TECHNIQUES AND INSTRUMENTATION

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1 FERTILITY AND STERILITY VOL. 78, NO. 3, SEPTEMBER 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. TECHNIQUES AND INSTRUMENTATION Lack of standardization in performance of the semen analysis among laboratories in the United States Brooks A. Keel, Ph.D., H.C.L.D., a Travis W. Stembridge, M.D., a Gilbert Pineda, b and Nicholas T. Serafy, Sr., M.A., H.C.L.D. b Women s Research Institute, University of Kansas School of Medicine-Wichita, Wichita, Kansas and American Association of Bioanalysts Proficiency Testing Service, Brownsville, Texas Received December 11, 2001; revised and accepted February 22, Presented at the 57th Annual Meeting of the American Society for Reproductive Medicine, Orlando, Florida, October 20 25, Reprint requests: Brooks A. Keel, Ph.D., H.C.L.D., Associate Vice President for Research, Florida State University, 109 Westcott Building, Tallahassee, Florida (FAX: ; bkeel@fsu.edu). a Department of Obstetrics and Gynecology, Women s Research Institute, University of Kansas School of Medicine- Wichita. b American Association of Bioanalysts Proficiency Testing Service /02/$22.00 PII S (02)03296-X Objective: To determine the level of standardization in performance of the semen analysis among clinical laboratories in the United States. Design: A survey was mailed to laboratories requesting information about the laboratory and performance of the semen analysis. Responses were received from 536 laboratories. Setting: Clinical laboratories enrolled in the American Association of Bioanalysts Andrology Proficiency Testing Program. Patient(s): None. Intervention(s): None. Main Outcome Measure(s): Agreement among laboratories. Result(s): Sixty-one percent of respondent laboratories were part of an assisted reproductive technology program. The laboratories perform less than 50 (53%), less than 10 (25%), or less than 5 (16%) andrology laboratory procedures per month. The laboratories routinely report sperm count (94% of laboratories), motility (95%), morphology (85%) and forward progression (69%), and semen volume (96%) as part of the semen analysis. Only 64% of laboratories routinely report abstinence, and 60% of laboratories indicate the criteria used for sperm morphology on the report form. The most common lower limits of normality for sperm count and motility were /ml (77% of laboratories) and 50% (59% of laboratories), respectively. Few laboratories performed quality control for sperm counts (29%), motility (41%), and morphology (41%). Conclusion(s): These data indicate a significant lack of standardization in the performance and reporting of semen analyses among laboratories in the United States. (Fertil Steril 2002;78: by American Society for Reproductive Medicine.) Key Words: Semen analysis, proficiency testing, andrology, clinical laboratory The semen analysis is perhaps the most important clinical laboratory test that is used in the diagnosis of male factor infertility. Although in theory the semen analysis is reasonably simple to perform, in practice the reported lack of standardization (1, 2), the wide variation among laboratories (3 7), and the urgent need for quality control (8 13) has led some to consider the semen analysis inaccurate and unreliable and to refer to it as the neglected test (1). The lack of standardization associated with the semen analysis has made it difficult for physicians to compare semen analysis results among laboratories. This is especially problematic when treating infertile couples who were referred from other clinics and who may have had fertility testing performed in other andrology laboratories. For example, because of disagreements between laboratories, a patient could very well be classified as normal by one laboratory and infertile by another (5). The World Health Organization (WHO), in response to a growing need for standardization of procedures for the examination of semen, has published a series of laboratory manuals to encourage the use of standard procedures to establish reference (i.e., normal ) values for the semen analysis (14 16). Although these manuals are widely recognized as the gold standard for semen testing worldwide, and several investigators have advocated the standard- 603

2 ization of the methods used in the andrology laboratory (1, 3, 8, 17) and have indicated the importance of quality control (9 13, 18), in many cases andrology testing is not comprehensive, technology and technical expertise is minimal, and quality is compromised (2, 3, 17). In this study, we surveyed more than 500 clinical laboratories performing semen analysis in the United States in an attempt to ascertain the consistency and variation in the performance of this test. MATERIALS AND METHODS The American Association of Bioanalysts (AAB) Proficiency Testing (PT) Service (Brownsville, TX) began offering comprehensive external quality control PT programs in In May 1996, the AAB PT Service made available PT programs for the clinical laboratory specialities of andrology (7) and embryology. In April 2000, a questionnaire survey (sample available upon request) was mailed to all laboratories participating in the Andrology Module of the AAB PT Service. The questionnaire was designed to obtain information on the type of laboratory and range of services offered, level of training of the testing personnel, testing methodology, normal ranges used for various semen and sperm