Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2019 / Article

Research Article | Open Access

Volume 2019 |Article ID 6472945 | https://doi.org/10.1155/2019/6472945

Juliana R. Pariz, Caroline Ranéa, Rosa A. C. Monteiro, Donald P. Evenson, Joël R. Drevet, Jorge Hallak, "Melatonin and Caffeine Supplementation Used, Respectively, as Protective and Stimulating Agents in the Cryopreservation of Human Sperm Improves Survival, Viability, and Motility after Thawing compared to Traditional TEST-Yolk Buffer", Oxidative Medicine and Cellular Longevity, vol. 2019, Article ID 6472945, 10 pages, 2019. https://doi.org/10.1155/2019/6472945

Melatonin and Caffeine Supplementation Used, Respectively, as Protective and Stimulating Agents in the Cryopreservation of Human Sperm Improves Survival, Viability, and Motility after Thawing compared to Traditional TEST-Yolk Buffer

Academic Editor: Alexandros Georgakilas
Received24 May 2019
Revised08 Aug 2019
Accepted07 Sep 2019
Published23 Oct 2019


Cryopreservation processes can damage spermatozoa and impair structural and functional cell characteristics. Plasma, nuclear membranes, and cellular organelles can suffer from the freeze and thaw process. This study evaluates the protective and stimulant effect of melatonin and caffeine supplementation on the functional characteristics of human spermatozoa before and after freezing. Thirty seminal samples from normozoospermic men aged 19–45 years old collected between October 2012 and May 2017 were included. Semen samples were supplemented with either 2 mM melatonin (MEL) prior to cryopreservation, 2 mM caffeine (CAF) in postthaw, or CAF and MEL (CM) in precryopreservation and postthaw, respectively. Kinetics and seminal parameters, mitochondrial activity, DNA fragmentation, and reactive oxygen species (ROS) levels were analyzed before and after cryopreservation. A significant reduction in sperm concentration, total and progressive motility, sperm kinetics, and mitochondrial activity, as well as a significant increase in DNA fragmentation and ROS production in postthaw samples compared to fresh samples, was identified. After administration of a caffeine and/or melatonin supplement, there was a significant increase in progressive motility in the CAF () and CM () groups, as well as mitochondrial activity in the CM group (). Cryopreservation has negative effects on overall sperm quality and increases ROS production. A combination of caffeine and melatonin in prefreeze and postthaw sperm samples has proven to be a very effective and simple way to improve semen quality. This will be particularly useful for initial low-quality semen samples, those which suffer the most from the freezing/thawing process.

1. Introduction

Sperm cryopreservation is recognized as an essential and relevant aid in the management of infertile patients, regardless of clinical diagnosis. It is of the utmost importance in a variety of clinical situations ranging from cancer to autoimmunity and its immunosuppressive therapeutic strategies to nonmalignant immunological disorders (e.g., lupus erythematous or rheumatoid arthritis) [17]. Sperm cryopreservation is also of great help when considering sperm donation and sperm banking, postmortem sperm retrieval, and saving sperm prior to vasectomy; after challenging sperm collection microsurgeries prior to assisted reproduction techniques (ARTs); or in people diagnosed with testicular dysgenesis syndrome or chronically exposed to gonadotoxic substances [4, 814].

One of the main disadvantages of sperm cryopreservation is the low postthaw viability, since 25 to 75% of recovered sperm have structural and/or functional damage or do not even survive the mechanical and osmotic stresses associated with the freezing/thawing process, potentially suffering lysis of the sperm cell membrane, specially in morphologically or functionally defective spermatozoa [15, 16]. This is evidenced by a dramatically decreased postthaw motility, which has been associated with higher risk of abortion and congenital malformations after ARTs [1720]. Besides the deleterious mechanical effects of the freezing/thawing process, most of the detrimental impact of cryopreservation on spermatozoa is linked to oxidative stress [21, 22]. As it is now well-described and accepted by both the clinical and scientific communities, oxidative lesions of spermatozoa have multiple visages affecting sperm structures including plasma membrane, mitochondria, nucleus, and DNA, compromising sperm functions (motility, gamete recognition, and its ability to capacitate as well as its ability to deliver an optimal paternal genetic material into the oocyte). All these consequently strongly affect spermatozoon fertilizing potential and reproductive success and increase the risk of adverse paternally inherited defects in the progeny [2339]. Therefore, a logical challenge is to attempt to repair/limit some of the damage caused by oxidative stress (OS) during the cryopreservation process in order to maximize gamete survival and function.

Many ingredients have been tested for their ability to reduce OS-mediated damage during cryopreservation of human semen and/or improve sperm mobility after thawing. For example, glutamine, catalase, and ascorbic acid had a protective action and preserved sperm motility when added to cryopreservation media while pentoxifylline supplementation after thawing improved sperm mobility and decreased ROS production [4043]. Taking advantage of the well-described pleiotropic antioxidant (AO) action of melatonin [4446] and the well-known stimulating effect of caffeine on sperm motility [4753], we have decided to evaluate the benefit of complementing our cryopreservation medium with melatonin or our postthaw medium with caffeine or a combination of the two. Thus, the aim of the present study was to investigate the effect of melatonin and caffeine supplementation on the functional characteristics of prefreeze and postthaw human spermatozoa.

2. Materials and Methods

2.1. Study Design and Patients

This prospective study included 30 normozoospermic samples from male patients aged 22 to 45 years who were treated at Androscience, Science and Innovation Center in Andrology and High-Complex Clinical and Andrology Laboratory, São Paulo, Brazil, from 2013 to 2017. Exclusion criteria included azoospermia, leukocytospermia, necrozoospermia, and other seminal abnormalities such as , , and a percentage of normal morphology <4%. This study was approved by the “Research Ethics Committee” of the University of São Paulo Medical School, Brazil (No. 031/13), and patients gave their informed consent in writing.

