Analysis of sperm concentration and motility in a microfluidic device

Transcription

1 Microfluid Nanofluid (2011) 10:59 67 DOI /s RESEARCH PAPER Analysis of sperm concentration and motility in a microfluidic device Yu-An Chen Zi-Wei Huang Fang-Sheng Tsai Chang-Yu Chen Cheng-Ming Lin Andrew M. Wo Received: 24 February 2010 / Accepted: 19 May 2010 / Published online: 10 June 2010 Ó Springer-Verlag 2010 Abstract A home-use device that allows rapid and quantitative sperm quality analysis is desirable but not yet fully realized. To aid this effort, this article presents a microfluidic device capable of quantifying sperm quality in terms of two critical fertility-related parameters motile sperm concentration and motility. The microdevice produces flow field for sperms to swim against, and sperms that overcame the flow within a specified time are propelled along in a separate channel and counted via resistive pulse technique. Data are compared to two control methods clinically utilized for sperm quality exam hemocytometer and the sperm quality analyzer. Results reveal the numbers of pulses generated by passage of sperms correlates strongly with the two control methods: pulse number from 0 to 335 corresponds to progressively motile sperm concentrations from 0 to / ml (hemocytometer) and Sperm Motility Index from 0 to 204 (sperm quality analyzer). The microdevice should be applicable to facilitate self-assessment of sperm quality at home. Keywords Microfluidics Sperm Infertility Resistive pulse technique 1 Introduction Microfluidics serves as a unique tool to investigate, manipulate, and test biological materials from cells to Y.-A. Chen Z.-W. Huang C.-Y. Chen C.-M. Lin A. M. Wo (&) Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan, ROC andrew@iam.ntu.edu.tw F.-S. Tsai Department of Urology, National Taiwan University Hospital, Taipei, Taiwan, ROC biomolecules (Sia and Whitesides 2003). Samples tested on microfluidic platform are required fewer and processed quicker than that in larger scale (Beebe et al. 2002; Whitesides et al. 2001). To focus on cells, microfluidics provides a microenvironment to deal with a number of critical issues, for example, cancer diagnosis (Lee et al. 2009), drug testing (Toh et al. 2009), cell manipulation (Lin et al. 2008; Manaresi et al. 2003), and electrophysiology (Chen et al. 2009; Ionescu-Zanetti et al. 2005; Li et al. 2006). In the last decade, human reproduction issues have been studied via microfluidic devices. Two types of cells sperms and eggs are the most important biological materials in reproduction. Sperm cells which are much different from other human cells feature rapid motion due to a flagellum. Egg cells, a kind of rare cell, mature only one egg in about a month. Many microfluidic devices have been proposed to deal with these two special kinds of cells, e.g., in vitro fertilization/embryo production (IVF/IVP) on chip (Koyama et al. 2006; Lopez-Garcia et al. 2008; Smith and Takayama 2007; Suh et al. 2005, 2006; Wheeler et al. 2007), motile sperm sorting (Cho et al. 2003; Horsman et al. 2005; Schuster et al. 2003; Seo et al. 2007; Shao et al. 2007; Wu et al. 2006), and sperm quality analysis (Kricka et al. 1993, 1997; McCormack et al. 2006). To deal with infertility due to male factor, knowledge of sperm quality is extremely important; yet, the means to acquire such could be improved upon. Currently, a male with such issue needs to provide fresh sample via out-patient clinic, suffering much embarrassment and anxiety in the process. Thus, products have been developed to allow sperm analysis in the comfort of the home, either in microscopic observation manner, such as Micra, or staining methods, e.g., FertilMARQ and Fertell. By observation of sperm quality under microscope, count, motility, and morphology

2 60 Microfluid Nanofluid (2011) 10:59 67 of sperm can be evaluated. However, training for users before using microscope to exam sperm quality is required. By staining methods, such as FertilMARQ, the count of sperms can be characterized but motility cannot be evaluated. Although the design of Fertell simultaneously exam sperm count and motility, the results only judge whether sperm quality is over or below a given criteria (concentration of progressively motile sperm greater or less than / ml as suggested by the World Health Organization). For a male in the process of treating for infertility, a quantitative readout to monitor long-term variation of sperm quality at home is important but is still difficult to realize fully. There are several nice pieces of research published. Kricka et al. (1993, 1997) showed a design of microchannels for evaluation of sperm motility in which sperm motility analysis conducted in a microenvironment took advantage of the confined sperm motions by microchannel for easier observation than traditional hemocytometer. Although motility of sperms can be measured easily by measuring the time taken for sperms to transverse a microchannel quantification of sperm concentration appears to be lacking. McCormack et al. (2006) developed a fluorescent labeling method for simultaneously analyzing motile sperm concentration and motility by monitoring fluorescent intensity of a