Significant improvement of sperm motility parameters through surface modification with Nano Diamond particles

The employment of Nanotechnology as a valuable tool could be beneficial to patients and also offer new alternatives for Assisted Reproductive Technology (ART). Surface modification by nanoparticles leads to the alteration of surface characteristics (e.g. roughness, elasticity, and surface charge). These alterations affect sperm behavior especially its motility since sperms exhibit wall following behavior and surface accumulation. Moreover, surface modification is an attempt towards mimicking the complex in vivo environment of the female tract (highly folded and ciliated) with continually changing surface topology and also its functions. In this paper, we present the results of investigating the interactions between sperm cells and surface modified substrates using Nano diamond particles. A combinational and low-cost method was used for modification. The results show that the sperm motility parameters are significantly improved from 5 to 85%. Also, the results indicate that in optimized surface modification condition, the sperms swim faster and straighter and the surface facilitates the swimming due to the inherent characteristics of ND particles. Taken together, the replacement of normal with modified surfaces in fertility-aid microfluidic devices can enhance their efficiency and further improve their outcomes.


Introduction
Motility of the sperms plays a key role in traversing the long path of the female tract and subsequently in their success of fertilization. The effects of different parameters on motility such as the flow [1][2][3], chemical environment [4][5][6], and temperature [7][8][9] have been studied so far. Among all, confinement and contact surface are known to be effective on sperm motility but their details remain unexplored.
When it comes to the surface, the wall following behavior and surface accumulation of sperms are highlighted. While near the wall, the hydrodynamic flow field loses its symmetry and results in surface accumulation [10]. Moreover, the interaction between the flagellar wave of human sperm and the surface, guides the swimming trajectory towards it being dependent on sperm distance from the surface and its orientation [11,12]. The surface can also influence the group behavior of the sperms leading to the synchronized tail beatings and increasing the swimming velocity [13].
Apart from hydrodynamic parameters that affect surface accumulation, geometrical constraints of the surface play the same role [14]. In this essence, the existence of nonplanar surfaces and surface roughness significantly changes sperm motion since the majority of sperms swim near the wall. This following behavior is observed at curvatures more than 150 micrometers otherwise rather sharp curvatures result in the repulsion of the sperms from the surface [15]. Many studies had focused on sperm motion in contact with flat surfaces, but in vivo geometries are more complex (highly folded and ciliated and continually changing) and relevant swimming behavior needs to be further addressed.
This calls for bio-inspired micro/nanotechnologies to enter the arena and fully unveil the effects of geometrical complexity on sperm motion especially through surface modification.
Surface modification leads to the alteration of surface characteristics such as roughness [16,17], mechanical properties [18,19], hydrophilicity [20,21], biocompatibility [22][23][24], and surface charge [25]. It can be achieved through different physical and chemical methods making the surface exhibit enhanced and even new characteristics with or without the use of external micro/nanoparticles. Expensive and substrate-specific methods exist in the former category as chemical vapor deposition (CVD) which limits the extensive usage of particles and their possible benefits. While, combinational modification methods could be helpful for their relatively lower cost and modification capability of various substrates. Finally, the combination of surface modification and microscopy provides an opportunity for understanding the interaction between modified surfaces and sperm motility.
Here, we used the combination of chemical etching and spin coating methods to modify the surface of widely-used microscope glass substrates with Nanodiamond (ND) particles for their unique biocompatibility and inherent potentials. The objective of the present study is to investigate the interaction of sperm cells with ND modified surfaces under the geometrical change in surface topology along with the ND coating itself.

Preparation of ND suspension
Commercially available Nanodiamond powder (Shenzhen Jingangyuan New Material Development Co., Ltd., China) with the purity of 95% percent and average size of 50 nm was purchased. The ND particles were then added to acetone (1:10 ratio). The mixture was sonicated in 50 cycles for 20 minutes with an ultrasonic homogenizer sonotrode supplied by 90 W power (FAPAN, Iran). TEM was used to characterize the morphology and particle size of the dispersed particles in the solution.
Finally, three different concentrations of the suspension as 1, 0.6, and 0.4 g/l v/v were prepared.

Substrate etching
The etching process of the microscope glass substrate included 6 steps. The procedure started with the immersion of the surfaces in a 50% v/v solution of H2SO4 (95%) and H2O2 (30%) for 3 days. The surfaces were rinsed after 3 days with distilled water until the stabilization of the solution pH. The surfaces were immersed in a solution of HNO3 (5 mol/l) for another 3 days. The rinsing was repeated on the fourth day. The procedure continued by the immersion of substrates in NaOH solution (4 mol/l) for 3 minutes with simultaneous heating at 55℃ on a hot plate. The excess NaOH was then removed from the substrates with distilled water and they were again immersed in HNO3 solution (5 mol/l) for 3 days. Having rinsed the surfaces on day 4 with distilled water, they were transferred to a furnace and dried at 60℃ for 8 hours. The procedure was adopted from [26].

Spin coating
The etched microscope glass substrates were first divided into 2. volume on the optimized surface was also investigated (50 and 100 µl). To ensure the repeatability of the results, the spin coating in each case was done on three different substrates.

Furnace heating
The marked spin coated substrates were then transferred to an electric furnace (Fan Azma Gostar, Iran) and heated for 3 hours at 100℃ and packed for testing.

2.2.Sperm sample preparation
Three discarded fresh routine semen analysis samples were consecutively collected from the Avicenna Infertility Clinic (Tehran, Iran). Collected semen specimens were liquefied and prepared by standard swim-up technique. In brief, 1 ml of whole semen was gently mixed with 1 ml of Ham's F10 medium (Sigma, USA), supplemented with human serum albumin (3%) (Sigma, USA) and centrifuged (330×g for 10 min). The supernatant was removed and 2 ml of "Ham's F-10" medium was added to the pellet. In this study, 3 µl of washed semen remnant was used for substrate testing.

