EP4634399A1 - Mammalian spermatozoa dna integrity testing via evaluation of its 8-oxodg content - Google Patents

Abstract

The present invention concerns the detection of DNA base oxidation via 8-OxodG immunoreactivity.

Description

MAMMALIAN SPERMATOZOA DNA INTEGRITY TESTING VIA EVALUATION OF ITS 8-OXOdG CONTENT

DESCRIPTION

Technical field of the invention

The present invention concerns the detection of DNA base oxidation via 8-OxodG immunoreactivity.

State of the art

The assessment of male semen quality, as recommended by WHO (WHO 2010), is limited to monitoring sperm count, morphology and gross motility. Although these sperm parameters are good indicators of the efficiency of spermatogenesis and the acquisition of certain functional sperm capacities (motility), they are not in themselves reliable indicators of male fertility which goes beyond these facts.

Successful reproduction is not limited to fertilization alone, but extends to the optimal development of the embryo and the birth of a viable, healthy individual. Looking at this broader picture, it is clear that the integrity of the female and male genetic material that will merge after fertilization to form the genetic makeup of all cells in the developing embryo is of primary importance. Any alteration in the DNA of the paternal and/or maternal gamete, if not properly corrected, can end-up increasing the mutational load of the next generation, contributing to its susceptibility to develop and transmit various pathologies.

If the oocyte is an active cell with all the protection and repair systems accumulated in its large cytoplasmic compartment, the picture is quite different for spermatozoa. In fact, mammalian evolution has chosen to produce millions of tiny spermatozoa that are practically devoid of any cytosol, harboring a very compact haploid nucleus, thus depriving them of cytosolic protectors and repair activities. Spermatozoa are thus exposed to high risks of DNA damage.

For somatic and germ cells, DNA damage has many faces and contributors, both intrinsic and extrinsic. For aerobic cells, the most trivial contributor to DNA damage is oxygen through the generation of reactive oxygen species (ROS) which, if not properly recycled, have the ability to attack all cell organic constituents (proteins, lipids, carbohydrates and nucleic acids). Specifically, DNA oxidation begins with base oxidation and, depending on the intensity of oxidation, can be followed by the generation of abasic sites, protein/DNA covalent cross-links, and ultimately DNA breaks (single and double stranded), all of which affect the optimal organization of cellular chromatin.

Sperm DNA fragmentation is now recognized as a critical factor in reproductive failure and several tests are commercially available to monitor it, including the TLINEL test, the Sperm Chromatin Structure Assay (SCSA), the Comet Test, and the Sperm Chromatin Dispersion (SCD) test. However, due to the lack of standardization and community consensus on which test is most predictive of reproductive success, WHO is still reluctant to mandate/recommend these tests as part of routine screening of infertile men. Few clinics, however, rely on these sperm DNA fragmentation tests to assess the integrity of the sperm nucleus and the burden that can be placed on the oocyte to successfully repair it.

While sperm DNA fragmentation could be the result of excessive oxidative stress, it may also have other origins such as: unrepaired meiotic breaks, persistence of apoptotic germ cells, and nuclear mechanical shearing during sperm iogenesis when histones are replaced by protamines. Therefore, a systematic link between sperm DNA fragmentation and sperm DNA oxidation cannot be established. Through the generation of transgenic mouse models in which we separated the two situations (sperm DNA oxidation without sperm DNA fragmentation), we helped to show that sperm DNA oxidation can occur independently of fragmentation and that it alone was sufficient to cause reproductive failure (Chabory et al., 2009). This implies that in order to have an accurate and complete assessment of paternal DNA integrity, a combination of tests that assess sperm DNA fragmentation and sperm DNA oxidation is needed. An assessment of sperm nuclear condensation would also be an asset to the diagnosis.

Working in that direction, over the past 15 years, we have helped to show that the mammalian sperm nucleus is particularly sensitive to DNA oxidation and that it affects specific nuclear domains of low compaction (still in nucleosomal organization, thus associated with persistent histones) and specific chromosomal regions (Kocer et al., 2015; Champroux et al., 2018). In animal models, sperm DNA oxidation has been shown to be associated with reproductive defects, including increased miscarriage, abnormal development and increased perinatal mortality, a series of defects also associated with infertility in the clinic. This prompted us to work on the development of a sperm DNA oxidation assay that could be used to monitor the level of oxidative damage to sperm nucleus DNA. The development of an 8-OxodG detection test was first carried out on the mouse model and then extended to humans as well as to farm animals (cattle, sheep, ...) as it could eventually be of interest to the artificial insemination (Al) industry.

An example of the suitability of such an assay for assessing the integrity of the human sperm nucleus is illustrated in the following publication, which aimed to assess the quality of sperm preparations using two different techniques, a conventional density gradient centrifugation (DGC) sperm preparation and a newly developed short electrophoresis coupled with filtration method (FELIX™; Memphasys, Australia). In this report, Figure 5 shows that the use of the DNA oxidation assay clearly distinguishes between the two sperm preparation methods, with sperm prepared by DGC showing a very high level of oxidative DNA damage compared to sperm prepared by FELIX™ as evaluated by the 8- OxodG assay.

