Stesura Seveso INTRODUCTION Semen analysis is a crucial and irreplaceable tool for evaluating male infertility and precise thresholds are needed. Since 1951, the scientific community recog- nized this concept and McLoad et al. (1, 2) indicated the cut-off values for sperm counts (> 20 x 106 /mL, total sperm count > 100 x 106) for the first time, to distin- guish fertility from subfertility. However the clinicians noted several times that some men with a sperm count below these ranges were able to conceive and this creat- 125Archivio Italiano di Urologia e Andrologia 2013; 85, 3 ORIGINAL PAPER Comparison between WHO (World Health Organization) 2010 and WHO 1999 parameters for semen analysis – interpretation of 529 consecutive samples Francesco Catanzariti, Ubaldo Cantoro, Vito Lacetera, Giovanni Muzzonigro, Massimo Polito Polytechnic University of Marche, Faculty of Medicine, Department of Odontostomatologic and Specialized Clinical Sciences, Urology Clinic, Department of General and Specialized Surgery, University Hospital, Ancona, Italy. Objective: To quantify how many men with normal semen according to WHO (WHO - World Health Organization) 1999 criteria, should be considered with abnormal semen according to 2010 criteria and vice versa; to study which parameter of volume, concen- tration, motility and morphology is the most responsible of this change. Materials and methods: We studied, using WHO 1999 parameters, 529 consecutive semen samples from 427 men, collected in our Department from January 2008 to December 2009, then we re-evaluated those results using WHO 2010 parameters; we also studied each parameter to understand how changed the classification from normal (defined normal by all parameters) to abnormal (defined abnormal by at least one parameter) using the two WHO criteria. Results: 3 men (0.56%) were azoospermic. Among the remaining 526 samples, 199 (37.83%) were considered normal and 246 (46.76%) abnormal both according to WHO 1999 and WHO 2010 cri- teria; we found that none of the samples classified normal according to the previous criteria was classified abnormal according the more recent criteria, while 82 (15.58%) evaluated as abnormal according 1999 criteria changed to normal according 2010 criteria. The concordance between 1999 and 2010 evaluation was 84.44%. Conclusions: In this study we noted that the changes from WHO 1999 to WHO 2010 criteria did not modify the interpretation of semen quality, because comparing the two classifications we demonstrated that there is a substantial agreement, considering the three parameters (count, motility and morphology) all together, and also considering each single parameter. Anyhow, almost 16% of the patients considered infertile according to the old criteria, should be evaluated normal by the new classification and they should not need any treatment for infertility. KEY WORDS: WHO 2010 parameters; Infertility; Semen analysis. Submitted 5 June 2013; Accepted 30 June 2013 No conflict of interest declared Summary ed an uncertainty in the clinical practice. In 1980 the World Health Organization (WHO) tried to clarify these doubts, by publishing the first (3) of 5 editions of guide- lines for semen analysis. The weak point of that edition was that its criteria had never been prospectively validat- ed by any study, because all the works present in the lit- erature of the following years used either a case-control design, comparing fertile and subfertile couples, or a cohort design among the first pregnancy planners (4, 5). DOI: 10.4081/aiua.2013.3.125 Archivio Italiano di Urologia e Andrologia 2013; 85, 3 F. Catanzariti, U. Cantoro, V. Lacetera, G. Muzzonigro, M. Polito 126 The ultimate edition published in 2010 (6) used the con- cepts of “percentile” and “confidence intervals”, for the first time, allowing the clinicians to evaluate the individual semen analysis values in the context of measurement error and indicated reference values for semen parameters based on data from fertile men above the 5th percentile. Furthermore, the results were generated in multiple labo- ratories using standardized procedures and based on real world data. However the last WHO guidelines for semen analysis radically changed the interpretation of semen analysis of the previous WHO 1999 (7) guidelines especially regarding the parameters of number (from 20 x 10^6/mL to 15 x 10^6/mL) and morphology (from 30% to 4%). This change mean that some of the patients, who were considered abnormal for the quality of their semen according to the old classification, would be considered normal, as