Papers in Press. Published January 8, 2016 as doi:10.1373/clinchem.2015.250282 The latest version is at http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2015.250282 Clinical Chemistry 62:3 000 – 000 (2016)
Automation and Analytical Techniques
Paper-Based Quantiﬁcation of Male Fertility Potential Reza Nosrati,1,† Max M. Gong,1,† Maria C. San Gabriel,2 Claudio E. Pedraza,2 Armand Zini,2 and David Sinton1*
BACKGROUND: More than 70 million couples worldwide are affected by infertility, with male-factor infertility accounting for about half of the cases. Semen analysis is critical for determining male fertility potential, but conventional testing is costly and complex. Here, we demonstrate a paper-based microfluidic approach to quantify male fertility potential, simultaneously measuring 3 critical semen parameters in 10 min: live and motile sperm concentrations and sperm motility. METHODS: The device measures the colorimetric change of yellow tetrazolium dye to purple formazan by the diaphorase flavoprotein enzyme present in metabolically active human sperm to quantify live and motile sperm concentration. Sperm motility was determined as the ratio of motile to live sperm. We assessed the performance of the device by use of clinical semen samples, in parallel with standard clinical approaches. RESULTS: Detection limits of 8.46 and 15.18 million/mL were achieved for live and motile sperm concentrations, respectively. The live and motile sperm concentrations and motility values from our device correlated with those of the standard clinical approaches (R2 ⱖ 0.84). In all cases, our device provided 100% agreement in terms of clinical outcome. The device was also robust and could tolerate conditions of high absolute humidity (22.8 g/m3) up to 16 weeks when packaged with desiccant. CONCLUSIONS: Our device outperforms existing commercial paper-based assays by quantitatively measuring live and motile sperm concentrations and motility, in only 10 min. This approach is applicable to current clinical practices as well as self-diagnostic applications.
© 2015 American Association for Clinical Chemistry
The global burden of infertility is high, affecting ⬎70 million couples (1, 2 ). Male infertility accounts for 40%–50% of infertility cases worldwide (3 ). Main causes of male infertility include low sperm count (azoospermia
Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada; 2 Urology Research Laboratory, Department of Surgery, McGill University and Research Institute, McGill University Health Centre, Montreal, Quebec, Canada. * Address correspondence to this author at: Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, Ontario, Canada, M5S 3G8. Fax 416-978-7753; e-mail [email protected]
and oligozoospermia), poor vitality (necrozoospermia), low motility (asthenozoospermia), low DNA integrity, and abnormal sperm morphology (teratospermia) (4, 5 ). Semen analysis to quantify these factors is the cornerstone of male infertility diagnosis (6 ) and also plays a critical role in monitoring male contraception after vasectomy to ensure permanent sterility (7 ). Conventional techniques for semen analysis include counting chambers, computer-assisted sperm analysis (CASA)3 (8 ), and vitality assays such as dye exclusion or hypotonic swelling (4, 9 ). Cell counting chambers, namely hemocytometers and Makler chambers, are the traditional tools for quantification of sperm concentration and motility, working via manual visual inspection under a microscope. CASA systems use advanced optical microscopy to assess sperm concentration and motility via automatic online tracking of sperm in a digital image sequence (8, 10 ). Dye exclusion methods use membrane-impermeable stains to selectively label dead cells (11 ). In hypotonic swelling, live sperm cells swell because of an influx of water to their cytoplasm from an induced osmotic pressure gradient (12 ). Both dye exclusion and hypotonic swelling vitality assays require manual microscopy inspection or flow cytometry to count the number of live and dead cells. All these current semen analysis techniques suffer from limitations that prevent their widespread application: testing procedures are long and complex and require expensive equipment, and the results are subjective, varying from clinician to clinician (13, 14 ). Compliance of male patients in providing clinical samples is also low because of embarrassment and anxiety (7 ). A low-cost and rapid test for semen analysis, suitable for both clinical and self-diagnosis, would have substantial implications for patient care. Microfluidic technologies have rapidly advanced diagnostic testing (15–17 ). In the context of fertility, traditional channel-based devices have been developed for sperm selection (18 –22 ), embryo development (23–25 ), and semen analysis (14, 26 –28 ). Recently, paper has emerged as a scalable diagnostic platform (29 –32 ). The home pregnancy test is the most widely used fertility-based diagnostic, en-
Received September 29, 2015; accepted December 11, 2015. Previously published online at DOI: 10.1373/clinchem.2015.250282 © 2015 American Association for Clinical Chemistry † R. Nosrati and M.M. Gong contributed equally to this work. 3 Nonstandard abbreviations: CASA, computer assisted sperm analysis; MTT, 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide; LOD, limit of detection.
