önela, , Sule Aylaa,e, İlknur Keskina,e, Cüneyd Parlayanb,e, Türkan Yiğitbaşıc, Bircan Kolbaşıa,e, Tuba Varlı Yelked, Tuğba Şenel Ustabaşd İstanbul Medipol University, School of Medicine, Department of Histology and Embryology, İstanbul, Turkey İstanbul Medipol University, Engineering and Natural Sciences, Department of Biomedical Engineering, İstanbul, Turkey İstanbul Medipol University, School of Medicine, Department of Medical Biochemistry, İstanbul, Turkey d Medipol International Health Center, In Vitro Fertilization Unit (IVF), İstanbul, Turkey e Regenerative and Restorative Medicine Research Center (REMER), İstanbul Medipol University, İstanbul, Turkey a
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A R T I C LE I N FO
A B S T R A C T
Keywords: Cryopreservation DNA fragmentation Leptin Motility Sperm
Purpose Radiotherapy, chemotherapy, various tumors and invasive surgery can result in ejaculatory dysfunction and testicular insufficiency. Sperm cryopreservation is the only method which can provide a baby for couples. Cryopreservation freezes tissues and cells, allowing them to be stored for future use by stopping all biological activities. Cryopreservation can cause some harmful changes in the structure and function of the sperm. Leptin molecule plays many roles in most biological processes including the satiety and cell renewal, proliferation, angiogenesis, modulation of energy expenditure and regulation of the neuroendocrine system. Leptin was also reported to be associated with spermatogenesis in several studies. Methods This study aims to use leptin molecule as a parameter for sperm motility and DNA fragmentation before and after the cryopreservation. In this study, semen samples were taken from 30 normospermic male volunteers. Each semen sample was examined for the same parameters before and after the cryopreservation. Samples were analyzed before and after cryopreservation in terms of sperm motility by morphological sperm analysis with spermac stain dye, DNA fragmentation by TUNEL assay, ultrastructural analysis with transmission electron microscopy (TEM), seminal leptin levels by ELISA method and reactive oxygen species (ROS) levels by colorimetric method. Results Decreased sperm motility, distribution of sperm morphology and increased DNA fragmentation were determined after cryopreservation. Similarly, seminal ROS and leptin levels were also increased significantly. There was a negative correlation between seminal leptin and sperm motility. Additionally, there was a positive correlation between seminal leptin and DNA fragmentation. Conclusion According to these results, leptin molecule can be used as a marker for sperm motility and DNA fragmentation before and after cryopreservation. We think that the results of this study can contribute to further studies in the clinical aspect.
1. Introduction Cryopreservation is a technique used in assisted reproductive techniques, aiming at preserving cells at very low temperatures without losing their viability and functionality. The sperms are exposed to physical and chemical stresses during cryopreservation, thus, the lipid structure of plasma membrane changes, and phosphatidylserine output takes place (Schiller et al., 2000). In spite of the recent advances that have been made in the field of sperm cryopreservation, the process has the potential to compromise sperm function and quality through generation of reactive oxygen species (ROS) and reduction in antioxidant
activity (Najafi et al., 2016). Reactive oxygen species (ROS) are a class of free radicals with highly reactive oxygenated agents. ROS production at high concentrations plays an important role in male infertility by affecting sperm metabolism, and consequently morphology, motility and fertility in a negative way causing lipid peroxidation and impaired membrane function (Cummins et al., 1994). Human spermatozoa are highly susceptible to oxidative stress (OS) damage due to low cytoplasmic antioxidant content and high polyunsaturated fatty acids (Najafi et al., 2016). Much evidence has shown that OS contributes to both direct and indirect cellular damage during the sperm cryopreservation process
Abbreviations: ROS, reactive oxygen species; TEM, transmission electron microscope; WHO, World Health Organization ⁎ Corresponding author at: Yeditepe University, Department of Histology and Embryology, School of Medicine, Turkey. E-mail addresses: [email protected], [email protected], [email protected] (T. Önel), [email protected] (S. Ayla), [email protected] (İ. Keskin), [email protected] (C. Parlayan), [email protected] (B. Kolbaşı), [email protected] (T.V. Yelke), [email protected] (T.Ş. Ustabaş). https://doi.org/10.1016/j.acthis.2018.10.006 Received 28 February 2018; Received in revised form 15 October 2018; Accepted 16 October 2018 0065-1281/ © 2018 Published by Elsevier GmbH.
Please cite this article as: Önel, T., Acta Histochemica, https://doi.org/10.1016/j.acthis.2018.10.006
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(Immotility;IM). (WHO, 2010) The individuals with the sperm number ranging between 20–200 million/ml. were included in the study. All these operations were performed in the room temperature of 20–24°.
(Agarwal et al., 2014). It has been reported that generation of ROS occurs during the freezing and/or thawing process of spermatozoa (Paoli et al., 2014). It seems likely that the sudden increase in oxygen consumption by spermatozoa during thawing results in ROS-induced membrane damage (Zubkova and Robaire, 2004). Leptin, known to be secreted by fat cells and any tissues, is a polypeptide that contains 167 amino acids, with 16 kD molecular weight, that maintains a specific blood level in the plasma, and is carried in blood freely and/or adhered to protein (Zhang et al., 1994). Leptin’s main mechanism of action is to inhibit the oscillation and expression of neuropeptide-Y, which is involved in the regulation of many organs while its main effect is to increase appetite from arcuate nucleus (Spitzweg and Heufelder, 1997). Although presence of leptin and its receptor has been demonstrated in spermatozoa, its role on spermatogenesis and sperm still needs to be clarified (Fontoura et al., 2017). Understanding that leptin is also synthesized by the placenta and that leptin receptors are also expressed in the placenta and ovaries (Hoggard et al., 1997; Karlsson et al., 1997) suggest that leptin may have important effects on the reproductive system. The aim of this study is to make comparisons of motility, morphology and DNA fragmentation in the sperm before and after cryopreservation, and to investigate the association of these parameters with leptin molecule levels. Through this, we aim to use leptin molecule level as a determinant in sperm motility and DNA fragmentation.
2.3. Sperm freezing and thawing procedure For freezing operation, semen sample and cryoprotectant (90128Irvine Scientific Freezing Medium) were mixed in 1: 1 ratio so as to result in a total volume of 1 ml. and sterile tubes were taken into ‘Cryo Vial, T3082A’. The tubes were placed in the liquid nitrogen tank (-196 °C) after being kept at room temperature for 8 min, and at nitrogen steam at height of 15 cm from the liquid nitrogen level for 20 min respectively. The thawing operation was carried out by the removal of the tubes from liquid nitrogen, and with hot water treatment at room temperature. After the sample was completely dissolved, eluent (HTF / HEPES ART-1023) was added, the tubes were centrifuged for 5 min at 2000 rpm and supernatant was expelled from cryoprotectan. The sperm samples obtained after the thawing operation were used for motility, morphology, DNA fragmentation, leptin and ROS level evaluations. 2.4. Analyses of sperm morphology Light microscopic morphological examination was carried out after staining with Spermac Stain kit (FP09 I21 R01 C.7- FertiPro N.V., Belgium). A smear preparation was made out of the sperm samples. After keeping in fixative as well as cationic and anionic dyes of the kit for one minute each, it was washed with distilled water and dried. At least 200 cells of each sample of individuals were analyzed with immersion oil at 1000x magnitude by using an Olympus BX51 (Tokyo, Japan) photomicroscope according to Kruger stick criteria (Menkveld et al 1991). All these operations were performed in the room temperature of 20–24°.