region where motile sperms were collected. After a certain time period, the intensity of fluorescence was utilized to quantify the motile sperm concentration. Furthermore, the motility of specimen also contributed to the increase in fluorescent signal. Hence, both motile sperm concentration and motility were taken into account by this design. However, fluorescent labeling might not be suitable for routine usage in hospitals or at home. Per discussion above, there is an overarching need for sperm quality test that can readily evaluate both concentration and motility information in the simplest possible manner, e.g., label-free and user friendly. This study aims to help meet this challenge via a microfluidic device. The device produces a flow field where label-free sperms from liquefied specimen swim upstream and leave impurities behind. The sperms overcoming the flow are flushed toward a region where resistive sensing is performed. This design may contribute to at home screening of male fertility and regular monitoring of sperm quality for infertile patients. 2 Experimental aspect 2.1 Conceptual design: male fertility screening in terms of sperm concentration and motility The approach proposed here evaluates fertility of human sperms in terms of motility and sperm concentration. Both quantities are critical since more sperms with good motility represent higher possibility of successful fertilization. Possibility of fertilization is not only dependent on sperm concentration or motility but also on both parameters. Hence, the method reported here attempts to exam fertility of sperms by taking these two parameters into account. More specifically, only semen with high sperm concentration and good motility will be judged as of high quality, whereas high concentration but poor motility or low concentration even with good motility might fall short in this method. The overall flow field is of prime importance in the device, as shown in Fig. 1a. The microchannel network is composed of three inter-connecting channels A, B, and C. Channel A directs a fast-flowing inlet stream into the device, then the flow divides into channel B and C at the junction. At start of test (t = 0), semen is loaded in reservoir B which connects to the end of channel B. Sperms enter channel B from the reservoir and travel through this channel if swimming fast enough to overcome the flow in channel B. The flow in channel B is set in a parabolic flow profile with the maximum flow velocity around 40 lm/s at the centerline. Sperms are challenged by this flow field in channel B and migrated upstream if motile enough. Since the swim velocity of sperms ranges from few to tens of microns per second, flow velocity in channel B was set within this range. The flow field in channel B serves as the first challenge for sperms to swim against. Thus, if the flow field were designed with velocity much faster than swim velocity of sperms, for example 500 lm/s at the centerline of channel B, no sperm would be able to swim against the flow along channel B. Conversely, flow velocity too slow might not be effective to evaluate the motility of sperms. The second challenge is the time limit for sperms to migrate through channel B to be counted. Only sperms which migrate through channel B within 12 min from start of test (t = 0) will be counted. In another words, sperms are challenged by (1) the flow field in channel B and (2) time constraint to travel through channel B for being counted. Hence, if there are more motile sperms in the tested sample, more sperms will be enumerated. Low sperm concentration or poor motility both will result in few sperms counted. Above conditions (flow field and time constraint) are consistent for testing different semen samples so that quality of samples can be compared and quantified by the number of sperms counted. The flow of channel A is sufficiently rapid (maximum velocity around 120 lm/s measured at the centerline of channel A) that motile sperms at the junction cannot swim upstream into channel A and, instead, being flushed through the aperture to be counted, as shown in Fig. 1b occasionally, a few sperms would swim upstream along the boundary of channel A, but the amount is substantially less than that

3 Microfluid Nanofluid (2011) 10: Fig. 1 Illustration of evaluation of sperm quality by the microdevice. a One stream in channel A divides into two streams at the junction: one flows toward channel B which sperms migrate along; another flushes sperms toward the aperture to be counted. Two electrodes immersed at two ends of channels A and C continuously measure voltage drop. b After a time period, sperms arrive at the junction and being flushed through the aperture. c The typical signal pattern includes a null period followed by pulses induced by sperms. d Compared to the pattern in c, the specimen in d shows poorer quality due to much less pulse number than that in c progressing toward channel C. The aperture is a feature in the microchannel (channel C in Fig. 2b) where a contraction of cross-sectional area is located. Motile sperms of sufficient strength would swim toward it and be counted electrically. Since the flow velocity near the aperture is high, the