Example: 8-OHdG testing allows a better discrimination of 2 sperm preparation technics

Since the advent of ART techniques, the preparation of optimal sperm samples has become a key objective for an industry in search of high performing spermatozoa.¹ Over the past 20 years, several sperm selection protocols and/or devices have been proposed^1-4, but to date, beside the swim-up and differential gradient centrifugation (DGC) preparations, none of them has reached the level of confidence necessary to be generalized to most infertility clinics. In consequence, in accordance with WHO guidelines^{4 5}, most infertility clinics worldwide rely on three basic protocols: 1 ) simple centrifugation of the raw semen sample with wash media to concentrate the sample and remove the seminal fluid, 2) preparation by swim-up and, 3) preparation by DGC.⁵ The last two techniques are the most commonly used semen preparation protocols, with DGC ranking first.⁶ In the case of swim-up preparations, the rationale is based on the ability of sperm to swim in a culture medium deposited on top of the raw semen sample (direct swim-up) or on a sperm pellet obtained after centrifugation of the sample (pellet swim-up). Following the principle that the most motile sperm cells will perform best, spermatozoa that reach the medium will be separated from those cells unable to do so. With DGC sperm preparation, which in some cases can be performed alone or in combination with swim-up preparation (DGC-SLI), the raw sperm sample is deposited on top of a two-layer discontinuous density gradient (usually 45%/80% or 40%/90%) which, after centrifugation (typically 300-400 g, 5-20 min), concentrates the highest quality spermatozoa as a pellet away from the cellular contaminants, debris, defective spermatozoa and seminal plasma.⁵ Additional centrifugation steps are then required to remove the gradient material. Spermatozoa prepared by DGC have been reported to have high progressive motility, optimal morphology, including highly condensed sperm chromatin, and to be free of contaminating epithelial, bacterial, and/or white blood cells.^7-9 The respective efficacy of both sperm preparation methods has been reported extensively.^10-12 Ultimately, it appears that both are equally efficient in achieving clinical pregnancies (Rao et al., 2022)¹³, with DGC offering a better sperm recovery rate particularly with asthenozoospermic, oligoasthenozoospermic (OA), and oligoasthenoteratozoospermic (OAT) specimens, the latter representing the vast majority of patients seen in infertility clinics.

Although DGC has convinced clinicians of its efficacy, it is thought to be associated with negative effects on spermatozoa, which are inherent to the technology used. The fact that it relies on several centrifugation steps, albeit at low speed and for short periods of time, may induce shear forces that could be detrimental to sperm structures and functions, especially in the case of fragile semen samples. In addition, prolonged handling of spermatozoa as well as immersion of spermatozoa in media containing xenobiotic compounds, may not be optimally supportive of sperm health. Indeed, several reports have highlighted that DGC could induce sperm damage that may involve disrupted integrity of the sperm plasma membrane, loss of mitochondrial efficiency, and especially loss of nuclear/paternal DNA integrity.^{14 18} At the crossroads of these alterations is oxidative damage due to the production of reactive oxygen species (ROS) throughout the sperm DGC preparation, whether it is due to the centrifugation, the prolonged handling, and/or the medium.¹⁹’²⁰

To reduce sperm handling time and mechanical damage due to centrifugation steps, alternative isolation techniques have been proposed.²¹ One of them relies on a short electrophoretic separation of raw semen samples based on the negative charge that these cells reach during their post-testicular maturation. In a recent improvement of this system, electrophoretic sperm separation was combined with a filtration system to separate small mature spermatozoa from contaminating cells (leukocytes, epithelial cells, immature sperm cells, apoptotic/necrotic sperm cells, bacteria). Prototypes of this sperm isolation device (Felix™, Memphasys Ltd, Sydney, Australia) have been tested in preclinical trials and have proven their ability to isolate spermatozoa with good fertilization potential, resulting in viable offspring without embryonic development problems.^{22 23}

Description of the invention

The study reported here evaluates the performance of this new sperm preparation protocol using the Felix™ device and to compare it to the classical DGC preparation method as well as to raw semen samples. In addition to monitoring classical sperm parameters (such as total and progressive sperm motility), special attention was paid to the evaluation of sperm DNA integrity using the Sperm Chromatin Structure Assay (SCSA®) and a unique proprietary sperm DNA oxidation assay (EVALSEM™, Clermont-Ferrand, France) the latter being the object of the present claim.

This report compares the quality of sperm prepared either by the conventional density gradient centrifugation technique or by a new electrophoretic device, Felix™ (Memphasys Ltd, Sydney, Australia). Felix™ combines short electrophoresis with proprietary size separation membranes and is an evolution of previously reported devices.^21-23 In practice, the main advantage of the Felix™ device over traditional preparations commonly used in infertility clinics (swim-up and DGC) is its speed of action, with the separation process completed in just over 5 minutes. This avoids long periods of sperm handling, multiple pipetting phases, centrifugation steps and immersion in various media. In addition, the Felix™ system relies on single-use separation cartridges that allow for a high degree of standardization, which is not the case with swim-up and DGC protocols that can change from clinic to clinic.

