News from the European Academy of Andrology Italian Pilot Study for an External Quality Control Scheme in Semen Analysis and Antisperm Antibodies Detection (2000)

Many difficulties in semen analysis stem from the composition of semen and from the particular nature of the sperm cell. To date, even the computer-aided sperm analysis (CASA) systems have been unable to fully account for biological reality and diversity. Difficulties are also found with antisperm antibody (ASA) detection techniques using the whole sperm cell as antigen. At present, the commercially available kits for ASA detect Ab not related with the patient’s fertility. In addition, many possible sources of variability exist in all the fields of seminology, such as the operator experience deriving from the number of semen samples analyzed daily, differences in the step-by-step execution of any single test, and differences in facilities, environmental conditions and technical instrumentation found in each laboratory. As a consequence of all the above, differences should be expected when comparing results obtained by different laboratories analyzing the same semen sample. To reduce variability around the world, the various editions of the WHO Guidelines (1999), have introduced the use of standardized protocols for the performance of semen analysis and allied procedures. This standardization has provided the basis for the introduction of both internal and external quality control (EQC) procedures. EQC programmes for semen analysis carried out worldwide in the last decade have confirmed that: (a) large differences can be found, even among experienced laboratories, in the assessment of sperm number, morphology, and motility (Neuwinger et al., 1990; Matson, 1995), and (b) difficulties exist in the implementation of an EQC programme in this field. The idea of an Italian pilot study started in 1995, when an EAA Subcommittee for Clinical Training Centre nomination visited the Seminology and Immunology of Reproduction Unit, a section of the Department of Andrology at the University of Rome. A suggestion was made to implement an EQC scheme for semen analysis in Italy. Two years later, the idea was reconsidered within the framework of a research project of the Istituto Superiore di Sanità (ISS) and, thanks to a contract between the University of Rome ‘La Sapienza’ and the ISS, a pilot EQC scheme was started for both semen analysis and ASA detection. The aim of the pilot project was to evaluate the feasibility of an EQC programme especially taking into account problems arising from the participation of distant laboratories and both their availability and interest in working together. After a period of study and planning, two EQC trials were carried out between 1997 and 1998. The participating laboratories, 20 for semen analysis and eight for ASA detection, were enrolled on a voluntary basis from public Departments of Andrology or Reproductive Medicine throughout Italy. Within each trial, formalin fixed semen, videotapes and serum samples were sent to each participant for the determination of sperm concentration and morphology, motility, and antibody determination, respectively. The coding and sample randomization were carried out by the ISS. Biological material for the EQC scheme was provided and prepared by the Seminology and Immunology of Reproduction Unit (the Reference laboratory). Semen samples, obtained from subjects attending the Reference laboratory, were pooled, and after formalin addition, divided into aliquots. The control samples obtained were stored in vials (0.5 mL) and finally cryopreserved in liquid nitrogen. Each laboratory received four control samples. The parameters to be examined were sperm concentration (×106/mL) and morphology (percentage of typical and atypical forms with a detailed description of the site of the altered morphology), leukocyte concentration (×106/mL) and presence/absence of germ cells. In the second trial, in order to assess within-batch variations, two control samples from the same pool were sent to each laboratory as unknown duplicates. For sperm motility assessment, a videotape containing four different recordings was sent to each laboratory. For each trial, ASA positive/negative sera were inactivated, tested (Gelatin Agglutination Test, Tray Agglutination Test, indirect Immunobead Test), titered, stored in vials (0.5 mL), and cryopreserved (–80 °C); in each trial eight samples were sent to the eight laboratories participating in the ASA detection EQC scheme. The Reference laboratory received, both for semen analysis and ASA detection, semen samples, videotapes and serum samples after the randomization of the ISS. The laboratories were asked to analyze the samples promptly and to perform tests under routine conditions. At the end of each trial, a statistical evaluation of data was performed and the presence of outliers was evaluated by means of the Dixon Test and the Huge error rule (Taylor, 1990). For each parameter a summary of the results obtained, including the distribution histogram, mean, standard deviation (SD), median, standard error of the median, and percentage coefficient of variation (CV%), was sent to participants. Some of the main results for the determination of sperm concentration, sperm morphology (considering the parameter Atypical forms percentage) and sperm motility (considering the parameter forward rapid and slow motility, Forward motility percentage) are reported in Table 1. For each sample and parameter, the mean, the SD, the range, and the CVs% are shown. The ranges of CVs% for Sperm concentration, Atypical forms percentage and Forward motility percentage were 29–52%, 17–26%, and 39–71%, respectively. The within-batch variation was estimated, from results obtained by participants in the second trial on unknown duplicates, as Pooled Standard Deviation (PSD=Σ(|result1-result2|)2/2n, where n=total number of duplicate samples). The PSDs were 8.8 and 9.2, for sperm concentration (×106/mL) and atypical forms (%), respectively. Concerning ASA detection, only 79 results on 128 samples were returned in the two trials. The ASA positive and the ASA negative results were 34 and 45, respectively; the percentage of consistent results were 76.5 and 93.3% for positive and negative samples, respectively. The need for quality control in laboratories undertaking semen analysis and sperm antibody detection is now widely recognized (Neuwinger et al., 1990; Matson, 1995), and EQC schemes have been developed all over the world (Neuwinger et al., 1990; Matson, 1995; Clavert et al., 1999; Cooper et al., 1999; WHO Guidelines, 1999). Some reports have shown how the operation of external quality control, together with internal quality control, can reduce the between-laboratory variation for semen analysis (Neuwinger et al., 1990; Cooper et al., 1999). In our pilot EQC scheme, results of semen analysis showed consistent between-laboratory variation in the assessment of both sperm concentration and morphology thus, confirming previous observations. These differences appear to be due, at least in part, to the use of different methodologies and criteria (for example in the definition of normal spermatozoa). When two different aliquots from the same semen pool were analyzed unknowingly in the same trial, quite different results were obtained. These data also suggest that systematic errors were occurring in the estimation of sperm concentration, morphology and motility. Regarding ASA detection, even though it is difficult to draw definitive conclusions on the data available, the percentage of consistent results (86.1%) seem to be satisfactory. In conclusion, our results and the difficulties encountered strongly indicate the need for the creation of a national EQC scheme and the establishment of a quality assurance working group in seminology. This should allow to co-ordinate a continuous programme with regional institutions and laboratories responsible for single areas and the creation of a national co-ordination in collaboration with a governmental institution, such as ISS. This programme in turn should be integrated into a European network by bringing together EAA Training Centers (as has recently been started) and the other groups working in this field. This could represent an opportunity for the scientific community to allow the definition of target values and maximum acceptable variability for single semen parameters and ASA methods, and to establishing regular training courses also in relation to the results of the quality control. This common effort would certainly help to avoid the present ‘Tower of Babel’ of data, which can only harm the cause of andrology. This work was supported by a Istituto Superiore di Sanità Research Project ‘Prevention and risk factors in Child and Maternal Health’.