Human Reproduction, Vol. 18, No. 5, 1018-1022,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Inter-individual variability in the morphological assessment of human sperm: effect of the level of experience and the use of standard methods
Service de Biologie de la Reproduction-CECOS, Hôpital Cochin, 75014 Paris, France
1 To whom correspondence should be addressed at: Laboratoire d’Histologie, Biologie de la Reproduction, CECOS, and Groupe d’Etude de la Fertilité Humaine (GREFH), Université Paris V, Pavillon Cassini, Hôpital Cochin, 123 Bd de Port-Royal, 75014 Paris, France. e-mail: jacques.auger{at}cch.ap-hop-paris.fr
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Abstract |
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BACKGROUND: The assessment of human sperm morphology is not devoid of methodological problems, making comparison among laboratories difficult. The main goal of this study was to assess how the experience and compliance to the recommended procedures can modulate it. METHODS: Sixty-two technicians and biologists of varying degrees of andrological experience classified the same 100 sperm in a blind study from images captured on a video microscope and projected onto a screen. David’s morphology classification which distinguishes normal and abnormal sperm and calculates a Multiple Anomalies Index (MAI) was used. These sperm were also classified by two trained and experienced biologists using videotaped images to provide reference values. RESULTS: The inter-participant coefficient of variation was 40% for normal sperm, lower for five defects and, 12% for MAI. Experienced participants had the closest results to the reference values. Moreover, participants not routinely using the recommended methodologies, regardless of their experience level, consistently showed marked differences compared with the reference values. CONCLUSIONS: The present data confirm the wide variability in the assessment of normal sperm and show a lower variability for several anomalies and MAI. They underscore the role that experience plays and the importance of following the recommended methodologies for relevant and comparable results.
Key words: multiple anomalies index/quality control/routine semen analysis
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Introduction |
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During the past three decades, multiple techniques of sample staining and systems of classification to assess human sperm morphology have been proposed [see Mortimer and Menkveld, (2001)
for review] without consensus on a method of a universal classification. Conscious of this problem, the editorial staff of the 4th edition of the World Health Organization (WHO) manual has recommended the use of a single method classifying essentially the morphologically normal sperm using strict criteria (Kruger et al., 1988; World Health Organization, 1999). Regardless of the morphological classification used, manual assessment of sperm cell morphology is visually subjective and creates intra- and inter- individual laboratory variability which can be assessed by internal and external quality control schemes (Neuwinger et al., 1990; Cooper et al., 1992; Matson, 1995; Auger et al., 2000; Franken et al., 2000). In the few external quality assessments (EQA) previously reported, samples of prepared semen were sent to the participating laboratories. In the present study, we invited technicians and biologists to participate in an EQA session to assess sperm morphology using high-resolution images of sperm projected directly from a video equipped microsocope. We could then evaluate their inter-individual variability in the recognition of the normal sperm and of the various sperm abnormalities according to the classification of David (David et al., 1975)—a classification used in the vast majority of French laboratories and proven useful from a clinical point of view (Jouannet et al., 1988; Auger et al., 2001; Slama et al., 2002)—and study the possible effect of their level of experience or of their compliance with the recommended technical procedures. |
Materials and methods |
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Sixty-two participants attended a Quality Control (QC) workshop on sperm morphology assessment at the University of Nancy, in the east of France. The participants were laboratory technicians, biologists or physicians from 40 French laboratories and two from Luxembourg. The participants had various experience levels and used various methods for sperm morphology assessment (Table IAA and IBB).
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A single semen sample with a high level of various morphological abnormalities was assessed. Briefly, the smear was prepared from a 10 µl drop, air-dried, fixed for 1 h with a mixture of absolute ethanol (2/3) and acetic acid (1/3), then stained by Shorr staining using an automatic stainer (Sakura DRS601, Bayer Diagnostics, Puteaux, France). The slide was put onto the stage of a BX50 microscope (Olympus, Rungis, France) equipped with a 100x objective and a XC-003P 3-CCD RGB colour camera (video output elements: 739 x 575; Sony, Clichy, France). The camera was connected to a VPL-FE110 video projector (resolution RGB: 1280 x 1024; Sony) giving a final high-resolution image. Before the workshop started, images were projected on the screen to check that the projected images of the sperm had adequate definition for their in-depth analysis and classification. It was found that a participant sitting in any of the first six rows of the hall and front of the screen had a proper angle of resolution and vision. The normal and abnormal sperm were classified according to the classification of David et al. (1975
), modified after the report of Jouannet et al. (1988). The details of the classification have been recently described (Auger et al., 2001). Briefly, normal sperm and abnormal sperm categories are defined by strict and specific criteria and all abnormalities of each sperm are recorded which makes it possible to calculate the Multiple Anomalies Index, MAI (Jouannet et al., 1988; World Health Organization, 1999), the mean number of anomalies per abnormal sperm. Two hours before the session started, a 1 h lecture using numerous slides and figures summed up the standardized procedure for David classification. Each participant received a form with a grid to record all of the anomalies observed for each sperm cell. A total of 100 sperm was assessed, each spermatozoon chosen being tagged with the projected light arrow of the microscope. Each sperm cell selected was projected for 45 s (delay necessary for a novice participant to assess a spermatozoon with multiple defects as determined in previous internal QC) and concomitantly recorded on a video tape inserted in a Panasonic professional video recorder (AG-7350 Panasonic, Saint Denis la Plaine, France). After the workshop and before data analysis, two trained biologists (F.E. and J.A.) jointly classified the videotaped sperm. For the rare sperm for which there was a disagreement between both biologists (mainly those having size defects), measurements were made to correctly classify them. The overall variability in the assessment of normal and abnormal sperm was studied. We then estimated the overall variability for the assessment of the same characteristics in reference to the level of training of the participants in two subgroups defined according to the number of assessments made per year (