An important clinical criterion for sperm preparation is the recovery rate as it will determine which samples can be processed and which type of ART can be performed afterwards. In our cohort, we observed that DGC allowed a better mean recovery rate (39.2% corresponding to a mean concentration of 31.6 ± 25.5 million spz/ml) than Felix™ (18.7% corresponding to a mean concentration of 9.2 ± 0.58 million spz/ml). This could have consequences on the choice of samples to be prepared, especially in the case of severe oligozoospermic samples, unless the aim is to prepare spermatozoa for IVF and IVF-ICSI. From our data, it appears that there is a trade-off between sperm recovery rate and sperm quality. The more sperm you retrieve, the lower the quality.

The effectiveness of DGC in preparing sperm of better morphology and higher motility has been proven and known for some time;^{29 30} a point we partly confirm in the present report as far as total motility is concerned. Regarding this specific parameter (total motility), Felix™ achieves a very similar level of efficiency (62.8% vs. 64.6% of total motile sperm cells after Felix™ and DGC preparations, respectively). However, it is interesting to note that for progressively motile spermatozoa, which are considered the best cells in terms of fertilization efficiency,^{31 32} the Felix™ preparation performs significantly better than the DGC (55,7% vs 47,1%, respectively). It is difficult to say whether this is due to the additional selection criteria of the Felix™ device (based on the negative surface charge of the spermatozoa because of their net sialic acid content reflecting the completion of epididymal maturation³³) or to the fact that sperm preparation by the Felix™ is very fast and thus prevents any loss of sperm motility, or possibly to both.

One of the most striking features reported here is the distinct impact of the two sperm preparation techniques on sperm DNA integrity. Because sperm DNA fragmentation (SDF) is nowadays a well-accepted marker of male fertility (for a recent review, see: Minhas et al., 2021 ), ³⁴ we used the Sperm Chromatin Structure Assay (SCSA®) to assess DNA integrity in raw and prepared sperm samples, respectively. SCSA®was chosen as the test to monitor SDF because it is routinely performed in our infertility clinic, which has the licensed SCSA® software to run the assay. In addition, SCSA® is currently the most widely used sperm DNA integrity test for which pathological thresholds have been determined. A pathological DNA fragmentation index DFI (DFI>20-25%) has been reported to occur in approximately 1 in 7 patients seen in infertility clinics.^{35 36} Our cohort (N=29) fell within this range with 5 samples with DFI >20 and 11 samples with moderate DFI (10>DFI<20). The average DFI of the crude semen samples in the cohort was around 15 and was not significantly improved (decreased) after DGC preparation. On the contrary, Felix™ was able to significantly decrease the DFI of the prepared fractions by almost 2-fold. Although Bungum et al 2008³⁷ reported that DFI assessed by SCSA® post DGC is not predictive of ART outcome, it is widely accepted that DFI is a good marker of male fertility and has been associated with an increased risk of miscarriage.³⁸ In the present cohort, sperm preparation by DGC did not significantly decrease the mean DFI. It is difficult to summarize all the reports in which DFI was evaluated after DGC or swim-up because SDF was rarely evaluated using the same test (SCD assay= sperm chromatin dispersion assay or Halo-assay, SCSA®, Comet, TUNEL, etc...) which in fine are known not to measure the same type of DNA damage. In agreement with our data, DGC has already been reported to be ineffective in reducing sperm DNA damage when assessed via SCSA®.³⁹ DGC has even been shown in some cases to increase the DFI of prepared samples,¹¹ leading to the suggestion that swim-up would be a better alternative prior to ART in the case of DNA-fragile sperm samples. The observation that DGC and Felix™ successfully and equally reduced the percentage of sperm with a decondensed nucleus (as revealed by the HDS parameter) is rather logical. DGC selects cells based on their morphology, so sperm cells with a well condensed nucleus will be enriched after DGC. Felix™ uses size-based filtration to remove poorly condensed sperm cells. For all these reasons, the use of a sperm preparation device such as the Felix™ that leads to a significant lower proportion of DNA- fragmented sperm cells could be a pertinent alternative.

Over the past decade, it has become clear that sperm DNA oxidation is another very common early DNA alteration that can eventually lead to SDF. If not properly corrected by the oocyte it could have adverse consequences for proper embryo development and potentially offspring health.¹⁹’²⁰⁴⁰ Sperm DNA oxidation is hypothesized to have a wide range of origins, whether caused by genetic disorders, inflammatory/infectious situations and/or environmental factors.^{41 42} Sperm handling procedures in ART represent another potential source of oxidative insults to sperm DNA due to lengthy protocols, suboptimal media exposures,¹⁸⁴³ non-physiological light/heat/cold exposures, and mechanical shearing associated with centrifugation steps. The data we present here illustrate this quite well, with DGC inducing a very high level of sperm DNA oxidation, regardless of the initial level of DNA oxidation in the raw semen sample. Whether this increase in sperm DNA oxidative damage induced by DGC is due to the multiple centrifugation steps or/and the medium and gradient used will need to be further investigated. On the contrary, sperm samples prepared via Felix™ harbor a significantly lower mean level of DNA oxidation even when compared with raw semen samples. We hypothesize that this is partly in relation with the speed at which the Felix™ preparation is carried out.