The samples were subdivided into two groups and cryopreserved by the slow freezing technique using modified human tubal fluid (modified HTF, Irvine Scientific, Santa Ana, CA, USA) without supplementation or with 2 mM melatonin (Sigma-Aldrich, Saint Louis, MO, USA). After thawing, each group was then subdivided into two samples, one of which was supplemented with 2 mM caffeine (1,3,7-trimethylxanthine; Sigma-Aldrich). The respective concentrations of MEL and CAF used in the course of this study were determined following a survey of the available literature. For MEL, it corresponds to the circulating human concentration. For CAF, we worked out the optimal concentration to be used in an earlier study [47] which roughly corresponds to a typical circulating concentration after the ingestion of one cup of coffee. Therefore, five groups were generated (see Figure 1): prefrozen samples; postthaw control samples without supplementation (CONT); postthaw samples with either melatonin (MEL), added before cryopreservation, or caffeine (CAF), used after thawing; or both additives: melatonin+caffeine (MC).

2.2. Semen Analysis and Sperm Functional Tests

Semen analysis and functional tests were performed on all precryopreservation and postthaw samples. Semen samples were collected by masturbation after a period of 2 to 5 days of ejaculatory abstinence. Sperm counts were made manually using a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel), and concentrations are expressed in million cells/ml. Sperm kinetic parameters including progressive (%PR) and total motility (%TM) [54], curvilinear velocity (VCL; μm/s), linear velocity (VSL; μm/s), mean velocity (VAP; μm/s), linearity (LIN; %), straightness (STR; %), wobble (WOB; %), and hyperactivity (HYP; %) were measured via the semiautomated platform “Sperm Class Analyzer” (SCA, MICROPTIC, Barcelona, Spain).

Mitochondrial activity was assessed by staining with 3,3-diaminobenzidine (DAB). Fifty μl of semen was added to a solution containing 1 mg/ml of DAB (Sigma-Aldrich) in an equal volume of PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4; Sigma-Aldrich) and incubated in the dark for 1 hour at 36.7°C. Two 10 μl smears were prepared on microscope slides and fixed in 10% formaldehyde for 10 minutes. About 200 sperm were observed under a phase contrast microscope (×100 oil-immersed objective—Eclipse E200, Nikon, Tokyo, Japan). Each sperm was classified as follows: Class I (DAB I; 100% of the colored centerpiece, with a prominent colored reaction showing the mitochondrial sheath as a compact cylinder), Class II (DAB II; more than 50% of the centerpiece colored but in a dispersed/patchy fashion), Class III (DAB III; less than 50% of the centerpiece colored), and Class IV (DAB IV; no coloration in the centerpiece) [55].

ROS levels were analyzed by light emission after stimulation with a luminal chemiluminescent probe [56, 57]. One aliquot of each sample (400 μl) was added to 20 μl of a luminol solution containing 5 mM luminol added to DMSO (Sigma-Aldrich) and evaluated using a luminometer (Berthold Technologies, Bad Wildbad, Germany). The results are expressed in 104 photons counted per minute (cpm) for spermatozoa [56, 57]. Evaluation of sperm DNA integrity was performed using the SCSA (Sperm Chromatin Structure Assay) method [58]. An aliquot fraction of the semen was diluted in a TNE buffer (0.15 M NaCl, 0.01 M Tris-HCl, 1 mM EDTA, ph = 7.4; Sigma-Aldrich) at a concentration of 1-2.106 sperm/ml. An acid detergent solution (400 μl; 0.08 M HCl, 0.15 M NaCl, and 0.1% Triton X-100 pH 1.2; Sigma-Aldrich) was then added to the samples. The cells were then stained with 1.2 ml of acridine orange solution (0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, and 0.15 M NaCl, pH 6.0; Sigma-Aldrich) containing 6 μg/ml acridine orange (Polysciences Inc., Washington, PA, USA). Five thousand sperm cells were evaluated using a flow cytometer (BD Biosciences, San Jose, CA, USA). Data was analyzed using SCSA software to determine the DNA fragmentation index (DFI%).

2.3. Sperm Cryopreservation

Seminal samples were subdivided into two aliquots of 1 ml each and processed using the Isolate method (Irvine Scientific). One ml two-layer density gradient (40%/80%; ISolate, Irvine Scientific) were constructed onto which were loaded 1 ml of seminal samples. Samples were centrifuged at 400g for 15 minutes at 36.6°C and supernatants carefully aspirated. One sperm pellet was resuspended in 1 ml of modified-HTF medium, and melatonin (2 mM in modified HTF) was added to the other pellet.

Cryopreservation by slow freezing cryopreservation initiates by addition of one volume of a glycerol-based cryoprotectant TEST-yolk buffer (TYB; Irvine Scientific) to each sample. All samples were subjected to the following sequential protocol: 8 min at -20°C, 1 hour in nitrogen vapor (-79°C), and finally immersion in liquid nitrogen (-196°C) for extended storage [59].

The thawing process included samples incubated at 25°C for 15 minutes and then at 36.7°C for 15 minutes [60]. After complete thawing, each sample was separated into two aliquots. Cryoprotectant solution was removed by centrifugation (400g for 10 minutes), and sperm pellet was resuspended in either HTF-modified or 2 mM solution of caffeine in modified HTF. Both samples were then incubated at 36.7°C for an additional 15 minutes [47, 61].

