*This work was funded by Chiang Mai IVF Center.
This study examined whether the addition of triple antioxidants (3A)—10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid—in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa.
We analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated.
The cryopreserved spermatozoa had significantly reduced percentages of motility (
Cryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.
Cryopreservation of human spermatozoa has been widely used in assisted reproductive technology (ART) for more than 60 years [
Reactive oxygen species (ROS) are formed as natural byproducts of cellular aerobic metabolism and function as signal molecules that regulate cell-to-cell communication [
In previous studies, antioxidants such as acetyl-L-carnitine (ALC), N-acetyl-L-cysteine (NAC), and α-lipoic acid (ALA) have been shown to exert protective effects individually on several tissues and might be beneficial in mammalian gametes [
In nature, various antioxidant systems act together in concert to provide protection against oxidative stress and promote repair. Previous studies that concentrated on the use of individual antioxidants did not replicate natural conditions. The supplementation of combinations of antioxidants should exert a synergistic effect and provide better protection against oxidative-induced injuries than single antioxidant supplements [
The rationale for combining the three antioxidants is as follows: ALC serves as a universal scavenger of free radicals and reduces DNA damage, while NAC is an important substrate for the synthesis of GSH, and ALA is capable of regenerating other antioxidants, including GSH, which plays a critical role in protecting cells from oxidative damage. We developed a simplified sucrose freezing medium for vitrification of human spermatozoa. In this study, we investigated the effect of triple antioxidant supplementation in freezing-thawing medium on both intracellular and extracellular ROS production, and we also evaluated SDF by imaging flow cytometry.
All chemicals were purchased from Sigma-Aldrich Chemicals (Sigma Chemical, St. Louis, MO, USA), unless otherwise stated.
Thirty normozoospermic semen samples from patients who had been referred to the
Liquefied semen samples were placed on the top of two layers (40% and 80% fractions) of Sil-Select Stock solution (FertiPro NV, Beemem, Belgium) and centrifuged at 350 ×
The sperm samples were cryopreserved by two different protocols (the SuV and liquid nitrogen vapor methods). In this study, the sucrose vitrification medium was phosphate buffered saline (PBS) solution containing 10% (w/v) bovine serum albumin and 0.5 M sucrose. For the sucrose vitrification method, each sperm sample (100 µL/aliquot) was diluted 1:1, with sucrose freezing medium supplemented with 10 µM (ALC; Abcam, Cambridge, UK), 10 µM NAC, and 5 µM ALA (SuV+3A) or without triple antioxidants (SuV). The concentrations of the three antioxidants in this study were based on a previous study by Truong and Gardner [
For the liquid nitrogen vapor method (Vapor), each sperm sample (100 µL/aliquot) was diluted with an equal volume of Spermfreeze medium (Fertipro, Beernem, Belgium) supplemented with (Vapor+3A) or without triple antioxidants (Vapor), as described in the previous paragraph. The mixtures were loaded into 0.25-mL straws and incubated at room temperature for 10 minutes. The straws were placed in a horizontal position at a distance of 5–7 cm above the level of liquid nitrogen for 15 minutes, and they were directly plunged into liquid nitrogen. The vitrified straws were left on liquid nitrogen for at least 1 week before subsequent experiments.
In the warming steps, the straws were thawed in 25°C water, washed in EBSS medium supplemented with or without triple antioxidants, and centrifuged at 200 ×
The post-thaw samples were immediately assessed for sperm motility and kinematic parameters using a computer-assisted semen analyzer (CASA; HTM IVOS II, Hamilton Thorne Biosciences, Beverly, MA, USA). Progressive motility (%), total motility (%), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), amplitude of lateral head displacement (ALH, µm), beat-cross frequency (Hz), straightness (STR, [VSL/VAP]×100), and linearity (LIN, [VSL/VCL]×100) were evaluated.