In conclusion, Felix™ proves to be a rather successful sperm preparation device, combining speed, an important practical issue for ART clinicians, with spermatozoa showing higher progressive motility and less DNA damage, as evidenced by lower DFI and oxidative DNA damage, two common alterations in paternal DNA known to impact reproductive success. On this basis, Felix™ sperm preparation for ART represents a convincing alternative to conventional sperm preparation protocols used worldwide, thus opening up new clinical perspectives.

This is an example of the suitability of the 8-OHdG assay for assessing the integrity of the human sperm nucleus.

Therefore an object of the present invention concerns a method for preparation of a sperm sample for 8-Oxo-7,8-dihydro-2’-deoxyguanosine (8- oxodG) detection, said method comprising the following steps : a) washing the sperm sample; b) decondensation of sperm DNA of the washed sample; c) fixation of the decondensed sperm DNA sample; d) saturation of aspecific sites of the sample from step c); e) 8-hydroxy-2’-deoxyguanosine (8-OHdG) labelling of the sample from step d); f) counterstaining of the labelled sample from step e); g) detection of the sample from step f) by fluorescence for instance.

According to a particular embodiment of the method of the present invention, step b) is carried out with dith iothreitol (DTT) in the dark. According to a particular embodiment of the method of the present invention, step e) is carried out with a FITC-coupled anti-8OHdG antibody.

According to a particular embodiment of the method of the present invention, step f) is carried out with propidium iodide (PI) in the dark.

Brief description of the figures

Figure 1 : Sperm cell recovery. A: Mean percentage of spermatozoa recovered after DGC or Felix™ preparation (N=29) compared to raw semen taken as 100%. B: Positive association between raw semen sperm counts and recovery rates after sperm preparations (Spearman’s rank order correlation rho=0.66; p < 0.001 ).

Figure 2: Sperm motility comparisons. Box plots for total motility (A), progressive motility (B) and percentage of immotile sperm cells (C) in raw samples and samples prepared by DGC or Felix™. N= 29. ** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001. The center horizontal line in each box represents the median, with the top and bottom edges of the box representing the 75^th and 25^th percentiles, respectively.

Figure 3: Comparisons of sperm DNA fragmentation (SDF) levels, measured by SCSA®. A: Box plots showing DFI between raw samples and samples prepared with either DGC or Felix™ (N=23). B: Box plots showing DFI on a sub-cohort consisting of samples for which the raw DFI was >10 (1X1=14). C: Box plots showing DFI on a sub-cohort consisting of samples for which the raw DFI was <10 (N=9). ** p < 0.01 ; *** p < 0.001. The center horizontal line in each box represents the median, with the top and bottom edges of the box representing the 75^th and 25^th percentiles, respectively.

Figure 4: Box plots showing High DNA Stainability (HDS) values in raw, DGC and Felix™-prepared samples. N=22. **** p < 0.0001. The center horizontal line in each box represents the median, with the top and bottom edges of the box representing the 75^th and 25^th percentiles, respectively.

Figure 5: Sperm DNA oxidation (OxiDNA) level, measured by evaluating the percentage of 8-OHdG positive cells. A: Box plot showing the percentage of spermatozoa with oxidized DNA between raw samples and samples prepared with either DGC or Felix™ (N=22). B: Box plot showing the percentage of DNA- oxidized spermatozoa on a sub-cohort consisting of samples for which the raw DNAox was >10 (N=12). C: Box plot showing the percentage of DNA-oxidized spermatozoa on a sub-cohort consisting of samples for which the raw DNAox was <10 (N=10). ** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001. The center horizontal line in each box represents the median, with the top and bottom edges of the box representing the 75^th and 25^th percentiles, respectively.

Figure 6: Box plot showing sample content in apoptotic bodies between the raw semen and DGC or Felix™ sperm preparation. N=22. * p-value<0.05; **** p- value<0.0001 . The center horizontal line in each box represents the median, with the top and bottom edges of the box representing the 75^th and 25^th percentiles, respectively.

Figure 7: Correlation analysis (A) and principal component analysis (B) between the parameters evaluated in the total cohort (N=58). Both analyses show the good correlation existing between the motility parameters as well as the rather good correlations existing between motility parameters and the DFI (r>0.6). On the contrary, in the current cohort, the oxidation of the sperm nucleus (DNAox) appears weakly correlated to all the parameters studied. See Materials & Methods for details.

Figure 8: 8-OxodG staining of human sperm cell (left sperm heads) versus unreactive (/.e. without DNA oxidation; right sperm heads).

EXAMPLES

EXAMPLE 1 : MATERIAL AND METHODS

Semen samples were obtained from a cohort of 29 patients admitted for fertility testing at the EUROFINS BIOMNIS infertility center (Clinique du Vai d'Ouest, Ecully, France). The remaining semen sample after completion of the planned analyses was used for the study with the patients' consent. The study was approved by the ethics committee of the institution.

