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A simple and inexpensive home sperm test could be of considerable value to couples attempting to conceive and to men curious about their fertility potential. A two-strip lateral flow immunochromatographic diagnostic device that allows men to evaluate their sperm count at low cost in the privacy of their own homes is described.
The ability of SpermCheck Fertility to predict sperm counts obtained using a hemacytometer procedure based on standard World Health Organization methodology was assessed. Test results obtained by lay users were also compared with those obtained by trained laboratory professionals, and the ease of use of the device was evaluated in consumer studies.
A total of 225 semen samples were analyzed in the method comparison, and the performance of SpermCheck Fertility was excellent with over 96% of all samples correctly classified as normozoospermic (≥2 × 107 sperm/ml), oligozoospermic (5 × 106–2 × 107 sperm/ml) or severely oligozoospermic (<5 × 106 sperm/ml). Consumer studies with 164 lay users demonstrated that SpermCheck Fertility was easy to use. Lay users and laboratory professionals agreed 95% of the time when reading the same test independently. Overall, the correct response rate on a 20-question survey about the test was over 97%.
SpermCheck Fertility is a simple and reliable immunodiagnostic test that can quickly inform men as to whether their sperm count is normal, low or very low. This home test can assist couples in deciding whether to seek comprehensive clinical evaluation of the fertility status of the male partner.
Infertility is experienced by 10–15% of couples, and a male factor contributes approximately half of the time (Thonneau et al., 1991; Bhasin et al., 1994). Although understanding of the complexity of infertility is increasing among lay couples, at least in developed countries, there remains room for improvement in the public's awareness of the prevalence of male factor infertility and the need for early evaluation of both partners.
Although male subfertility is difficult to define and often cannot be diagnosed on the basis of sperm count alone, a simple diagnostic sperm test that men could perform in the privacy of their own home could be useful to couples concerned about their fertility. Herr et al. (1999) described an immunodiagnostic device capable of detecting low numbers of sperm. This lateral flow immunochromatographic test was named SpermCheck and employs monoclonal antibodies that recognize the spermatid and sperm-specific acrosomal protein SP-10.
Based upon several anatomical, genetic and biochemical characteristics, SP-10 has been validated as a selective analyte for measuring sperm concentrations. SP-10 mRNA and protein are expressed only in the testis where they appear after meiosis only in spermatids and in sperm (Kurth et al., 1991; Kurth et al., 1993) and are not found in other organs (Freemerman et al., 1994). The specificity of SP-10 gene [ARCV1] expression was also demonstrated in transgenic mice in which SP-10-GFP was detected only in testes (Reddi et al., 1999). The SP-10 protein is present in every male studied (Herr et al., 1992), is readily released from the sperm's acrosomal compartment with mild detergents (Herr et al., 1990; Foster et al., 1994) and remains soluble in aqueous solutions, making this analyte amenable to detection with pairs of monoclonal antibodies that capture the SP-10 protein (Shen et al., 1993). Using monoclonal immunoreagents, a direct relationship exists between sperm number and signal strength in ELISA assays that measure SP-10 concentrations (Herr et al., 1999), indicating that an immunodiagnostic device could be developed using the SP-10 analyte.
The present report describes the optimization as well as clinical and consumer testing of SpermCheck Fertility, a convenient, easy to use, rapid home test useful for detecting different levels of sperm in semen samples.
The SpermCheck Fertility test is a rapid qualitative test designed to detect the presence of sperm in human semen. The test employs solid-phase chromatographic immunoassay technology and consists of two immunochromatographic strips in a single cassette. One test strip is calibrated to give a positive result if the sperm concentration is 2 × 107 sperm/ml or greater, and the other strip gives a positive result at concentrations of 5 × 106 sperm/ml and above. The operation of the test is similar to the recently described SpermCheck Vasectomy test (Klotz et al., 2008) and is illustrated in Fig. 1. The devices employ a pair of monoclonal antibodies which bind distinct, non-overlapping epitopes on the sperm acrosomal antigen SP-10. Following a 20 min semen liquefaction period, a defined volume of semen (100 µl) is sampled with a calibrated pipette and mixed with a detergent containing buffer. The detergent solubilizes the sperm's acrosomal membranes releasing the SP-10 protein into the buffer. Four drops of the buffer solution are then applied to sample wells on each side of the device. The solution rehydrates colloidal gold-conjugated monoclonal antibody present on an absorbent pad inside the cassette, so that it can bind to the solubilized SP-10 antigen. Next, the solution migrates along nitrocellulose strips by capillarity. A second monoclonal antibody, dried onto the nitrocellulose membrane at the test line (T in Fig. 1), captures the gold-antibody–SP-10 complexes as they flow along the membrane, resulting in the appearance of a visible reddish color at this position. The test results are read 7 min after the addition of diluted semen to the sample wells. Representative results and their interpretation are presented in Fig. 2.