sperms being flushed to channel C would proceed further downstream and not return. The aperture serves as an electrical detector based on resistive pulse technique, which provides a change in resistance across a restriction filled with conductive media Fig. 2 The microdevice. a The microdevice hardware consists of three main components: the glass substrate, the PDMS bulk with microchannels in relief, and three containers each holding a liquid column at different heights driving the entire flow field. b Micrograph of the microchannel with key features shown. c Dimensions of microchannel network due to the insertion of an insulated object (DeBlois and Bean 1970). Hence, when a current/voltage source is applied across that restriction, the resultant voltage/current will alter accordingly owing to the partial displacement of the space by an insulated object. In our case, a constant current source was applied at far ends of channels A and C. Thus, one sperm being dragged through the aperture would cause one voltage pulse as shown in Fig. 1c. Figure 1c and d demonstrates typical signal patterns representing specimens with good and poor quality, respectively. Figure 1c shows the pattern with no pulse in the early period (from the beginning while sperm was placed at the end of channel B) since sperms must spend time to travel through channel B. Then, the following period shows pulses generated because of sperm passage across the aperture. To enable comparison of quality of sperms, a time limit for data acquisition was set (t : t* = 12 min). During this time limit, the number of pulses induced can reflect fertilization ability of sperms. Only sperms that successfully migrate through channel B, and then being flushed across the aperture in this time limit would be taken into account. Hence, pulse number is function of sperm concentration and motility rather than only a single parameter. In other words, more sperms with progressive swimming capability should be expected to result in more pulses generated. Figure 1d shows the signal pattern obtained by specimen with quality poorer than that shown in Fig. 1c. In a given time period t*, pulses in Fig. 1d are much less than those in Fig. 1c because of fewer motile sperms with good motility in Fig. 1d than that in Fig. 1c.

4 62 Microfluid Nanofluid (2011) 10: Microfluidic design Figure 2a shows the microdevice in some detail. The microdevice is composed of three parts: glass slide for the substrate of the microchannel, polydimethylsiloxane (PDMS) with microchannel in relief structure, and three reservoirs for holding liquid columns at different heights. The cross-sectional area of the reservoirs was chosen to be as large as possible in order to reduce the variation of the liquid column heights at each loading (5% variation at most). The driving force of the flow field resulted from the differences in heights of three liquid columns, *7 mm buffer solution in reservoir A, *2 mm pure semen in B, and *1 mm buffer solution in C. The enlarged photo of the junction is shown in Fig. 2b, where channels B and C are connected with channel A intersected at the angle 45. The aperture is located at the end of channel C and serves as an electrical detector to count sperm passage. Channel B for sperms to swim upstream was designed as 5 mm long and 500 lm wide. In order to obtain high signal to noise ratio, the amplitude of the pulses should be as large as possible while minimizing the noise. Large amplitude signal can be achieved if the size of aperture is reduced as small as possible but still allows the passage of sperms (DeBlois and Bean 1970). Other details of dimensions are given in Fig. 2c. Flow field consistency in each test is important to assure fair examination for each specimen. The pressure differences applied by three liquid columns were obtained by trial and error to produce the designed flow field. The height of the medium in reservoir A is the highest one among three reservoirs to force fluid through the entire device. Furthermore, since the aperture increases the flow resistance of channel C, the height of the fluid in reservoir C was loaded as the lowest one. Thus, the height of fluid column in reservoir B was set in between. Since the driving force of flow field is difference in pressure applied by liquid columns, density of liquids are critical to assure flow consistency in each test. Density of human semen ranges from 1.03 to 1.06 g/cm 3 (20 samples). Thus, the small difference in density of semen samples, around 3%, can be ignored. Moreover, since the buffer solution is a mixture of RPMI 1640 with small amount of seminal plasma, the density of buffer solution is similar to that of RPMI The steadiness of flow field even with varying column heights in the reservoirs was confirmed as follows. The flow rate in channel A ( ll/s) was approximated by considering the maximum flow velocity 120 lm/s as mean velocity in fully developed flow. At this flow rate, only 0.29 ll would be reduced in reservoir A during the experiment. The hydrostatic pressure at the inlet of channel A would drop by only 0.04%. Meanwhile, the increase in pressure at the ends of channel B and C would be only 0.1 and 0.06%. Consequently, the