parameters, and quality control issues. The identity of the specific laboratory and personnel submitting the survey was kept completely anonymous throughout the study. Of the 621 laboratories enrolled in this program, 536 laboratories returned the completed survey (86% return rate). In some cases, laboratories were grouped for comparison according to the type of laboratory or the number of routine semen analyses performed per month. These groups were then compared statistically using Fisher s exact test or G-test as appropriate. RESULTS One-third of the respondents characterized their clinical laboratory services as being general clinical laboratory (32%), one-third as andrology and embryology conjoint (31%), with the remainder being andrology alone (14%), andrology and embryology separate (13%), embryology alone (6%), and other (4%). The respondents further characterized their respective laboratories as being either a physician office (34%), general hospital (19%), independent (16%), hospital-based andrology/embryology (14%), officebased andrology/embryology (5%), or other (12%). Sixty-one percent of respondents indicated that their laboratory was an integral part of a program that performed assisted reproductive technology (ART) procedures. Most (94%) of the respondents indicated that their laboratories were accredited (2% not accredited and 4% not stated), and of these, 35% were accredited by their state s Department of Health or Health Care Financing Administration, 29% by the TABLE 1 Average andrology laboratory procedures performed by the participating laboratories. Procedure No. of labs Procedures performed per month Mean SE Median Range Routine semen analysis ,200 IUI preparation ,000 Morphology only Sperm count only ,944 Wet preparation All procedures combined ,775 College of American Pathologists, 16% by the Joint Commission on Accreditation of Healthcare Organizations, 5% by the Commission on Office Laboratory Accreditation, and 15% by multiple agencies. On average, each laboratory employed 3.44 individuals (range, 1 25) to perform semen analysis. Most of these testing personnel possessed at least a bachelor s degree (61%), while others possessed as the highest degree a high school diploma (1%), associate s degree (15%), master s degree (10%), doctoral degree (10%), or other degree (3%). Seventy-seven percent of the testing personnel performing semen analysis in this study were board certified, with the majority of these being certified medical technologists (53%) or medical laboratory technicians (15%). The number of andrology laboratory procedures performed each month by the participating laboratories ranged widely from as few as one procedure per month to more than 2,000 per month (Table 1). However, most laboratories (53%) reported performing fewer than 50 combined andrology laboratory procedures per month, while 25% of laboratories reported performing less than 10 procedures per month, and 16% of laboratories performed less than 5 procedures per month. We next examined the percentage of laboratories reporting various parameters on a routine semen analysis. Greater than 90% of participating laboratories indicated that they routinely report sperm motility, sperm count, and semen volume as part of the semen analysis, while 85% of laboratories routinely report sperm morphology (Table 2). Although 69% of laboratories routinely evaluate and report forward progression, only 11% routinely report velocity (Table 2), in part because of the relatively low percentage of participants (16%) who incorporate computer-assisted semen analysis into their laboratories. Seminal ph, time required for complete liquefaction of semen, and the assessment of seminal viscosity were routinely reported by 63%, 73%, and 82% of laboratories, 604 Keel et al. Lack of standardization in semen analysis Vol. 78, No. 3, September 2002

3 TABLE 2 Parameters reported on routine semen analysis. Parameters Laboratories reporting No. % Motility Count Total motile count Velocity Morphology Viability Forward progression Volume ph Liquefaction Viscosity Abstinence Time of collection Time of analysis respectively. More than 85% of laboratories indicated that they report the time of specimen collection and the subsequent time of semen analysis. Seventy-six percent of participating laboratories reported that they always provided patients with written instructions on specimen collection, compared with 10% who never and 10% who sometimes provided instructions (4% did not specify). Approximately one-third (37%) of laboratories reported routinely using the hemocytometer to quantitate sperm, while another one-third (35%) used the Makler counting chamber (Selfi-Medical Instruments, Haifa, Israel). An additional 4% of laboratories used the Cell-Vu (Millenium Sciences Inc., New York, NY) and 15% used the Micro-Cell (Conception Technologies, San Diego, CA) disposable counting chambers. Four percent of laboratories used other, less common counting chambers, and 5% of laboratories reported routinely using more than one type of counting chamber. As presented above, 85% of laboratories indicated that they perform morphology assessments as part of the routine semen analysis. Of the laboratories performing ART procedures, less than one-half (46%) indicated that they perform morphology assessments on the ejaculate processed for in vitro insemination of oocytes. Most laboratories (83%) evaluated 100 sperm per slide or less when assessing sperm