Academic Article
A biomimetic sperm selection device for routine sperm selection
by Steven A. Vasilescu, Dale M. Goss, Kathryn H. Gurner, Rebecca L. Kelley, Maria Mazi, Fabrice K. De Bond, Jennifer Lorimer, Fabrizzio Horta, Farin Y. Parast, David K. Gardner, Reza Nosrati and Majid E. Warkiani
Abstract
Research question: Can a biomimetic microfluidic sperm sorter isolate motile sperm while minimizing DNA damage in comparison with density gradient centrifugation (DGC)?
Design: This was a two-phase study of 61 men, consisting of a proof-of-concept study with 21 donated semen samples in a university research laboratory, followed by a diagnostic andrology study with 40 consenting patients who presented at a fertility clinic for semen diagnostics. Each sample was split to perform DGC and microfluidic sperm selection (one-step sperm selection with 15 min of incubation) side-by-side. Outcomes evaluated included concentration, progressive motility, and DNA fragmentation index (DFI) of raw semen, and sperm isolated using DGC and the microfluidic device. Results were analysed using Friedman’s test for non-parametric data (significant when P < 0.05). DFI values were assessed by sperm chromatin dispersion assay.
Results: Sperm isolated using DGC and the microfluidic device showed improved DFI values and motility compared with the raw semen sample in both cohorts. However, the microfluidic device was significantly better than DGC at reducing DFI values in both the proof-of-concept study (P = 0.012) and the diagnostic andrology study (P < 0.001). Progressive motility was significantly higher for sperm isolated using the microfluidic device in the proof-of-concept study (P = 0.0061) but not the diagnostic andrology study. Sperm concentration was significantly lower for samples isolated using the microfluidic device compared with DGC for both cohorts (P < 0.001).
Conclusion: Channel-based biomimetic sperm selection can passively select motile sperm with low DNA fragmentation. When compared with DGC, this method isolates fewer sperm but with a higher proportion of progressively motile cells and greater DNA integrity.
Keywords: sperm selection; microfluidics; density gradient centrifugation; DNA fragmentation
We kindly thank the researchers at University of Technology Sydney for this collaboration, and for sharing the results obtained with their system.
Introduction
Infertility affects approximately 15% of couples worldwide, with approximately 55% of those having a male contributing factor (WHO, 2023). The use of medical intervention in the form of assisted reproductive technology (ART) is growing annually, yet the success rate of assisted reproduction cycles per embryo transfer has stagnated at approximately 33% per cycle, and the proportion of live births has plateaued at approximately 26% per cycle over the last two decades (Adamson et al., 2018; Nosrati et al., 2017). Many factors play a role in the success of a cycle, and one crucial aspect of all methods is sperm selection, where sperm quality can have a direct effect on outcomes (Donnelly et al., 1998; Tandara et al., 2014).
An increased DNA fragmentation index (DFI) is prevalent in infertile men and in men aged >40 years, and is even higher in those with abnormalities in conventional semen parameters such as motility, morphology and concentration (Moskovtsev et al., 2009). Furthermore, couples with idiopathic infertility and recurrent pregnancy loss, where conventional sperm parameters lie within healthy norms, show a higher incidence of DNA fragmentation (Esteves et al., 2021; Tan et al., 2019). Studies have shown, using the sperm chromatin structure assay (SCSA), that approximately 28% of men from infertile couples have moderate (>20%) or high (>30%) DFI values (Erenpreiss et al., 2008), while in couples with ‘unexplained infertility’, the percentage of men with moderate-to-high DFI values is 26.1% (Oleszczuk et al., 2013); this incidence increases with age. High levels of DNA fragmentation (>30%) in sperm have been shown to increase the risk factors involved in IVF by reducing embryo quality, lowering implantation rates, and increasing the chance of miscarriage up to 3.9 times that of patients using sperm with low DFI values (Borges Jr et al., 2019; Duran et al., 2002; Robinson et al., 2012; Sedó et al., 2017).