slight change in pressure difference across the microchannel during the experiment contributes to negligible change in flow velocity in all three channels. Flow velocities in each channel were measured by analyzing the trajectories of 1 lm dia. beads at the centerlines of channels A and B, where the maximum flow velocity occurs. Both measurements were conducted where the flows were fully developed, i.e., several millimeters downstream of the inlet of channel A and hundreds of microns far from the end of channel B. Flow velocities were computed by tracing bead trajectories recorded by CCD at 30 fps. 2.3 Fabrication Fabrication process is composed of two techniques: photolithography and soft lithography. Photolithography was utilized to fabricate the master mold for repeated molding of the microchannel. Fabrication of the master began with cleaning of a substrate (microscope glass slide). Afterward, photoresist (AZ P4620, AZ Electronic Materials) was coated on the glass surface by a spin coater to a thickness of about 17 lm. Then, a hot plate set at 90 C was used to cure the film of photoresist. Then, pattern of the microchannel network on Cr mask was transferred onto the photoresist by overlaying it with the Cr mask, followed by exposure to UV light ( nm, 18 s at 16 mw/cm 2, ORIEL). After exposure, the entire glass was immersed into a mixture of AZ 400 K (AZ Electronic Materials) and water (in 1:4 ratio) to develop with the microchannel pattern onto the photoresist, forming the master mold for subsequent replication of microchannel. The following steps utilized replica molding technique of soft lithography. Two reagents A and B of PDMS were mixed in a ratio of 10:1 (Sylgard 184, Dow Corning) then poured over the master, allowed for bubble removal, and cured at 80 C. After solidification, PDMS bulk was peeled off and three holes at each end of channels were punched serving as fluid interface. After air plasma treatment, the PDMS bulk with the microchannel in relief was irreversibly sealed with a glass substrate. The final step was to attach three PMMA reservoirs onto the chip. Figure 2a shows a photograph of the device which is composed of a glass substrate, a PDMS bulk, and three PMMA reservoirs. 2.4 Sperm and buffer solution preparation Eight samples used in the experiments were acquired from patients in the National Taiwan University Hospital with approval by the NTUH Institute Review Board. All specimens were allowed to liquefy then measured by the Sperm Quality Analyzer (SQA-IIB, Medical Electronic Systems)

5 Microfluid Nanofluid (2011) 10: to establish one benchmark Sperm Motility Index (SMI) for comparison with results from our microdevice. SMI is a parameter which reflects both motile sperm concentration and average velocity. Martinez et al. (2000) demonstrated a good correlation of this parameter with concentration of progressively motile sperm which highly relates to fertility. According to SMI, sperm quality can be divided into three grades: less than 80 is regarded as poor, between 80 and 160 is medium, higher than 160 is considered as good. The SMI of eight samples collected covers these three grades from 0 to 204. Another benchmark was provided by traditional cell counting method measuring progressively motile sperm concentration on hemocytometer under a microscope (BX-51, Olympus). To assure freshness of sperm quality, all experiments were done within 8 h after retrieval from patients. Buffer solution was prepared by mixing RPMI 1640 (Cat. No , Gibco) with seminal plasma in ratio of 4:1. The purpose of adding seminal plasma into RPMI 1640 is to prevent sperms from adhering to glass substrate (bottom surface of channel). One micron diameter polystyrene beads (4009a, Duke Scientific) were added in the buffer solution to monitor flow field before running each test. After semen loading in reservoir B, a few seconds would be taken to check flow field consistency by observing bead motion. Overall flow pattern in the channel network was visualized by fluorescent beads (G900, Duke Scientific) added in the solution. Streamlines of the flow field were visualized by combining several fluorescent images grabbed with long exposure time of 2 s. In theory, 1 lm dia. polystyrene beads diluted in the buffer solution would also cause resistive variation of the aperture during passage. However, prior researches have demonstrated that the change of resistance owing to the insertion of an insulated sphere in a cylindrical tube is proportional to cube of the sphere diameter (DeBlois and Bean 1970). Although the aperture geometry in our case is not a cylindrical tube (instead, a nozzle-diffuser shape), the fact that resistive pulse is proportional to cube of bead diameter is still valid. Based on this argument, the amplitude of the pulse induced by 1 lm bead is estimated to be less than 1 mv, while sperms generated pulses about 20 mv in average. Besides, experimental results confirmed this estimation no observable pulse was produced as the beads cross the aperture. 