Copyright (C) 2016 by The American Association for Clinical Chemistry
Fig. 1. Schematic of the paper-based semen analysis device and protocol. (A), Exploded view of the 3D device. (B) Schematic view of the assembled device and images of devices before and 10 min after applying a semen sample. (C), In the concentration spot, the semen sample is pipetted directly on the dried MTT in paper to generate a colorimetric signal. (D), In the motility spot, sperm must swim through the highly viscous buffer and relatively narrow (8-μm) pores within the membrane ﬁlter (inset) to reach the reaction spot and generate a colorimetric signal.
abling women to perform self-testing at their own discretion. Lateral flow assays have also been developed for home semen analysis; for example, SpermCheck (ContraVac) and FertilMARQ (Wisconsin Pharmacal) use colorimetric signals to semiquantitatively determine total sperm concentration within a sample (i.e., whether sperm concentration is ⬎20 million/mL) (33, 34 ). Additionally, the Fertell assay (Genosis) measures motile sperm count by allowing sperm to swim up a fluid-filled reservoir toward a nitrocellulosebased immobilization region (35 ). Although these microfluidic technologies are effective and have shown promise for self-screening of male fertility potential, they suffer from some limitations, including prolonged multistep processing, lack of quantification, reliance on the interpretation of the end user, and high cost (13 ). Additionally, current tests measure only 1 semen parameter. Here, we present a low-cost and rapid paper-based microfluidic approach for quantifying male fertility potential that simultaneously measures 3 critical semen parameters in 10 min: live sperm concentration, motile sperm concentration, and sperm motility. Materials and Methods DEVICE FABRICATION
The device consists of 3 layers: 1 laminate layer on the bottom and 2 paper layers patterned with wax on top. An 2
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exploded view of the device is shown in Fig. 1A. We designed the device patterns in AutoCAD to be printed on paper (No. One Chromatography Paper, SigmaAldrich) with a solid wax printer (ColorQube 8570N, Xerox Canada). Printed sheets of paper were then heated in an oven at 125 °C for 5 min to allow the wax to penetrate through the thickness of the paper. We used a hole punch to punch 2 holes of 4-mm diameter (unless otherwise indicated) in layer 1, as access ports to spots in layer 2. A transparent adhesive laminate layer (selfadhesive sheets, Felloes) was directly applied to the back side of layer 2 to limit evaporation and facilitate imaging. On each of the 4-mm-diameter background (B), concentration (C), and motility (M) spots in layer 2, 3 L of 5-g/L 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich), prepared in distilled water, was dried. We used double-sided tape to bond layers 1 and 2 such that the through-holes in layer 1 were aligned with the reaction spots for concentration and motility in layer 2. Before bonding, a 5 ⫻ 5-mm membrane filter with 8-m pore size (Nuclepore track-etched polycarbonate membrane filter, Whatman–GE Healthcare Life Science) was manually aligned on top of the motility spot and sandwiched between layers 1 and 2 with double-sided tape. In addition to devices with 4-mm reaction spot diameter, we designed, fabricated, and tested devices with 3- and 5-mm reaction spot diam-
Paper-Based Semen Analysis
eters, and spots both with and without patterned dots, to characterize the operating parameters of the device. HUMAN SEMEN PREPARATION
We purchased cryogenically frozen human semen (50 million/mL concentration and 40% motility) in 1-mL vials from ReproMed and stored in liquid nitrogen. All donors provided consent for research participation in accordance with regulations of the Assisted Human Reproduction Act. Frozen semen vials were thawed for 5 min in a 37°C water bath before experiments. Fresh human semen samples from patients (n ⫽ 5) and healthy donors (n ⫽ 12) were obtained by masturbation after 2– 4 days of sexual abstinence at the Urology Research Laboratory, Royal Victoria Hospital, Canada. All donors and patients signed informed consent, and the information for this study remains confidential within the institution. This study was approved by the ethics review board at McGill University. Samples were incubated at 37 °C for 30 min to allow liquefaction. This liquefaction step was