2. Method and material 2.1. Experimental groups In our study, samples were taken from 30 patient (Table 1). Samples were collected from volunteers who attended the Unit of In Vitro Fertilization (IVF), Medipol International Health Center, Turkey in 20102011. The inclusion criteria for the non-smoker group were: age 20–40 years, not having suffered from any inflammatory disease in childhood, systemic disease, or other diseases that could affect reproductive functions such as testicular trauma; not using any drugs or substances (such as antidepressants, medicine for renal diseases, tension pills, and alcohol), never having smoked and being normospermic according to criteria of the WHO laboratory manual for the examination and processing of human semen (World Health Organization, 2010). Semen samples were evaluated in two ways, before freezing and after freezingthawing operations. The semen samples were taken into sterile containers through masturbation method after 3–5 days of sexual abstinence. For the study, the Ethics Committee Approval of Noninvasive Clinical Researches of İstanbul Medipol University (10840098604.01.01-E.2304) was received.
2.5. Detection of DNA fragmentation DNA fragmentation was examined by using the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) kit (FP09 I21 R01 C.7- in situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's guidelines. Briefly, the air-driedsmeared slides were fixed in 4% paraformaldehyde at room temperatureand rinsed in phosphate buffer (PBS), pH 7.4, and then permeabilizedwith 2% Triton X-100. The TdT-labeled nucleotide mixture wasadded to each slide and incubated in a humidified chamber at37 °C for 60 min in the dark. Slides were rinsedtwice in PBS and counterstained with 10 μl DAPI II (Abbott Molecular, USA, 06J50-001) and covered with coverslip. At least 200 cells of each sample were analyzed with a confocal microscope (Zeiss LSM 780 NLO). Each spermatozoon was assigned to contain either normal (blue nuclear fluorescence due to DAPI II) or fragmented DNA (green nuclear fluorescence). The final percentage of sperm with fragmented DNA was referred to as % TUNEL positive.
2.2. Motility and concentration Sperm motility and number were assessed before and after freezing operation using the Makler counting chamber (Sefi Medical Instr.). The each sperm movement was assessed in accordance with the WHO laboratory guidelines as forward movement (Progressive Motility; PR), in-motion (Nonprogressive Motility; NP) and immobility
2.6. Electron microscopic examination The semen samples that had been collected for electron microscopic examination were added washing solution after liquefaction, centrifuged at 2000 ×g for 5 min and supernatant was removed. The acquired pellet was applied an immersion fixation in 2.5% 0.1 M PBS buffered (pH 7.2) glutaraldehyde fixative at 4 °C for 4 h, and after washing it was applied a post-fixation with 1% OsO4 for 1 h. It was dehydrated with acetone series (70%, 90%, 96%, and 100%), treated with acetone and incubated with Epon 812 (#45359-Sigma - Aldrich, USA) in an incubator at 60 °C. The semi-thin sections (1 μm) received in ultramicrotome (Leica EM UC7) were stained with toluidine blue. Approximately 60 nm-thick thin sections were placed on copper grids
Table 1 Demographic characteristics of patients (mean of patient age, semen volüme, sperm concentrations, sperm motility and normal morphology). Mean Patient age Semen volüme Sperm concentrations Sperm progressive motility Sperm normal morphology
31,5 3,2 ml. 50 % 13 % 2%
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and contrasted with uranyl acetate and lead citrate. At least 10 sperm cells of the each sample of individuals prepered contrasted grids were examined in terms of changes occurring in head, neck and tail parts, and photographed with JEOL SX TEM (Tokyo, Japan).
Table 2 Correlation of seminal leptin levels with sperm DNA fragmentation (TUNEL) and motility rates. Leptin Motility
2.7. Leptin analyzes with ELISA Leptin levels of semen was examined by using the ELISA kit (Human Leptin ELISA Kit, Shanghai Yehua Biological Technology Co., Ltd., Shanghai, China). All reagents and non-cryopreserved / cryopreserved seminal plasma samples with sperm were brought to room temperature (18–25 °C) before use. 100 μl of each standard and sample were added into appropriate wells. Then, wells were covered and incubated for 2.5 h at room temperature or overnight at 4 °C with gentle shaking. The solution was discarded and washed 4 times with 1x wash solution. After the last wash, any remaining wash buffer was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels. 100 μl of 1× prepared Biotinylated Detection Antibody was added into each well and incubated for 1 h at temperature with gentle shaking. The solution was discarded and washed like previously described above. 100 μl prepared HRP-Streptavidin solution was added into each well and incubated for 45 min at room temperature with gentle shaking. The solution was discarded and washed again. 100 μl of ELISA Colorimetric TMB Reagent was added into each well and incubated for 30 min at room temperature in the dark with gentle shaking. 50 μl of Stop solution was added into each well and read at 450 nm immediately.
TUNEL
Pearson Correlation Sig. (2 -tailed) N Pearson Correlation Sig. (2-tailed) N
−,358** 0,005 60 ,572** 0 60
in the sperm observed prior to freezing operation. While significant reduction was observed in the number of sperm with normal morphology that are thawed after freezing operation; many sperm with head anomalies, with ruptured or curled tail, and sperm with cytoplasmic droplets were also observed. While the ratio of sperm with normal morphology was determined as 48.50 ± 22.08% before freezing operation, it was determined as 16.93 ± 14.04% after freezing -thawing operation. A statistically significant decrease (p < 0.05) was observed in the morphological damage rates between the groups compared prior freezing and after freezing -thawing operation (Fig. 6 and Table 2). 3.2. DNA fragmentation Before the freezing operation, TUNEL positive cells in a few sperm were seen as green fluorescence. Regarding the sperm thawed after 1 month of freezing, an increase was observed in the rate of TUNEL positive sperm, compared to the prior of the freezing operation (Fig. 1) Fig. 2. While the TUNEL positive sperm rate was 21.00 ± 8.22% before the freezing operation, it increased to 47.00 ± 9.01% in the sperm thawed 1 month after the freezing operation (Fig. 3). A statistically significant increase (p < 0.05) was determined in the comparison of the rates of DNA fragmentation (TUNEL positive sperm) between the groups before and after freezing-thawing operation (Fig. 7 and Table 2).