Density gradient centrifugation (DGC) and swim-up methods are the most common techniques used for processing semen samples and selecting sperm for use in assisted reproduction. However, in recent studies, these methods have been implicated in the increase in sperm DNA fragmentation, purportedly due to the generation of reactive oxygen species (Aitken et al., 2014; Muratori et al., 2016; Stevanato et al., 2008). As it is not yet possible to assess sperm DNA fragmentation non-invasively prior to use in IVF or intracytoplasmic sperm injection (ICSI), innovative methods are required to select and isolate sperm with low DFI values for use in IVF or ICSI at appropriate concentrations. There have been several attempts to create an alternative sperm selection method, including the MACS ART Annexin V System (Miltenyi Biotec, Australia), Physiological Intracytoplasmic Sperm Injection dish (CooperSurgical, USA) and the DGC-zeta potential method (Hasanen et al., 2020; Karimi et al., 2020), and many innovative approaches have originated from the application of microfluidics (Keskin et al., 2022; Quinn et al., 2018; Suh et al., 2006; Villeneuve et al., 2023).
Microfluidic devices have been developed to select high-quality sperm using flow or sperm migration behaviour, without the need for active input such as centrifugation or electrophoretic fields. These devices purport to reduce exposure to oxidative stress and subsequent DNA fragmentation (Dadkhah et al., 2023; Nosrati et al., 2014; Simchi et al., 2021). However, the clinical translation and adoption of many of these technologies have been limited (Nosrati, 2022), largely due to their complexity of workflow, operational instability and/or inconsistent results. Without an intuitive user interface, many devices have not seen further side-by-side clinical testing to evaluate their performance in the hands of clinical scientists. An effective sperm selection platform must not only provide high-quality sperm in a timely manner, but must also be simple to use and consistent in its performance over a range of sperm motility levels (Nosrati et al., 2017; Rappa et al., 2016).
This study aimed to test a novel microfluidic channel-based biomimetic sperm selection device against the gold standard, DGC, to isolate motile sperm from a range of semen samples. This radial microfluidic device selects sperm based on their preference to follow boundaries in a static fluid environment mimicking the geometries of the female reproductive tract (Figure 1). This device consists of a radial array of hundreds of media-filled channels, whereby semen interfaces with the entrances to these channels, selecting sperm by their ability to traverse corners, boundaries and troughs, mimicking the endometrium and epithelium of the oviduct, towards a central outlet port. It was postulated, based on the authors’ previous insights into motile sperm behaviour in confined geometries and channels (Eamer et al., 2016; Nosrati et al., 2014; Vasilescu et al., 2023), that the boundary-following tendencies of sperm will lead to the isolation of a sample with high motility and low DFI values.
Materials and Methods
Device fabrication
The microfluidic device contained a semen reservoir designed to hold 1 ml of raw semen. The semen reservoir was connected to a sperm trapping and collection area via a radial array of microchannels (Figure 1 and Supplementary Video 1). Microfluidic devices were fabricated via a moulding process using three-dimensional-printed moulds adapted from Shrestha et al. (2019). The moulds were fabricated using a digital light processing three-dimensional printer (MiiCraft Ultra 50; MiiCraft, Taiwan) and a photopolymer resin (BV-007; Creative CADWorks, Canada). The microfluidic device design was created in Solidworks 2019 (SolidWorks Corp., USA) and sliced at 25 µm. The device was made from two separate moulds comprising the top and bottom layers of the device. After printing, each mould was washed with isopropanol, and dried with an air gun to remove any excess liquid resin from the microchannels. Washing with isopropanol was repeated three times, and the moulds were cured in an ultraviolet curing box for 2 min prior to being placed in a 70% ethanol bath for 2 h. The moulds were subsequently dried with an air gun and treated with oxygen plasma (Basic Plasma Cleaner PDC-002; Harrick Plasma, USA) for 2 min, followed by salinization using trichloro (1H, 1H, 2H-perfluoro-octyl) silane (Sigma-Aldrich, USA) in a desiccator under vacuum for 1.5 h. The moulds were cast with polydimethylsiloxane (PDMS) (Sylgard 184; Dow Corning, USA), a non-toxic polymer (Colucci et al., 2018), prepared using a mixture of base and curing agents in a ratio of 1:10. The mixture was degassed to remove all bubbles before casting on to the moulds and cured in a hot air oven (75°C) for 2 h. After curing, the PDMS layers were gently peeled off the moulds; holes were punched for the semen inlet, overflow reservoir and sperm collection outlet; and then oxygen plasma treatment was used for bonding.