necessary in all semen analysis techniques to break the thick semisolid form of the freshly ejaculated semen sample into thinner liquid with more homogeneous sperm distribution (4 ). We used HEPES-buffered salt solution (135 mmol/L NaCl, 5 mmol/L KCl, 12 mmol/L D-glucose, 25 mmol/L HEPES, and 0.75 mmol/L Na2HPO4兩 䡠 兩2H2O, Sigma-Aldrich) supplemented with 1 g/L polyvinyl alcohol (Sigma-Aldrich) and 0.5% methylcellulose (M0512; Sigma-Aldrich) to prepare different dilutions of semen and also as buffer in the motility assay. This buffer was selected as the medium for sperm motility assessment because it mimics the natural properties of mucus in vivo (22 ). All experiments were conducted at room temperature and within 10 min of liquefaction/thawing. HUMAN SEMEN ANALYSIS
We analyzed semen samples for concentration and motility with a CASA system (Penetrating Innovations) equipped with an Olympus BH2 Microscope, Minitub Accupixel camera, and Sperm Vision HR software (version 1.0.5, 2008), obtaining standard semen parameters in accordance with WHO guidelines (4 ). Semen samples were analyzed for vitality with a dye exclusion assay. Briefly, 10 L eosin-nigrosin stain (0.65% eosin Y and 10% nigrosin in distilled water) was mixed with 10 L semen sample, and the suspension was smeared on glass slides and allowed to air-dry. We used manual visual inspection under a microscope to count live and dead sperm. Sperm were scored as live if luminous or unstained and dead if stained pink or red. At least 200 cells were counted, and percentage vitality was calculated. LIVE AND MOTILE SPERM CONCENTRATION ASSAYS
To operate the device, we pipetted 3 L high-viscosity buffer on the motility spot to saturate the paper and the
membrane filter below, forming a buffer reservoir in layer 1. We then pipetted 3 L semen sample on the concentration and motility spots (unless otherwise indicated). After 10 min (unless otherwise indicated), the back side (laminate side) of the device was scanned with a lettersized scanner (CanoScan 9000F, Canon) for quantification of colorimetric signals (Fig. 1B). In the concentration spot, the semen sample directly reacted with the dried MTT to generate a colorimetric signal (Fig. 1C). In the motility spot, sperm must swim vertically through the viscous buffer in layer 1 and then through the narrow 8-m pore membrane filter to generate a colorimetric signal (Fig. 1D). The high-viscosity buffer simulates in vivo fluid in the female tract and minimizes mixing and flow in the motility spot. The differences between color intensities of the concentration and motility spots with respect to the color intensity of the background spot quantify the live and motile sperm concentrations, respectively. To characterize the operating parameters for the paper-based semen analysis device, we also tested sample volumes of 1 and 5 L and reaction times of 5, 20, and 30 min. DATA ANALYSIS
For characterization experiments, devices were scanned before and 10 min after applying the sample. In ImageJ software (36 ), we obtained the color intensity (representing the grayscale image intensity) of the background spot (IB), concentration spot (IC), and motility spot (IM) for each device. We used the difference in color intensity between the background and concentration spots, IB ⫺ IC, to quantify live sperm concentration; the difference in color intensity between background and motility spots, IB ⫺ IM, to quantify motile sperm concentration; and the ratio of motile sperm concentration to live sperm concentration to quantify motility. Data quantification was completed in Microsoft Excel. Results and Discussion The paper-based semen analysis device is a multilayer porous composite that simultaneously tests for live and motile sperm concentration and sperm motility. The simple design and fabrication process of the device required minimal reagents and equipment, with a total material cost of approximately US$0.05 per device (approximately US$0.02 for paper, laminate, and tape and approximately US$0.03 for reagents). Readout from the device is achieved with the enzymatic colorimetric MTT assay. Briefly, the yellow MTT tetrazolium converts to purple formazan upon removal of the bromide by diaphorase flavoprotein enzyme present in metabolically active human sperm (37–39 ). This colorimetric signal from the MTT assay has been shown to be strongly correlated with established sperm vitality assays such as Clinical Chemistry 62:3 (2016) 3