The morphology of sperm was assessed by staining with Diff-Quick (Arnaparn, Nonthaburi, Thailand) and analyzed by an HTM IVOS II CASA equipped with a Dimensions II Strict Morphology software system using Kruger’s strict criteria. A total of 200 spermatozoa were analyzed in each slide at ×400 magnification.
Sperm viability was assessed using 0.5% (w/v) eosin-Y dissolved in 0.9% NaCl. A 10-µL sperm suspension was mixed with 10 µL of 0.5% eosin-Y. Then, the mixture was placed on a glass slide and covered with a coverslip. The samples were immediately assessed for sperm viability using a compound microscope (Olympus, Tokyo, Japan). A total of 200 spermatozoa were analyzed in each slide [
The extracellular ROS level was assessed by a chemiluminescence technique, using a Glomax 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The result was presented as relative light units (RLU) of counted photons per minute or mV/s. Briefly, 10 µL of sperm samples from each aliquot were diluted with 400 µL of PBS and mixed with 10 µL of luminol reagent (5-amino-2,3 dihydro-1,4 phthalazinedione). Then, each sample was measured twice, the average value of RLU/sec was corrected by dividing with the sperm concentration, and the final value of extracellular ROS was expressed in units of RLU/sec/106. A final extracellular ROS value lower than 20 RLU/sec/106 was classified as normal [
The intracellular sperm ROS level was evaluated using cell-permeable 2’7’-dichlorofluorescein diacetate (DCFH-DA), which was oxidized by the free intracellular H2O2 molecules into green fluorescence dichlorofluorescein (DCF). A total amount of 100 µM DCFH-DA and 2.5 µM propidium iodide (PI) was separately added to a concentration of 5 ×106 sperm/mL from each sample, followed by incubating at 37oC in 5% CO2 for 10 and 2 minutes, respectively. After incubation, the samples were washed with PBS and analyzed using an imaging flow cytometer (Amnis-Merck, Seattle, WA, USA) equipped with a charge-coupled device (CCD) camera, and a laser operated at 20 mW as a light source. At least 5,000 events were collected for each sample and analyzed by FlowSight (Amnis-Merck). Sperm populations were identified by plotting the forward scatter and side scatter, excluding other debris. Green fluorescence (DCF) was evaluated between 500 and 530 nm, while red fluorescence PI was evaluated between 580 and 630 nm (excitation 488 nm; emission, 530 nm in the FL-2 channel and 632 nm in the FL-5 channel). The percentage of viable DCF-positive cells (DCF+, PI–) and the mean fluorescence were calculated using image analysis software (IDEAS, Amnis-Merck).
The sperm chromatin structure assay (SCSA) is a flow cytometric assay that relies on the fact that abnormal sperm chromatin is highly susceptible to physical induction of partial DNA denaturation in situ [
All data, the mean numbers of sperm motility and kinematics, sperm viability, normal morphology, ROS levels, and percentage of DNA fragmentation were presented as mean±standard error of the mean and compared by one-way analysis of variance (ANOVA). The percentage data were arcsine-transformed to obtain a normal distribution before analysis with one-way ANOVA using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). The differences were compared by the post-hoc Fisher’s protected least significant difference test. Significant differences were defined as a
The average age of the 30 donors was 34.5±0.8 years. The semen parameters, including semen volume, concentration, and motility before sperm preparation were 2.8±0.2 mL, 57.5±5.6 ×106 sperm/mL, and 71.7%±2.5%, respectively.