The World Health Organization (1999) has defined a reference value of 2 × 107 sperm/ml or more for a ‘normal’ semen analysis. This concentration was therefore selected for the detection limit of one of the test strips in SpermCheck Fertility. Because the risk of male infertility and/or subfertility increases further with decreasing sperm count (Guzick et al., 2001; van Zyl and Menkveld, 2006), a home sperm test that gave additional information about sperm counts below the WHO reference value would provide more value to the consumer. Therefore, a test that would inform men not only as to whether they are normospermic or oligospermic, but also inform those men who are oligospermic as to the severity of their oligospermia was developed. Although there is no consensus definition of severe oligospermia, a sperm concentration of 5 × 106/ml was chosen as the detection limit for the second test strip on the basis of a detailed review of the recent literature on the evaluation of male fertility. Many recent publications have defined severe oligospermia as a sperm concentration of <5 × 106/ml (Mahajan et al., 1999; Yao et al., 2001; Gupta and Kumar, 2002; Ishikawa et al., 2008; Su et al., 2008; Zhang et al., 2008; Ng et al., 2009). A review of the past 10 years of literature on male fertility shows this to be the most commonly used definition of the term ‘severe oligospermia’. The literature thus supports the use of 5 × 106 sperm/ml as a reasonable definition of a very low sperm count even if some authors employ slightly differing definitions.
The literature also demonstrates the usefulness of distinguishing between low and very low sperm counts. Several recent publications note that significant numbers of fertile men have sperm concentrations <2 × 107/ml, and illustrate that the likelihood of infertility increases as sperm concentration decreases further below the WHO reference level (Ombelet et al., 1997; Zinaman et al., 2000; Gunalp et al., 2001; Guzick et al., 2001; Menkveld et al., 2001; Nallella et al., 2006). The predictive value of sperm concentration increases when the sperm count is extremely low (Check, 2006). Thus, authors are increasingly suggesting lower thresholds for sperm concentration in the clinical evaluation of male fertility status (Ombelet et al., 1997; Gunalp et al., 2001; Guzick et al., 2001). In fact, Guzick et al. (2001) recommend three classifications—fertile, subfertile and indeterminate. In summary, although the current WHO reference value is useful in establishing general categories of normospermia and oligospermia, further information on the extent of oligospermia is valuable to users of a home sperm count test, and therefore SpermCheck Fertility was designed as a two-strip test to tell the user whether his sperm count is normal, low or very low.
Semen samples were collected from healthy normal men and vasectomized subjects. Sperm from normal donor semen were counted using a Neubauer Hemacytometer (see below) and pooled. Pooled seminal fluid from confirmed azoospermic vasectomized donors was used to dilute the normal semen to obtain test samples over a range of sperm concentrations, which were then used to determine the sensitivity of SpermCheck Fertility test devices. When parameters for producing devices with the desired sensitivity had been established, production-scale lots were manufactured. Sensitivity and precision studies confirmed that the test strips of these devices gave appropriate positive or negative results with semen samples ranging in sperm concentration from < 1 × 106 to > 3 × 107/ml (data not shown). Potential cross-reactivity and interference by bacteria, other body fluids and other possible contaminants of semen samples was ruled out by testing positive and negative samples that had been spiked with these substances at concentrations well in excess of those likely to be found in the semen of men using the test (Supplementary Table SI).