2.4. Statistical Analysis

The normality of semen parameters was verified and evaluated using the Kolmogorov-Smirnov test. The Student paired -test was chosen to compare data between two groups (prefrozen samples and CONT). ANOVA with analysis of repeated measurements and the Holm-Sidak post hoc test were used to compare CONT with the CAF, MEL, and CM groups. In addition, an interaction test was performed to evaluate synergistic interactions and the combined effects of caffeine and melatonin supplementation. All analyses were performed using the SPSS version 19.0, and a value of was considered statistically significant.

3. Results

Thirty normozoospermic seminal samples from patients aged 22 to 45 years were included in this study (). Patients were referred for a variety of andrological reasons, including fertility assessment (50.0%), prevasectomy evaluation (16.7%), infertility (10.0%), premature ejaculation (6.7%), erectile dysfunction (6.7%), testicular pain (3.3%), postvasectomy reversal (3.3%), and varicocele (3.3%).

3.1. Initial Seminal Analysis after Freezing/Thawing

The main parameters of the precryopreservation (fresh) and postthaw samples without supplementation (CONT) were analyzed (Table 1). The classic negative effects of cryopreservation on sperm motility were clearly observed. In particular, we recorded a significant decrease in progressive and total motility in postthaw sperm samples (). Thanks to the detailed SCA analysis, we recorded a decrease in almost all kinetic parameters including VCL, VSL, VAP, LIN, STR, and WOB () with the exception of hyperactivity (HYP; ). In addition, a significant reduction in mitochondrial activity after thawing was observed in the DAB I and DAB II groups (). Finally, as expected too, semen samples after thawing showed an increase in ROS levels (, with important increase in postthaw samples and biological relevance) and DFI% () when compared to the precryopreservation (CONT) group.


Concentration (million/ml)
 Mean; SD62.48; 47.3218.13; 11.94<0.001
Progressive motility (%)
 Mean; SD50.22; 10.967.50; 2.71<0.001
Total motility (%)
 Mean; SD66.09; 8.9416.13; 12.27<0.001
Curvilinear velocity (VCL) (μm/s)
 Mean; SD55.69; 10.8929.47; 20.27<0.001
Straight-line velocity (VSL) (μm/s)
 Mean; SD19.45; 7.707.61; 8.970.002
Average path velocity (VAP) (μm/s)
 Mean; SD30.66; 8.1214.12; 11.43<0.001
Linearity (LIN) (%)
 Mean; SD34.54; 9.5320.34; 15.010.005
Straightness (STR) (%)
 Mean; SD62.27; 9.5021.86; 18.63<0.001
Wobble (WOB) (%)
 Mean; SD54.70; 6.6641.90; 15.620.009
Hyperactivity (%)
 Mean; SD7.97; 7.462.47; 6.830.073
DNA fragmentation index (%)
 Mean; SD36.86; 15.2947.93; 18.090.046
DAB I (%)
 Mean; SD26.19; 15.619.43; 7.23<0.001
DAB II (%)
 Mean; SD47.62; 17.3951.48; 12.180.418
 Mean; SD11.67; 5.5421.19; 7.84<0.001
DAB IV (%)
 Mean; SD12.95; 8.9218.04; 10.940.106
ROS level (104 cpm/ sperm)
 Mean; SD0.92; 0.763.22; 4.940.052

DAB: mitochondrial activity grade; ROS: radical oxygen species. Values in bold are statistically significant () based on Student’s paired -test. slightly >0.05 not statistically significant but is biologically relevant.
3.2. Melatonin and Caffeine Supplementation

As shown in Table 2, there was a significant increase in progressive motility () in the CAF and MC samples compared to the CONT samples. However, no statistically significant improvement was recorded by examining each specific kinetic parameter individually via the SCA platform. Combined supplementation (CM) resulted in an increase in mitochondrial activity, as evidenced by the higher proportion of DAB I sperm () and the concomitant decrease in the proportion of DAB IV sperm (). No statistically significant effects were observed on DFI% and ROS concentrations under the tested conditions.


Concentration (million/ml)
 Mean; SD18.13; 11.9418.19; 10.8221.05; 14.8321.62; 14.900.6620.4970.166
Progressive motility (%)
 Mean; SD7.50; 2.7113.27; 2.629.49; 8.2816.54; 13.250.0050.9800.048
Total motility (%)
 Mean; SD16.13; 12.2720.24; 13.0219.94; 11.8424.80; 15.080.4660.8130.078
Curvilinear velocity (VCL)(μm/s)
 Mean; SD29.47; 20.2741.53; 20.7137.81; 18.4346.38; 21.870.9991.0000.626
Straight-line velocity (VSL) (μm/s)
 Mean; SD7.61; 8.9710.67; 6.167.80; 5.4816.62; 19.031.0000.9610.729
Average path velocity (VAP) (μm/s)
 Mean; SD14.12; 11.4319.47; 10.3016.85; 10.1426.09; 18.091.0000.9890.462
Linearity (LIN) (%)
 Mean; SD20.34; 15.0124.44; 5.0018.51; 5.1630.81; 23.281.0000.8990.693
Straightness (STR) (%)
 Mean; SD21.86; 18.6352.69; 7.0443.45; 6.2854.38; 19.851.0000.9170.926
Wobble (WOB) (%)
 Mean; SD41.90; 15.6245.68; 5.5442.17; 6.3752.25; 14.850.9460.8660.669
Hyperactivity (%)
 Mean; SD2.47; 6.832.37; 1.800.78; 0.901.23; 1.610.9950.9290.966
DNA fragmentation (%)
 Mean; SD47.93; 18.0943.67; 18.0045.07; 22.5644.13; 23.260.9010.9950.988
DAB I (%)
 Mean; SD9.43; 7.2313.18; 10.3511.68; 10.6019.14; 13.440.1550.5260.001
DAB II (%)
 Mean; SD51.48; 12.1852.82; 11.0751.45; 13.6553.23; 12.880.9021.0000.945
 Mean; SD21.19; 7.8419.45; 8.0319.54; 10.7417.77; 6.620.8460.9670.230
DAB IV (%)
 Mean; SD18.04; 10.9416.36; 10.2617.31; 5.5910.00; 5.510.5800.489<0.001
ROS level (104 cpm/ sperm)
 Mean; SD3.22; 4.942.95; 4.431.81; 2.172.52; 2.571.000.8490.998