The cryoprotective effects on motility parameters of vitrified spermatozoa are illustrated in
The levels of ROS in the sperm suspension were measured by a chemiluminescence assay. The extracellular ROS levels in sperm suspensions were significantly higher after the freeze-thawed process (SuV, 0.74±0.09; SuV+3A, 0.82±0.16; Vapor, 1.05±0.21; Vapor+3A, 1.07±0.20 RLU/sec/106) than in fresh control group (0.22±0.03 RLU/sec/106,
To evaluate the protective effect of antioxidants on sperm DNA integrity, the DNA fragmentation rate of spermatozoa was assessed by flow-based SCSA after the freezing-thawing process. As shown in
Sperm cryopreservation, which is routinely utilized in human ART programs, is closely associated with the use of permeable CPA or a combination of permeable and non-permeable CPAs [
Previous studies have focused on other motility parameters as crucial for the prediction of male fertility [
There is evidence that cryopreservation can cause cellular damage by different pathways. The excessive production of ROS is known to play an important role in this regard [
Recent studies have suggested that the use of antioxidants such as cysteine [
In our study, the use of triple antioxidants did not decrease the levels of extracellular ROS. Supplementation of NAC (a substrate for the synthesis of GSH), and ALA (stimulator of GSH synthetase) was probably not a good choice because sperm cells, unlike other cells, shed most of their cytoplasm during maturation. As a result, intracytoplasmic enzymatic antioxidant defense mechanisms could be lost or markedly decreased. The findings that SDF had a tendency to decrease without a concomitant reduction in ROS levels could imply that other mechanisms were involved. Although ROS are among the most studied reactive molecules, there are at least three other groups of such species, designated by their reactive heteroatom as reactive nitrogen species, reactive sulfur species, and reactive halogen (chlorine and bromine) species [
Flow cytometry is a useful tool to identify sperm populations with dysfunctional ability due to intracellular ROS generation [
Flow-based SCSA is currently the gold standard for DNA fragmentation screening in infertile men to predict fertility outcomes [
The present study indicated that vitrification had an adverse effect on SDF during cryopreservation. This result is consistent with previous studies showing that the number of sperm with fragmented DNA was associated with a freezing-thawing procedure [
In summary, a simplified vitrification medium, consisting of sucrose, compared favorably with the conventional liquid nitrogen vapor freezing protocol. Triple antioxidants in this study, aimed at increasing the activity of the enzymatic antioxidant pathways inside the sperm cytoplasm, did not have significant effects on improving sperm motility, viability, and DNA fragmentation. In future studies, extracellular antioxidants should be considered instead of those that rely on the endogenous enzymatic pathway, as mature sperm contain a scant amount of cytoplasm. The commonly used method of flow cytometric measurements of ROS production based on DCFH-DA is probably inappropriate for sperm because of their unique structure. ROS might not be the only reactive radicals involved in sperm damage after cryopreservation. Clinical outcomes, such as sperm motility, viability, DNA fragmentation, fertilization, and live birth, might be better indicators than ROS production.
This work was funded by Chiang Mai IVF Center. Tayita Suttirojpattana and Theesit Juanpanich are employees of Chiang Mai IVF Center. No other potential conflicts of interest relevant to this article were reported.
Conceptualization: TV, TS, TJ. Data curation: TJ, TS. Formal analysis: TS. Methodology: TJ, TV. Project administration: TV, RP. Visualization: TS, TJ. Writing–original draft: TS, TJ. Writing–review & editing: all authors.
The author would like to thank the Korat Health Center for their kind assistance with this study.
Determination of human sperm DNA fragmentation by imaging flow cytometry. On the left panel, yellow to red-stained cells indicate DNA fragmentation and green-stained cells indicate intact DNA in sperm, respectively. On the right panel, the sperm DNA fragmentation was evaluated individually by the intensity of acridine orange using imaging flow cytometry. High to moderate DNA fragmentation is shown in red and yellow colors, respectively. Normal to low DNA fragmentation is shown in green. SCSA, sperm chromatin structure assay; HDS, high DNA stainability; DFI, DNA fragmentation index; BF, bright field; AO, acridine orange; Mod, moderate.