2.3.Sperm Imaging
The witness and spin coated substrates were incubated for 10 min at 37℃ prior to imaging. A 3 l  droplet of the same sperm sample was applied to the center of the glass substrates followed by adding a coverslip. An optical microscope (Olympus CX21, China) was used for imaging with magnification of 40x. A 2MP Microscope Imager Digital USB Camera (Eyescope, NXM-EPA200) was mounted on the microscope eye and used to capture image sequences with 16 Hz frame rate. The imaging of each substrate was performed for 1 min.

2.4.Sperm motion analysis
Quantitative motion analysis was performed using freely available ImageJ software. Manual tracking was utilized to calculate sperm position in the XY plane. Thereafter, a custom-written Matlab code [27] was used to derive motility parameters according to computer-aided sperm analysis (CASA) standards along with swimming trajectories. The motility parameters included Curvilinear velocity (VCL), Average path velocity (VAP), Straight line velocity (VSL), Linearity (LIN), Mean curvature (MCR), and The amplitude of lateral head displacement (ALH) according to [27]. 12 sperms were tracked and followed for at least 40 frames in each case. The values were averaged over each trajectory and the trajectory-wise mean over the number of trajectories was considered as the final value.

2.5.Atomic Force Microscopy (AFM)
AFM technique was used to visualize and compare the topography of witness and spin coated surfaces. The AFM z (x, y) maps were used to estimate some texture parameters allowing for surface roughness characterization, respectively and were defined as:

2.7.Contact Angle Measurement
Contact angle measurements (sessile drop technique) was also carried out on the witness and the best modified surface with bi-distilled water. The static sessile drop contact angle between the surface and the tangent line starting from the three-phase point was measured using a contact angle goniometer using an optical subsystem to capture the profile of a pure liquid on a solid surface (Jikan CAG-10, Iran). Furthermore, the contact angle magnitude was measured using the complemented software designed for drop shape analysis.

Results and Discussion
The ND particles were successfully spin coated and stabilized on the glass substrates. Fig. 2a shows the TEM image that characterizes the dispersed nanoparticles in terms of size showing a cluster of the particles with approximate size of 50 nm. Fig. 2b shows the XRD image taken from one of the modified surfaces spin coated with 1 g/l concentration of ND particles. Diamond peaks were detectable in the modified spectra (green line) with different 2 positions matching the first peak of the base spectra (blue line) at 44.12° corresponding (111) lattice plane which proves the existence of the ND particles on the surface. However, the peak intensity is not high (one order of magnitude lower than pure diamond) due to the low concentration of ND particles in the prepared suspension. Modified XRD spectra (green line) for the spin coated substrate with 1 g/l concentration of ND particles in the prepared suspension.
Matching of the first diamond peak at 44.12° corresponding (111) lattice with the base spectra (blue line) plane proves the existence of the ND particles on the surface.  Linearity (LIN) was higher in most substrates by 9% in average. Among the substrates, only the 2000-c1 case that acquired the highest rank in velocity enhancement, did not show significant increase being almost equal to the witness (Fig 3(d)). The increase in linearity implies that sperm follow straighter trajectories on the modified surfaces. The amplitude of lateral head displacement (ALH) increased significantly, from 1.33 µm for the witness to 2.71 µm for the 2000-c1 case (P<0.001 with a t-test) and 19% in average (Fig 3(e)). Regarding the MCR of the instantaneous sperm trajectory, it was decreased considerably for all the modified surfaces ranging from 23% to 71% as compared with the witness (Fig. 3(f)), indicating an increase in symmetry of the flagellar wave. This significant reduction in oscillations may contribute to the increased velocity and LIN experienced on NDmodified surfaces.

3.1.Sperm motion analysis on ND-coated surfaces
Results indicate that the sperm motility is generally enhanced on ND-modified substrates with 1 g/l and 2000 rpm case exhibiting the highest enhancements as compared to other cases. This can be explained with the higher concentration of ND particles used for spin coating. Moreover, compared to 3000 rpm, it shows that the lower rotational speed improves the distribution of ND particles on the surface rather than splashing of the applied droplet.
To further investigate the effect of droplet volume, six substrates were modified based on 2000-c1 case, three of which spin coated with 50µl droplet volume and the remaining with 100µl. Table 1 summarizes the key motility parameters for human sperm swimming on both substrates. Comparing the motility parameters shows that as the droplet volume increases, the better distribution of ND particles occurs on the surface leading to significant enhancement in VSL, VCL, and VAP. It also shows 5% improvement in LIN, 6% increase in BCF and the reduction of mean curvature by 60%. However, the WOB and ALH were almost constant for both substrates being insensitive to droplet volume.
Taken together, these observations indicate that sperm swim faster with improved linear progressive motility on ND-modified surface as compared with witness. When considering assisted reproduction, progressive motility of sperm is essential to ensure the arrival to the fertilization site and thus fertilize the ovum. The faster swimming speed in 2000-c1 case indicates that ND particles encourage hyperactivated swimming which negatively affects progressive movement. In addition, the ability of sperms to swim straighter also reduces sperm exhaustion nominating them for motility-based sperm selection [28].

Conclusion
The female reproductive tract is known to facilitate migration of sperms and micro/nanotechnologies offer the opportunity of mimicking the function of reproductive tract in vitro. Here we demonstrated that ND modified glass substrates improve the sperm motility from 5 to 85%. The results indicated that sperms swim straighter and with enhanced speed on the modified surfaces which in turn prevents sperm exhaustion. This behavior can root in rather ROS-free, friction-decreasing or less hydrophilic ND-modified surfaces.