DAB: mitochondrial activity grade; ROS: radical oxygen species. Values in bold are statistically significant () based on ANOVA and the Holm-Sidak post hoc test.

4. Discussion

This study evaluated the effect of MEL and CAF addition on the functional characteristics of normozoospermic sperm samples before cryopreservation and postthawing, using the slow freezing method and glycerol as a cryoprotectant.

As expected, the cryopreservation process has had its panel of associated adverse effects reducing sperm motility and mitochondrial activity while increasing sperm ROS levels and DNA fragmentation [15, 17, 27, 39, 6268]. In the present study, sperm motility went down from 50.22% in fresh semen to 7.5% postthawing. Also, a decrease in total sperm count was expected due to the known severity of the cryopreservation-thawing processes and increased sperm cell membrane fragility in defective spermatozoa submitted also to seminal processing with further loss due to cell lysis [16]. Most individual sperm velocity parameters (VCL, VAP, LIN, STR, and WOB) were significantly decreased after cryopreservation in total agreement with earlier reports (as an example, see [17]), a situation that has been associated with the low success rate of ART procedures such as IUI (intrauterine insemination) when cryopreserved sperm samples are used [67]. In addition, completing the classical picture, sperm mitochondria activity was significantly reduced after cryopreservation and was associated with an increase in ROS and its associated effect (i.e., higher DNA fragmentation index) revealing alteration in the integrity of the sperm nucleus [21, 27, 62, 63, 68].

The addition of CAF during thawing resulted in a significant increase (almost twice as high) in the percentage of progressive motile sperm, a situation that was slightly reinforced when MEL was also added to the cryopreservation step without providing a very convincing additive/synergistic effect. It is interesting to note that when both additives were used, a significant improvement in sperm mitochondrial activity was recorded, whereas this was not the case when the additives were used separately. This result may suggest a synergistic action of MEL and CAF in protecting and stimulating, respectively, the structure and function of mitochondria. In fact, CAF can act as an inhibitor of the enzymatic activity of phosphodiesterase, which is responsible for the degradation of cyclic adenosine monophosphate (cAMP) resulting in an increase in intracellular cAMP concentration. CAF can also act as a protein kinase A (PKA) stimulator by enhancing cytochrome c oxidase (COX) activity and oxidative phosphorylation (Figure 2) [4750, 53]. In addition, the protective action of MEL on sperm mitochondria may explain the lower levels of ROS recorded in the different samples tested (although this was just a trend). Melatonin is a known powerful antioxidant that also plays a role in stimulating mitochondrial respiration and ATP synthesis, as well as increasing the activity of respiratory chain complexes I and IV (oxidative phosphorylation) [45]. Thus, the protective action of MEL on sperm can also explain the higher concentrations of viable sperm after thawing recorded in MEL and MC samples (although again a strong statistical significance was not achieved) ultimately preventing sperm from passing into the apoptotic pathway [41, 51, 53].

Our data are in agreement with an earlier study that has shown in 43 human semen samples that melatonin addition resulted in an increase in sperm motility and vitality and in reduced ROS generation and lipid peroxidation [69]. They are also in line with in vitro studies in large domestic animals that have shown that treatment of semen samples with melatonin significantly increased sperm motility [70, 71], reduced sperm membrane lipid peroxidation [24, 72], prevented sperm capacitation and apoptosis [73], and protected spermatozoa against high levels of ROS [51]. Our data also concur with the observation that men with higher levels of MEL (or its downstream metabolites) have better seminal parameters including sperm concentration, motility, and normal morphology [71]. It also makes sense with the observation that infertile men exhibit lower levels of MEL when compared to fertile ones [71].

Regarding CAF addition to postthaw sperm samples, reports are scarce and mostly concern large domestic animals for which IUI with frozen sperm is commonly used. As we report in this study, it was shown that addition of CAF to postthaw bull sperm resulted in an increase in sperm motility among other parameters that were also ameliorated including sperm capacitation and acrosome reaction, leading to better gestation rates [74, 75]. In humans, the effect of CAF was mainly evaluated on spermatogenesis resulting in the observation that high CAF intake (>800 mg/day) was associated with reduced sperm concentration [50]. One study conducted with fertile men did show that the consumption of more than 6 coffee cups per day resulted in an increase in sperm motility [48, 76]. As energy expenditure is one of the major factors involved in the loss of fertilization capacity after sperm cryopreservation [77], the potential CAF-mediated increase in intracellular energy production should be investigated further.