2.8. Reactive oxygen species (ROS) determination in semen Reactive oxygen species were measured colorimetrically in serum by the method developed by Erel (Erel, 2005). In this experiment Fox solution was prepared using 140 mM NaCl and 25 mM H2SO4 after this 150 mM D-Sorbitol and 25 μM X-orange were added into 225 ml of Fox solution to prepare reactive solution 1. Next, to prepare reactive solution 2, 10 mM 4-Hydroxybenzoic acid, 5 mM Ammonium Fe2 and SO4 were added into 25 ml of Fox solution. 20 μM H2O2 was prepared as standard. The solutions and seminal plasma samples were placed into 96-well plate and were measured spectrophotometrically at 658 nm through Spectra Max Microplate Spectrofluorometric device (KonicaMinolta CM 3600 A, Japan)
3.3. Transmission Electron microscopic findings The spermatozoa were examined for the ultrastructure of head, neck and tail areas before freezing and after thawing (Fig. 1). Typical head shapes, intact cell membranes, homogenously distributed acrosomes and nuclei as well as spermatozoa with normal morphology were observed before the freezing procedure. Intact internal and external acrosome membranes and electron-dense acrosome structures were also observed beneath the plasma membrane. In addition, there were also spermatozoa with disturbed head morphology as well as those containing cytoplasmic residue in the neck. In addition to reduced number of spermatozoa with normal morphology, a large number of spermatozoa with disturbed acrosome structures, subacrosomal swelling, acrosomal loss and vesicle formation, separation and loss in plasma membrane and chromatin condensation disorders were observed thawed after freezing (Fig. 1).
2.9. Ethics All patients signed an informed consent and the information for this study remained confidential within the institution. For the study, the Ethics Committee Approval of Noninvasive Clinical Researches of İstanbul Medipol University (10840098-604.01.01-E.2304) was received. 2.10. Statistical analysis The statistical analysis was performed using GraphPad Prism 7.0 program. The comparisons of motility, morphology, DNA fragmentation, ROS and leptin levels were performed using Two Sample Paired ttest before and after freezing operation. The correlation between seminal leptin levels and semen parameters was performed using Pearson and Linear regression test p < 0.05 was considered significant.
3.4. Leptin analysis by ELISA The semen samples were analyzed by ELISA method in terms of the leptin levels prior to the freezing and after freezing –thawing operation. While the seminal leptin level before freezing was 347, 58 ± 11,112 μg/ml, after freezing-thawing operation took place, it was 505,58 ± 62,43 μg/ml (Paired t-test). A statistically significant increase (p < 0.05) in leptin levels of the groups (N = 30) were determined when compared prior to the freezing and after freezing –thawing operation (Fig. 4). The correlation between sperm motility ratio and leptin levels was shown. (−,358**) (Fig. 6). A statistically significant (p < 0.01) negative correlation was observed between the
3. Results 3.1. Light microscopic findings In addition to many normal sperm, a small number of sperm with acrosomal disorder, neck fracture and tail anomalies were also observed 3
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Fig. 1. Sperm morphology, DNA fragmentation and structure before and after cryopreservation was compared A) normal morphology sperm (→) B) Head (▲) and neck abnormality (*) of sperm. C) before cryopreservation of sperm TUNEL nagative; blue color, D) after cryopreservation of sperm TUNEL positive; green color. E) Normal spermatozoon with intact head, acrosome (→), membranes and mid piece structure and cytoplasmic droplet in mid-piece (▲) F) disintegration and vesiculation of the acrosome(→), fragmentation of nucleus (*), loss of plasma membrane and irregular sequence of mitochondria (▲). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
freezing –thawing operation (Fig. 5).
groups (Pearson and Linear regression test). The correlation between DNA fragmentation (TUNEL positive sperm) ratio and leptin levels were shown (,572**) (Fig. 7). A statistically significant (p < 0.01) positive correlation was observed between the groups (Pearson and Linear regression test).
3.6. Discussion and conclusion In our study, the sperm were analyzed in terms of sperm parameters, DNA fragmentation, sperm fine structure, ROS and leptin levels before and after freeze-thawing operation. A decrease in sperm motility, and an increase in morphological damage, DNA fragmentation, ROS and leptin levels were observed after freezing-thawing operation. The harmful effects of cryopreservation process on sperm parameters have arisen many researchers’ interest. It has been proven in some studies that cryopreservation leads to a number of harmful changes in sperm structure and function (Thomson et al., 2009). These
3.5. ROS analysis While the ROS level was 15.61 ± 4.07 μmol H2O2 Eq./L before the freezing operation, it was 18.29 ± 3.70 μmol H2O2 Eq./L after the freezing-thawing operation (Paired t-test). A statistically significant increase (p < 0.05) was determined when the ROS levels were compared between the groups (N = 30) prior to the freezing and after 4
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Fig. 2. Sperm motility rates before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 5. Reactive oxygen levels (ROS) before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 6. Correlation of seminal leptin levels with sperm motility rates by Pearson correlation test.
Fig. 3. DNA fragmentation rates before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 7. Correlation of seminal leptin levels with sperm DNA fragmentation (TUNEL) rates by Pearson correlation test. Fig. 4. Leptin levels before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
shown that freezing damage can alter plasma membrane structure and integrity (Aydin et al., 2013). Ice crystals can cause damage to plasma membranes as well as organelle membranes. When this kind of mechanical damage occurs in the mitochondrial membrane, oxidative phosphorylation is adversely affected from this situation and ROS is released into the cell. We showed by our study that the ROS levels after freezing –thawing operation significantly increased compared to those prior to the freezing operation. Several studies have shown that sperm motility, vitality and normal morphology sperm rates decrease after cryopreservation and that DNA fragmentation rates increase (Ngamwuttiwong and Kunathikom, 2007; Petyim and Choavaratana, 2006). During our study, when we thawed the sperms after 1 month of freezing, we have seen an increase in the rate of multiple morphological damage compared to the pre-freezing. While sperm that have a morphology closer to normal were observed before the freezing operation;
changes can be basically summarized as the decrease of motility, morphological changes, the loss of membrane integrity and fluidity (Mazzilli et al., 1995) and DNA fragmentation (Paoli et al., 2014). Similarly, a statistically significant difference was observed in sperm motility and morphology, DNA fragmentation and leptin levels in our study. The ice crystals-related structural damages, membrane and organelle damages and DNA fragmentation were observed in the sperm after cryopreservation process. The intracellular and extracellular ice crystals, that occurred as a result of the uncontrolled cooling and improper thawing temperatures, lead to dysfunction causing structural damages in cells and organelle membranes by mechanical influence (Nallella et al., 2004). This leads to a decrease in the vitality and fertilization capacity and in mobility rate of sperm (Paoli et al., 2014). It has been 5
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concentrations exhibits statistically significant negative correlation with decreasing motility rates, and exhibit a positive correlation with increasing DNA fragmentation rates after freezing-thawing operation. This result suggests that leptin may be a marker for sperm motility and DNA fragmentation. The determination of leptin-related mechanisms will make important contributions to the studies that will be done on male fertility.