2.5 Experimental procedures Experimental procedures are as follows. Microchannel network was first filled with buffer solution and then liquid columns were applied into reservoirs A and C to specified heights. Then, two stainless steel electrodes were immersed into the liquid columns in reservoirs A and C to inject the constant current by wiring a typical op-amp current source (one op-amp LF411 with one transistor 2N3906 wired by few resistors and powered by a DC power supply, GPC- 3030D, GW) (Horowitz and Hill 1989). This current flows from the electrode in reservoir A, through the aperture, and drains at the electrode in reservoir C. Digital multimeter (GDM-8145, GW Instek) was serially connected to the microdevice for real-time current monitoring. After injecting steady current, 200 ll pure semen was loaded into reservoir B and voltage recording began. Data acquisition module (PCI-6250, SHC68-68-EPM Shielded Cable, and SCB-68 screw terminals, National Instruments) was employed to record the voltage drop across two electrodes for 12 min at the sampling rate of 1 khz. Electric field distributed over the channel network was numerically estimated. Since current flows along channel A followed by crossing the aperture, and then continues along channel C, electric field in channel B remains lower than 100 V/m. Most area in overall channel network is lower than 1 kv/m except the region close to the aperture. By comparing with electrophoretic operation on sperms, up to 10 4 V/m (Engelmann et al. 1988), influence on sperm motility owing to much lower electric field can be ignored. Acquired voltage traces were processed offline via LabVIEW programs. Butterworth filters handled the raw data at low-pass cutoff frequency of 30 Hz. Besides, baseline of the recorded voltage trace was obtained by median filter. By subtracting from the baseline, the DC level of the voltage trace was leveled to zero so that pulses with amplitude over a given threshold 4 mv were counted (noise was around 1 mv). Number of pulses was counted by the function Peak Detector in LabVIEW program. Threshold was set to be 4 mv so that pulses with amplitude over this threshold were counted. Moreover, manual counting of the sperms passing through aperture was also performed under microscope as comparison. Only 9% difference in the counts obtained by these two methods indicated high fidelity of the detection system. The difference primarily resulted from errors in manual counting, for example, the throughput of sperms passing the aperture up to several per second could cause minor error in manual counting. Moreover, few unexpected electrical spikes were also observed to cause errors in pulse number. 3 Results and discussions 3.1 Visualization and characterization of flow field Figure 3a shows the flow field visualized by green fluorescent beads. The streamlines are presented by bead

6 64 Microfluid Nanofluid (2011) 10:59 67 trajectories, and stagnant green spots are beads adhered to channel wall. The flow coming from channel A separated into two streams at the junction: one stream turning into channel B and the other toward channel C. As illustrated in Fig. 3b, a streamline terminating at the stagnation point on channel wall divides the flow field into these two zones. In zone 1 shown at the right-hand side, the flow entering into channel B slowed down rapidly from about 120 lm/s maximum velocity to about 40 lm/s owing to the enlarged cross-section. In zone 2, the stream toward channel C decelerated at the end of channel A and then accelerated near the aperture. The nozzle-diffuser shape geometry forces this stream to speed up to the flow velocity over 100 lm/s at the aperture followed by deceleration in the diffuser. The segmental fluorescent bead trajectories visualized in channel C implied relatively low flow velocity compared to that in channels A and B. 3.2 Interaction of sperm with channel geometry The most dominant tendency of sperms interacting with geometry of microchannel is probably the behavior of swimming along channel walls. Micrograph in Fig. 4 shows high percentage of sperms found to migrate along the channel wall. Sperms labeled by red circles are those swimming next to the channel wall and by blue circles are in the central region. Around 34% sperms migrating along the wall in this case is attributed to the fact that sperms kept swimming along the walls after initial contact. This observation is consistent with the finding of Lopez-Garcia et al. (2008). By studying motion of sperms in microchannels with different types of constrictions, their results also showed the preference to travel along wall and contour of the device. Seo et al. (2007) presented another tendency of sperms to swim upstream: results show that bull and mouse sperms also traversed the microchannel along the wall, which implied not only human but mammalian sperms probably behave similarly. Region R2 of Fig. 4 shows that sperms arriving at the neighborhood of the aperture would be dragged by the flow through the aperture. Some sperms in channel C tend to swim back but could not succeed since increased flow velocity through the aperture stops their approach, as illustrated in Region R Sperm quality analysis Sperms that arrive at the neighborhood of the aperture would be flushed through and raised the resistance temporally. Figure 5 shows the typical pattern of