The performance of SpermCheck Fertility was compared with the generally accepted standard procedure of determining sperm concentration microscopically with a hemacytometer (the predicate device). The study used semen samples from healthy male donors, and the protocol was reviewed and approved by the University of Virginia Institutional Review Board for Health Sciences Research. A diverse cohort of men was recruited for the study, with particular attention to the inclusion of men who either were in couples who had experienced difficulty conceiving, knew of or suspected subfertility for other reasons, or expressed an interest in knowing their sperm count.
After giving informed consent to participate in the study, subjects were provided with a semen collection cup and advised to abstain from ejaculating for a minimum of 48 h and a maximum of 7 days. These men produced semen samples at home and brought these to the study site within 1 h, where they were tested with SpermCheck Fertility by a laboratory professional. Previous studies had established that storage of semen samples with a wide range of sperm concentrations at room temperature for up to 6 h had no effect on SpermCheck Fertility test results (data not shown). Testing for this study was completed in well under 6 h from the time when the sample was produced by the donor. The sperm concentration of each semen sample was also determined by counting with a hemacytometer following a standard operating procedure (see below). The study was blinded in that neither the person doing the hemacytometer count nor the person performing the SpermCheck test had any knowledge of the result of the other procedure.
In order to increase the representation of negative samples in the study cohort, 12 subjects who had undergone vasectomy years earlier were included. An additional 33 de-identified post-vasectomy clinical specimens were obtained from the Urology Clinic at the University of Virginia Medical Center for inclusion in the method comparison. These clinical samples were treated exactly as the donor samples in the study. Since these semen samples came from men who had undergone vasectomy only 2–4 months earlier, it was expected that a subset would not be azoospermic and would thus provide a fair test of the effectiveness of the left (5 × 106/ml) test strip in obtaining correct negative results for samples with very low, but non-zero sperm counts. Indeed, 12 of these samples had sperm present on microscopic examination, with counts ranging from ~2000 to 200 000/ml (data not shown).
The methodology employed to accurately determine sperm concentration in semen samples is based on WHO guidelines (1999) and has been described in detail elsewhere (Klotz et al., 2008). Briefly, wet preps of neat semen samples were prepared and examined microscopically to estimate approximate sperm concentrations.
Samples with relatively abundant spermatozoa were appropriately diluted with bicarbonate/formalin buffer, which immobilized the sperm, and two independent counts (with at least 200 sperm in each count) were performed using both chambers of an Improved Neubauer Hemacytometer (Hausser Scientific, Horsham, PA, USA). If the two counts were not within ±5% of their average, the count was not considered valid and was repeated with a new dilution of the sample. To calculate the concentration of sperm in the sample expressed as spermatozoa per milliliter, the average number of sperm in a 1 mm square was first determined by dividing the total number of sperm counted by the number of squares counted. This quotient was multiplied by the dilution factor, if any, and the result was multiplied by 104.
Semen samples with low sperm concentrations were not centrifuged before counting and were diluted either minimally or not at all (if all sperm present were already immotile). The hemacytometer was placed in a humidified chamber for 10 min to allow sperm to settle. Two independent counts were performed and the sperm concentration was calculated as above. If fewer than 200 sperm were counted in all 1 mm squares on both sides of a hemacytometer (18 squares total) and the sample had not been diluted, then the sperm concentration was <111 000/ml. Counts this low must be considered to be only approximate. However, knowledge that a sample contains <111 000 sperm/ml is more than adequate to assess the performance of a test device with a decision level of 5 × 106/ml.
Three independent consumer field evaluations of the SpermCheck Fertility test device were conducted to determine the test performance when used by unassisted lay users following the instructions provided in the package insert (see Supplementary data). The first study assessed the performance of the consumer compared with the trained professional, using the consumer's own semen sample. Each subject recruited to participate in this study received a semen collection cup and was asked to produce a semen sample at home after abstaining from ejaculation for a minimum of 48 h and a maximum of 7 days. Subjects returned to the test site within 1 h of producing the sample, where they were provided with a SpermCheck Fertility kit and where they performed the test. After completing the test, the consumer answered a questionnaire about the use of the kit and the interpretation of the results. The participant's test result was read simultaneously by a professional so that a lay versus professional comparison of the test result reading could be made. The laboratory professional and the consumer read and recorded the test results independently, and test results were not discussed until after the consumer had completed the test and answered the questionnaire.