5. Conclusions

Although preliminary and perfectible, this study illustrates that there is room for improvement of the postthaw performance of frozen human semen samples via the addition of a protectant or/and stimulant. This could prove to be useful with seminal samples of poor initial quality whatever the cause. In our example, CAF or CAF+MEL (MC) resulted in an improvement of postthaw sperm motility. In addition, and solely when CAF+MEL was used, sperm motility improvement was associated with a healthier mitochondrial status. In practice, these simple actions could logically translate into higher ART success rate, an issue that remains to be evaluated.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflict of interest.


Dr. Pariz was a recipient of a doctorate scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). Prof. Dr. Hallak is a recipient of a Scientific Productivity scholarship from the Brazilian National Council for Scientific and Technological Development (CNPq) (process number 301373/2013-2). The authors would like to acknowledge Androscience, Science and Innovation Center in Andrology and High-Complex Clinical and Andrology Laboratory, for supporting this study, and SCSA Diagnostics, Brookings, SD, USA, for providing the SCSA analysis.


  1. K. Oktay, B. E. Harvey, A. H. Partridge et al., “Fertility preservation in patients with cancer: ASCO clinical practice guideline update,” Journal of Clinical Oncology, vol. 36, no. 19, pp. 1994–2001, 2018. View at: Publisher Site | Google Scholar
  2. A. K. Nangia, S. A. Krieg, and S. S. Kim, “Clinical guidelines for sperm cryopreservation in cancer patients,” Fertility and Sterility, vol. 100, no. 5, pp. 1203–1209, 2013. View at: Publisher Site | Google Scholar
  3. N. Sermondade, N. Massin, F. Boitrelle et al., “Sperm parameters and male fertility after bariatric surgery: three case series,” Reproductive Biomedicine Online, vol. 24, no. 2, pp. 206–210, 2012. View at: Publisher Site | Google Scholar
  4. D. H. Williams, “Fertility preservation in the male with cancer,” Current Urology Reports, vol. 14, no. 4, pp. 315–326, 2013. View at: Publisher Site | Google Scholar
  5. M. Lambertini, L. del Mastro, M. C. Pescio et al., “Cancer and fertility preservation: international recommendations from an expert meeting,” BMC Medicine, vol. 14, no. 1, p. 1, 2016. View at: Publisher Site | Google Scholar
  6. C. A. A. Silva, J. Hallak, F. F. Pasqualotto, M. F. Barba, M. I. Saito, and M. H. B. Kiss, “Gonadal function in male adolescents and young males with juvenile onset systemic lupus erythematosus,” The Journal of Rheumatology, vol. 29, no. 9, pp. 2000–2005, 2002. View at: Google Scholar
  7. P. M. F. Soares, E. F. Borba, E. Bonfa, J. Hallak, A. L. Corrêa, and C. A. A. Silva, “Gonad evaluation in male systemic lupus erythematosus,” Arthritis and Rheumatism, vol. 56, no. 7, pp. 2352–2361, 2007. View at: Publisher Site | Google Scholar
  8. A. Agarwal, P. Ranganathan, N. Kattal et al., “Fertility after cancer: a prospective review of assisted reproductive outcome with banked semen specimens,” Fertility and Sterility, vol. 81, no. 2, pp. 342–348, 2004. View at: Publisher Site | Google Scholar
  9. F. F. Pasqualotto, B. P. Sobreiro, J. Hallak, E. B. Pasqualotto, and A. M. Lucon, “Induction of spermatogenesis in azoospermic men after varicocelectomy repair: an update,” Fertility and Sterility, vol. 85, no. 3, pp. 635–639, 2006. View at: Publisher Site | Google Scholar
  10. J. Hallak, A. Mahran, J. Chae, and A. Agarwal, “Poor semen quality from patients with malignancies does not rule out sperm banking,” Urological Research, vol. 28, no. 4, pp. 281–284, 2000. View at: Publisher Site | Google Scholar
  11. I. Craft, M. Tsirigotis, V. Bennett et al., “Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia,” Fertility and Sterility, vol. 63, no. 5, pp. 1038–1042, 1995. View at: Publisher Site | Google Scholar
  12. G. R. Dohle, L. Ramos, M. H. Pieters, D. D. Braat, and R. F. Weber, “Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia,” Human Reproduction, vol. 13, no. 3, pp. 620–623, 1998. View at: Publisher Site | Google Scholar
  13. M. Meseguer, N. Garrido, J. Remohí et al., “Testicular sperm extraction (TESE) and ICSI in patients with permanent azoospermia after chemotherapy,” Human Reproduction, vol. 18, no. 6, pp. 1281–1285, 2003. View at: Publisher Site | Google Scholar
  14. A. W. Pastuszak, W. S. Lai, T.-C. Hsieh, and L. I. Lipshultz, “Posthumous sperm utilization in men presenting for sperm banking: an analysis of patient choice,” Andrology, vol. 1, no. 2, pp. 251–255, 2013. View at: Publisher Site | Google Scholar
  15. J. Hallak, R. K. Sharma, A. J. Thomas, and A. Agarwal, “Why cancer patients request disposal of cryopreserved semen specimens posttherapy: a retrospective study,” Fertility and Sterility, vol. 69, no. 5, pp. 889–893, 1998. View at: Publisher Site | Google Scholar
  16. E. E. Noiles, K. A. Thompson, and B. T. Storey, “Water permeability, Lp, of the mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton,” Cryobiology, vol. 35, no. 1, pp. 79–92, 1997. View at: Publisher Site | Google Scholar