Fig. 2. Characterization of the operating parameters of the paper-based semen analysis device. (A), ΔC as a function of sample volume. (B), ΔC as a function of reaction time. (C), ΔC color intensity as a function of reaction spot diameter and patterned dots. Each data point is the mean of 4 independent measurements, with error bars as 1 SD. The values of ΔC were calculated by taking the difference between the concentration and background spots. a.u., arbitrary units.
eosin-nigrosin and hypoosmotic swelling (37, 40 ). The paper-based semen analysis device tests simultaneously for live sperm concentration and motile sperm concentration in 2 distinct spots in 10 min. In the concentration spot, the semen sample is pipetted directly on the dried MTT to generate a colorimetric signal (Fig. 1C). In the motility spot, sperm must swim vertically through the viscous buffer and then through the membrane filter to generate a colorimetric signal. The differences between color intensities of the concentration and motility spots with respect to the color intensity of the background spot quantify the live and motile sperm concentrations, respectively. Fig. 2 shows the characterization of the operating parameters for the paper-based semen analysis device with human semen samples. With respect to sample volume, the difference in color intensity compared to the background spot (⌬C) increased ⬎350%, from 12.2 for 1-L sample volume to 56.6 for 3-L sample volume, and plateaued thereafter (Fig. 2A) in devices with 4-mmdiameter reaction spots and 10-min reaction time. With regard to reaction time, ⌬C increased about 11% by increasing the reaction time from 5 to 10 min (Fig. 2B), in devices with 4-mm-diameter reaction spots and 3-L sample volume. However, ⌬C decreased about 40% by further increasing the reaction time to 20 –30 min. We attributed this drop in color intensity over long reaction times to evaporation of liquid, which consistently caused discoloration in this manner. Devices were fabricated and tested with 3-, 4-, and 5-mm reaction spot diameters and with and without patterned dots, as shown in Fig. 2C. The results indicated that both ⌬C and associated error (inset in Fig. 2C) decreased with increasing reaction spot diameter. Dots can improve readout by pinning the interface during evaporation (41 ). Here, however, devices without dots had 43%–56% higher ⌬C than devices with patterned dots. On the basis of this characterization, the following operating parameters were selected for all sub4
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sequent experiments: 4-mm diameter reaction spots, no patterned dots, 3-L sample volume, and 10-min reaction time. CLINICAL ASSESSMENT WITH HUMAN SEMEN SAMPLES
The paper-based semen analysis device was tested with raw human semen samples at the Royal Victoria Hospital in Montreal, Canada. Current clinical practices (CASA and dye exclusion assay) were used to assess concentration, vitality, and motility of each sample in parallel with the device. Fig. 3A shows the calibration curve for live sperm concentration, ranging from 7.27 to 359 million/mL (with total concentrations and viabilities ranging from 8.56 to 381 million/mL and 52% to 94%, respectively). Fig. 3B shows the calibration curve for motile sperm concentration, ranging from 3.73 to 315 million/mL (with motility ranging from 9% to 87%). The limits of detection (LODs) for the live and motile sperm concentration assays were calculated as 3 times the SD of the signal from a blank device with zero sperm concentration (i.e., applying only buffer on the concentration and motility spots and scanning after 10 min). These values were 8.46 million/mL for live sperm concentration and 15.18 million/mL for motile sperm concentration. On the basis of reference values for human semen characteristics from the WHO, the lower reference limits for live sperm concentration and motile sperm concentration are 8.7 and 6 million/mL, respectively (resulting from respective threshold values of 15 million/mL for total concentration, 58% for vitality, and 40% for motility) (4 ). Considering these limits and the LOD of our device, a detectable color change in the concentration and motility spots indicates that the patient has sufficient fertility potential. In contrast, if no detectable color change is observed, then that patient may be at high risk for azoospermia, oligozoospermia, necrozoospermia, or asthenozoospermia.