Semen analysis

Semen samples were produced on the clinic site by masturbation into a sterile container following a recommended period of sexual abstinence of at least 3 days. Semen samples were allowed to liquefy at room temperature prior to be subjected to a routine semen analysis which was performed as recommended by WHO guidelines.⁵ For sperm DNA integrity evaluation, the SCSA® and its corresponding software (licenced to BIOMNIS, Lyon, France) and DNA oxidation assay (OxiDNA, EVALSEM™, Clermont-Ferrand, France) were used. Sperm motility assessment, total motility, progressive motility and the percentage of immotile cells were determined using a computer-assisted sperm analyzer (CASA) system (Sperm Class Analyser, SCA®, Microptic, Barcelona, Spain).⁵ Concentration and morphology were also evaluated via the SCA® platform. Following the initial evaluation of the raw semen sample, 1 ml of raw semen was prepared via DGC and 1 ml via the Felix™ device. To avoid any operator- mediated bias a single lab technician was involved in handling the samples throughout the study.

Sperm preparation by Density Gradient Centrifugation (DGC)

DGC was performed by discontinuous PureSperm (Nidacon, Gothenburg, Sweden) gradient. Briefly, semen samples (1 ml) were layered upon a 45% / 90% PureSperm density gradient and processed by centrifugation at 400 g for 20 min. The recovered 90% pellet (0.5ml) was resuspended in 3 ml of Sperm Washing medium (Fujifilm, Tockyo, Japan), centrifuged for 7 min at 800 g to eliminate remaining colloidal particles, and finally resuspended in 1 ml of sperm preparation medium (Origio, Versailles France).

Sperm preparation by Felix™

Sperm preparation using the Felix™ device was performed as recommended by the supplier (Memphasys Ltd, Sydney, Australia). Briefly, the separation cartridge was equilibrated with 4m I of G-Rinse™ medium (Vitrolife, Sweden) into each chamber (sample and harvest chambers). One ml of G-IVF™ PLUS medium (Vitrolife, Sweden) was added to the harvest chamber. Then, 1 ml of the liquefied semen sample was loaded into the sample chamber. A 6 minute-long electrophoretic migration was then conducted. At the end of the cycle, 0.3 to 0.5 ml of the isolated sperm suspension was collected from the harvest chamber and analyzed.

Sperm DNA integrity assays Sperm Chromatin Structure Assay (SCSA®)

The SCSA® evaluates the susceptibility of sperm DNA to acid-induced DNA injury revealed by staining with the fluorescent dye acridine orange (AO).^{24 25} Briefly, semen samples are diluted in Earle’s balanced salt solution (Sigma-Aldrich, France) to a sperm concentration of ~1 -2*10⁶ /ml and then treated with an acidic (pH 1 .2) buffer (0.15M NaCI, 0.08M HCI, 0.1 % triton-X 100) for 30 s to open the DNA strands at sites of DNA strand breaks. The sperm are then stained with acridine orange (AO) by adding 2 volumes of a solution consisting of: 0.2M Na2HPO4, 1 mM EDTA, 0.15M NaCI, 0.1 M citric acid, 6pg AO/ml staining buffer pH 6. AO intercalates into double-strand DNA and fluoresces green (515-530 BP filter) and stacks on single-strand DNA that fluoresces red (630 LP filter) upon excitation from a 488 nm laser. The extent of single and double DNA strand breaks (DNA fragmentation index, %DFI) and level of excess nuclear histones (high DNA stainable sperm, %HDS) are simultaneously measured in individual sperm using a SCSA-programmed Flow cytometer (Programme CXP; Cytomics FC500, Beckman Coulter Life Sciences, Villepinte, France).

Sperm DNA oxidation assay (Oxi DNA)

PBS-washed samples were incubated for 45 min in the dark in a decondensation buffer comprising 2 mM dithiothreitol (DTT), 0.5% Triton X-100 in 1 x PBS, as previously described²⁶²⁷, followed by fixation with 4% paraformaldehyde. Spermatozoa were then incubated in 1 .5% normal goat serum saturation solution in 1X PBS. Incubation with a FITC-conjugated anti-8-OHdG monoclonal antibody (15A3, 1 :200; Abeam Inc, Toronto, ON, Canada) was conducted overnight at 4°C. The samples were then washed and stained with PI (propidium iodide, 5 pM in 1X PBS) for 5 min at room temperature in the dark. This makes it possible to distinguish apoptotic bodies from spermatozoa, as previously described.²⁸ The percentage of labelled cells was analyzed by flow cytometry (Attune TM NxT, Acoustic Focusing Cytometer, Thermo Fisher Scientific, Inc, Villebon/Yvette, France). The forward-scattered light and side-scattered light were detected on a linear scale and used to gate sperm cells. Oxidized cells were detected by recording the percentage of cells emitting green and red fluorescence (number of events recorded: 10⁵ sperm cells). The Attune TM NxT Software 3.1.1243.0 was used. Nuclear labeling was confirmed by microscopy (not shown). Detailed protocole for the preparation of human spermatozoa for 8-Oxo-7,8- dihydro-2’-deoxygua nosine (8-OxodG) detection:

- Disperse per tube (Eppendorf 1.5 ml) the volume of sperm to have approximately 5.10⁶ spermatozoa.