resulted voltage trace continuously acquired for 12 min. From the beginning to the first pulse, this time period was taken by sperms to migrate through channel B. Four repetitive tests show the swimming velocity of the first sperm reaching the aperture was about 17 lm/s (with respect to a fixed reference frame). Comparing to the sperm-swimming velocity in a hydrostatic fluid, which ranges from few to tens of microns per second, the swimming velocity of sperms in channel B is decreased due to increased drag from the background flow. Subsequent voltage traces show the pulses generated are due to sperms being dragged through the aperture one by one, with each pulse induced representing one sperm passage. Pulse amplitudes from few to tens of millivolts in comparison with the noise level (less than 1 mv) show high signal to noise ratio, which clearly identify the sperms passage. Some sperms being flushed through the aperture turned around and approach the aperture as Fig. 3 The flow field within the device. a Fluorescence beads image showing flow from channel A divides into two streams: one enters channel B and the other passing the aperture and enters channel C. The flow velocity at the centerline of channel B roughly 40 lm/s establishes the threshold for sperm to progress forward. The flow velocity of 120 lm/s at channel A is designed to prevent sperms from entering channel A. b Two flow zones 1 and 2 are separated by the streamline terminated at a stagnation point

7 Microfluid Nanofluid (2011) 10: Fig. 4 Interaction of the sperms with the channel geometry. Region R1: sperms prefer to swim along channel walls. Micrograph shows high percentage of sperms traveling along channel wall (25 sperms along channel wall vs. 48 in central region). Region R2: Sperms are flushed through the aperture by the flow. Region R3: after being flushed through the aperture, some sperms tend to reverse in direction to approach the aperture, but unable to swim pass the aperture Fig. 5 Typical voltage trace can be viewed as two parts. The trace signal represents waiting period as sperms progress along channel B. Then, pulses are recorded as sperms pass through the aperture described in R3 of Fig. 4. While those sperms were few, the sperms could not approach the aperture due to the fastflowing fluid. Gradually, quantity of these sperms increases and they move closer to the aperture. Eventually, the signal would be influenced by these sperms since they were very close to the aperture. The reason for their success in approaching the aperture is likely due to eventual accumulation of sperms there, whereby increasing flow resistance around the aperture causing reduction in flow velocity. Hence, as the flow velocity decreases, the sperms could get close to the aperture and even inducing false pulses, i.e., the pulses induced by sperms approaching from channel C rather than from channel B. The time period of recording the voltage, 12 min, was chosen to avoid this situation. Patients specimens were used to scrutinize performance of the device. Figure 6 compares the traces of three samples, B, D, and H, which had motile sperm concentrations of 17, 7, and /ml (total sperm concentration 54, 15, /ml), and corresponding SMI of 148, 51, and 0, respectively. Three captured micrographs reveal significant difference in sperm concentration. According to the motile sperm concentrations measured by hemocytometer and SMI from the Sperm Quality Analyzer, these three samples show distinguishable qualities. The results of these samples obtained by the microdevice presented large difference in the three voltage traces. Sample B with the highest motile sperm concentration and SMI among the three samples exhibited the most number of pulses. Compared to sample B, sample D with the middle quality produced much less pulses than sample B. For sample H, no motile sperms could be found under microscope so that no pulse was detected. Results from the microdevice demonstrated consistency with the two benchmarks. Eight samples were tested on the microdevice with progressively motile sperm concentrations ranging from 0 to /ml (total sperm concentration from 0 to /ml), as shown in Fig. 7. The samples could be divided into two groups by number of the pulses acquired. The first group including samples F, G, and H with the lowest motile sperm concentrations and resulted in only three pulses detected in sample G, and none in both F and H. The three pulses recorded were considered as noise since real-time observation of the sample G revealed no event of sperm passage through the aperture. In this group, although only sample H had no motile sperm, the other samples F and G were still relatively low in the motile sperm concentrations 2 and /ml, respectively, and could be regarded as infertile by the WHO standard, progressively motile sperm concentration at least /ml (the threshold given by timing /ml total sperm concentration by 50% motility). Low motile sperm concentrations caused no sperms to migrate through channel B and induce pulse within a limited time. The situation was that the motile sperms initially placed at the end of channel B were very few, comparing to those samples with high motile sperm concentrations. In addition, not all motile sperms but the sperms swimming fast enough to overcome the flow and traverse channel B in the