head, neck and tail damages were observed after freezing-thawing operation, and this situation was considered as multiple morphological damage. Sperm DNA integrity is important not only for the successful transfer of genetic material to future generations, but also for proper fertilization, quality embryo development and gestation. The recent studies show that sperm DNA damage is inversely associated with fertilization potential (Twigg et al., 1998), pregnancy rates and live birth rates. Oxidative stress is one of the basic mechanisms that potentially affect DNA fragmentation in sperm. The researches have shown that cryopreservation process, and following, thawing operation leads to caspase activation, and apoptosis is induced by this mechanism (Lewis, 2014). In our study, we determined the DNA fragmentation and apoptosis rates prior to the freezing operation and after 1 month of freezing then thawing operation by TUNEL method. DNA fragmentation was observed at low rates prior to the freezing operation. When the thawing operation was implemented 1 month after the freezing operation, a statistically significant increase was observed in DNA fragmentation rates compared to the pre- freezing operation. We think that this situation was caused by the deterioration of cell membrane structure, associated with the physical and chemical stress. The studies have suggested that leptin may play a role in the initiation of puberty in humans (Roemmich and Rogol, 1999). Fontoura et al. showed that leptin also potentiated reduction of DNA apoptosis in frozen-thawed capacitated samples in relation to sperm capacitation. The reduction of DNA apoptosis after leptin incubation could be explained by increased antioxidant defense, minimizing deleterious effects of oxidative stress during freezing and thawing processes (Fontoura et al., 2017). It has been explored by a study conducted on rats that varicocele-associated spermatogenic dysfunction is associated with increased leptin and leptin receptor hormone. Von Sobbe et al. showed by their study conducted in 2003 that semen leptin concentration and serum leptin concentration go parallel with each other, and testicular dysfunction levels and leptin levels in patients with infertility increase proportionately (von Sobbe et al., 2003). Leptin has been reported to be associated with spermatogenesis in several studies, but the association between leptin and sperm function and motility has not been found yet (Guo et al., 2014). Guo et al. showed that seminal leptin levels increase in the patients with idiopathic asthenozoospermia, and there is a negative correlation between seminal leptin levels and sperm motility (Guo et al., 2014). We showed by our study that there was a significant increase in seminal leptin levels after freezing–thawing operation and increased seminal leptin levels correlated negatively with sperm motility. Regarding leptin resistance, Vendramini et al. have shown that increased leptin levels and DNA fragmentation increase are parallel to each other (Vendramini et al., 2014). We showed by our study that increased seminal leptin concentrations and increased DNA fragmentation rates after freezing –thawing operation are statistically positively correlated with each other. Many detailed information such as chromatin structure, plasma structure and acrosome membrane structure, the arrangement of the mitochondria located in the neck can be obtained from the sperms examined by electron microscopy. Aydın et al. showed in the study conducted over the control and smoking group in 2012 that as well as the existence of a number of sperm with normal morphology; those including the disruption of the acrosome structure, blistering, separation and loss in the plasma membrane in neck region occurred in many sperm, and tail ruptures in some sperm after freezing and thawing operation (Aydin et al., 2013). Similarly, as well as a small number of normal sperm, the sperm having the findings of apoptosis such as the deterioration and swelling in membrane integrity, blistering, vacuole increment, the deterioration of chromatin condensation, and with damage at neck morphology, mitochondrial alignment, and tail and dynein structures were observed in our study. Consequently, it has been shown that increasing seminal leptin
Conflict of interest The authors state that they do not have any potential conflict of interest that would compromise the validity of the research conducted. Acknowledgments This study was supported by the Research Fund of Medipol University (BAP 86770134-604/13)–İstanbul/Turkey as Msc. thesis. We thank Dr. M. Şerif Aydın for contributions in the laboratory. References Agarwal, A., Virk, G., Ong, C., du Plessis, S.S., 2014. Effect of oxidative stress on male reproduction. World J. Mens Health 32 (1), 1–17. Aydin, M.S., Senturk, G.E., Ercan, F., 2013. Cryopreservation increases DNA fragmentation in spermatozoa of smokers. Acta Histochem. 115 (4), 394–400. Cummins, J.M., Jequier, A.M., Kan, R., 1994. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol. Reprod. Dev. 37 (3), 345–362. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38 (12), 1103–1111. Fontoura, P., Mello, M.D., Gallo-Sa, P., Erthal-Martins, M.C., Cardoso, M.C., Ramos, C., 2017. Leptin improves sperm cryopreservation via antioxidant defense. J. Reprod. Infertil. 18 (1), 172–178. Guo, J., Zhao, Y., Huang, W., Hu, W., Gu, J., Chen, C., Zhou, J., Peng, Y., Gong, M., Wang, Z., 2014. Sperm motility inversely correlates with seminal leptin levels in idiopathic asthenozoospermia. Int. J. Clin. Exp. Med. 7 (10), 3550–3555. Hoggard, N., Hunter, L., Duncan, J.S., Williams, L.M., Trayhurn, P., Mercer, J.G., 1997. Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc. Natl. Acad. Sci. U. S. A. 94 (20), 11073–11078. Karlsson, C., Lindell, K., Svensson, E., Bergh, C., Lind, P., Billig, H., Carlsson, L.M., Carlsson, B., 1997. Expression of functional leptin receptors in the human ovary. J. Clin. Endocrinol. Metab. 82 (12), 4144–4148. Lewis, S.E., 2014. Sperm DNA fragmentation and base oxidation. Adv. Exp. Med. Biol. 791, 103–116. Mazzilli, F., Rossi, T., Sabatini, L., Pulcinelli, F.M., Rapone, S., Dondero, F., Gazzaniga, P.P., 1995. Human sperm cryopreservation and reactive oxygen species (ROS) production. Acta Europaea fertilitatis 26 (4), 145–148. Najafi, A., Asadi, E., Moawad, A.R., Mikaeili, S., Amidi, F., Adutwum, E., Safa, M., Sobhani, A.G., 2016. Supplementation of freezing and thawing media with brainderived neurotrophic factor protects human sperm from freeze-thaw-induced damage. Fertil. Steril. 106 (7), 1658–1665 e1654. Nallella, K.P., Sharma, R.K., Allamaneni, S.S., Aziz, N., Agarwal, A., 2004. Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants. Fertil. Steril. 82 (4), 913–918. Ngamwuttiwong, T., Kunathikom, S., 2007. Evaluation of cryoinjury of sperm chromatin according to liquid nitrogen vapour method (I). J. Med. Assoc. Thailand 90 (2), 224–228. Paoli, D., Lombardo, F., Lenzi, A., Gandini, L., 2014. Sperm cryopreservation: effects on chromatin structure. Adv. Exp. Med. Biol. 791, 137–150. Petyim, S., Choavaratana, R., 2006. Cryodamage on sperm chromatin according to different freezing methods, assessed by AO test. J. Med. Assoc. Thailand 89 (3), 306–313. Roemmich, J.N., Rogol, A.D., 1999. Role of leptin during childhood growth and development. Endocrinol. Metab. Clin. North Am. 28 (4), 749–764 viii. Schiller, J., Arnhold, J., Glander, H.J., Arnold, K., 2000. Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy – effects of freezing and thawing. Chem. Phys. Lipids 106 (2), 145–156. Spitzweg, C., Heufelder, A.E., 1997. More clues from fat mice: leptin acts as an opponent of the hypothalamic neuropeptide Y system. Eur. J. Endocrinol. 136 (6), 590–591. Thomson, L.K., Fleming, S.D., Schulke, L., Barone, K., Zieschang, J.A., Clark, A.M., 2009. The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation. Fertil. Steril. 92 (3), 991–1001. Twigg, J., Fulton, N., Gomez, E., Irvine, D.S., Aitken, R.J., 1998. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod. 13 (6), 1429–1436. Vendramini, V., Cedenho, A.P., Miraglia, S.M., Spaine, D.M., 2014. Reproductive function
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Leptin in sperm analysis can be a new indicator ⁎