Semen collection and patient demographics
Human semen samples were obtained through ejaculation after 2–4 days of sexual abstinence as recommended by the World Health Organization (WHO, 2021). Raw semen samples were kept on a shaker at room temperature for 15–20 min to allow for full liquefaction. The proof-of-concept study was approved by Monash University Human Research Ethics Committee (ID 26713, approval date 9 December 2020), and the diagnostic andrology study was approved by Melbourne IVF Human Research Ethics Committee (88-22-MIVF, approved 10 May 2022). Samples were obtained from donors for the proof-of-concept study, and from patients presenting for semen analysis for the diagnostic andrology study. Specific age information is not available for the proof-of-concept study, but these donors were students between the ages of 18 and 24 years. Patients in the diagnostic andrology study were between 26 and 54 years of age (mean 36.3 years) (Table 1). Differences were found between the two populations; specifically, the average semen volume was greater in the diagnostic andrology group (P = 0.012). All participants provided informed consent for their inclusion as completely de-identified participants in this study.
The exclusion criteria for both groups were azoospermia, severe oligozoospermia (<1 million/ml sperm concentration) or absolute asthenozoospermia (0% motile sperm). Additionally, participants providing samples <1 ml volume, and those designated for cryopreservation in the diagnostic andrology study were excluded. Finally, patients who reported active infection or had samples with signs of active infection or inflammation (presence of many round cells) were also excluded.
Experimental procedure
In total, 61 patient and donor samples were processed across two separate studies. Although the two studies were performed at different sites and by different personnel (research embryologists in the proof-of-concept study, and clinical embryologists and andrologists in the diagnostic andrology study), both studies followed the same approach whereby all samples were split into three groups – raw semen, and sperm isolated using DGC and the microfluidic device – and processed in parallel. In cases where the semen volume was <2.1 ml, the unprocessed sample was diluted to 2.1 ml in G-MOPS Plus medium (Vitrolife, Sweden) to enable parallelized processing (nine of 61 samples), as 1 ml was required for both DGC and the microfluidic device, and 100 μl of raw semen was required as the control.
To perform sperm selection with the microfluidic device (see Figure 1Ai–iv), the device was pre-filled with Sydney IVF Gamete Buffer (Cook Medical, USA) at room temperature for the proof-of-concept study, or G-MOPS Plus medium (Vitrolife) for the diagnostic andrology study by injecting 1.5 ml through the central outlet using a filled 3-ml plastic syringe (Becton, Dickinson and Company, USA). These two media are the sperm processing buffers used routinely at each site. A strip of AS-110 acrylic medical grade adhesive tape (AR Care Ltd, UK) was placed over the central outlet to create an airtight seal for channel stability, preventing undesired flow of fluids. The device was then left for 5–10 min on a warm plate (37°C) to equilibrate. Next, 1 ml of liquified semen was injected into the device using a 1-ml plastic syringe, and the device was left undisturbed at 37°C (on a heated stage) for 15 min. After incubation, the tape was removed, and 200 µl of sperm suspended in media was collected from the central outlet using a 200-µl pipette and transferred to a final tube for analysis. The migration of sperm through various stages of the device can be seen in Supplementary Video 1.
In the proof-of-concept study, DGC was performed using Sydney IVF 80/40 gradients (Cook Medical, USA). First, 1 ml of 40% gradient was placed in a conical 15-ml tube and underlaid with 80% gradient. Next, 1 ml of semen was layered carefully on top of the 40% density gradient layer using a 1-ml pipette. The solution was centrifuged at 500 x g for 10 min, and the pellet was aspirated directly from the bottom of the tube by collecting 200 µl of fluid. The pellet was resuspended in 3 ml of Sydney IVF Gamete Buffer, centrifuged for 5 min at 500 x g, the subsequent pellet was aspirated from the bottom by collecting 200 µl of sperm in Sydney IVF Gamete Buffer, followed by transfer to a final tube for analysis.