A second study evaluated the ability of a separate group of consumers to perform the test using provided semen samples of known concentration. Samples with the desired test concentrations were prepared by diluting a pool of normospermic semen samples with a pool of seminal fluid from vasectomized donors. The concentrations tested were 1.25 × 106, 7.5 × 106, 3 × 107 and 6 × 107/ml, such that negative, weak positive and strong positive results were expected in the test windows. Subjects performed the SpermCheck Fertility test at the study site without assistance, recorded the results and answered the same questionnaire used in the first consumer study. The test samples were coded so that the subjects had no knowledge of what test results to expect.
The third consumer study was designed solely to assess the ease of use of the SpermCheck Fertility test kit in the home setting. Thirty-two subjects who had not taken part in the other consumer testing received a SpermCheck Fertility test kit. These men were asked to produce a semen sample at home after abstaining from ejaculation for a minimum of 48 h and a maximum of 7 days, and to perform the SpermCheck Fertility home test according to the supplied instructions. After completing the test, each consumer answered a questionnaire about the use of the kit and the interpretation of the results. The subjects then returned the completed questionnaires with the test results to the test site.
The questionnaire included eight statements about the ease of use of the kit. Subjects were asked to indicate their agreement or disagreement with each statement on a scale of 1–5, with 5 indicating strong agreement and 1 indicating strong disagreement. There were also six multiple choice questions about the testing procedure, and six true or false questions about reading and interpreting the results. Participants in all three studies varied in age, education levels, occupation, race and ethnicity. Given that the primary purpose of this study was to see if the lay person could perform the home test correctly without any assistance or guidance from trained health care professionals, participants did not receive a demonstration of the use of the kit.
The data from the first two consumer studies were analyzed to determine whether untrained, unassisted lay users could obtain acceptable results with the test, and the questionnaire responses were used to assess the ease of use of the kit and the adequacy of the test instructions.
There were 194 men who gave their informed consent to participate in the study. Two of the samples were excluded from the analysis, one because the subject did not observe the required 48 h abstinence period and one because the volume of the ejaculate was insufficient for testing. As noted above, 12 of the study participants had undergone vasectomy and 3 reported clinically diagnosed subfertility or infertility. Another 11 participants reported that they and their partners had experienced difficulty conceiving, with the periods of unprotected intercourse ranging from a few months to several years. Of these men, seven had consulted physicians or other healthcare providers about their fertility concerns and four had not. There were five other men who suspected that they may have a fertility issue for reasons such as varicocele, viral illness, testicular cancer or trauma. One study participant was using self-administered testicular heating to reduce his viable sperm count. The remaining semen donors had no specific knowledge of their current fertility status, although 34 had fathered children in the past. Of the test subjects, 35 reported that they chose to participate in the study primarily because they were curious about their sperm count. Thus, the study cohort was representative of men who might be expected to use an over-the-counter home sperm count test as a first step in assessing their fertility status. With the addition of 33 de-identified clinical post-vasectomy semen samples, a total of 225 specimens were analyzed in this study.
Agreement between the hemacytometer counts and the SpermCheck results was excellent, whether analyzed in terms of the performance of each of the two test strips independently or in terms of the overall accuracy of SpermCheck Fertility in predicting whether the sperm count was normozoospermic (i.e. ≥20 × 106/ml), oligozoospermic (<20 × 106/ml) or severely oligozoospermic (<5 × 106/ml). As shown in Table I, the left (5 million/ml) test strip of the SpermCheck Fertility test demonstrated 98% agreement when compared with counting by hemacytometer. All of the five discrepant samples were false positives. That is, a test line was seen on the SpermCheck Fertility device for a sample with a sperm concentration <5 × 106/ml. Table I shows that the right (20 million/ml) test strip of the SpermCheck Fertility test also demonstrated 98% agreement when compared with counting by hemacytometer. Again, all of the four discrepant samples were false positives (a test line was seen for a sample with a sperm concentration < 20 × 106/ml). Thus, SpermCheck was 100% effective in identifying semen samples with sperm counts above the WHO reference value for a normal semen analysis (Table II).
