Study design and procedures
The decision was made to evaluate the sperm DNA damage not on neat semen but after density gradient centrifugation. This should allow to get results within the potential available population of spermatozoa intended to be used in assisted reproductive techniques (ART). After density gradient centrifugation, sperm DNA damage was measured in sperm samples by both the SCD test and the TUNEL assay. Since the SCD test is a non-automated and subjective method, inter-slide reliability for readings of the same sperm sample and the intra- and inter-observer reliabilities of the same slide were assessed. Afterwards, we assessed the inter-method reliability between the SCD test and TUNEL/FCM for the measurements of sperm DNA damage (Fig. 1).
Sperm collection and preparation
This study was performed on 25 men attending Clermont-Ferrand (France) University Hospital’s Center for Reproductive Medicine for fertility issues. All 25 patients attended the Center because of fertility issues, and no sperm donor was included. One semen sample was collected for each of them.
The andrology laboratory has implemented a quality management system based on the International Standard ISO 15189.
Semen samples were collected by masturbation into sterile containers after a period of 2–3 days of sexual abstinence. After semen liquefaction for 30 min at 37 °C, basic semen analysis was performed according to World Health Organization guidelines [13], except for sperm morphology assessment, which was done according to David morphological classification [14].
To isolate sperm cell populations, a two-step discontinuous Sperm Filter® (Cryo Bio System, Rambouillet, France) gradient (90–45%) diluted in Sperm Preparation Medium® (Origio, Limonest, France) was applied on the sperm samples. The purified sperm population was recovered from the 95% layer, washed in Sperm Prep Medium® (750 g, 8 min) and suspended in a suitable volume of phosphate buffer saline (PBS, Sigma-Aldrich, Lyon, France) supplemented with 1% (v/v) Bovine Serum Albumin (Sigma-Aldrich, Lyon, France) according to the manufacturer’s protocol for SCD tests (Halotech DNA, Spain) to reach a final concentration of 5 to 10.106 spermatozoa.mL− 1.
Sperm DNA damage
SCD test (Halosperm® kit)
The SCD test was performed using the Halosperm® kit based on the manufacturer’s protocol (Halotech DNA, Spain). The Eppendorf tubes of low-melting point agarose provided in the kit were placed in a water bath at 90–100 °C for 5 min. At the same time, the pre-coated slides were placed on a tray at 4 °C for 5 min. From this point, the protocol was applied twice in a row for each prepared sperm sample in order to get two slides in the end (50 slides for 25 sperm samples). The melted agarose was quickly added with 60 μL of each sperm sample and mixed. Sixty μL were thus pipetted twice in a row for each sperm sample and put in two different Eppendorf tubes. The chilled pre-coated slides were pipetted with 20 μL of the cellular suspensions, immediately covered (22 × 22 mm coverslip), then held at 4 °C for 5 min. Once the gel formed with the spermatozoa embedded inside, the coverslips were gently removed and the denaturation solution provided in the kit (containing hydrochloric acid) was applied for 7 min at room temperature. The slides were then placed in the lysing solution (Triton X-100, Dithiothreitol) for 25 min, and washed with distilled water for 5 min at room temperature. After dehydration by successive increasing concentrations of ethanol (70, 90 and 95%), the slides were dried and readied for bright-field microscopy by staining for 15 min with Wright staining solution (Merck 1.01383.0500, Darmstadt, Germany) and PBS (1:1, Merck 1.07294.1000, Darmstadt, Germany). These staining solutions are not provided in the kit, but are used by Fernández et al. [15]. The slides were mounted using Eukitt® mounting medium (O. Kindler GmbH & Co, Germany), a colorless medium with crystal-clear optics, which does not change color nor structure of mounted material (according to the technical data sheet). The slides were then stored in the dark at room temperature. This approach thus made it possible to take different readings at different times.
Positive controls were performed for each measurement. After incubation in permeabilization solution (0.1% sodium citrate, 0.1% Triton X-100) for 30 min at room temperature, spermatozoa were treated by DNAse I (Roche Diagnostics GmbH, Germany) at a final concentration of 3 IU.mL− 1 at 37 °C for 30 min and washed in PBS/BSA 1% (v/v) before measuring DNA damage by a SCD test as detailed above.
As described previously [15], the observed spermatozoa were scored in five patterns. A total 200 spermatozoa were scored per slide and per observer.