In the diagnostic andrology study, DGC was performed using PureSperm 80/40 gradient (Nidacon, Sweden). With aseptic techniques, 1 ml of 40% PureSperm density gradient was pipetted into a 15-ml conical tube. Using a new pipette, 1 ml of PureSperm 80% density gradient was underlaid carefully to avoid mixing the two layers. After layering 1 ml of semen sample carefully on to the gradient, without disrupting the density gradient, the gradient tube was centrifuged at 500 x g for 10 min. After centrifugation, the gradient tube was removed carefully from the centrifuge to avoid mixing the layers. The pellet was removed using a clean Pasteur pipette, transferred to a clean 15-ml blue-capped Falcon conical tube containing 8 ml of G-MOPS Plus media, and centrifuged at 500 x g for 5 min. Finally, the pellet was transferred to the final tube with 200 µl of G-MOPS Plus media. The same media were used at both sites for both the microfluidic device and the wash centrifugation step of DGC processing.
Sperm chromatin dispersion assay and motility analysis
DFI was assessed with a modified sperm chromatin dispersion (SCD) test, using the HT-HSG2 kit (Halotech DNA, Spain) as reported previously (Vasilescu et al., 2021). Sperm DNA fragmentation was obtained for the raw semen sample and for sperm isolated using the microfluidic device and DGC. Briefly, 80 μl of sperm suspension was added to 80 μl of pre-aliquoted warmed agarose in a 2-ml Eppendorf tube (Eppendorf, Germany). Thereafter, 10 μl of the semen–agarose mixture was pipetted on to pre-coated slides and covered with a coverslip. The slides were placed on a cold plate at 4°C for 5 min to allow the agarose to set. The coverslips were then slid gently off the slides, and the slides were immediately immersed horizontally in an acid solution and incubated for 7 min with a new coverslip placed on top. Next, slides were gently tilted vertically to allow the acid solution to run off. Slides were then immersed horizontally in the lysing solution for 20 min, washed with distilled water for 5 min, and then dehydrated in increasing concentrations of ethanol (70% and 100%) for 2 min each, air-dried, and stored at room temperature in the dark. Sperm were categorized into one of five groups during counting following SCD: (i) sperm with a halo width equal to or larger than the minor diameter of the core; (ii) sperm with a small halo, similar to or smaller than one-third of the minor diameter of the core; (iii) sperm with a medium halo, between the size of small and large halos from Groups i or ii; (iv) sperm with no halo; and (v) sperm with a degraded halo. Sperm with a small, degraded or absent halo contained fragmented DNA (Groups ii, iv and v). DFI values were recorded as the percentage of sperm cells with fragmented DNA. Three hundred sperm were counted per sample, and each sample was counted twice; counts were considered to be accurate if the difference in DFI value was within 5%. Seven samples were removed from the DFI results due to inadequate staining for reliable counting.
Motility and concentration for each group were assessed using the WHO guidelines (WHO, 2021), Sperm were classified as either progressively motile, non-progressive or immotile, with a minimum of 200 sperm assessed for motility. This method for assessing sperm concentration and motility was performed consistently at both sites to ensure accurate comparisons and minimize confounding variables. For assessing sperm concentration, a 1:10 dilution of raw semen with gamete buffer (Sydney IVF Gamete Buffer for the proof-of-concept study; G-MOPS Plus for the diagnostic andrology study) was required for 10 of the 50 samples. Concentration was assessed under a phase contrast microscope (Olympus CKX53; Evident, Japan) using a haemocytometer at 200 X, and a duplicate count was performed between two scientists, with the counts repeated if the difference between the two counts exceeded 5%. All sperm were counted in the four corner squares and the centre square, and the total number of sperm was multiplied by the dilution factor for each sample where applicable. For motility assessment, samples were assessed under similar conditions after liquefaction of semen within 1 h of collection, and immediately after processing with DGC and the microfluidic device. Sperm were also assessed by two scientists, and the assessment was repeated if the difference between counts exceeded 5%.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, USA). The normality of distribution was assessed using the Shapiro–Wilk test. The significance of differences between values for demographic data (Table 1) was assessed using the Mann–Whitney U-test. Experimental analysis (Table 2) was assessed using Friedman’s test for non-parametric data to account for repeated measures from the same patient after Dunn’s multiple comparisons test to correct for multiple comparisons. Pearson’s correlation test was performed to assess the linear relationship between DFI values of sperm isolated using the microfluidic device and DGC. Pearson’s correlation coefficient (r-value) was calculated to quantify the strength and direction of the linear association between these two variables. Data are presented as median and interquartile range (IQR) and mean ± SEM. P < 0.05 was considered to indicate significance.