Tuğçe Önela, , Sule Aylaa,e, İlknur Keskina,e, Cüneyd Parlayanb,e, Türkan Yiğitbaşıc, Bircan Kolbaşıa,e, Tuba Varlı Yelked, Tuğba Şenel Ustabaşd İstanbul Medipol University, School of Medicine, Department of Histology and Embryology, İstanbul, Turkey İstanbul Medipol University, Engineering and Natural Sciences, Department of Biomedical Engineering, İstanbul, Turkey İstanbul Medipol University, School of Medicine, Department of Medical Biochemistry, İstanbul, Turkey d Medipol International Health Center, In Vitro Fertilization Unit (IVF), İstanbul, Turkey e Regenerative and Restorative Medicine Research Center (REMER), İstanbul Medipol University, İstanbul, Turkey a
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A R T I C LE I N FO
A B S T R A C T
Keywords: Cryopreservation DNA fragmentation Leptin Motility Sperm
Purpose Radiotherapy, chemotherapy, various tumors and invasive surgery can result in ejaculatory dysfunction and testicular insufficiency. Sperm cryopreservation is the only method which can provide a baby for couples. Cryopreservation freezes tissues and cells, allowing them to be stored for future use by stopping all biological activities. Cryopreservation can cause some harmful changes in the structure and function of the sperm. Leptin molecule plays many roles in most biological processes including the satiety and cell renewal, proliferation, angiogenesis, modulation of energy expenditure and regulation of the neuroendocrine system. Leptin was also reported to be associated with spermatogenesis in several studies. Methods This study aims to use leptin molecule as a parameter for sperm motility and DNA fragmentation before and after the cryopreservation. In this study, semen samples were taken from 30 normospermic male volunteers. Each semen sample was examined for the same parameters before and after the cryopreservation. Samples were analyzed before and after cryopreservation in terms of sperm motility by morphological sperm analysis with spermac stain dye, DNA fragmentation by TUNEL assay, ultrastructural analysis with transmission electron microscopy (TEM), seminal leptin levels by ELISA method and reactive oxygen species (ROS) levels by colorimetric method. Results Decreased sperm motility, distribution of sperm morphology and increased DNA fragmentation were determined after cryopreservation. Similarly, seminal ROS and leptin levels were also increased significantly. There was a negative correlation between seminal leptin and sperm motility. Additionally, there was a positive correlation between seminal leptin and DNA fragmentation. Conclusion According to these results, leptin molecule can be used as a marker for sperm motility and DNA fragmentation before and after cryopreservation. We think that the results of this study can contribute to further studies in the clinical aspect.
1. Introduction Cryopreservation is a technique used in assisted reproductive techniques, aiming at preserving cells at very low temperatures without losing their viability and functionality. The sperms are exposed to physical and chemical stresses during cryopreservation, thus, the lipid structure of plasma membrane changes, and phosphatidylserine output takes place (Schiller et al., 2000). In spite of the recent advances that have been made in the field of sperm cryopreservation, the process has the potential to compromise sperm function and quality through generation of reactive oxygen species (ROS) and reduction in antioxidant
activity (Najafi et al., 2016). Reactive oxygen species (ROS) are a class of free radicals with highly reactive oxygenated agents. ROS production at high concentrations plays an important role in male infertility by affecting sperm metabolism, and consequently morphology, motility and fertility in a negative way causing lipid peroxidation and impaired membrane function (Cummins et al., 1994). Human spermatozoa are highly susceptible to oxidative stress (OS) damage due to low cytoplasmic antioxidant content and high polyunsaturated fatty acids (Najafi et al., 2016). Much evidence has shown that OS contributes to both direct and indirect cellular damage during the sperm cryopreservation process
Abbreviations: ROS, reactive oxygen species; TEM, transmission electron microscope; WHO, World Health Organization ⁎ Corresponding author at: Yeditepe University, Department of Histology and Embryology, School of Medicine, Turkey. E-mail addresses: [email protected], [email protected], [email protected] (T. Önel), [email protected] (S. Ayla), [email protected] (İ. Keskin), [email protected] (C. Parlayan), [email protected] (B. Kolbaşı), [email protected] (T.V. Yelke), [email protected] (T.Ş. Ustabaş). https://doi.org/10.1016/j.acthis.2018.10.006 Received 28 February 2018; Received in revised form 15 October 2018; Accepted 16 October 2018 0065-1281/ © 2018 Published by Elsevier GmbH.
Please cite this article as: Önel, T., Acta Histochemica, https://doi.org/10.1016/j.acthis.2018.10.006
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(Immotility;IM). (WHO, 2010) The individuals with the sperm number ranging between 20–200 million/ml. were included in the study. All these operations were performed in the room temperature of 20–24°.
(Agarwal et al., 2014). It has been reported that generation of ROS occurs during the freezing and/or thawing process of spermatozoa (Paoli et al., 2014). It seems likely that the sudden increase in oxygen consumption by spermatozoa during thawing results in ROS-induced membrane damage (Zubkova and Robaire, 2004). Leptin, known to be secreted by fat cells and any tissues, is a polypeptide that contains 167 amino acids, with 16 kD molecular weight, that maintains a specific blood level in the plasma, and is carried in blood freely and/or adhered to protein (Zhang et al., 1994). Leptin’s main mechanism of action is to inhibit the oscillation and expression of neuropeptide-Y, which is involved in the regulation of many organs while its main effect is to increase appetite from arcuate nucleus (Spitzweg and Heufelder, 1997). Although presence of leptin and its receptor has been demonstrated in spermatozoa, its role on spermatogenesis and sperm still needs to be clarified (Fontoura et al., 2017). Understanding that leptin is also synthesized by the placenta and that leptin receptors are also expressed in the placenta and ovaries (Hoggard et al., 1997; Karlsson et al., 1997) suggest that leptin may have important effects on the reproductive system. The aim of this study is to make comparisons of motility, morphology and DNA fragmentation in the sperm before and after cryopreservation, and to investigate the association of these parameters with leptin molecule levels. Through this, we aim to use leptin molecule level as a determinant in sperm motility and DNA fragmentation.
2.3. Sperm freezing and thawing procedure For freezing operation, semen sample and cryoprotectant (90128Irvine Scientific Freezing Medium) were mixed in 1: 1 ratio so as to result in a total volume of 1 ml. and sterile tubes were taken into ‘Cryo Vial, T3082A’. The tubes were placed in the liquid nitrogen tank (-196 °C) after being kept at room temperature for 8 min, and at nitrogen steam at height of 15 cm from the liquid nitrogen level for 20 min respectively. The thawing operation was carried out by the removal of the tubes from liquid nitrogen, and with hot water treatment at room temperature. After the sample was completely dissolved, eluent (HTF / HEPES ART-1023) was added, the tubes were centrifuged for 5 min at 2000 rpm and supernatant was expelled from cryoprotectan. The sperm samples obtained after the thawing operation were used for motility, morphology, DNA fragmentation, leptin and ROS level evaluations. 2.4. Analyses of sperm morphology Light microscopic morphological examination was carried out after staining with Spermac Stain kit (FP09 I21 R01 C.7- FertiPro N.V., Belgium). A smear preparation was made out of the sperm samples. After keeping in fixative as well as cationic and anionic dyes of the kit for one minute each, it was washed with distilled water and dried. At least 200 cells of each sample of individuals were analyzed with immersion oil at 1000x magnitude by using an Olympus BX51 (Tokyo, Japan) photomicroscope according to Kruger stick criteria (Menkveld et al 1991). All these operations were performed in the room temperature of 20–24°.