As the aim of the study was to assess the reliability of the SCD test, the study design (see below) was planned such that each sperm sample (once migrated) was split by preparing two slides. Each slide was read independently by two different blinded readers in random order. The coding of the slides had been done by a third person. Each reader ignored the value of the measure obtained by the other reader and each reader performed a double reading, also in random order. Slides had been re-coded before any reassessment by the same observer.
TUNEL assay
The TUNEL assay was performed with flow cytometry as previously described before [16] to select the population of spermatozoa and to discard the debris and round cells. DNA fragmentation was detected with the “in situ cell death detection kit” according to the manufacturer’s protocol (Roche, Meylan, France). Briefly, 1.5 × 106 washed spermatozoa were fixed with 2% paraformaldehyde for 30 min at room temperature. The spermatozoa were then rinsed and incubated for 3 min in permeabilization solution containing 0.1% Triton X-100 (v/v) in 0.1% citrate (w/v) on ice. After washing, the spermatozoa were labeled with 50 μL labeling solution containing dUTP and 50 μL terminal deoxynucleotidyl transferase (TdT). The incubation lasted 60 min at + 37 °C in a humidified atmosphere in the dark. After counterstaining with 2 mg.mL− 1 propidium iodide (PI), measurement was performed by flow cytometry.
For each sample, we ran a negative control by omitting the TdT enzyme and a positive control by incubating the spermatozoa with 3 IU DNase I for 15 min at 37 °C in Tris-HCl buffer before labeling. Flow cytometry was performed on an Epics XL cytometer (Beckman-Coulter, USA). A minimum of 20,000 spermatozoa were examined for each assay. Spermatozoa obtained in the plots of CMF were gated by using side-angle light scatter (SSC) and forward-angle light scatter (FSC). This was done to put out of the gate, debris and other cells than spermatozoa. An additional figure gives more details about flow cytometry measurements (see Additional file 1).
An argon laser delivered a 488 nm excitation wavelength. Green fluorescence (TUNEL-positive cells) was detected with FL1 (using a 525-nm band-pass filter) and red fluorescence (PI-positive cells) with FL3 (using a 620-nm band-pass filter). Both fluorescence signals were recorded after logarithmic amplification. Rate of labeled cells was calculated by the flow cytometer software.
Statistical analyses
All analyses are based on the same sperm samples from 25 patients. The first focus of the study was the reliability of SCD test in measuring sperm DNA damage. We tested for the following potential factor effects: the effect of preparing several slides from the same sperm sample (referred to as “slide effect”), the effect of involving several readers for the same slide (referred to as “reader effect”), and the effect of one reader reading the same slide several times (referred to as “reading effect”). Thus, regarding the SCD test, each sperm sample was split into two slides, each slide was read by two readers (the same pair of trained observers for the whole study), and each reader read each slide twice. The reliability of SCD was assessed using a hierarchical frame following the same scheme for each factor. First, the factor effect was tested through a discordance test using a paired Student t-test or a non-parametric signed-rank test if differences showed non-normal distribution (assessed by a Shapiro–Wilk test). When the tests found no significant discordance on a factor, the concordance between the two modalities of this factor was estimated using the intraclass correlation coefficient (ICC) [17]. In cases of non-discordant values and very good to almost perfect concordance (ICC at 0.8 or more), the two available values were lumped together by computing their mean. Once a factor was assessed, analyses moved on to focus on the next factor, following the same scheme. Analyses followed a hierarchical schedule, first testing the “reading effect”, then the “reader effect” and finally the “slide effect”, according to the average differences which were expected to sort in ascending order from difference between readings (see Fig. 2), then between readers (see Fig. 3), and lastly between slides (see Fig. 4). Scatterplots and Bland–Altman plots were graphed for each factor analysis [18] (see Figs. 2 to 4).
If, as expected, the quantifications of DNA damage measured by SCD were sufficiently reliable and reproducible, inter-method reliability between SCD and TUNEL was assessed following the same experimental design in these same 25 patients. Finally, we assessed the relationship between sperm parameters and DNA damage (through both SCD test and TUNEL assay) by performing non-parametric Spearman correlation coefficient tests.
All statistical analyses were performed using SAS v9.4 for windows (SAS Institute Inc., Cary, NC) with a double-sided type I error set at 0.05.