  17. J. Hallak, P. N. Kolettis, V. S. Sekhon, A. J. Thomas, and A. Agarwal, “Cryopreservation of sperm from patients with leukemia: is it worth the effort?” Cancer, vol. 85, no. 9, pp. 1973–1978, 1999. View at: Publisher Site | Google Scholar
  18. S. I. Moskovtsev, J. Willis, J. White, and J. B. M. Mullen, “Sperm DNA damage: correlation to severity of semen abnormalities,” Urology, vol. 74, no. 4, pp. 789–793, 2009. View at: Publisher Site | Google Scholar
  19. C. Ortega, G. Verheyen, D. Raick, M. Camus, P. Devroey, and H. Tournaye, “Absolute asthenozoospermia and ICSI: what are the options?” Human Reproduction Update, vol. 17, no. 5, pp. 684–692, 2011. View at: Publisher Site | Google Scholar
  20. M. Hessel, J. C. M. Robben, K. W. M. D'Hauwers, D. D. M. Braat, and L. Ramos, “The influence of sperm motility and cryopreservation on the treatment outcome after intracytoplasmic sperm injection following testicular sperm extraction,” Acta Obstetricia et Gynecologica Scandinavica, vol. 94, no. 12, pp. 1313–1321, 2015. View at: Publisher Site | Google Scholar
  21. A. W. Wang, H. Zhang, I. Ikemoto, D. J. Anderson, and K. R. Loughlin, “Reactive oxygen species generation by seminal cells during cryopreservation,” Urology, vol. 49, no. 6, pp. 921–925, 1997. View at: Publisher Site | Google Scholar
  22. N. Zribi, N. Feki Chakroun, H. el Euch, J. Gargouri, A. Bahloul, and L. Ammar Keskes, “Effects of cryopreservation on human sperm deoxyribonucleic acid integrity,” Fertility and Sterility, vol. 93, no. 1, pp. 159–166, 2010. View at: Publisher Site | Google Scholar
  23. R. J. Aitken, D. S. Irvine, and F. C. Wu, “Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility,” American Journal of Obstetrics and Gynecology, vol. 164, no. 2, pp. 542–551, 1991. View at: Publisher Site | Google Scholar
  24. C. M. B. da Silva, B. Macías-García, A. Miró-Morán et al., “Melatonin reduces lipid peroxidation and apoptotic‐like changes in stallion spermatozoa,” Journal of Pineal Research, vol. 51, no. 2, pp. 172–179, 2011. View at: Publisher Site | Google Scholar
  25. M. A. Baker and R. J. Aitken, “Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility,” Reproductive Biology and Endocrinology, vol. 3, no. 1, p. 67, 2005. View at: Publisher Site | Google Scholar
  26. S. E. M. Lewis and R. J. Aitken, “DNA damage to spermatozoa has impacts on fertilization and pregnancy,” Cell and Tissue Research, vol. 322, no. 1, pp. 33–41, 2005. View at: Publisher Site | Google Scholar
  27. B. T. Ji, X. O. Shu, W. Zheng et al., “Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers,” Journal of the National Cancer Institute, vol. 89, no. 3, pp. 238–243, 1997. View at: Publisher Site | Google Scholar
  28. J. F. Crow, “The origins, patterns and implications of human spontaneous mutation,” Nature Reviews Genetics, vol. 1, no. 1, pp. 40–47, 2000. View at: Publisher Site | Google Scholar
  29. A. Sipos, F. Rasmussen, G. Harrison et al., “Paternal age and schizophrenia: a population based cohort study,” BMJ, vol. 329, no. 7474, article 1070, 2004. View at: Publisher Site | Google Scholar
  30. R. J. Aitken, G. N. De Iuliis, and R. I. McLachlan, “Biological and clinical significance of DNA damage in the male germ line,” International Journal of Andrology, vol. 32, no. 1, pp. 46–56, 2009. View at: Publisher Site | Google Scholar
  31. T. Wainstock, A. Walfisch, I. Shoham-Vardi et al., “Fertility treatments and pediatric neoplasms of the offspring: results of a population-based cohort with a median follow-up of 10 years,” American Journal of Obstetrics and Gynecology, vol. 216, no. 3, pp. 314.e1–314.e14, 2017. View at: Publisher Site | Google Scholar
  32. B. F. da Silva, M. Borrelli Jr., R. M. Fariello et al., “Is sperm cryopreservation an option for fertility preservation in patients with spinal cord injury-induced anejaculation?” Fertility and Sterility, vol. 94, no. 2, pp. 564–573, 2010. View at: Publisher Site | Google Scholar
  33. N. Kemal Duru, M. Morshedi, and S. Oehninger, “Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa,” Fertility and Sterility, vol. 74, no. 6, pp. 1200–1207, 2000. View at: Publisher Site | Google Scholar
  34. R. M. Fariello, J. R. Pariz, D. M. Spaine, A. P. Cedenho, R. P. Bertolla, and R. Fraietta, “Association between obesity and alteration of sperm DNA integrity and mitochondrial activity,” BJU International, vol. 110, no. 6, pp. 863–867, 2012. View at: Publisher Site | Google Scholar
  35. J. G. Alvarez and B. T. Storey, “Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation,” Journal of Andrology, vol. 13, no. 3, pp. 232–241, 1992. View at: Publisher Site | Google Scholar
  36. P. Gharagozloo and R. J. Aitken, “The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy,” Human Reproduction, vol. 26, no. 7, pp. 1628–1640, 2011. View at: Publisher Site | Google Scholar
  37. N. Sukcharoen, J. Keith, D. Stewart Irvine, and R. John Aitken, “Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination,” Fertility and Sterility, vol. 63, no. 6, pp. 1293–1300, 1995. View at: Publisher Site | Google Scholar