Paper-Based Semen Analysis
Fig. 3. Calibration curves. (A), Calibration curve for live sperm concentration by use of ΔC between C (concentration) and B (background) spots. The LOD for live sperm concentration is 8.46 million/mL. (B), Calibration curve for motile sperm concentration by use of ΔC between M (motility) and background spots. The LOD for motile sperm concentration is 15.18 million/mL. Each data point is the mean of 4 independent measurements, with error bars as 1 SD. a.u., arbitrary units.
Fig. 4 compares live sperm concentration, motile sperm concentration, and motility measured by both the device and standard clinical approaches. For each clinical sample, 4 devices were tested in parallel, and motility values from the device were calculated by dividing the motile sperm concentration with the live sperm concentration. Results from our paper-based semen analysis strongly correlated with clinical data from CASA and dye exclusion vitality assays run in parallel (with respective R2 values of 0.84, 0.91, and 0.92 for live sperm concentration, motile sperm concentration, and motility, as shown in Supplemental Fig. 1, which accompanies the online version of this article at http://www.clinchem.org/ content/vol62/issue3). Our device provided 100% agree-
Fig. 4. Clinical assessment of the paper-based semen analysis device. Direct comparison of live sperm concentration (A), motile sperm concentration (B), and motility (C) values measured with the device vs those measured with standard clinical approaches. For each sample, 4 devices (white bars) were tested in parallel with the clinical testing (gray bars). Live and motile sperm concentration values for tested devices were calculated by use of the linear calibration equations in Fig. 3. Motility values for tested devices were calculated by dividing live sperm concentration by motile sperm concentration. Dashed lines indicate the WHO lower reference limits for human semen parameters.
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ROBUSTNESS IN DEMANDING CONDITIONS
Table 1. Robustness of the paper-based semen analysis device after 8 weeks of storage in different humidity and temperature conditions.
Absolute humidity, g/m3
ment in terms of clinical outcome for patients. In addition, the device provided quantitative information related to sperm motility, which is not possible with existing commercial paper-based devices. A lower reference limit of 40% is defined by WHO for motility (4 ), which is used in clinics as a threshold value for poor motility and a diagnostic threshold for male-factor infertility. As shown in Fig. 4C, sample number 4, while indicating appropriate live and motile sperm concentrations, would result in an assisted reproduction recommendation on the basis of both device and clinical motility results. Collectively, our paper-based semen analysis device reliably measured 3 critical male fertility parameters. The simple operation of the device provides an opportunity for application in self-screening of male fertility potential. In particular, a visual readout can be provided with the device, resulting in straightforward and semiquantitative analysis of semen parameters.