- Provide 2 tubes of controls, positive (optional) and negative (see preparation of these tubes below).

1 . Washing of the samples

- Add 1 ml of 1X PBS to the volume of sperm

- Centrifuge at 1090 g, 6 minutes, at room temperature (RT)

- Remove the supernatant (200 pl pipette)

- Gently resuspend and wash the pellet with 1 ml o 1X PBS

- Centrifuge at 1090 g 6 minutes, RT, then remove the supernantant

2. Decondensation of sperm DNA

Prepare the decondensation solution (to be prepared extemporaneously and keep in the dark)

Weight 3 mg of dithiothreitol (DTT) (Euromedex - Ref EU0006-B)

Add 10 mL of 1X PBS

Add 50 pL of T riton X-100 (Euromedex - Ref 2000-B)

- Add 500 pL I tube of decondensation solution

- Incubate for 45 min at RT, protected from light (resuspend the pellet well by gentle finger tilting every 15 min)

- Centrifugation 1090 g, 6 min, then remove the supernatant

- Resuspend and wash with 500 pL of 1X PBS (resuspend the pellet gently with a Pipetman P200)

- Centrifugation 1090 g, 6 min, then discard the supernatant

3. Fixation with 4% paraformaldehyde (PFA) in tube

Prepare the 4% PFA solution (to be prepared extemporaneously)

10 mL PFA 16% (Electron Microscopy Sciences - Ref 15710)

30 mL of 1X PBS

Aliquot into 1.5 mL tubes by freezing at -20°C (do not refreeze an already thawed tube)

- Add 200 pL of 4% PFA I tube, gently resuspend the pellet

- Incubate for 20 minutes at 4°C - Centrifuge 1090 g, 6 minutes, RT

- Remove the supernatant (with a 200 pL Pipetman) into a PFA waste garbage can

- Wash and resuspend with 200 pL of 1X PBS (resuspend the pellet well)

- Centrifugation 1090 g, 6 minutes

- Resuspend in 500 pL of 1X PBS and store at 4°C (if necessary)

4. Saturation of aspecific sites

Preparation of the saturation solution 1X PBS 0.1 % Triton X-100 1.5 % normal goat serum (NGS)

Add 1 mL of 100% Triton X-100 to 1 L of 1X PBS (shake well with magnetic bar steering)

Add 150 pL of NGS (Sigma - Ref G9023) in 10 mL of 1X PBS Triton X-1000.1 %

- Resuspend the spermatozoa in 300 pL of 1X Triton X-100 0.1 % NGS 1.5%

PBS solution

- Incubate 1 h at RT in the dark

- Centrifuge 1400 g, 2 min and discard the supernatant

5. 8-hydroxy-2’-deoxyguanosine (8-OHdG) labelling

Negative control (C-) o Addition of 100 pL

1/200 dilution of Ig2b control isotype antibody (ABCAM - Ref 91368) in the saturation solution o Addition of 5% Trisodium citrate (Euromedex - Ref 6132-04-3)

Samples and positive control (C+) o Addition of 100 pL

1/200 dilution of FITC-coupled anti8-OHdG (ABCAM - Ref 18393) in the saturation solution o Addition of 5% Trisodium citrate

Incubate O/N at 4°C

Next day

- Centrifugation 1400 g, 2 min and remove the supernatant

- Transfer the pellet to 200 pL of 1X PBS

- Centrifuge 1400 g, 2 min and discard the supernatant

6. Counterstain - Add 200 pL of propidium iodide (PI) (Invitrogen - Ref 27011 ) at 5 pM diluted in 1X PBS

- Incubation 5 min at RT in the dark

7. Slide (for fluorescence microscopy detection)

- Dry the “washed” slides beforehand

Slides “superfrost” (VWR: ref 631-0909), washed in acetone/EtOH and kept at -20°C

- Filter the samples with filter 50 pm (BD - Ref 340631 )

- Deposits in concentric circle

- Dry for about 30 min in a fume hood, in the dark

- Mowiol assembly (Electron Microscopy Sciences - Ref 17977-150)

8. FC detection ATTUNE Nxt (ThermoFisher Scientific

Use of the CMF ATTUNE Nxt blue laser (488 nm)

- emission filters: BL1 , 530/30 (PMT voltage =) detection of fluorochrome FITC

BL2, 590/40 (PMT voltage =) detection of PI fluorochrome

- collection of fluorescence in Log mode

The results of this detailed protocole for sperm sample preparation and 8-oxodG detection are shown in Figure 8.