a result of the new classification. Our study tried to quantify this change and to understand the per- centage of concordance between the two classifications in the assessment of a sample, considering all three parameters at the same time (sperm count, motility and morphology) and analyzing each parameter individually to understand which of them is most responsible for the shift from normospermia to dyspermia. MATERIALS AND METHODS We studied 529 consecutive semen samples from 427 men collected in our Department from January 2008 to December 2009. Semen analysis was performed 2 or more times in 74 patients during this period. This group was composed of healthy men and men affected by dif- ferent diseases (infertility, infections, varicocele, and other pathologies), Semen analysis was performed after at least 3 days of sexual abstinence. Semen samples were collected at the hospital by masturbation directly into a 120 mL sterile jar. Semen samples were analyzed within 1 hour of ejaculation. After liquefaction, semen volume was measured in a graded syringe with 0.1-mL accuracy. Sperm concentration was counted and motility assessed in a Makler counting chamber at a magnification of x 200. All semen parameters were classified first, according to the 1999 WHO criteria, then retrospectively using WHO 2010 parameters; we also studied each parameter to understand how the results changed from normal (defined as all parameters normal) to abnormal (defined with at least one parameter abnormal) using the two criteria. RESULTS The mean age of participants was 30.26 (18-60). Sperm characteristics are summarized in Table 1. Between the 529 samples, we detected 3 (0.56%) cases of azoospermia. In analysing the specimens using the old criteria (Figure 1) we found 199 (37.62%) cases of nor- mospermia, 140 (26.47%) cases of asthenozoospermia and 83 (15.69%) cases of oligoasthenozoospermia, while using WHO 2010 criteria (Figure 2) we found slightly different rates of prevalence: 283 (53.50%) cases of nor- mospermia, 105 (19.85%) cases of asthenozoospermia and 85 (16.07%) cases of oligoasthenozoospermia. When we considered only the number (Figures 3, 4) we observed that 355 (67.49%) patients had a normal num- ber according to both WHO 1999 and WHO 2010 crite- ria, 138 (26.24 %) patients were considered to have oligozoospermia by both classifications and 33 (6.27%) patients were considered to have a normal number according to WHO 2010 criteria and to have oligo- Mean 5th-95th Percentile Age (y) 30.26 18-45 Mediana 5th-95th Percentile Volume (ml) 3 1-6 Concentration (10^6/ml) 40 0.28-163.60 Total Motility (%) 46 10-80 Normal Morphology (%) 52 20-73 Table 1. Characteristics of age and semen quality of the patients (n: 529 men). Figure 1. Number of patients in each category (WHO 1999). Figure 2. Number of patients in each category (WHO 2010) zoospermia according to the WHO 1999 classification. We found that some patients who had an abnormal num- ber according to old criteria were not considered abnor- mal according the new one. Therefore the concordance between the two classifications for number was 93.73%. Considering only the parameter of motility (Figures 5, 6) we found that 248 (47.15%) cases had normal motility both according to WHO 1999 and WHO 2010 criteria, 192 (36.50%) patients were considered to have astheno- zoospermia according to both classifications and 86 (16.35%) patients were considered to have a low motili- ty according to WHO 1999 parameters and to have a normal motility according to the new parameters. We found that some patients who had an abnormal motility by the old criteria were not considered abnormal accord- ing to the new one. The concordance according to the two criteria about motility was 83.65%. Studying only morphology (Figures 7, 8) we observed that 469 (89.16%) cases presented normal morphology according to both classifications, 2 (0.38%) cases had teratozoospermia according to both WHO 1999 and WHO 2010 classification and 55 (10.46%) patients were considered to have abnormal morphology according to the old classification and to have normal morphology according to the new one. We found that patients who had an abnormal morphology by the old criteria were not considered abnormal according to the new one. Therefore the concordance between the two classifica- tions of morphology was 89.54%. Considering the three parameters together (Figures 9, 10) 199 (37.83%) patients were considered normal according 127Archivio Italiano di Urologia e Andrologia 2013; 85, 3 Comparison between WHO (World Health Organization) 