The robustness of the paper-based semen analysis device was tested in challenging conditions. Specifically, high humidity and air temperature can both discolor paper and degrade dried reagents in paper-based devices (29 ). Table 1 summarizes the results over 8 weeks of storage as a function of temperature and humidity conditions. The color intensity of each stored device was compared with devices fabricated the same day (“fresh”). Fig. 5A shows color intensity as a function of humidity. Devices stored in an absolute humidity ⬍3.38 g/m3, regardless of the storage temperature, showed ⬍10% change in the color intensity of the reaction spots— deemed a “pass” in Table 1. The resulting signal from testing the passed devices with human semen is shown in Fig. 5B, with output comparable to otherwise identical tests with newly fabricated devices. At absolute humidities ⬎3.38 g/m3, the color intensity of the reaction spots decreased with increasing humidity. For these devices, the change of color in the reaction spots from yellow to purple suggests that in such highly humid environments, MTT is converted to insoluble formazan over time. This humidity-dependent instability of the device, when stored in humid conditions, can be resolved by packaging the device with desiccant in a sealed plastic bag. Fig. 5C shows color intensity of the reaction spots over time for devices stored with and without desiccant in environments with 21.8 and 22.8 g/m3 absolute humidities (the highest humidities tested in our 8-week robustness test). Results indicate that devices stored with desiccant showed ⬍10% decrease in the color intensity of the reaction spots after 16 weeks of storage. However, devices stored without desiccant showed a ⬎12% decrease in color intensity after only 2 weeks of
Fig. 5. Robustness of the paper-based semen analysis device. (A), Color intensity of the reaction spots as a function of humidity of the storage environment after 8 weeks. (B), ΔC of the devices that passed the storage test in comparison with the signals from fresh devices fabricated that day (dashed line). (C), Color intensity of the reaction spots over time for devices stored with and without desiccant in environments with 21.8 and 22.8 g/m3 humidity. Each data point is the mean of 4 independent measurements, with error bars as 1 SD. The values of ΔC were calculated by taking the difference between the concentration and background spots. a.u., arbitrary units.
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Paper-Based Semen Analysis
storage and a ⬎60% decrease after 18 weeks. The color intensity for devices stored at 22.8 g/m3 absolute humidity started to decline after 16 weeks, the point at which the desiccant reached its moisture capacity. Altogether, these results indicate that packaging the device with desiccant maintains its functionality for up to 4 months in humid conditions. In summary, we demonstrate here a low-cost and rapid paper-based microfluidic approach for quantifying male fertility potential, simultaneously measuring 3 critical semen parameters in 10 min: live sperm concentration, motile sperm concentration, and sperm motility. We clinically assessed the performance of the device with raw human semen samples at the Royal Victoria Hospital. Detection limits of 8.46 and 15.18 million/mL were achieved for live sperm concentration and motile sperm concentration, respectively. Our paper-based semen analysis device provided clinical outcomes identical to those of the conventional CASA and dye exclusion vitality assay (R2 ⬎ 0.84). In particular, the device provides direct visual readout with straightforward and semiquantitative analysis of semen parameters. The device outperforms existing commercial paper-based devices by providing quantitative information related to sperm concentration, vitality, and most importantly motility, in only 10 min. The device is also robust and can tolerate a range of temperature conditions without losing functionality. It can withstand high absolute humidity conditions (22.8 g/m3) for ⬎16 weeks when packaged with desic-
cant. Our paper-based technology is an attractive alternative to conventional laboratory testing, with additional potential for self-screening of male fertility potential.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: D. Sinton, University of Toronto, Department of Mechanical and Industrial Engineering. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: The University of Toronto is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC I2IPJ469164-14, NSERC RGPIN-2015-06701), Canadian Institutes of Health Research (CIHR 139088), and Grand Challenges Canada (0005-02-02-01-01). R. Nosrati, Ontario Graduate Scholarship; M.M. Gong, Alexander Graham Bell Canada Graduate Scholarship-Doctoral from NSERC. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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