Cooperative roles of biological flow and surface topography in guiding sperm migration revealed by a microfluidic model

Abstract

Successful reproduction in mammals requires sperm to swim against a fluid flow and through the long and complex female reproductive tract before reaching the egg in the oviduct. Millions of them do not make it. Despite their clinical importance, the roles played in sperm migration by the diverse biophysical and biochemical microenvironments within the reproductive tract are largely unknown. In this article, we present the development of a double layer microfluidic device that recreates two important biophysical environments within the female reproductive tract: fluid flow and surface topography. The unique feature of the device is that it enables one to study the cooperative roles of fluid flow and surface topography in guiding sperm migration. Using bull sperm as a model system, we found that microfluidic grooves embedded on a channel surface facilitate sperm migration against fluid flow. These findings suggest ways to design in vitro fertilization devices to treat infertility and to develop non-invasive contraceptives that use a microarchitectural design to entrap sperm.

Fig. 1. A double layer microfluidic device for modelling the biophysical environment of sperm in the female reproductive tract

A. Image of a PDMS device bonded onto a PDMS coated glass slide (1″ × 3″), with a port on the left for sperm seeding, and a port on the right for flow input. B. A close up image of the 6 sets of channels in the centre, four with grooves (noted with G) and two without (noted with F). C. A close up image of a G channel showing a set of nine 20 μm wide and 20 μm deep grooves. The G channel is 300 μm wide and 120 μm in height. D. A 3D drawing illustrating the cross section of the G channel. Drawing not to scale. E. A micrograph of a frozen tissue section of the bovine cervix, stained with PAS/hematoxylin. Micrometer size grooves are seen along the main channel wall. The white arrows indicate one of the microgrooves. Detailed methods in Suarez et al.

Fig. 2. Experimental and numerical flow characterization in the microfluidic model

A. Computed flow speed profile within a microfluidic channel (300 μm × 120 μm) with 20 μm × 20 μm micro-fabricated grooves on the upper surface. Note that the flow speeds in the micro-fabricated grooves are greatly reduced. B. Flow speed profile within one groove as indicated in the white box in A. C. Measured flow speed at the centre of the main channel (x in A) using fluorescent particle tracking method is validated against the calculated speed of the same location at various pumping flow rates. D. Calculated flow speed at the centre of the grooves (e. g. red x in B) at various pumping flow rates. E. Simulation of the shear stress distribution in the microfluidic channel with micro-fabricated grooves. F. Shear rate at the bottom and along the mid line (as indicated by the red x shown in E) at various pumping rates.

Fig. 3. Surface topographies guide sperm migration. A-C

Trajectories of 50 sperm swimming near a flat surface (A), toward a sidewall (B), and within 20 μm × 20 μm grooves (C). Each coloured line is a trajectory of 1.78 s long in A and C, and 1.24 s long in B. The black dots mark both the starting and end points of each trajectory. The coordinate (0,0) marks the starting point of all the tracks in (A,C), and marks the point where the cell hits the side wall in (B). The cells continue move along the sidewall (or along the horizontal axis) once they hit the sidewall. The inset is a graph of the incident (θ_in) and outgoing angles (θ_out) of the trajectories shown in B. D. Directional persistence is significantly higher for cells travelling within the micro-fabricated grooves than on a flat surface. E. Sperm travelling in the grooves are slower than those on a flat surface. Statistical significances are determined by one-way ANOVA with Bonferroni post test. ***: p-value < 0.005, ****: p-value < 0.001. Points found outside the mean ± 3 SD were considered outliers and are not shown (outliers were no more than one point in each data set). F. Speed distribution of sperm swimming on a flat surface and in grooves.

Fig. 4. Sperm swim against the flow

A–B. Trajectories (N=50) of sperm swimming along a flat surface with no flow (A), and a 3 μL min⁻¹ flow (B) within the microfluidic channel. C. Persistence along x-axis direction (opposite to the flow direction) was enhanced in the presence of the high flow (3 and 5 μL min⁻¹). *: p-value < 0.05, ****: p-value < 0.001. D. Sperm speed was reduced in the presence of the high flow (3 and 5 μL min⁻¹).

Fig. 5. Micro-fabricated grooves facilitate sperm migration against the flow

A. Time lapse images of a bull sperm swimming in a 20 μm × 20 μm groove (A1) and on the bottom surface of the microfluidic channel (A2) with a flow rate of 3 μL min⁻¹. In A2, the cell eventually leaves the surface (becomes out of focus) and is swept downstream. Red circles are used to mark the positions of the sperm head. B. Percentage of cells that were swept away by the flow for sperm swimming on flat surfaces or within grooves. C. Trajectories of 50 sperm swimming in 20 μm × 20 μm grooves in the absence (C1) and presence (C2) of the flow. The starting point of each trajectory along the x-axis is placed randomly along the y-axis for the ease of visualization of the track direction within the groove. Sperm swim toward both directions nearly equally with no flow, and with a 3 μL min⁻¹ flow, sperm primarily swim against the flow. D. Flow influences on sperm swimming speed. Instantaneous sperm speed distribution sampled at 8.17 Hz on flat surface (D1) and within grooves (D2–3) at various pumping rates. The dots are experimental data computed from 50 tracks and each track is 1.78 s long and the solid lines are fits to Gaussian functions. D4 shows the flow influences on average speed of sperm when swimming on flat surfaces and within micro-fabricated grooves.