Comparison of extracellular reactive oxygen species levels between fresh and freeze-thawed spermatozoa supplemented with or without the use of triple antioxidants. ROS, reactive oxygen species; RLU, relative light units; SuV, sucrose vitrification; 3A, triple antioxidants; Vapor, liquid nitrogen vapor. a),b)Bars with different superscripts differ significantly (
Quantitative intracellular H2O2 generation was evaluated by the measurement of dichlorofluorescein (DCF) fluorescence intensity using imaging-flow cytometry. Flow-cytometric histograms show the amount of intracellular H2O2 generation H2O2 in sperm. (A) Fresh nonfrozen group, (B) SuV group, (C) SuV+3A group, (D) Vapor group, and (E) Vapor+3A group. Values are presented as mean±standard error of the mean. Significant differences were defined as
CASA motility, kinetic parameters, viability, and morphology of fresh and frozen-thawed human spermatozoa supplemented with or without the use of triple antioxidants (n=30)
Parameter | Fresh control | Freeze-thawed spermatozoa |
|||
---|---|---|---|---|---|
SuV | SuV+3A | Vapor | Vapor+3A | ||
Motility (%) | 95.3±0.5 |
73.3±2.0 |
76.9±1.7 |
71.7±2.3 |
74.7±2.1 |
Progressive fraction (%) | 91.1±0.9 |
65.8±2.1 |
69.4±1.8 |
64.6±2.5 |
66.8±2.1 |
VAP (µm/sec) | 61.0±2.1 |
46.0±1.4 |
50.0±1.2 |
50.7±1.2 |
50.2±1.4 |
VSL (µm/sec) | 39.1±1.9 |
35.5±1.4 |
39.3±1.2 |
36.2±1.2 |
37.1±1.1 |
VCL (µm/sec) | 127.5±4.3 |
94.5±3.0 |
101.0±2.9 |
111.4±3.1 |
109.1±3.7 |
ALH (µm) | 7.2±0.2 |
5.2±0.2 |
5.5±0.2 |
6.1±0.2 |
7.5±1.6 |
BCF (Hz) | 26.1±0.7 |
26.7±0.5 |
27.0±0.4 |
27.9±0.6 |
28.2±0.5 |
STR (%) |
65.0±1.6 |
73.6±1.2 |
74.8±1.0 |
69.6±0.9 |
71.7±0.8 |
LIN (%) |
33.2±1.2 |
38.5±1.1 |
39.5±1.0 |
34.0±0.8 |
35.6±0.8 |
Eosin viability (%) | 91.8±2.0 |
69.4±2.7 |
74.0±2.5 |
69.8±2.2 |
71.3±2.2 |
Normality (%) | 16.4±1.5 | 17.2±1.7 | 18.7±1.7 | 20.0±1.6 | 19.7±1.6 |
Values are presented as mean±standard error of the mean.
CASA, computer-assisted sperm analysis; SuV, sucrose vitrification; 3A, triple antioxidants; Vapor, liquid nitrogen vapor; VAP, average path velocity; VSL, straight line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; STR, straightness; LIN, linearity.
Different superscript letters within a row indicate significant differences,
STR: (VSL/VAP)×100;
LIN: (VSL/VCL)×100.
Comparison of DNA fragmentation test between fresh and freeze-thawed spermatozoa with or without triple antioxidant supplementation (n = 30)
Parameter | Fresh control | Frozen-thawed spermatozoa | |||
---|---|---|---|---|---|
SuV | SuV+3A | Vapor | Vapor+3A | ||
DNA fragmentation (%) | 7.3±1.2 | 15.3±4.1 |
8.4±1.6 | 14.0±3.0 |
9.5±1.8 |
High DNA stainability (%) | 1.2±0.2 |
1.5±0.2 |
1.7±0.2 |
2.2±0.3 |
2.3±0.3 |
Values are presented as mean±standard error of the mean.
SuV, sucrose vitrification; 3A, triple antioxidants; Vapor, liquid nitrogen vapor.
Different superscript letters within a row indicate significant differences,
SuV and Vapor showed a non-significant tendency to be higher than the fresh control (