Results
The radial microfluidic device selects sperm based on their preference to follow boundaries in a confined space within a static fluid environment, as shown in Figure 1Bii and iii. Table 2 shows the sperm quality metric values for DFI, motility and concentration for the three groups processed in parallel (raw semen, DGC and microfluidic device) for both the proof-of-concept study and the diagnostic andrology study.
Proof-of-concept study
The proof-of-concept study showed that sperm isolated using the microfluidic device had significantly lower median DFI values compared with sperm isolated using DGC (0.7% versus 4.1% respectively, P = 0.0012) (Figure 2A). Sperm isolated using the microfluidic device also showed consistently lower DFI values than sperm isolated using DGC in all 21 samples, as the distribution of DFI values compared between split samples shows in Figure 2B. There is a positive correlation with a slope <1 and r = 0.81, showing that increases in DFI values for sperm isolated using DGC were accompanied by increases in DFI values for sperm isolated using the microfluidic device, although the latter increased at a lower rate. Sperm isolated using the microfluidic device yielded a 92.2% decrease in the average DFI value (calculated as the percentage reduction in DFI value compared with the raw semen sample), outperforming DGC which reduced the average DFI value by 57.4% (P < 0.0001). A significantly higher percentage of sperm isolated using the microfluidic device were progressively motile compared with sperm isolated using DGC (92.8% versus 77.7%, respectively; P = 0.0061), with the former showing improvement in 20 of 21 samples (Supplementary Figure 1 and Figure 2C). This represents a two-fold average increase in progressive motility in sperm isolated using the microfluidic device compared with DGC (61.1% for microfluidic device, 30.5% for DGC). Microfluidic selection reduced sperm concentration significantly compared with DGC selection (2.9 × 106 sperm/ml versus 61.0 × 106 sperm/ml, respectively; P < 0.0001) (Figure 2D). The concentration difference between raw semen and sperm isolated using DGC was not significant. Each selection method resulted in a 200-µl suspension of sperm in gamete buffer. Results for before and after microfluidic device processing can be seen in Figure 2E and F, respectively.
Diagnostic andrology study
The diagnostic andrology study repeated trends observed in the proof-of-concept study in a population of patients presenting for infertility. Of the 33 samples processed for DFI values, the sperm isolated using the microfluidic device had significantly lower median DFI values compared with sperm isolated using DGC (1.0% versus 3.9%, respectively; P < 0.001), and a reduction was seen in 30 of 33 samples assessed (Supplementary Figure 2 and Figure 3A,B). Although the DFI values of sperm isolated using the microfluidic device and DGC were significantly reduced compared with the raw semen sample (P < 0.0001 and P = 0.0022, respectively), use of the microfluidic device yielded an 82.9% average improvement (calculated as the percentage reduction in DFI value compared with the raw semen sample), significantly outperforming DGC (P < 0.001) which reduced the DFI value by an average of 44.4%. Furthermore, irrespective of the DFI value of raw semen, DFI values of sperm isolated using the microfluidic device were consistently reduced to <10%; in comparison, the use of DGC resulted in 10 of 33 samples with DFI values >10% (Figure 3B). Although a weak positive correlation was found, with a slope < 1 and r = 0.44, a less pronounced increase in the DFI values of sperm isolated using the microfluidic device was observed compared with sperm isolated using DGC. Unlike the proof-of-concept study, there was no significant difference in progressive motility of sperm isolated using the microfluidic device compared with DGC, and both the microfluidic device and DGC improved progressive motility significantly compared with the raw semen sample (Figure 3C, both P < 0.0001). Furthermore, the sperm concentration reflected similar results as the proof-of-concept study, with samples isolated using the microfluidic device yielding a lower median sperm concentration compared with samples isolated using DGC (2.0 × 106 sperm/ml versus 20.0 × 106 sperm/ml, respectively; P < 0.0001) (Table 2 and Figure 3D).