morphology (5%, 5%, and 6% of laboratories evaluated 200, 300, and 400 or more sperm per slide, respectively). A myriad of different morphology stains were reported, with the Diff Quick (Allegiance Healthcare Corp., McGraw Park, IL) being the most common (30% of laboratories). Similarly, more than 20 different criteria were used for sperm morphology assessment, with strict criteria being the TABLE 3 Lower limits of normality for sperm morphology reported by participating laboratories. % Normal Laboratories reporting No. % most common (33% of laboratories), followed by WHO criteria (23%) and American Society of Clinical Pathologists (ASCP) (19) criteria (10%). Interestingly, 28% of participants either made no response to this question or indicated that they were unaware of the morphology criteria employed by the laboratory. Furthermore, only 60% of laboratories indicated the criteria used on the actual semen analysis report. As might be expected from the variety of morphology criteria employed, a wide range of cutoffs were reported for normal morphology values (Table 3). The lower limits of normality for sperm count and motility reported by the participating laboratories are shown in Table 4. Most laboratories (77%) used /ml sperm as the cutoff for normal sperm count, while 59% of laboratories used 50% as the cutoff for normal sperm motility. However, 62 reporting laboratories used values as high as /ml for the lower limit of normal sperm counts, and 35 reporting laboratories used 70% 80% as the lower limit for normal sperm motility. TABLE 4 Lower limits of normal for sperm count and motility reported by participating laboratories. Sperm count ( 10 6 /ml) Laboratories Laboratories No. % % Sperm motility No. % FERTILITY & STERILITY 605

4 TABLE 5 Percentage of participating laboratories performing internal quality control (QC) procedures. Count Motility Morphology Did not specify Does not perform QC Performs QC Daily QC Weekly QC Monthly QC To determine if the observed variation in accepted cutoff ranges for normal sperm count and motility could be explained by the inclusion of nonspecialized or inexperienced laboratories performing semen analysis, we compared the results obtained from andrology and/or embryology laboratories (i.e., ART laboratories) with all other laboratories (i.e., non-art laboratories). We observed no statistical differences between the number of ART and non-art laboratories using /ml as the cutoff for normal sperm count. There was a statistical difference between these two groups in the number of laboratories using 60% as the cutoff for normal sperm motility. However, a greater proportion of ART laboratories used values 60% as the cutoff for normal sperm motility, compared with non-art laboratories. Lastly, we compared the results from laboratories performing 5 routine semen analyses per month with laboratories performing 5 per month regardless of the type of laboratory. We observed no statistical differences between these two groups in the proportion of laboratories choosing /ml or 60% as the cutoff for normal sperm count and motility, respectively. Just over half of the reporting laboratories considered ml to be the lower limit of normal for semen volume. Most laboratories considered the lower limit of normal seminal ph to range between 7.1 and 7.3, while 66% considered an upper limit of normal seminal ph to range from 7.8 to 8.0. The percentage of participating laboratories performing internal quality control (QC) procedures is shown in Table 5. Only 29% of laboratories performed QC for sperm counts; 41% performed QC for motility, and 41% for sperm morphology. Approximately one-half of laboratories performing QC carried out this procedure daily for sperm count and monthly for sperm motility and morphology, respectively. Thirty-four percent of participating laboratories indicated that they either were not familiar with the WHO laboratory manual (14 16) or did not have a copy of the manual in their laboratories, while 33% and 22% reported owning the third (15) or fourth edition (16), respectively. DISCUSSION Recently we reported the results obtained from participation in the AAB national PT program in andrology (7). Reported sperm concentrations among participating laboratories varied by as much as two orders of magnitude; for example, compare a reported sperm concentration of /ml in one laboratory with /ml in another for the same sample (7). Furthermore, a high degree of variation among laboratories participating in the morphology PT program was also discovered, with coefficients of variation ranging from 15% to 93% (7). This study (7) supports the urgent plea for standardization of semen analysis methodologies expressed by others (1, 2, 8, 11) and the recommendation that laboratories performing the semen analysis reduce the variation observed by adhering to accepted standards. In the study presented herein, we have extended our earlier observations by assessing the practices and methods employed by these laboratories in the performance of the routine semen analysis on their patients. Several other studies have attempted to ascertain various levels of standardization in the performance of semen analysis by surveying laboratories performing this test. Chong et al. (1) analyzed the actual semen analysis reports from 64 laboratories in the United States, Baker et al. (17) randomly surveyed 129 acute care community hospitals in the United States, and Ombelet et al. (2) questioned 