2010 and WHO 1999 parameters for semen analysis Figure 3. Concordance between WHO 1999 and WHO 2010 about number. Figure 4. Concordance between WHO 1999 and WHO 2010 about number. Legend NNNN: Percentage of men with normal number according to both WHO 1999 cri- teria and WHO 2010 criteria. NNO: Percentage of men with normal number according to WHO 1999 criteria and oligozoospermia according to WHO 2010 criteria. ONN: Percentage of men with oligozoospermia according to WHO 1999 criteria and normal number according to WHO 2010 criteria. OO: Percentage of men with oligozoospermia according to both WHO 1999 criteria and WHO 2010 criteria. Figure 5. Concordance between WHO 1999 and WHO 2010 about total motility. Figure 6. Concordance between WHO 1999 and WHO 2010 about total motility Legend NMNN: Percentage of men with normal motility according to both WHO 1999 cri- teria and WHO 2010 criteria. NMA: Percentage of men with normal motility according to WHO 1999 criteria and asthenozoospermia according to WHO 2010 criteria. ANM: Percentage of men with asthenozoospermia according to WHO 1999 criteria and normal motility according to WHO 2010 criteria. AA: Percentage of men with asthenozoospermia according to both WHO 1999 crite- ria and WHO 2010 criteria. Archivio Italiano di Urologia e Andrologia 2013; 85, 3 F. Catanzariti, U. Cantoro, V. Lacetera, G. Muzzonigro, M. Polito 128 to both WHO 1999 and WHO 2010 classification, 246 (46.77%) cases were considered abnormal according to both classifications and 82 (15.59%) cases were evaluated abnormal according to the 1999 parameters and normal according to those of 2010. We found that patients who had all three parameters abnormal by the old criteria were not considered abnormal according to the new one, so that the concordance between 1999 and 2010 interpreta- tion was 84.44%. DISCUSSION In literature there are few studies that analyze the changes in the interpretation of semen analysis from WHO 1999 to WHO 2010 criteria. Murray et al. (8) recently did a multi-institutional retro- spective study involving 387 infertile men, with the aim to understand how many semen samples of patients who were classified as infertile by timeline and previous semen analysis criteria would change classifications to be in the normal fertile range based on the 2010 WHO lower reference limits. They observed that overall, 43 (11.1%) patients who had one or more abnormal param- eters in the original analysis would be converted to hav- ing all parameters within normal parameters and the most important changes in the interpretation of the data were in motility and morphology. These results are simi- lar to our (11.1% vs 15.59%) Another recent study by Zou et al. (9) indirectly analyzed this change of interpretation. In his work Zou examined Figure 7. Concordance between WHO 1999 and WHO 2010 about morphology. Figure 8. Concordance between WHO 1999 and WHO 2010 about morphology. Legend NmNm: Percentage of men with normozoospermia according to both WHO 1999 criteria and WHO 2010 criteria. NmT: Percentage of men with normozoospermia according toWHO 1999 criteria and teratozoospermia according to WHO 2010 criteria. TNm: Percentage of men with teratozoospermia according to WHO 1999 criteria and normozoospermia according to WHO 2010 criteria. TT: Percentage of men with teratozoospermia according to both WHO 1999 criteria and WHO 2010 criteria. Figure 9. Total concordance between WHO 1999 and WHO 2010. Figure 10. Total concordance between WHO 1999 and WHO 2010. Legend FF: Percentage of men considered fertile according to both WHO 1999 criteria and WHO 2010 criteria. FI: Percentage of men considered fertile according to WHO 1999 criteria and infer- tile according to WHO 2010 criteria. IF: Percentage of men considered infertile according to WHO 1999 criteria and fer- tile according to WHO 2010 criteria. II: Percentage of men considered infertile according to both WHO 1999 criteria and WHO 2010 criteria. the determinants of semen quality in a large sample of military personnel from different geographical areas of the People's Republic of China. Among 1194 patients he found that 88.3% had at least one semen parameter below the normal values according to World Health Organization (WHO) recommendations (1999), and 62.5% according to WHO recommendations (2010). Therefore, this study also demonstrated that the new classification created a little shift in the patients from infertile to fertile. Metha et al. (10) instead, evaluated the improvement in semen parameters and serum testosterone (T) in their study, following