  38. M. Hansen, J. J. Kurinczuk, C. Bower, and S. Webb, “The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization,” The New England Journal of Medicine, vol. 346, no. 10, pp. 725–730, 2002. View at: Publisher Site | Google Scholar
  39. D. T. Carrell, L. Liu, C. M. Peterson et al., “Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss,” Archives of Andrology, vol. 49, no. 1, pp. 49–55, 2003. View at: Publisher Site | Google Scholar
  40. P. Renard, G. Grizard, J. F. Griveau, B. Sion, D. Boucher, and D. le Lannou, “Improvement of motility and fertilization potential of postthaw human sperm using glutamine,” Cryobiology, vol. 33, no. 3, pp. 311–319, 1996. View at: Publisher Site | Google Scholar
  41. Z. Li, Q. Lin, R. Liu, W. Xiao, and W. Liu, “Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation,” Journal of Andrology, vol. 31, no. 5, pp. 437–444, 2010. View at: Publisher Site | Google Scholar
  42. S. C. Esteves, D. M. Spaine, and A. P. Cedenho, “Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method,” Brazilian Journal of Medical and Biological Research, vol. 40, no. 7, pp. 985–992, 2007. View at: Publisher Site | Google Scholar
  43. P. Stanic, Z. Sonicki, and E. Suchanek, “Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa,” International Journal of Andrology, vol. 25, no. 3, pp. 186–190, 2002. View at: Publisher Site | Google Scholar
  44. K. Kleszczyński, L. H. Hardkop, and T. W. Fischer, “Differential effects of melatonin as a broad range UV-damage preventive dermato-endocrine regulator,” Dermato-Endocrinology, vol. 3, no. 1, pp. 27–31, 2011. View at: Publisher Site | Google Scholar
  45. S. Succu, F. Berlinguer, V. Pasciu, V. Satta, G. G. Leoni, and S. Naitana, “Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner,” Journal of Pineal Research, vol. 50, no. 3, pp. 310–318, 2011. View at: Publisher Site | Google Scholar
  46. R. J. Aitken and S. D. Roman, “Antioxidant systems and oxidative stress in the testes,” Adv. Exp. Med. Biol., vol. 636, pp. 154–171, 2009. View at: Publisher Site | Google Scholar
  47. J. R. Pariz and J. Hallak, “Effects of caffeine supplementation in post-thaw human semen over different incubation periods,” Andrologia, vol. 48, no. 9, pp. 1049–1054, 2016. View at: Publisher Site | Google Scholar
  48. B. P. Sobreiro, A. M. Lucon, F. F. Pasqualotto, J. Hallak, K. S. Athayde, and S. Arap, “Semen analysis in fertile patients undergoing vasectomy: reference values and variations according to age, length of sexual abstinence, seasonality, smoking habits and caffeine intake,” São Paulo Medical Journal, vol. 123, no. 4, pp. 161–166, 2005. View at: Publisher Site | Google Scholar
  49. H. Klonoff-Cohen, J. Bleha, and P. Lam-Kruglick, “A prospective study of the effects of female and male caffeine consumption on the reproductive endpoints of IVF and gamete intra-fallopian transfer,” Human Reproduction, vol. 17, no. 7, pp. 1746–1754, 2002. View at: Publisher Site | Google Scholar
  50. T. K. Jensen, S. H. Swan, N. E. Skakkebaek, S. Rasmussen, and N. Jorgensen, “Caffeine intake and semen quality in a population of 2,554 young Danish men,” American Journal of Epidemiology, vol. 171, no. 8, pp. 883–891, 2010. View at: Publisher Site | Google Scholar
  51. X. X. Li, X. G. Yang, Y. Q. Lu et al., “Protective effects of melatonin against oxidative stress in flow cytometry-sorted buffalo sperm,” Reproduction in Domestic Animals, vol. 47, no. 2, pp. 299–307, 2012. View at: Publisher Site | Google Scholar
  52. J. Carré, N. Gatimel, J. Moreau, J. Parinaud, and R. Léandri, “Does air pollution play a role in infertility?: a systematic review,” Environmental Health, vol. 16, no. 1, p. 82, 2017. View at: Publisher Site | Google Scholar
  53. F. Zhang, L. Zhang, Y. Qi, and H. Xu, “Mitochondrial cAMP signaling,” Cellular and Molecular Life Sciences, vol. 73, no. 24, pp. 4577–4590, 2016. View at: Publisher Site | Google Scholar
  54. WHO, World Health Organization, WHO Laboratory Manual for the Examination and Processing of Human Semen, WHO Press, Geneva, 5th edition, 2010.
  55. F. Hrudka, “Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress,” International Journal of Andrology, vol. 10, no. 6, pp. 809–828, 1987. View at: Publisher Site | Google Scholar
  56. R. J. Aitken, D. W. Buckingham, and K. M. West, “Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence,” Journal of Cellular Physiology, vol. 151, no. 3, pp. 466–477, 1992. View at: Publisher Site | Google Scholar
  57. K. A. McKinney, S. E. Lewis, and W. Thompson, “Reactive oxygen species generation in human sperm: luminol and lucigenin chemiluminescence probes,” Archives of Andrology, vol. 36, no. 2, pp. 119–125, 1996. View at: Publisher Site | Google Scholar
  58. D. P. Evenson, P. J. Higgins, D. Grueneberg, and B. E. Ballachey, “Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea,” Cytometry, vol. 6, no. 3, pp. 238–253, 1985. View at: Publisher Site | Google Scholar
  59. J. K. Sherman, “Current status of clinical cryobanking of human semen,” in Andrology: Male Fertility and Sterility, L. Paulson, J. D. Negro-Vilar, A. Lucena, and E. Martini, Eds., pp. 517–549, Academic Press, 1st edition, 1986. View at: Google Scholar