170 specialized fertility centers and ART programs from 40 countries around the world. Our study population was obtained from laboratories participating in an andrology PT program (7). Each of these studies exhibits potential sample bias owing to the type of laboratory surveyed (i.e., specialized fertility centers vs. general clinical hospital laboratories), which may reflect the type of clinical services offered and the level of expertise provided by the participating laboratory. In our study, for example, 64% of participating laboratories characterized their services as andrology, embryology, or both, while the remainder characterized themselves as general clinical laboratories. It should also be pointed out that the laboratories surveyed in the study herein were chosen from laboratories that have volunteered to participate in a nationwide PT program, which at least suggests a heightened awareness of QC, quality assurance, and other federal standards governing clinical laboratories performing moderate- and high-complexity testing in the United States (20). Specific differences in results among the four studies referred to above (1, 2, 7, 17) could be related to the uniqueness of the laboratories surveyed. However, we observed no clear relationship between our findings and the level of experience (i.e., laboratories performing andrology and/or embryology procedures) or the number of procedures performed. Regardless of the source of the information, or the characteristics of the laboratory, all of the above studies observed wide variation in the reporting of semen analysis 606 Keel et al. Lack of standardization in semen analysis Vol. 78, No. 3, September 2002

5 data and a lack of standardization in the performance of this test. Arguably, the most important parameters reported on a routine semen analysis are sperm concentration, motility, and percentage of normal morphological forms. In our study, 85% or more of the participating laboratories indicated that they evaluate these parameters on their routine semen analysis. In contrast, only 47%, 81%, and 78% of participating laboratories reported sperm count, motility, and morphology, respectively, in the Baker et al. (17) study, while Chong et al. (1) reported that 48% of laboratories reported motility and 53% of laboratories reported morphology. Therefore, there appears to be little consensus among laboratories as to what parameters constitute a routine semen analysis. Greater than 85% of participating laboratories herein reported that they included on the semen analysis report information related to the time of specimen collection and time of analysis, which can provide valuable information related to the evaluation of sperm motility and forward progression. Time of specimen collection was reported in only 28% of participating laboratories in the Chong et al. (1) study. It is also well known that the period of sexual abstinence can have profound effects on semen parameters (21), yet only 64% of laboratories reported this information on the routine semen analysis. Chong et al. (1) reported that only 19% of laboratories gave patients written instructions, while 76% of laboratories participating in our study indicated that they always provided written instructions to patients on semen collection procedures. Thus, providing patients with adequate written instructions on collection procedures may help to improve the quality of the specimen received and the accuracy of the resulting report (22). Although there has been reported a divergence of opinion as to what can be considered as normal semen (23), perhaps the most widely accepted standards are those published by WHO (14 16). In our study, most participating laboratories used standards equivalent to WHO recommendations for reporting sperm count ( /ml) and motility ( 50% motile) (77% and 59% of laboratories, respectively). Of concern, however, is the number of laboratories that consider values far outside these standardized limits as the cutoff for normal. Seventy-two of the participating laboratories considered a normal sperm count to be greater than /ml, while 62 of the laboratories routinely used /ml as the cutoff for normal sperm count. Furthermore, in two of the participating laboratories, a sperm count of less than /ml would have been considered abnormal, a value four-fold higher than the cutoff suggested by WHO (15). Ninety-four of the participating laboratories considered a normal sperm motility to be greater than 60%, while 35 laboratories used 70% as the cutoff for normal sperm motility. In six of the laboratories, a sperm motility of anything less than 80% would have been reported as abnormal. Thus, a semen analysis reporting a sperm count of /ml and 65% motility would be considered completely normozoospermic in one laboratory while severely abnormal in another, making it very difficult, if not impossible, for physicians to compare semen analysis results between different laboratories. It is apparent from these data that many laboratories reporting semen analysis in the United States do not adhere to WHO standards. Moreover, our study indicates that as many as 34% of laboratories performing this important test either have never heard of the WHO manual or do not have a copy of this manual in their laboratory, making it impractical or impossible for the laboratory personnel performing the semen analysis to use or refer to these standards. Ombelet et al. (2) reported wide and complex