Statistics

Data are presented as box plots (Raw, DGC, Felix™). Due to rejection of hypotheses on normality of distribution and homogeneity of variance, the comparisons employed a non-parametric Kruskall-Wallis test. When Kruskall- Wallis test was significant (p<0.05), pairwise Wilcoxon-Mann-Withney tests were carried out. Significance levels were indicated on the graphs using ****, ***, ** and * corresponding to p<0.0001 , p<0.001 , p<0.01 and p<0.05, respectively. For the correlation analysis (Fig. 6), for the purpose of meeting the assumption on normality, relevant transformation of data on numeric variables was performed. Pair plots were done to visualize the distribution of single variables as well as relationships between two variables. The Pearson correlation coefficient was indicated for each pair of variables giving the measure of the linear relationship between two variables. The test for association between paired samples was based on Pearson's product moment correlation coefficient. Significance levels for the correlation test were indicated on the graphs using ****, ***, ** and * corresponding to p<0.0001 , p<0.001 , p<0.01 and p<0.05, respectively. Principal component analysis (PCA) was done using correlations between variables. PCA Eigen values were used to determine how many factors to retain (Table 3). The correlation circle showing a projection of original variables in the principal component space PC1 (Dim 1 ) and PC2 (Dim 2) was constructed (Fig 6B). The proximity of the lines indicates the degree of correlation between the variables. All analyses were performed with the application of the statistical packages Rstatix, GGally, BestNormalize, FactoMineR, Factoextra in the R environment (version 4.2.1 - R Core Team [2022]: R Foundation for Statistical Computing, Vienna, Austria).

EXAMPLE 2 : RESULTS

This evaluation study took place over a 6-month period from May 2022 to October 2022. All samples were processed in an identical manner. Any abnormal liquefaction steps in terms of time required and sample viscosity were duly noted and in that case the sample was excluded. Sample processing was performed directly after the liquefaction step and the time since ejaculation was noted (and did not exceed 2 hours). The presence of leukocytes, cellular debris, and artifacts in the samples was recorded and classified as low, medium, or high. All samples from the cohort were qualified “low”. Samples that did not allow us to divide them into two 1 ml aliquots, each to be loaded onto the DGC and Felix™ device, respectively, were not included in the evaluation program. It should be noted that severe oligozoosperm ic samples could not be included due to the selected flow cytometry-assisted DNA integrity assays (SCSA® and OxiDNA) carried out, each of which requires starting with at least 3 to 5 x 10⁶ spermatozoa. A final cohort of 29 samples was analyzed. In the evaluated cohort, the mean age of patients was 36.72 ± 5.98 years and ranged from 25 to 47 years. The mean volume of samples was 5.8 ± 1.93 ml and ranged from 2.9 ml to 10.7 ml. The mean sperm concentration of the samples was 57.8 ± 43.9 x 10⁶ spermatozoa/ml and ranged from 11.1 x 10⁶ spermatozoa/ml to 178 x 10^s spermatozoa/ml. Most of the processed samples matched what is considered normozoospermic samples according to WHO standard values for motility, morphology and vitality.⁵ Tables 1 & 2 below provide the detailed data for all the measured parameters. Table 1 : Sample characteristics

N= sample number, Sv= semen volume, SC= sperm count, RR=recovery rate, TM=total motility, PM=progressive motility, IM=immotile.

Table 2: Sample characteristics (Nuclear/DNA integrity parameters and presence of apoptotic bodies).

N= sample number, DFI=DNA fragmentation index, HDS=high DNA stainability, AB= apoptotic bodies

Recovery rate

The average sperm recovery rates after DGC and Felix™ preparation were calculated and were 39.2% and 18.7%, respectively, compared to the 100% raw semen sample (Fig 1A). This rate was largely sample dependent, ranging from 13% to 76% and 7.3% to 44% for DGC and Felix™ preparations, respectively. As it could be expected, there is a mild but significant correlation (r = 0.66; p < 0.01 ) between the sperm concentration of the raw samples and the recovery rate (Fig. 1 B).

Sperm motility

When examining sperm motility (Fig 2A), the mean percentage of total motile spermatozoa was significantly improved, in a rather equal manner, by both sperm preparations (64.6% and 62.8% total motile sperm cells in DGC and Felix™ preparations, respectively) compared to the mean percentage of total motile spermatozoa of the raw sample (52.4%). Interestingly, when examining the mean percentage of progressively motile sperm cells (Fig 2B), the Felix™ sperm preparation showed significantly greater efficiency in selecting progressive motile sperm cells (55.7%) than DGC (47.1 %). It should be noted that the DGC preparation did not significantly increase the mean percentage of progressive motile spermatozoa compared to the raw semen samples (44.8%), in the tested cohort. Confirming the ability of both sperm preparation methods to improve the selection of motile spermatozoa, Fig 2C shows that DGC and Felix™ preparations contain significantly fewer immotile sperm cells than in the raw semen.