  60. M. A. Henry, E. E. Noiles, D. Gao, P. Mazur, and J. K. Critser, “Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function,” Fertility and Sterility, vol. 60, no. 5, pp. 911–918, 1993. View at: Publisher Site | Google Scholar
  61. C. Almiñana, M. A. Gil, C. Cuello et al., “Adjustments in IVF system for individual boars: value of additives and time of sperm–oocyte co-incubation,” Theriogenology, vol. 64, no. 8, pp. 1783–1796, 2005. View at: Publisher Site | Google Scholar
  62. H. Awad, F. Halawa, T. Mostafa, and H. Atta, “Melatonin hormone profile in infertile males,” International Journal of Andrology, vol. 29, no. 3, pp. 409–413, 2006. View at: Publisher Site | Google Scholar
  63. J. Hallak, B. N. Hendin, A. J. Thomas, and A. Agarwal, “Investigation of fertilizing capacity of cryopreserved spermatozoa from patients with cancer,” The Journal of Urology, vol. 159, no. 4, pp. 1217–1220, 1998. View at: Publisher Site | Google Scholar
  64. J. Hallak, R. K. Sharma, C. Wellstead, and A. Agarwal, “Cryopreservation of human spermatozoa: comparison of TEST-yolk buffer and glycerol,” International Journal of Fertility and Women's Medicine, vol. 45, no. 1, pp. 38–42, 2000. View at: Google Scholar
  65. J. Hallak, A. Mahran, J. Chae, and A. Agarwal, “The effects of cryopreservation on semen from men with sarcoma or carcinoma,” Journal of Assisted Reproduction and Genetics, vol. 17, no. 4, pp. 218–221, 2000. View at: Publisher Site | Google Scholar
  66. J. Verstegen, M. Iguer-Ouada, and K. Onclin, “Computer assisted semen analyzers in andrology research and veterinary practice,” Theriogenology, vol. 57, no. 1, pp. 149–179, 2002. View at: Publisher Site | Google Scholar
  67. R. Luboshitzky, Z. Shen-Orr, R. Nave, S. Lavi, and P. Lavie, “Melatonin administration alters semen quality in healthy men,” Journal of Andrology, vol. 23, no. 4, pp. 572–578, 2002. View at: Publisher Site | Google Scholar
  68. U. Paasch, R. K. Sharma, A. K. Gupta et al., “Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa,” Biology of Reproduction, vol. 71, no. 6, pp. 1828–1837, 2004. View at: Publisher Site | Google Scholar
  69. M. Karimfar, F. Niazvand, K. Haghani, S. Ghafourian, R. Shirazi, and S. Bakhtiyari, “The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm,” International Journal of Immunopathology and Pharmacology, vol. 28, no. 1, pp. 69–76, 2015. View at: Publisher Site | Google Scholar
  70. H. Y. Jang, Y. H. Kim, B. W. Kim et al., “Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development,” Reproduction in Domestic Animals, vol. 45, no. 6, pp. 943–950, 2010. View at: Publisher Site | Google Scholar
  71. A. Casao, N. Mendoza, R. Pérez-Pé et al., “Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate,” Journal of Pineal Research, vol. 48, no. 1, pp. 39–46, 2010. View at: Publisher Site | Google Scholar
  72. A. Ortiz, J. Espino, I. Bejarano et al., “High endogenous melatonin concentrations enhance sperm quality and short-term in vitro exposure to melatonin improves aspects of sperm motility,” Journal of Pineal Research, vol. 50, no. 2, pp. 132–139, 2011. View at: Publisher Site | Google Scholar
  73. S. Yamaguchi and H. Funahashi, “Effect of the addition of beta-mercaptoethanol to a thawing solution supplemented with caffeine on the function of frozen-thawed boar sperm and on the fertility of sows after artificial insemination,” Theriogenology, vol. 77, no. 5, pp. 926–932, 2012. View at: Publisher Site | Google Scholar
  74. S. Yamaguchi, H. Funahashi, and T. Murakami, “Improved fertility in gilts and sows after artificial insemination of frozen-thawed boar semen by supplementation of semen extender with caffeine and CaCl2,” The Journal of Reproduction and Development, vol. 55, no. 6, pp. 645–649, 2009. View at: Publisher Site | Google Scholar
  75. C. H. Ramlau-Hansen, A. M. Thulstrup, J. P. Bonde, J. Olsen, and B. H. Bech, “Semen quality according to prenatal coffee and present caffeine exposure: two decades of follow-up of a pregnancy cohort,” Human Reproduction, vol. 23, no. 12, pp. 2799–2805, 2008. View at: Publisher Site | Google Scholar
  76. R. J. Aitken, A. Mattei, and S. Irvine, “Paradoxical stimulation of human sperm motility by 2-deoxyadenosine,” Reproduction, vol. 78, no. 2, pp. 515–527, 1986. View at: Publisher Site | Google Scholar
  77. D. L. Garbers, N. L. First, J. J. Sullivan, and H. A. Lardy, “Stimulation and maintenance of ejaculated bovine spermatozoan respiration and motility by caffeine,” Biology of Reproduction, vol. 5, no. 3, pp. 336–339, 1971. View at: Publisher Site | Google Scholar

Copyright © 2019 Juliana R. Pariz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Related articles

No related content is available yet for this article.
 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

No related content is available yet for this article.

Article of the Year Award: Outstanding research contributions of 2020, as selected by our Chief Editors. Read the winning articles.