Because the SpermCheck Fertility device is designed to let men determine whether their semen sample is normozoospermic, oligozoospermic or severely oligozoospermic, it is useful to examine not only the performance of each test strip independently, but also the performance of the device as a whole in achieving this overall goal. As seen in Table III, of the 225 semen samples included in this study, 156 had hemacytometer-determined sperm counts of ≥20 million/ml and 56 had sperm counts <5 million/ml. The remaining 13 samples had sperm counts between 5 million and 19.9 million/ml. SpermCheck Fertility accurately classified all 156 normozoospermic samples, 10 of the 13 oligozoospermic samples and 51 of the 56 severely oligozoospermic samples. Thus, the overall accuracy of the test was 96%.
There were 61 participants who tested their own semen samples while being observed by a laboratory professional and obtained the following SpermCheck Fertility results: 52 normal, 5 intermediate and 3 very low. One test was invalid (no control line was seen, indicating that the test had not run properly and the result could not be interpreted). There was 95.1% (58/61) agreement between the results obtained by the lay users and those obtained by laboratory professionals reading the same test. There were 41 subjects who tested prepared semen samples with pre-determined sperm concentrations and obtained the expected test result in all cases. Since each participant tested four coded semen samples, this represents a total of 164 correct tests. There were 32 men who participated in the home use study, and the SpermCheck Fertility results for these subjects were 27 normal, 3 intermediate and 2 very low (data not shown).
The combined responses to the questionnaire of all three consumer studies (134 subjects total) are presented in Table IV. There was very good to excellent agreement for all questions, with at least 93% correct responses or agreement. The data presented here support the conclusion that lay users can correctly follow the labeling instructions, obtain acceptable test results and interpret the meaning of these results when using SpermCheck Fertility.
SpermCheck Fertility was highly accurate in predicting whether sperm count was high (≥2 × 107/ml), very low (<5 × 106/ml) or intermediate between these two threshold values. There were no false-negative test results observed for either the 20 or the 5 million/ml test strips. The hemacytometer-determined sperm counts of the five semen samples that gave false-positive results with the 5 million/ml strip were 4.76 × 106, 4.60 × 106, 2.50 × 106, 2.24 × 106 and 5.96 × 105/ml. Thus, two of the false positives were very close to the decision level of the test strip. It is to be expected that samples with sperm counts this close to the sensitivity level of the device could give either positive or negative test results. The hemacytometer-determined sperm counts of the four semen samples that gave false-positive results on the 20 million/ml test strip were 4.76 x 106, 9.98 × 106, 1.08 × 107 and 1.38 × 107/ml. One possible explanation for false-positive results with an immunochromatographic test could be that degenerate sperm or immature forms could contain quantities of SP-10 antigen that contribute signal in the assay while not being recognized as spermatozoa in the microscopic counts. Nonetheless, the overall accuracy of the device was over 96% and it is noteworthy that SpermCheck Fertility correctly identified 100% of the semen samples that had sperm counts greater than or equal to the WHO reference value for a normal semen analysis.
The evaluation of a man's fertility potential is a complex problem since it is impacted by numerous factors. Fertility or infertility obviously cannot be predicted on the basis of sperm count alone. Nonetheless, a home diagnostic that provides accurate and reasonably precise information on sperm count is of value to couples concerned about their ability to conceive. This is why a two-strip test was developed to not only inform men as to which side of a single reference value their sperm count falls on, but to provide additional data as to the severity of oligozoospermia in those men whose counts are <2 × 107/ml. Since other sperm abnormalities may be more likely as sperm count declines, it is important to alert men with very low sperm counts to the need for a comprehensive semen analysis and clinical evaluation.