2. Method and material 2.1. Experimental groups In our study, samples were taken from 30 patient (Table 1). Samples were collected from volunteers who attended the Unit of In Vitro Fertilization (IVF), Medipol International Health Center, Turkey in 20102011. The inclusion criteria for the non-smoker group were: age 20–40 years, not having suffered from any inflammatory disease in childhood, systemic disease, or other diseases that could affect reproductive functions such as testicular trauma; not using any drugs or substances (such as antidepressants, medicine for renal diseases, tension pills, and alcohol), never having smoked and being normospermic according to criteria of the WHO laboratory manual for the examination and processing of human semen (World Health Organization, 2010). Semen samples were evaluated in two ways, before freezing and after freezingthawing operations. The semen samples were taken into sterile containers through masturbation method after 3–5 days of sexual abstinence. For the study, the Ethics Committee Approval of Noninvasive Clinical Researches of İstanbul Medipol University (10840098604.01.01-E.2304) was received.
2.5. Detection of DNA fragmentation DNA fragmentation was examined by using the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) kit (FP09 I21 R01 C.7- in situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's guidelines. Briefly, the air-driedsmeared slides were fixed in 4% paraformaldehyde at room temperatureand rinsed in phosphate buffer (PBS), pH 7.4, and then permeabilizedwith 2% Triton X-100. The TdT-labeled nucleotide mixture wasadded to each slide and incubated in a humidified chamber at37 °C for 60 min in the dark. Slides were rinsedtwice in PBS and counterstained with 10 μl DAPI II (Abbott Molecular, USA, 06J50-001) and covered with coverslip. At least 200 cells of each sample were analyzed with a confocal microscope (Zeiss LSM 780 NLO). Each spermatozoon was assigned to contain either normal (blue nuclear fluorescence due to DAPI II) or fragmented DNA (green nuclear fluorescence). The final percentage of sperm with fragmented DNA was referred to as % TUNEL positive.
2.2. Motility and concentration Sperm motility and number were assessed before and after freezing operation using the Makler counting chamber (Sefi Medical Instr.). The each sperm movement was assessed in accordance with the WHO laboratory guidelines as forward movement (Progressive Motility; PR), in-motion (Nonprogressive Motility; NP) and immobility
2.6. Electron microscopic examination The semen samples that had been collected for electron microscopic examination were added washing solution after liquefaction, centrifuged at 2000 ×g for 5 min and supernatant was removed. The acquired pellet was applied an immersion fixation in 2.5% 0.1 M PBS buffered (pH 7.2) glutaraldehyde fixative at 4 °C for 4 h, and after washing it was applied a post-fixation with 1% OsO4 for 1 h. It was dehydrated with acetone series (70%, 90%, 96%, and 100%), treated with acetone and incubated with Epon 812 (#45359-Sigma - Aldrich, USA) in an incubator at 60 °C. The semi-thin sections (1 μm) received in ultramicrotome (Leica EM UC7) were stained with toluidine blue. Approximately 60 nm-thick thin sections were placed on copper grids
Table 1 Demographic characteristics of patients (mean of patient age, semen volüme, sperm concentrations, sperm motility and normal morphology). Mean Patient age Semen volüme Sperm concentrations Sperm progressive motility Sperm normal morphology
31,5 3,2 ml. 50 % 13 % 2%
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and contrasted with uranyl acetate and lead citrate. At least 10 sperm cells of the each sample of individuals prepered contrasted grids were examined in terms of changes occurring in head, neck and tail parts, and photographed with JEOL SX TEM (Tokyo, Japan).
Table 2 Correlation of seminal leptin levels with sperm DNA fragmentation (TUNEL) and motility rates. Leptin Motility
2.7. Leptin analyzes with ELISA Leptin levels of semen was examined by using the ELISA kit (Human Leptin ELISA Kit, Shanghai Yehua Biological Technology Co., Ltd., Shanghai, China). All reagents and non-cryopreserved / cryopreserved seminal plasma samples with sperm were brought to room temperature (18–25 °C) before use. 100 μl of each standard and sample were added into appropriate wells. Then, wells were covered and incubated for 2.5 h at room temperature or overnight at 4 °C with gentle shaking. The solution was discarded and washed 4 times with 1x wash solution. After the last wash, any remaining wash buffer was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels. 100 μl of 1× prepared Biotinylated Detection Antibody was added into each well and incubated for 1 h at temperature with gentle shaking. The solution was discarded and washed like previously described above. 100 μl prepared HRP-Streptavidin solution was added into each well and incubated for 45 min at room temperature with gentle shaking. The solution was discarded and washed again. 100 μl of ELISA Colorimetric TMB Reagent was added into each well and incubated for 30 min at room temperature in the dark with gentle shaking. 50 μl of Stop solution was added into each well and read at 450 nm immediately.
TUNEL
Pearson Correlation Sig. (2 -tailed) N Pearson Correlation Sig. (2-tailed) N
−,358** 0,005 60 ,572** 0 60
in the sperm observed prior to freezing operation. While significant reduction was observed in the number of sperm with normal morphology that are thawed after freezing operation; many sperm with head anomalies, with ruptured or curled tail, and sperm with cytoplasmic droplets were also observed. While the ratio of sperm with normal morphology was determined as 48.50 ± 22.08% before freezing operation, it was determined as 16.93 ± 14.04% after freezing -thawing operation. A statistically significant decrease (p < 0.05) was observed in the morphological damage rates between the groups compared prior freezing and after freezing -thawing operation (Fig. 6 and Table 2). 3.2. DNA fragmentation Before the freezing operation, TUNEL positive cells in a few sperm were seen as green fluorescence. Regarding the sperm thawed after 1 month of freezing, an increase was observed in the rate of TUNEL positive sperm, compared to the prior of the freezing operation (Fig. 1) Fig. 2. While the TUNEL positive sperm rate was 21.00 ± 8.22% before the freezing operation, it increased to 47.00 ± 9.01% in the sperm thawed 1 month after the freezing operation (Fig. 3). A statistically significant increase (p < 0.05) was determined in the comparison of the rates of DNA fragmentation (TUNEL positive sperm) between the groups before and after freezing-thawing operation (Fig. 7 and Table 2).