Site comparison
Results from each site were compared (Supplementary Figures 3A–C), and both progressive motility (P < 0.0001) and concentration (P = 0.0015) of sperm isolated using DGC were significantly lower in the proof-of-concept study compared with the diagnostic andrology study. No other significant differences were observed between the sites.
Discussion
This study demonstrated that passive biomimetic microfluidic channel-based processing of semen, from healthy donors as well as patients attending an IVF clinic for diagnostic andrology, enables the selection of a high proportion of progressively motile sperm with significantly lower DFI values compared with conventional DGC. A biomimetic mode of sperm selection offers consistent results and can be performed with minimal training, with the operation of the microfluidic device requiring only a syringe, a pipette, and a heated stage or incubator to operate the device (Figure 1A,B). During the incubation time, only motile sperm make their way from the semen reservoir down the microchannels via boundary-following behaviour towards the collection chamber, and in doing so, are resuspended in the gamete buffer (Figure 1Aiii). A sharp decrease in height and a gradual reduction in the channel height towards the centre of the device effectively limits the chance for sperm to exit the 200-µL collection zone. Passive sperm selection may also minimize iatrogenic DNA damage by avoiding any centrifugal forces, and selects sperm based on previously reported boundary-following behaviour which correlates with reduced DNA fragmentation (Denissenko et al., 2012; Eamer et al., 2016).
The present research group has tested a similar device against swim-up sperm selection on a smaller cohort of donors, which harnessed MACS ART Annexin V beads (Miltenyi Biotec) and opposing neodymium magnetic plates (AMF Magnetics, Australia) to negatively select apoptotic sperm (Vasilescu et al., 2023). This previous device was more complex in operation, required multiple reagents, was fabricated from a different material (three-dimensional-printed photopolymer resin), and was designed with different internal geometry. The current device uses a simpler, more accessible approach aimed at routine use. To test this microfluidic device, donor samples were used to compare DGC against the microfluidic method of sperm selection, and the latter allowed selection of sperm with higher DNA integrity and progressively motile sperm from this cohort. While conventional semen processing with DGC did result in an average improvement in motility and DFI values compared with unprocessed semen, it did so with a higher level of variability in sperm quality. Specifically, three of 21 samples processed via DGC showed an increase in DFI values, and many samples only showed an incremental reduction (<10%) (Supplementary Figure 1). Conversely, all samples processed with the microfluidic device showed a significant improvement, irrespective of the starting DFI value and motility. The average DFI value of sperm isolated using the microfluidic device was <1%, demonstrating that this method of sperm selection, when applied to motile sperm populations, is effective regardless of the starting DFI value. Similarly, the motility of sperm isolated using the microfluidic device was consistently higher compared with the motility of sperm isolated using DGC (Figure 2C). These results were consistent with previous studies indicating a high level of variance in recovered sperm motility when using DGC (Malvezzi et al., 2014; Muratori et al., 2016).