8 66 Microfluid Nanofluid (2011) 10:59 67 Fig. 7 Correlation of motile sperm concentration measured by hemocytometer with pulse number by our microdevice. Eight samples tested by the microdevice show that pulse number correlates well with motile sperm concentration. The SMI values for these samples were also proportional to pulses numbers. The two control methods the motile sperm concentrations by hemocytometer and the SMI values by the Sperm Quality Analyzer both verified evaluation of sperm quality by the microdevice Fig. 6 Three samples B, D, and H with motile sperm concentrations 17, 7, and /ml from hemocytometer respectively were tested in the microdevice. Three micrographs (top 3 figures) show obvious difference in sperm concentrations among these samples. The three voltage signals below quantified these sperm qualities, respectively. Also shown are corresponding Sperm Motility Index 148, 51, and 0. The benchmarks hemocytometer and Sperm Motility Index agree well with signal from the device limited time could be counted. Besides, the SMI values of these three samples are 0, no record, and 46 for H, G, and F, respectively. No record can be regarded as a value very close to 0 according to experienced judgment. The SMI values all below 80 discloses that three samples are poor in motile sperm concentration and motility. In other words, the samples could be poor in fertility. Examination by the microdevice shows agreement with the motile sperm concentrations measured by hemocytometer and SMI value by the Sperm Quality Analyzer. The second group contains samples A, B, C, D, and E, with corresponding motile sperm concentrations of 19, 17, 14, 7, and /ml, respectively. Each sample during test produced 335, 238, 173, 59, and 19 pulses which highly correlated to their motile sperm concentrations. The correlation suggests that motile sperm concentration could be the factor linearly correlating to pulse number. The SMI assayed for these samples were 204, 148, 110, 51, and 60 for samples A to E, respectively. It means that qualities of samples D and E were determined as poor, qualities of B and C is medium, and sample A is good. The SMI values being proportional to the pulse numbers also verify the analysis of sperm quality by the microdevice except the mismatch between samples D and E. The SMI of samples D and E were possibly attributed to the imprecision of the SMI measurement. The wide range of pulse numbers from 19 to 335, corresponding to SMI from 51 to 204, indicates that pulse number was sensitive to the quality of sperm. Two control methods, motile sperm concentration provided by hemocytometer and the SMI by the Sperm Quality Analyzer, both proved the accurate evaluation of sperm quality by the microdevice. Moreover, the plateau at low motile sperm concentrations followed by the increase of pulse numbers shows that both motile sperm concentration and motility are critical to pulse generation. 4 Conclusions This study presents a microfluidic design for evaluating sperm quality in terms of motile sperm concentration and motility. The flow field generated within the device enables quantification of sperm quality via resistive pulse detection. Output signals from the device are in good agreement with that from the two controls motile sperm concentration

9 Microfluid Nanofluid (2011) 10: provided by hemocytometer and SMI by the Sperm Quality Analyzer. Operationally, there are several pros and cons of the device that deserve further comments. Label-free detection allows substantial cost saving of biomarkers and ease of operation in a home situation. Moreover, the fact that semen sample remains in the reservoir while sperms swim against the flow ensures much debris in the sample will not at all affect detection outcome, thus completely eliminated the need for very difficult pre-sample treatment techniques. One drawback of the use of resistive pulse measurement is the necessity of a small aperture which sperms must pass through to provide a sufficiently distinctive signature and the aperture is prong to clog due to fine debris in the buffer solution (in reservoir A). Some form of filtering should be employed to alleviate this problem but would impose additional pressure drop due to added flow resistance, which must be accounted for in order to achieve a specified flow velocity within the sperm-swimming channel. Application wise, the use of the device for home monitoring allows real-time information of sperm quality. This should be useful in many scenarios, e.g., during family planning stage and infertility treatment process, among others. Recorded signals can serve as feedback to urologists to better monitor progress, which might be difficult to do otherwise. Acknowledgments Funding from the National Science Council (Grants NSC M and NSC E ) is gratefully acknowledged. Professors Ju-Ton Hsieh and Hong-Chiang Chang in the Department of Urology, National Taiwan University Hospital are acknowledged for their helpful discussion throughout this study. Miss M.M. 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