2.8. Reactive oxygen species (ROS) determination in semen Reactive oxygen species were measured colorimetrically in serum by the method developed by Erel (Erel, 2005). In this experiment Fox solution was prepared using 140 mM NaCl and 25 mM H2SO4 after this 150 mM D-Sorbitol and 25 μM X-orange were added into 225 ml of Fox solution to prepare reactive solution 1. Next, to prepare reactive solution 2, 10 mM 4-Hydroxybenzoic acid, 5 mM Ammonium Fe2 and SO4 were added into 25 ml of Fox solution. 20 μM H2O2 was prepared as standard. The solutions and seminal plasma samples were placed into 96-well plate and were measured spectrophotometrically at 658 nm through Spectra Max Microplate Spectrofluorometric device (KonicaMinolta CM 3600 A, Japan)
3.3. Transmission Electron microscopic findings The spermatozoa were examined for the ultrastructure of head, neck and tail areas before freezing and after thawing (Fig. 1). Typical head shapes, intact cell membranes, homogenously distributed acrosomes and nuclei as well as spermatozoa with normal morphology were observed before the freezing procedure. Intact internal and external acrosome membranes and electron-dense acrosome structures were also observed beneath the plasma membrane. In addition, there were also spermatozoa with disturbed head morphology as well as those containing cytoplasmic residue in the neck. In addition to reduced number of spermatozoa with normal morphology, a large number of spermatozoa with disturbed acrosome structures, subacrosomal swelling, acrosomal loss and vesicle formation, separation and loss in plasma membrane and chromatin condensation disorders were observed thawed after freezing (Fig. 1).
2.9. Ethics All patients signed an informed consent and the information for this study remained confidential within the institution. For the study, the Ethics Committee Approval of Noninvasive Clinical Researches of İstanbul Medipol University (10840098-604.01.01-E.2304) was received. 2.10. Statistical analysis The statistical analysis was performed using GraphPad Prism 7.0 program. The comparisons of motility, morphology, DNA fragmentation, ROS and leptin levels were performed using Two Sample Paired ttest before and after freezing operation. The correlation between seminal leptin levels and semen parameters was performed using Pearson and Linear regression test p < 0.05 was considered significant.
3.4. Leptin analysis by ELISA The semen samples were analyzed by ELISA method in terms of the leptin levels prior to the freezing and after freezing –thawing operation. While the seminal leptin level before freezing was 347, 58 ± 11,112 μg/ml, after freezing-thawing operation took place, it was 505,58 ± 62,43 μg/ml (Paired t-test). A statistically significant increase (p < 0.05) in leptin levels of the groups (N = 30) were determined when compared prior to the freezing and after freezing –thawing operation (Fig. 4). The correlation between sperm motility ratio and leptin levels was shown. (−,358**) (Fig. 6). A statistically significant (p < 0.01) negative correlation was observed between the
3. Results 3.1. Light microscopic findings In addition to many normal sperm, a small number of sperm with acrosomal disorder, neck fracture and tail anomalies were also observed 3
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Fig. 1. Sperm morphology, DNA fragmentation and structure before and after cryopreservation was compared A) normal morphology sperm (→) B) Head (▲) and neck abnormality (*) of sperm. C) before cryopreservation of sperm TUNEL nagative; blue color, D) after cryopreservation of sperm TUNEL positive; green color. E) Normal spermatozoon with intact head, acrosome (→), membranes and mid piece structure and cytoplasmic droplet in mid-piece (▲) F) disintegration and vesiculation of the acrosome(→), fragmentation of nucleus (*), loss of plasma membrane and irregular sequence of mitochondria (▲). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
freezing –thawing operation (Fig. 5).
groups (Pearson and Linear regression test). The correlation between DNA fragmentation (TUNEL positive sperm) ratio and leptin levels were shown (,572**) (Fig. 7). A statistically significant (p < 0.01) positive correlation was observed between the groups (Pearson and Linear regression test).
3.6. Discussion and conclusion In our study, the sperm were analyzed in terms of sperm parameters, DNA fragmentation, sperm fine structure, ROS and leptin levels before and after freeze-thawing operation. A decrease in sperm motility, and an increase in morphological damage, DNA fragmentation, ROS and leptin levels were observed after freezing-thawing operation. The harmful effects of cryopreservation process on sperm parameters have arisen many researchers’ interest. It has been proven in some studies that cryopreservation leads to a number of harmful changes in sperm structure and function (Thomson et al., 2009). These
3.5. ROS analysis While the ROS level was 15.61 ± 4.07 μmol H2O2 Eq./L before the freezing operation, it was 18.29 ± 3.70 μmol H2O2 Eq./L after the freezing-thawing operation (Paired t-test). A statistically significant increase (p < 0.05) was determined when the ROS levels were compared between the groups (N = 30) prior to the freezing and after 4
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Fig. 2. Sperm motility rates before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 5. Reactive oxygen levels (ROS) before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 6. Correlation of seminal leptin levels with sperm motility rates by Pearson correlation test.
Fig. 3. DNA fragmentation rates before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
Fig. 7. Correlation of seminal leptin levels with sperm DNA fragmentation (TUNEL) rates by Pearson correlation test. Fig. 4. Leptin levels before and after cryopreservation (N = 30). Significant differences determined by Paired t-test (p < 0.0001).
shown that freezing damage can alter plasma membrane structure and integrity (Aydin et al., 2013). Ice crystals can cause damage to plasma membranes as well as organelle membranes. When this kind of mechanical damage occurs in the mitochondrial membrane, oxidative phosphorylation is adversely affected from this situation and ROS is released into the cell. We showed by our study that the ROS levels after freezing –thawing operation significantly increased compared to those prior to the freezing operation. Several studies have shown that sperm motility, vitality and normal morphology sperm rates decrease after cryopreservation and that DNA fragmentation rates increase (Ngamwuttiwong and Kunathikom, 2007; Petyim and Choavaratana, 2006). During our study, when we thawed the sperms after 1 month of freezing, we have seen an increase in the rate of multiple morphological damage compared to the pre-freezing. While sperm that have a morphology closer to normal were observed before the freezing operation;
changes can be basically summarized as the decrease of motility, morphological changes, the loss of membrane integrity and fluidity (Mazzilli et al., 1995) and DNA fragmentation (Paoli et al., 2014). Similarly, a statistically significant difference was observed in sperm motility and morphology, DNA fragmentation and leptin levels in our study. The ice crystals-related structural damages, membrane and organelle damages and DNA fragmentation were observed in the sperm after cryopreservation process. The intracellular and extracellular ice crystals, that occurred as a result of the uncontrolled cooling and improper thawing temperatures, lead to dysfunction causing structural damages in cells and organelle membranes by mechanical influence (Nallella et al., 2004). This leads to a decrease in the vitality and fertilization capacity and in mobility rate of sperm (Paoli et al., 2014). It has been 5
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concentrations exhibits statistically significant negative correlation with decreasing motility rates, and exhibit a positive correlation with increasing DNA fragmentation rates after freezing-thawing operation. This result suggests that leptin may be a marker for sperm motility and DNA fragmentation. The determination of leptin-related mechanisms will make important contributions to the studies that will be done on male fertility.