It is important to note that SpermCheck Fertility is an over-the-counter home test rather than a clinical test. It is intended as a front-line self-diagnostic to direct men to the fertility specialist. In this light, it would not have been appropriate to limit the clinical testing of the device to men who have already attended a fertility clinic and had a comprehensive workup including multiple laboratory semen analyses, since such individuals are among those least likely to use a home sperm test. Rather, SpermCheck Fertility is intended for use by men of all ages who have no present knowledge of their fertility status. Some users may be men in committed relationships who have experienced difficulty fathering a child. Others may suspect infertility for various reasons including, but not limited to, a prior medical history. In addition, users of SpermCheck Fertility are also expected to include, for example, young men who do not suspect infertility for any reason but simply are understandably curious about whether they have normal sperm levels, and older men who have fathered children in previous relationships but wish to know whether their fertility status may have changed. Thus, the expected users of the SpermCheck Fertility device are a broad group, much wider than just the subset of men who are partners in a couple that is already having difficulty conceiving. It is highly likely that many users of SpermCheck Fertility will be men in couples who have not yet tried to have children, or have not yet tried for long enough to be considered to have ‘experienced difficulty’ according to current clinical guidelines which define infertility only after one full year of unprotected intercourse. It is also anticipated that many users will be men who have no a priori reason to suspect that they may be subfertile, but simply wish to confirm that their sperm count is within the normal range. Clearly, there will be many relevant situations where individuals or couples feel a need to test for male fertility beyond the subset of those seeking assistance from a fertility clinic.
In consideration of the above, a wide range of participants were sought when selecting the SpermCheck Fertility study cohort. As anticipated, a large number of the study participants chose to enroll specifically because they were interested in knowing their own sperm count. Indeed, 116 subjects listed a desire to know their sperm count as a reason for joining the study, and 35 of these men listed it as a major reason for participating. Five of these ‘curious’ men suspected infertility even though they had not experienced difficulty conceiving, and listed medical histories such as varicocele, viral illness, testicular cancer and trauma as the reasons for their suspicion. These subjects are representative of many otherwise normal men in the general population who may suspect that their sperm counts are depressed for these reasons and others including, but not limited to, a past history of disease, alcohol or drug abuse, steroid use, or toxin exposure (Emanuele and Emanuele, 1998; Pasqualotto et al., 2004; Dorfman, 2008). However, it is also noteworthy that 27 of the 35 men in the study cohort who listed curiosity about their sperm counts as their major reason for enrolling did not report any previous history of difficulty in conceiving or any knowledge or suspicion of a fertility problem. Of these men, six reported that they had previously fathered children, including one who had consulted a physician before his wife became pregnant after 5 months of unprotected intercourse (without any reported fertility treatments). This couple is typical of those who are likely to avail themselves of an over-the-counter sperm test.
Finally, it is also noteworthy that sperm counts of <2 × 107/ml were found in 11.2% (15/134) of the study participants who did not report any difficulty conceiving, disclose any knowledge or suspicion of subfertility, or express a major interest in knowing their sperm count. This rate of oligozoospermia is somewhat lower than that found among the 46 subjects who did report difficulty conceiving, disclose knowledge or suspicion of subfertility, or list curiosity about their own sperm count as a major reason for enrolling in the study (9/46 = 19.6%). However, the unsuspected cases of oligospermia demonstrate that even men who may not perceive a need to use a product such as SpermCheck Fertility can nonetheless have sperm counts below the WHO reference level for a normal semen analysis and underscore the fact that the study cohort is representative of the intended users of a home sperm test in terms of the distribution of actual sperm counts.
Other home sperm tests are available (Bjorndahl et al., 2006). However, SpermCheck Fertility offers advantages in being a simple and inexpensive test based on the exquisitely spermatid and sperm-specific biomarker SP-10 (Foster et al., 1994; Freemerman et al., 1994; Herr et al., 1990, 1992; Kurth et al., 1991, 1993; Shen et al., 1993; Herr, 1999; Reddi et al., 1999). The SpermCheck Fertility test also offers an additional advantage of monitoring the severity of oligospermia with a two-strip readout.
This research was supported by National Institutes of Health Grant U54 29099, Fogarty International Center Grant D43 TW/HD 00654 and a grant from the Commonwealth of Virginia Technology Research Fund as well as research contracts from ContraVac, Inc. (to J.C.H.) with manufacturing and development expenses underwritten by Princeton BioMeditech Corporation.
We would like to gratefully acknowledge all of the individuals who participated as research subjects in these studies and as semen donors for the basic scientific work that led to the development of the SpermCheck test.
Conflict of interest: M.A.C., K.L.K., K.-a.K., H.Y.C., J.K., S.S.H., C.J.F. and J.C.H. have a financial interest in or other relationship with ContraVac, Inc. or Princeton BioMeditech Corporation.