Point‐of‐care semen analysis of patients with infertility via smartphone and colorimetric paper‐based diagnostic device

Abstract Male infertility affects millions of males worldwide and is rising in prevalence due to social and environmental conditions. However, men often feel too embarrassed to receive a semen analysis in the hospital due to social stigmas. To overcome this problem, we developed a 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide test strip to distinguish semen samples with low total motile sperm concentration from those with normal motile sperm concentration. This is a point‐of‐care colorimetric semen analytical method with a one‐step, inexpensive, equipment‐free evaluation process, and adequate accuracy validated in a 42‐sample clinical trial. In this study, results were evaluated visually and with a smartphone application. Using visual observation methods, the area under the curve (AUC) was 0.71 (95% of confidence interval = 0.55–0.86; p = 0.021), sensitivity was 41%, specificity was 95%, positive predictive value was 90%, negative predictive value (NPV) was 59.4%, and accuracy was 67%. Using a smartphone recording and analytical system, AUC was 0.766 (95% of confidence interval = 0.612–0.92; p = 0.003), sensitivity was 96%, specificity was 65%, PPV was 75%, NPV was 92.9%, and accuracy was 80.9%. This work demonstrated a screening tool that could elevate semen analysis to the level of routine healthcare and provide for private, in‐home self‐assessment.


| INTRODUCTION
Infertility is defined as a disease characterized by the failure to establish a clinical pregnancy after 12 months of regular sexual intercourse. 1 The concept of describing infertility as a disease is part of a conscious effort to arouse public attention regarding the issue. The increasing prevalence of infertility has been associated with trends in delayed marriage, obesity, and stress. 2,3 Currently, nearly 200 million people worldwide suffer from infertility. 4 Male infertility was found to be responsible for 20%-30% of all infertility cases and involved in 50% of cases overall. 5 Furthermore, some researchers have discovered a significant decreasing trend in sperm quality over recent decades. [6][7][8] Unfortunately, men often feel too embarrassed to seek help such as a semen analysis when encountering infertility issues, which sometimes leaves women to bear the burdens and pressures of infertility and infertility testing. 9 Even though the infertility examination for men (semen analysis) is much easier and less invasive than that for women (ovulatory function test, hysterosalpingogram, and saline infusion sonohysterography, etc.), less men seek medical assistance with infertility than women. 10 This imbalance may be attributable to cultural stigmas and men's' fears over loss of social status. 11,12 For all of these reasons, it is important to develop a point-of-care (POC) diagnostic device that allows men to evaluate sperm health in the privacy of their own homes.
The current diagnostic methods for examining male infertility are primarily rely on microscope-based examination and computerassisted semen analysis (CASA) systems described in the World Health Organization (WHO) Laboratory manual for the examination and processing of human semen. 13 Standard semen analysis comprises examinations of semen volume, pH, sperm concentration, count, motility, morphology, leukocyte count, and an immunobead test. Although standard methods could provide a holistic approach for determining sperm quality, such approaches require bulky and costly microscopes, equipment, and well-trained technicians. These requirements are expensive, often costing hundreds of USD. They also limit accessibility to semen analysis in resource-limit areas and push the process into the category of nonroutine health examinations. To meet the need for rapid, convenient, and easy-to-handle semen analysis tools, technologies for home-based semen analysis have recently emerged. Most of the current home-based semen analytical devices have relied on an image magnifier and an image interpretation application (APP) that can only return a few analytical parameters, for example, concentration, TMS, and motility. Furthermore, the plane image presentation of both the standard microscopic-based diagnostic methods and the smartphone-based evaluations are not useful for accurately tracking the three-dimensional spatial motion of spermatozoa because they lack z-axis observation capacity. 14 The relatively high price of smartphone-based analysis (usually >50 USD, but varied depending on economic environment) also presents an obstacle for continuous monitoring. Chemical-based semen analysis tools, for example, the FertilityScore kit and Sperm check, offer a relatively inexpensive and useful alternative testing option. The FertilityScore kit is a colorimetric kit based on the reduction of blue resazurin dye by motile spermatozoa, and the Sperm check tool is a lateral flow assay based on an antigen-antibody interaction. 15,16 It is worth noting that the physical-based methods usually detect the parameters of sperm concentration and motility separately while the chemical-based methods usually detect motile sperm concentration. 17 Here, we introduce a novel strategy that differs from those mentioned above. This approach, extremely inexpensive and easy-to-use, is a paper-based diagnostic device capable of detecting total motile sperm concentration (TMSC) based on the mitochondrial activity of motile spermatozoa. It can be used to distinguish low TMSC semen samples from those with normal TMSC levels.
Mitochondrial activity and functionality are the keys to sperm functionality and play a critical role in many sperm functions including basic motility, acrosome reaction, and final fertilization. 18 Studies have shown that defects in sperm mitochondria functionality may result in reduced sperm quality and male infertility. 19,20 Moreover, the activity of succinate dehydrogenase (SDH), one of the mitochondrial respiratory chain enzymes, was found to be highly correlated with sperm quality, including sperm concentration, motility, and vitality. [21][22][23] Therefore, testing mitochondrial functionality, especially the activity of SDH, may be a potential approach for determining sperm quality. In a previous study, we developed a paper-based diagnostic device for evaluating the mitochondrial activity of spermatozoa based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and used it to successfully examine the mitochondrial activity of porcine spermatozoa. 24,25 MTT is a yellow-colored tetrazolium salt that can be reduced into purplecolored formazan by SDH within the mitochondria of metabolically active cells. The amount of formazan produced is directly proportional to the metabolic activity of viable cells, indicating that MTT can be used as a powerful tool for providing information regarding basic sperm quality. Recent studies have shown that TMSC, which is calculated by multiplying the sperm volume by the percentage of sperm motility (% of grade a/b), may be a better parameter for determining male infertility than the parameters introduced by the WHO (2010). 26,27 Our MTT test strips determine TMSC based on mitochondrial functionality within spermatozoa, and may provide a more relevant determinant of sperm health than volume, concentration, or motility.
The goal of this study was to develop a truly POC diagnostic semen analysis device to distinguish semen samples with low TMSC from those with normal TMSC levels in order to assist men suffering from infertility issues. Here, we present a rapid, inexpensive, user-friendly, colorimetrically based POC sperm health testing device that can be used with low sample volumes. This device was used to test fresh, unprocessed clinical semen samples, and results were compared to those from traditional, clinical, microscope-based analysis. Based on our findings, we further demonstrate the potential for the development of an all-in-one semen analysis device that can simultaneously detect multiple parameters quickly and easily.  Table 1.