head, neck and tail damages were observed after freezing-thawing operation, and this situation was considered as multiple morphological damage. Sperm DNA integrity is important not only for the successful transfer of genetic material to future generations, but also for proper fertilization, quality embryo development and gestation. The recent studies show that sperm DNA damage is inversely associated with fertilization potential (Twigg et al., 1998), pregnancy rates and live birth rates. Oxidative stress is one of the basic mechanisms that potentially affect DNA fragmentation in sperm. The researches have shown that cryopreservation process, and following, thawing operation leads to caspase activation, and apoptosis is induced by this mechanism (Lewis, 2014). In our study, we determined the DNA fragmentation and apoptosis rates prior to the freezing operation and after 1 month of freezing then thawing operation by TUNEL method. DNA fragmentation was observed at low rates prior to the freezing operation. When the thawing operation was implemented 1 month after the freezing operation, a statistically significant increase was observed in DNA fragmentation rates compared to the pre- freezing operation. We think that this situation was caused by the deterioration of cell membrane structure, associated with the physical and chemical stress. The studies have suggested that leptin may play a role in the initiation of puberty in humans (Roemmich and Rogol, 1999). Fontoura et al. showed that leptin also potentiated reduction of DNA apoptosis in frozen-thawed capacitated samples in relation to sperm capacitation. The reduction of DNA apoptosis after leptin incubation could be explained by increased antioxidant defense, minimizing deleterious effects of oxidative stress during freezing and thawing processes (Fontoura et al., 2017). It has been explored by a study conducted on rats that varicocele-associated spermatogenic dysfunction is associated with increased leptin and leptin receptor hormone. Von Sobbe et al. showed by their study conducted in 2003 that semen leptin concentration and serum leptin concentration go parallel with each other, and testicular dysfunction levels and leptin levels in patients with infertility increase proportionately (von Sobbe et al., 2003). Leptin has been reported to be associated with spermatogenesis in several studies, but the association between leptin and sperm function and motility has not been found yet (Guo et al., 2014). Guo et al. showed that seminal leptin levels increase in the patients with idiopathic asthenozoospermia, and there is a negative correlation between seminal leptin levels and sperm motility (Guo et al., 2014). We showed by our study that there was a significant increase in seminal leptin levels after freezing–thawing operation and increased seminal leptin levels correlated negatively with sperm motility. Regarding leptin resistance, Vendramini et al. have shown that increased leptin levels and DNA fragmentation increase are parallel to each other (Vendramini et al., 2014). We showed by our study that increased seminal leptin concentrations and increased DNA fragmentation rates after freezing –thawing operation are statistically positively correlated with each other. Many detailed information such as chromatin structure, plasma structure and acrosome membrane structure, the arrangement of the mitochondria located in the neck can be obtained from the sperms examined by electron microscopy. Aydın et al. showed in the study conducted over the control and smoking group in 2012 that as well as the existence of a number of sperm with normal morphology; those including the disruption of the acrosome structure, blistering, separation and loss in the plasma membrane in neck region occurred in many sperm, and tail ruptures in some sperm after freezing and thawing operation (Aydin et al., 2013). Similarly, as well as a small number of normal sperm, the sperm having the findings of apoptosis such as the deterioration and swelling in membrane integrity, blistering, vacuole increment, the deterioration of chromatin condensation, and with damage at neck morphology, mitochondrial alignment, and tail and dynein structures were observed in our study. Consequently, it has been shown that increasing seminal leptin
Conflict of interest The authors state that they do not have any potential conflict of interest that would compromise the validity of the research conducted. Acknowledgments This study was supported by the Research Fund of Medipol University (BAP 86770134-604/13)–İstanbul/Turkey as Msc. thesis. We thank Dr. M. Şerif Aydın for contributions in the laboratory. References Agarwal, A., Virk, G., Ong, C., du Plessis, S.S., 2014. Effect of oxidative stress on male reproduction. World J. Mens Health 32 (1), 1–17. Aydin, M.S., Senturk, G.E., Ercan, F., 2013. Cryopreservation increases DNA fragmentation in spermatozoa of smokers. Acta Histochem. 115 (4), 394–400. Cummins, J.M., Jequier, A.M., Kan, R., 1994. Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress? Mol. Reprod. Dev. 37 (3), 345–362. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38 (12), 1103–1111. Fontoura, P., Mello, M.D., Gallo-Sa, P., Erthal-Martins, M.C., Cardoso, M.C., Ramos, C., 2017. Leptin improves sperm cryopreservation via antioxidant defense. J. Reprod. Infertil. 18 (1), 172–178. Guo, J., Zhao, Y., Huang, W., Hu, W., Gu, J., Chen, C., Zhou, J., Peng, Y., Gong, M., Wang, Z., 2014. Sperm motility inversely correlates with seminal leptin levels in idiopathic asthenozoospermia. Int. J. Clin. Exp. Med. 7 (10), 3550–3555. Hoggard, N., Hunter, L., Duncan, J.S., Williams, L.M., Trayhurn, P., Mercer, J.G., 1997. Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc. Natl. Acad. Sci. U. S. A. 94 (20), 11073–11078. Karlsson, C., Lindell, K., Svensson, E., Bergh, C., Lind, P., Billig, H., Carlsson, L.M., Carlsson, B., 1997. Expression of functional leptin receptors in the human ovary. J. Clin. Endocrinol. Metab. 82 (12), 4144–4148. Lewis, S.E., 2014. Sperm DNA fragmentation and base oxidation. Adv. Exp. Med. Biol. 791, 103–116. Mazzilli, F., Rossi, T., Sabatini, L., Pulcinelli, F.M., Rapone, S., Dondero, F., Gazzaniga, P.P., 1995. Human sperm cryopreservation and reactive oxygen species (ROS) production. Acta Europaea fertilitatis 26 (4), 145–148. Najafi, A., Asadi, E., Moawad, A.R., Mikaeili, S., Amidi, F., Adutwum, E., Safa, M., Sobhani, A.G., 2016. Supplementation of freezing and thawing media with brainderived neurotrophic factor protects human sperm from freeze-thaw-induced damage. Fertil. Steril. 106 (7), 1658–1665 e1654. Nallella, K.P., Sharma, R.K., Allamaneni, S.S., Aziz, N., Agarwal, A., 2004. Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants. Fertil. Steril. 82 (4), 913–918. Ngamwuttiwong, T., Kunathikom, S., 2007. Evaluation of cryoinjury of sperm chromatin according to liquid nitrogen vapour method (I). J. Med. Assoc. Thailand 90 (2), 224–228. Paoli, D., Lombardo, F., Lenzi, A., Gandini, L., 2014. Sperm cryopreservation: effects on chromatin structure. Adv. Exp. Med. Biol. 791, 137–150. Petyim, S., Choavaratana, R., 2006. Cryodamage on sperm chromatin according to different freezing methods, assessed by AO test. J. Med. Assoc. Thailand 89 (3), 306–313. Roemmich, J.N., Rogol, A.D., 1999. Role of leptin during childhood growth and development. Endocrinol. Metab. Clin. North Am. 28 (4), 749–764 viii. Schiller, J., Arnhold, J., Glander, H.J., Arnold, K., 2000. Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy – effects of freezing and thawing. Chem. Phys. Lipids 106 (2), 145–156. Spitzweg, C., Heufelder, A.E., 1997. More clues from fat mice: leptin acts as an opponent of the hypothalamic neuropeptide Y system. Eur. J. Endocrinol. 136 (6), 590–591. Thomson, L.K., Fleming, S.D., Schulke, L., Barone, K., Zieschang, J.A., Clark, A.M., 2009. The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation. Fertil. Steril. 92 (3), 991–1001. Twigg, J., Fulton, N., Gomez, E., Irvine, D.S., Aitken, R.J., 1998. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod. 13 (6), 1429–1436. Vendramini, V., Cedenho, A.P., Miraglia, S.M., Spaine, D.M., 2014. Reproductive function
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