variation in different sperm morphology classification systems employed in laboratories performing semen analysis. Our results would also indicate a wide variation in methodologies used, with as many as 20 different criteria employed by the participating laboratories. Of concern was the fact that 28% of laboratories indicated that they were unaware of the criteria used in their laboratories and 40% of laboratories did not indicate the criteria used on the semen analysis report. Therefore, in many cases, it is virtually impossible for referring physicians to interpret sperm morphology data reported on the routine semen analysis because of uncertainty of the normal reference ranges, methods employed, and criteria used. Several authors have stressed the importance of QC procedures in the andrology laboratory (9 12, 17, 18, 24). Baker et al. (17), in their survey of acute care community hospital laboratories, reported that less than 3% of laboratories performing semen analyses used controls. Ombelet et al. (2) reported that approximately 57% of laboratories participating in their survey performed internal controls. In our report, internal QC procedures were performed for sperm count, motility, and morphology determinations by less than half of the participating laboratories. Clearly, these data support a need for increased use of internal QC procedures in the semen analysis laboratory. Our findings indicate a significant degree of variation in the performance of the semen analysis and support the urgent plea for standardization of semen analysis methodologies expressed by others (1 3, 7, 8, 11, 17). It is strongly recommended that laboratories performing the semen analysis adopt accepted standards such as those proposed by WHO (14 17). Hopefully, adherence to such standards, employing proper internal QCs, and participation in national external PT programs (7), will significantly improve the accuracy, reliability, and comparability of the semen analysis. However, until such time as standardization of the semen analysis becomes more widespread, physicians must use caution when interpreting and comparing results between laboratories and should thoroughly evaluate the procedures, FERTILITY & STERILITY 607

6 criteria, and normal ranges employed by referring laboratories before relying on their results. References 1. Chong P, Walters CA, Weinrieb SA. The neglected laboratory test. The semen analysis. J Androl 1983;4: Ombelet W, Pollet H, Bosmans E, Vereecken A. Results of a questionnaire on sperm morphology assessment. Hum Reprod 1997;12: Jequier AM, Ukombe EB. Errors inherent in the performance of a routine semen analysis. Br J Urol 1983;55: Ayodeji O, Baker HW. Is there a specific abnormality of sperm morphology in men with varicoceles? Fertil Steril 1986;45: Neuwinger J, Bejre HM, Nieschlag E. External quality control in the andrology laboratory: an experimental multicenter trial. Fertil Steril 1990;54: Walker RH. Pilot surveys for proficiency testing of semen analysis. Arch Pathol Lab Med 1992;116: Keel BA, Quinn P, Schmidt CF Jr, Serafy NT Jr, Serafy NT Sr, Schalue TK. Results of the American Association of Bioanalysts national proficiency testing programme in andrology. Hum Reprod 2000;15: Mortimer D, Shi MA, Tan R. Standardization and quality control of sperm concentration and sperm motility counts in semen analysis. Hum Reprod 1986;1: Dunphy BC, Kay R, Barratt CLR, Cooke ID. Quality control during the conventional analysis of semen. An essential exercise. J Androl 1989; 10: Cooper TG, Neuwinger J, Bahrs S, Nieschlag E. Internal quality control of semen analysis. Fertil Steril 1992;58: Mortimer D, ed. Practical laboratory andrology. New York: Oxford University Press, Clements GN, Cooke ID, Barratt CLR. Implementing comprehensive quality control in the andrology laboratory. Hum Reprod 1995;10: Cooper TG, Atkinson AD, Nieschlag E. Experience with external quality control in spermatology. Hum Reprod 1999;14: World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 2d ed. Cambridge: Cambridge University Press, World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3d ed. Cambridge: Cambridge University Press, World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 4th ed. Cambridge: Cambridge University Press, Baker DJ, Paterson MA, Klaasen JM, Wyrick-Glatzel J. Semen evaluations in the clinical laboratory. How well are they being performed? Lab Med 1994;25: Muller CH. The andrology laboratory in assisted reproductive technologies program. J Androl 1992;13: Adelman MM, Cahill EM, eds. Atlas of sperm morphology. Chicago: American Society of Clinical Pathologists (ASCP) Press, Keel BA. The assisted reproductive technology laboratories and regulatory agencies. Infert Reprod Med N Am 1998;9: Schwartz A, Laplanche A, Jouannet P, David G. Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence. J Reprod Fert 1979;57: Keel BA. The semen analysis. In: Keel BA, Webster BW, eds. Handbook of the laboratory diagnosis and treatment of infertility. Boca Raton, FL: CRC Press, 1999; Helmerhorst FM, Guid Oei S, Bloemenkamp KWM, Keirse JNC. Consistency and variation in fertility investigations in Europe. Hum Reprod 1995;10: Coetzee K, Kruger TF, Lombard CJ, Shaughnessy D, Oehninger S, Ozgur K, et al. Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne Research integrated visual optical semen analyzer. Fertil Steril 1999;71: Keel et al. Lack of standardization in semen analysis Vol. 78, No. 3, September 2002