Device Geometry and Operation
Figure 1 illustrates the workflow of our device whereby sperm can migrate from a semen reservoir into microchannels preloaded with sperm buffer. Each microchannel contains layers of grooves and ridges to facilitate double the number of avenues for sperm to follow corners when compared to conventional straight channels. The channels themselves also provide a larger entry space compared to previous iterations of the device so more sperm find their way into the channels for selection. The geometry of the channels was optimized first by device stability and then by motile sperm yield. The width of the grooves and the height of each channel was incrementally increased to a point where the device consistently yielded over 60% more sperm than the original geometry without allowing semen to penetrate the channels during the semen injection step. Furthermore, we also present a second iteration of this microfluidic platform where sperm, having been pre-mixed with AAV coated magnetic nanoparticles, are sorted based on motility before being introduced to a magnetic field amplified by paramagnetic microparticles, effectively trapping phosphatidylserine positive sperm. Lastly, sperm are collected from the center outlet.
Sperm DFI and Conventional Quality Metrics
Sperm DFI, concentration, vitality, and motility values were compared before and after SU and microfluidic sperm selection methods. Samples greater than 2.1 mL were split, without dilution, between SU and microfluidic selection methods, while samples between 1.1 and 2.1 mL were split to give 0.1 mL of neat semen for raw sample analysis, and the remaining volume split 50/50 by volume and diluted up to 1 mL before selection. Samples less than 1.1 mL were not included in the study. Among the 33 volunteers who participated, 25 had semen volumes equal to or greater than 2.1 mL, with 5 of the donated classified as infertile according to WHO 5th Edition criteria. The average DFI value of all raw samples was 12.19% (± 5.59) and ranged from 3.0–25.12% (Fig. 2). The results show that DFI values of semen samples prepared through the microfluidic method (1.44% ± 1.4) were significantly lower than those prepared using the SU method (7.92% ± 4.72, P = 0.0176). This represents an average DFI reduction of 88.2% and 35.0% for MSSP and SU selected sperm, respectively.
For each experiment, 150 µL of isolated sperm were recovered from the microfluidic device. The microfluidic method represents a significant time improvement from conventional SU-based techniques. Sperm concentrations from the device increased with incubation time and ranged from an average of 0.61 million sperm/mL after 5 minutes of semen incubation to 1.54, 5.46, and 7.4 million sperm/mL after 10, 15, and 20 minutes, respectively. For 15 minutes of microfluidic device incubation, the average number of sperm collected from the device was 825,000 sperm, sufficient for droplet-based IVF and more than enough to select an individual sperm for intracytoplasmic sperm injection (ICSI). In addition, the vitality of sperm from the devices was assessed to confirm no adverse effects on the viability of cells was present from the materials being used in the device. Sperm vitality remained above 97% for all incubation times and was consistently greater than the raw semen (48.1% ±15.6) and SU (86% ±11.6) method.
Sperm motility was also assessed for each method and compared to the raw semen. This was done across four different incubation times for the device (5, 10, 15, and 20 minutes). The average progressive motility from the microfluidic device (93.5% at 15 minutes) saw a statistically significant increase when compared to raw semen (37%, P = 0.001) and SU method (74.1%, P = 0.0181) (Fig. 3A). Considering the high yield, vitality, and motility of sperm from the microfluidic device at 15 minutes, 15 minutes was chosen as the ideal time for DFI-based experiments, cryopreservation experiments, and apoptotic selection-based experiments. The OpenCASA plugin in ImageJ was used to quantify sperm motility parameters. The VSL, VCL, and VAP for sperm separated via microfluidics at 36ºC (44.2 ± 10.4, 73.3 ± 6.7, and 54.2 ± 15.2 µm/s) each showed a considerable average increase from the raw semen (28.17 ± 3.78, 59.47 ± 3.68, and 41.48 ± 4.76 µm/s) and from the SU method (30.2 ± 2.5, 73.8 ± 7.1, and 46.2 ± 3.6 µm/s) (Fig. 3B). Both room temperature (22°C) and body temperature were compared for microfluidic sperm selection (at 15 min incubation) to compare the differences in sperm motility behavior upon collection, which may affect their level of hyperactivity, capacitation, and suitability for conventional IVF. The room temperature microfluidic sperm separation showed a similar increase in VSL (as the 36°C incubated device) but was comparable to the SU method in terms of VCL and had a higher range of values in VAP. Similarly, the LIN and WOB showed average increases with microfluidic sperm selection when compared to the SU method and the neat semen (Fig. 3C). However, ALH and BCF were largely unchanged between all groups. Interestingly, the SU method demonstrated a loss in LIN, WOB, and STR from the neat semen while the RT microfluidic separation gave the largest increases exceeding that of body temperature incubated microfluidic separation.