When performing a study on 40 consenting patients undergoing diagnostic andrology, similar trends were observed. Despite being a more clinically diverse cohort, notable improvements in DFI values were observed in 30 of 33 samples of sperm isolated using the microfluidic device compared with DGC (two samples had identical DFI values for both groups). This consistency highlights the usability and standardization achievable with a biomimetic device. Additionally, although overall improvements in DFI values were observed in sperm isolated using DGC (44.4% average), three samples had increased DFI values, possibly due to the iatrogenic damage caused by centrifugation on particularly susceptible samples, but this would require further investigation (Supplementary Figure 2). This was not observed when using the biomimetic device, which showed an average improvement in DFI values of 82.9%, with only one sample showing a reduction in DFI value <60%. Another noteworthy observation made in both studies was that, although DFI values were reduced in most samples isolated using DGC, the average reductions in DFI values of 57.4% and 44.4% for the proof-of-concept study and diagnostic andrology study, respectively, were inefficient compared with the average reductions observed in the samples isolated using the biomimetic device (92.2% and 82.9%, respectively). Compared with DGC, use of the microfluidic device increases the chance of selecting sperm with high DFI values, and this creates a population of sperm for fertilization which has lower DNA damage and may provide clinical benefit within IVF workflows by reducing the incidence of miscarriage and failed implantation, as suggested in the literature (Borges Jr et al., 2019; Duran et al., 2002; Robinson et al., 2012; Sedó et al., 2017).
The microfluidic device performed consistently between sites for all three key parameters measured (Supplementary Figures 3A–C). Although differences were observed in progressive motility and concentration between the DGC groups, these differences can be attributed to multiple factors, including operator experience between research scientists at the university research laboratory for the proof-of-concept study versus clinical embryologists in the diagnostic andrology study. Although there were differences in the density gradients used at the two sites, both gradients were a 40% and 80% gradient solution combination, and were silane-coated silica-based.
The average DFI values in sperm isolated using DGC vary in the literature, and depend largely on sample populations. Some studies have indicated an increase in total DNA fragmentation (Muratori et al., 2019), and others have suggested an average improvement in DFI values, with a subpopulation of samples experiencing an increase in DFI values or no improvement in DFI values, which is consistent with the results of this study (Rappa et al., 2016; Stevanato et al., 2008). DNA fragmentation in sperm is commonly attributed to oxidative stress, plausibly induced by repetitive centrifugation used in conventional sperm selection methods (Henkel et al., 2005; Hernández-Silva et al., 2021; Nabi et al., 2014). High DNA fragmentation is associated with pregnancy loss in conventional IVF and ICSI, as well as lower implantation rates and a reduction in average embryo quality (Benagiano et al., 2017; Coughlan et al., 2015; Larson-Cook et al., 2003; Robinson et al., 2012). What is perhaps more concerning is that sperm DNA fragmentation has no obvious effect on fertilization, but becomes apparent during blastocyst development by reducing the generation of good-quality blastocysts and ability to achieve successful implantation (Newman et al., 2022; Seli et al., 2004). As a result, the risk of using compromised sperm remains present in clinical practice, and highlights the need for the selection of sperm with high DNA integrity. Importantly, this is of relevance when considering that advanced reproductive age has an increased negative effect on sperm DNA damage (Horta et al., 2020). Furthermore, male ageing has been linked to a significant increase in miscarriage rate, and a decrease in live birth rate, with a larger impact in women of advanced reproductive age (Horta et al., 2019).
A clear limitation of the output of the current microfluidic device, and arguably of microfluidic motility-based sperm selection in general, is the smaller number of sperm isolated when using the microfluidic device when compared with DGC. In conventional IVF, 50,000 sperm are typically required per oocyte, and an average of 10–12 oocytes are harvested per stimulated cycle (Oseguera-López et al., 2019). However, it has been shown that high fertilization and cleavage rates are possible with as few as 2000–4000 sperm per oocyte (Fiorentino et al., 1994). The average number of sperm isolated using the microfluidic device was approximately 720,000, which may be too few for many conventional IVF cases if clinics were to adhere to the requirement of approximately 50,000 motile sperm per oocyte (Li et al., 2018). Logically speaking, sperm selected using passive biomimetic selection, such as the device in this study, may have higher fertilization efficiency than those selected using active measures such as centrifugation; therefore, fewer sperm may be required for conventional IVF, similar to that of in-vivo fertilization whereby only approximately 200 sperm fertilize the oocyte (Chaffey, 2003). Nevertheless, in future prototypes of this biomimetic device, improvement in the yield of motile sperm for high-responder women for whom many oocytes are collected will improve the potential for clinical adoption, as the yield and high selectivity of this device is better suited for ICSI cases which require fewer sperm for insemination, and ICSI is often prescribed for cases where the male partner has a high DFI value. The form factor of the device also enables the adherance of an 18 mm x 36 mm automatic witnessing tag and patient label for seamless clinical integration.