varicocelectomy in those men consid- ered abnormal according to the 1999 WHO criteria yet normal by the new 2010 criteria. They analysed 152 patients in total, that met the inclusion criteria (sperm concentration 15-20 million/mL, motility 40-50%, or morphology 4-14%): 111 patients (73%) underwent bilateral varicocelectomy, while 41 (27%) underwent a left side varicocelectomy. Overall, sperm concentration and serum testosterone (T) improved following surgery. Among men who met the inclusion criteria for sperm concentration, only sperm concentration was significant- ly increased (17.8 vs. 38.0 million/mL, p = 0.03). They concluded that microsurgical varicocelectomy in the subset of men considered to have normal semen param- eters according to the 2010 WHO reference ranges, but abnormal according to the 1999 reference ranges lead to a significant improvement in serum T, sperm concentra- tion, and, in some cases, sperm motility. Metha and his colleagues underlined that even among patients considered normal for the new classification but abnormal for the old one, microsurgical varicocelectomy should be performed because of its improvement of fer- tility potential as well as T levels. Our study evaluated if there were any changes in the eval- uation of the semen quality according to WHO 1999 cri- teria and WHO 2010 criteria in order to understand if there are cases, which can change from abnormal to nor- mal, and vice versa using the two classifications. We con- cluded that, considering each parameter by itself and all three parameters together, the concordance is very high. Concordance was high according to the number parame- ter (93.73%) that was slightly changed, from 20 x 10^6/mL (WHO 1999) to 15 x 10^6/mL (WHO 2010), but also according to morphology parameter (89.54%) that was radically changed from 30% (WHO 1999) to 4% (WHO 2010). Another interesting observation from our results is that there are not cases considered normal by the old classification that change to abnormal by the new one, while there are patients that resulted abnormal by the old criteria and normal according to new WHO 2010 parameters. This change is more consistent for motility (16.35%) than morphology (10.46%), as we expected. This implies that none of the patients that were previously considered nor- mal changed to abnormal, according to the new classifi- cation, but some patients, about 15%, changed from abnormal to normal by the new classification. So thanks to the new criteria, the rate of patients that do not need any treatment for infertility is now reduced because they are no more considered infertile. ACKNOWLEDGEMENTS The Authors would like to thank all of the patients for their contribution to this study and all the Urologists of the Department of Urology of the Polytechnic University of Marche in Ancona, who contributed to this study, with their data collection and sharing. REFERENCES 1. MacLeod J, Gold RZ. The male factor in fertility and infertility. II. Sperm counts in 1000 men of known fertility and in 1000 cases of infertile marriage. J Urol. 1951; 66:436. 2. MacLeod J, Gold RZ. The male factor in fertility and infertility. VI. Semen quality and other factors in relation to ease of conception. FertilSteril. 1953; 4:10. 3. World Health Organization.WHO laboratory manual for the examination of human semen and semen-cervical mucus interac- tion. Singapore: Press Concern, 1980. 4. Bonde JP, Ernst E, Jensen TK, et al. Relation between semen qual- ity and fertility: a population-based study of 430 first-pregnancy planners. Lancet. 1998; 352:1172-7. 5. 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Impact of the revised who semen analysis reference limits on selection criteria for micro- surgical varicocelectomy. J Urol. 2012; 187:4S. 129Archivio Italiano di Urologia e Andrologia 2013; 85, 3 Comparison between WHO (World Health Organization) 2010 and WHO 1999 parameters for semen analysis Correspondence Francesco Catanzariti, MD (Corresponding Author) Resident in Urology fracatanzariti@libero.it Ubaldo Cantoro, MD Resident in Urology ubaldocantoro@tiscali.it Vito Lacetera, MD Urologist vlacetera@gmail.com Giovanni Muzzonigro, MD Professor of Urology, Chief Department of Urology g.muzzonigro@univpm.it Massimo Polito, MD Urologist, Chief Department of Uro-Andrology max_polito@virgilio.it Polytechnic University of Marche, Faculty of Medicine Department of Odontostomatologic and Specialized Clinical Sciences Urology Clinic, Department of General and Specialized Surgery University Hospital of Ancona Via Conca 71 - I-60020 Ancona, Italy