Sperm DNA integrity

Sperm DNA fragmentation in fresh and DGC or Felix™ prepared semen samples, assessed by SCSA® technology, showed that DGC did not significantly improve (ie. decrease) the mean percentage of sperm cells with DNA fragmentation compared to raw semen (Fig 3A). On the contrary, the Felix™ preparation significantly decreased the mean percentage of spermatozoa with DNA fragmentation. Overall, compared to raw and DGC-prepared semen samples, the Felix™ preparation decreased the mean percentage of sperm cells with DNA fragmentation by approximately 2-fold. Figures 3B and 3C illustrate that after an arbitrary separation of the cohort (N=24) into 2 sub-cohorts containing, on the one hand, raw semen samples (N=15) for which the DFI was moderate to high (DFI>10) and, on the other hand, raw semen samples (N=9) for which the DFI was low ( D F I < 10), it appears that DGC did not significantly improve the mean level of sperm DNA fragmentation, whereas Felix™ did. When samples had a low level of sperm DNA fragmentation (Fig. 3C), no preparation was effective in further decreasing this level, although the Felix™ preparation did still show a tendency to do so. Flow cytometry-assisted SCSA technology provides access to a second parameter, namely high DNA stainability (HDS), which takes into account the fluorescence intensity of the cells being assessed. It is accepted that HDS assesses the level of sperm nuclear compaction.²⁴²⁵ Figure 4 shows that DGC and Felix™ preparations significantly decrease the mean percentage of HDS spermatozoa in the prepared fractions compared to raw semen samples.

Because semen preparations have been suspected of potentially inducing oxidative damage, this prompted us to monitor sperm DNA oxidative damage using our proprietary sperm DNA oxidation assay.²⁷ The data presented in Fig 5A show that the mean percentage of spermatozoa with oxidative DNA damage (estimated via the content of oxidized guanine residues, the so-called 8-oxodG residue) increased dramatically in the DGC-prepared sperm fractions, from 14.3% in the raw sperm sample to 63.9%. On the contrary, the semen fraction prepared by Felix™ did not show such an increase in oxidative DNA damage (Fig. 5A), as the mean percentage of sperm cells with oxidized DNA in the total cohort (N=26) was significantly lower than that of the raw semen samples (10.4% vs. 14.3%, respectively; p < 0.05). As was done with DNA fragmentation analysis, when separating the cohort into 2 sub-cohorts with on the one hand the raw samples above 10% oxidized cells (N=11 ) and on the other hand, the raw samples below 10% (N=15) oxidized sperm cells (Fig. 5B & 5C), it confirms that the DGC preparation oxidizes equally both cohorts whereas Felix™ does not. It also confirms within the sub-cohort of spermatozoa with oxidized DNA that Felix™ preparation significantly (p < 0.01) decreases the percentage of sperm cells with oxidized DNA (Fig. 5B).

Figure 8 confirms that the claimed method is clearly able to distinguish spermatozoa with oxidized DNA (left) from spermatozoa without oxidized DNA (right) via 8-OxodG immunoreactivity.

Presence of apoptotic bodies in the prepared sperm fractions. Since our flow cytometry-assisted sperm oxiDNA assay uses propidium iodide (PI) staining in parallel with the assessment of 8-OHdG content in order to get rid of contaminating apoptotic structures that may interfere with the assessment of 8-OHdG level (M450 bodies)²⁸, this provided us with a way to estimate how well the two methods addressed this aspect. Figure 6 shows that both sperm preparations significantly reduced the percentage of these contaminating structures. DGC preparation was however significantly better than Felix™ at reducing the number of these specific contaminant bodies.

Correlation analysis.

Fig 7A presents a correlation analysis between the parameters evaluated in this particular cohort. It shows without surprise that the 3 motility parameters investigated (total motility, progressive motility, immotility) are well correlated. Sperm DNA fragmentation appears mildly correlated with motility parameters (especially with immotility). Sperm DNA oxidation does not appear to be correlated with any of the other parameters monitored. Of interest, is the observation that despite the proposal¹⁹ that sperm DNA fragmentation has mainly an oxidative origin, in this particular cohort sperm DNA oxidation was not correlated with sperm DNA fragmentation. These elements are rather well illustrated in the principal component analysis (PCA) shown in figure 7B. PCA generated 7 principal components. The first three components explained 84.32% of the total variance of the data studied, and having a variance greater than 0.7 (eigenvalue greater than 0.7); therefore, they were considered significant PCs (Table 3). Figure 7B presents only the two first PC (PC1/Dim 1 & PC2/Dim2).

Table 3: Principal components (PCs), eigenvalues, percentage of variance explained by the PCs (% VPC) and cumulative percentage of variance explained by the PCs. List of references

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Claims

1. Method for preparation of a sperm sample for 8-Oxo-7,8-dihydro-2’- deoxyguanosine (8-oxodG) detection, said method comprising the following steps : a) washing the sperm sample; b) decondensation of sperm DNA of the washed sample; c) fixation of the decondensed sperm DNA sample; d) saturation of aspecific sites of the sample from step c); e) 8-hydroxy-2’-deoxyguanosine (8-OHdG) labelling of the sample from step d); f) counterstaining of the labelled sample from step e); g) detection of the sample from step f).

2. Method according to claim 1 , wherein step b) is carried out with dithiothreitol (DTT) in the dark.

3. Method according to claim 1 or 2, wherein step e) is carried out with a FITC- coupled anti-8OHdG antibody.

4. Method according to any of claims 1 to 3, wherein step f) is carried out with propidium iodide (PI) in the dark.