Semen processing using DGC requires several manual interventions during sample handling, each with the potential for human error. Passive devices such as that used in this study, as well as ZyMōt (ZyMōt Fertility, USA) and Lenshooke CA0 (Hamilton Thorne, USA), limit human interaction in semen to sample injection and sperm collection, usually without centrifugation (Shirota et al., 2016). The microfluidic device used in this study, with a simple three-step operation, will reduce the clinical workload while offering a greater reduction in DFI value after processing. While many studies have investigated the impacts of commercialized microfluidic devices, and these have been reviewed systematically (Ferreira Aderaldo et al., 2023), the present biomimetic device takes a different approach to sperm selection by leveraging the boundary-following behaviour of sperm to perform selection, and requiring sperm to travel several millimetres to a collection zone. Reductions in DNA fragmentation and the simplicity of this device are comparable to existing commercial devices such as ZyMōt and LensHooke (Parrella et al., 2019; Hsu et al., 2023), both of which exploit sperm motility via membrane filtration. In a recent study comparing DGC, ZyMōt and LensHooke CA0, Hsu et al. (2023) demonstrated progressively motile sperm counts of 80.6%, 85.6% and 90.8%, respectively, and DFI values of 11.8%, 3.7% and 2.4%, respectively, in normospermic samples (Hsu et al., 2023). Further comparative studies are now required to determine whether the use of a biomimetic device that leverages boundary-following behaviour in sperm will lead to improved outcomes.
This study has several limitations which can be addressed in larger follow-up studies. Firstly, limited access to samples with high DFI values (>25%) prevented a robust testing approach on extreme cases, which are perhaps those which would benefit the most from a reduction in DFI value. Secondly, to prove the clinical usefulness of this approach, clinical outcomes are required when assessing the device on a range of patients, whereby the effect of each sperm selection method on fertilization and embryo development is evaluated thoroughly. Ideally, a randomized controlled trial or sister oocyte study (whereby half the oocytes are inseminated with sperm isolated using conventional methods, and half the oocytes are inseminated with sperm isolated using the microfluidic device) will better display the clinical utility of this microfluidic device in IVF workflows. The prototype in its current format does show utility for ICSI cases, for which lower numbers of high-quality sperm are sufficient. This format suits a side-by-side study for an ICSI cohort, but may not suit an IVF insemination side-by-side comparison with DGC. Thirdly, the method of DNA fragmentation assessment, SCD, only identifies single-stranded DNA breaks, and has limitations in the subjective nature of the assessment. Future studies and validation are needed using SCSA for a more robust assessment of DFI values by detecting double-stranded DNA breaks. SCD is also susceptible to human error and sperm concentration restraints during the preparation and staining of samples, as shown by seven of 40 patients with inadequate staining for SCD assessment in this study. Finally, the current biomimetic prototype does limit the output of sperm by only processing 1 ml of semen, whereas conventional methods process the entire ejaculate. The purpose of this was to enable side-by-side testing against DGC; however, further studies are currently evaluating a larger volume platform capable of processing an entire semen sample to maximize the sperm yield for use in IVF and intrauterine insemination.
This study reports a novel, highly selective biomimetic method for sperm selection in a simple-to-use, single-use chip format for isolating highly motile sperm with minimal DNA fragmentation, without the need for centrifugation or other active mechanisms. Considering the limitations of this study, this proof-of-concept test shows that highly selective, lower output sperm isolation, such as channel-based microfluidic selection in its current form, may prove to be a practical alternative for ICSI cycles if higher motile concentrations for larger oocyte numbers are preferred for conventional IVF. As many patients with high DFI values tend to be prescribed ICSI as a method of fertilization, this device does have the potential to address these cases, considering its ability to reduce DFI values consistently compared with DGC. The novel selectiveness of mimicking the female reproductive system provides a high-quality population of sperm for use in treatments. Clinical studies have now been initiated to validate the proposed benefits of this selection mechanism.