Motile Sperm Recovery Following Sperm Cryopreservation
To investigate the effect of sorting on motile sperm recovery following sperm cryopreservation and the influence of different separation methods on sperm recovery, sperm cryopreservation was performed on raw semen and both SU and MSSP sorted sperm. Sperm selections were performed side-by-side and subsequently cryopreserved for 7 days before thawing alongside an aliquot of raw semen. As shown in Fig. 4A, sperm processed through microfluidics (on average) showed a significant 75% improvement in motile sperm recovery when compared to the raw conventionally from sperm (36.7–64.2%). SU processed sperm showed a non-significant average improvement of 43.9% (36.7–52.8%). The recovery of live sperm post-thawing on the other hand, showed no significant difference between each group with a higher averaged value for MSSP (Fig. 4B). Sperm frozen from a raw semen dilution had an average recovery of 66.5% while the SU and MSSP processed sperm had an average recovery of 49.4% and 67.0%, respectively. OpenCASA revealed differences in the post-thaw velocity parameter of sperm from different groups (Supplementary Fig. 3A and 3B). SU processed sperm showed the largest and most consistent decrease in velocity across VSL, VCL, and VAP, while unprocessed and MSSP sperm showed very minor changes in velocity in all except VAP where raw sperm showed an increase.
Characterization of Apoptotic Sperm and Negative Selection of Apoptotic Sperm from the Hybrid MSSP (H-MSSP).
To investigate the incidence rate of sperm apoptosis caused by each selection method and to reduce the number of apoptotic sperm at collection, sperm apoptosis was measured for raw, SU, motility only and hybrid MSSPs. For the removal of apoptotic sperm from collected sperm samples a magnetic sperm selection approach was used to trap pre-labelled apoptotic sperm using a magnetic field amplified by super-paramagnetic microbeads (Fig. 5A). Raw, SU, MSSP only, and IVFM with magnetic separation were compared between 5 samples of larger volume (> 2.5 mL). The percentage of apoptosis was assessed via flow cytometry using PI and AAV-FITC double staining. The percentage of apoptotic sperm (Fig. 5B) showed a large average increase in SU sorted sperm when compared to the raw sperm (8.5–26.5%). While motile sperm selection via the MSSP only group showed no average reduction in apoptosis, the hybrid MSSP showed a near 50% reduction in apoptotic sperm versus the MSSP only sperm. Necrotic or late apoptotic sperm were reduced in all sperm selection methods (Table 1). Figure 5C shows example distribution of the relative decrease in alive (AAV-/PI-), dead (AAV-/PI+), necrotic (AAV+/PI+) and apoptotic (AAV+/PI-) sperm populations between each method from the raw sample (with between 1% and 25% total apoptotic sperm).
Table 1
Sperm assessments before and after SU and Microfluidic Sperm Selection at 15 minutes
Sperm Metric
|
Raw Semen
|
Swim Up
|
MSSP
|
Hybrid MSSP
|
Concentration (X10^6)
|
87.7 (± 39.2)
|
29.4 (6.6)
|
5.5 (0.15)
|
7.83 (0.23)
|
Total Motility (%)
|
45.4 (± 14.3)
|
82.2 (± 10.2)
|
95.5 (± 3.1)
|
97.6 (± 2.5)
|
Progressive Motility (X10^6)
|
42.2 (± 15.0)
|
74.1 (± 9.6)
|
93.5 (± 4.5)
|
97.0 (± 2.9)
|
Sperm Vitality (%)
|
48.1 (± 15.6)
|
86.0 (± 11.6)
|
97 (± 2.1)
|
98.5 (± 1.1)
|
DNA fragmentation (%)
|
12.2 (± 5.6)
|
7.9 (± 4.7)
|
1.4 (± 1.4)
|
NA
|
Apoptotic Sperm (%)
|
10.8 (± 9.9)
|
26.5 (± 2.7)
|
10.8 (± 2.8)
|
5.66 (± 3.0)
|
Necrotic/Late Apoptotic Sperm
|
23.6 (± 14.2)
|
13.31(± 1.5)
|
9.94 (± 3.5)
|
5.85 (± 3.2)
|
Cryosurvival
(% motile)
|
36.7 (± 6.1)
|
52.8 (± 14.3)
|
64.2 (± 10.5)
|
NA
|
Cryosurvival
(Vitality %)
|
66.5 (± 9.0)
|
49.4 (± 7.8)
|
67.0 (± 8.4)
|
NA
|