| MTT test strip semen analysis
The mechanism and colorimetric change features of the MTT test strip are illustrated in Figure 1. The large number of mitochondria    We used the ColorPicker APP to transform the colorimetric changes of the test strip into RGB scale. After adjusting for the "blank" color,   Figure 2(e). The sensitivity, specificity, PPV, NPV, and accuracy were calculated as 96%, 65%, 75%, 92.9%, and 80.9%, respectively. These data were also presented in the confusion matrix ( Figure 2(f)).  Figure 3.

| DISCUSSION
An estimated 15% of all couples suffer from infertility, 30 Table 3. The MTT test strip we developed is a cotton-based diagnostic tool that appears to be the most inexpensive and easy-to-use semen analytical device, T A B L E 2 Predictive value of the parameters mentioned in Table 1 Age ( [48][49][50][51] The poor prognostic power of the current diagnostic criteria may result from their lack of functional consideration of spermatozoa characteristics including DNA integrity and mitochondrial activity, which are key to successful fertilization. [52][53][54] In  Abbreviations: MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; SC, sperm concentration; TMSC, total motile sperm count.
our study, we could only choose the most correlated parameter, that is, TMSC, which examined multiple parameters to increase the potential for determining fertilization. 26

| Semen preparation
The semen samples were provided via patient masturbation following 3 to 5 days of sexual abstinence. The semen samples were ejaculated into an aseptic plastic cup and shelved for 30 min at room temperature for semen liquefaction. 13 All semen samples were obtained using sterilized cups and analyzed using our MTT test strips immediately following completion of clinical semen analysis. The time span between ejaculation and both the manual semen analysis and the MTT strip test was less than 1 h. and Spearman correlation analysis was used for the nonnormal distributed data. For results with significant correlation, multivariate logistic regression analysis was used to assess the predictive probability of male infertility using our MTT-test strip, and results were demonstrated using a ROC curve with AUC and a 95% confidence interval.

| MTT test strip fabrication
Based on the ROC curve, the Youden's J statistic was used to calculate the maximum potential effectiveness of the MTT test strip. The results were presented as a confusion matrix, wherein we calculated sensitivity, specificity, PPV, NPV, and accuracy.

| CONCLUSIONS
This study demonstrates a rapid, inexpensive, sensitive, equipmentfree, and easy-to-use POC semen analytical tool for assessing male fertility. Men can easily test their semen quality with a one-step evaluation process at home using the MTT test strip, which costs only $0.03 per test. Our results were clinically validated by comparing them to TMSC values from the medical records of 42 patients. As the problem of infertility becomes an increasingly pressing issue, implementation of a tool such as our MTT test strip could alleviate burdens and increase the efficiency of fertility testing and planned pregnancy, speeding the process by as much as an entire year compared to the generally accepted protocol.