520 per year, n = 45 versus >520 per year, n =17). We have also investigated if the compliance with the recommended methodologies (World Health Organization,1992) for making smears, staining and routinely assessing sperm morphology could have any influence on the results of the current assessment. Therefore, we have studied the differences between participants and reference values for the normal and abnormal sperm cells for three groups of participants. The first group was composed of participants assessing more than 520 slides per year for at least 2 years and routinely using the WHO recommended methodologies (World Health Organization, 1992). The second group was composed of participants with less experience (<2 years, whatever the number of assessments per year) but who routinely used the recommended methodologies as for the first group. The third group was composed of the other participants who do not use routinely the recommended methodologies, irrespective of their experience level. Inter-participant variability in the assessment of sperm morphology was expressed as the coefficient of variation: CV (%) = 100 x standard deviation/mean value. The equality of means values of the characteristics studied among the three subgroups selected on their experience level and compliance with the WHO guidelines was tested by a one-way analysis of variance. When rejecting the null hypothesis, the Bonferroni test was used for pair wise mean comparisons. Other paired comparisons were made using the Mann–Whitney-test. All statistics were run using the BMDP statistical software (Dixon, 1988). |
Results |
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The mean and median values of the percentage of normal sperm, of each anomaly recorded and of the MAI, the corresponding overall inter-participant CV and the reference results are presented in Table II. The lowest CV were observed for the MAI (12%), the percentages of bent midpieces (23%), of absent tails (25%) and abnormal or absent acrosomes (26%). The overall CV for the percentage of normal sperm was found to be equal to 40% with a very broad range (6–39%), a marked difference with the low dispersion of values found for the MAI. The mean and median values of the percentage of normal sperm, MAI and each anomaly recorded and the corresponding overall inter-participant CV in two groups of participants selected according to their experience are presented in Table III. Experienced participants found significantly less normal sperm, and more multiple heads, abnormal acrosomes, thin midpieces and irregular calibres of the tail. Figure 1 summarizes the results of the three groups of participants studied according to their level of experience and compliance with the recommended methodologies. Participants who did not routinely use the recommended methodologies, regardless of their experience level, had the highest differences compared with the reference values. In addition, their results differed significantly from those obtained in the group of experienced participants following routinely the WHO guidelines.
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Discussion |
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A noticeable amount of variability in the assessment of human sperm morphology was found in the present study. This variability is obviously prejudicial to the comparison of the results in the same laboratory or from one laboratory to another. Moreover, it can also strongly impair the management of infertile couples because sperm morphology is a major determinant of human fertility in vivo (Guzick et al., 2001
; Slama et al., 2002) and in vitro (Kruger et al., 1988). The results of the EQA reported herein are based on a large group of biologists or technicians with different levels of experience and practice. In this respect, they can be considered representative of the baseline variability in the assessment of human sperm morphology in a developed country. The overall CV for the percentage of normal sperm was found to be equal to 40%, a result similar to what was previously found in several EQA (Matson et al., 1995; Ombelet et al. 1998; Keel et al., 2000). Interestingly, the variability for various anomaly categories and the MAI was markedly lower. The inter-participant variability for visual pattern defect recognition (for example, multiple head) was lower than for the defect categories requiring a visual assessment of the head or tail size (for example, tapered heads), see Table II, a result previously reported (Baker and Clarke, 1987). However, continuous quality controls in our laboratory have indicated that training with the help of an eyepiece equipped with a measuring reticle strongly improves the classification of size defects which in turn decreases markedly the intra- and inter-individual variability in assessment. The better concordance in assessment of several categories of abnormal sperm together with their clinical value (Jeulin et al., 1986; Jouannet et al., 1988; Menkveld et al., 1996; Slama et al., 2002), suggest the relevance of determining the amount of morphological defects or the MAI besides the percentage of normal sperm. There are obvious sources of variability in the assessment of sperm morphology, for example, the current use of several systems of classification [strict criteria, Düsseldorf, WHO 87 and 92; American Society for Clinical Pathology (ASCP), Adelman and Cahill, 1989] or David classifications, (Ombelet et al., 1997). Table IV, summarizing the schemes and results of various EQA, illustrates the wide variation in human sperm morphology assessment whatever the classification method used (inter-individual CVs ranging between 28 and 65%). These marked variations point to other putative sources of variability such as the observer level of experience or the procedures used (Dunphy et al., 1989; Menkveld et al., 1990; Neuwinger et al., 1990; Davis and Gravance, 1993; Meschede et al., 1993). It was found here that participants regularly assessing high numbers of samples for sperm morphology scoring had stricter evaluations of the normal (and the abnormal sperm as illustrated by the MAI value) than novice participants, the former having the mean values closest to the reference values although with some differences. Such differences could be that the trained biologists have assessed the sperm after the workshop, with the possibility to discuss together and to size the cells (which was not the case during the EQA since no means of measuring was offered to the participants). The role of repeated testing on reproducibility and convergence with reference values has been reported (Mortimer, 1994; Franken et al., 2000). It was shown that the level of experience has clearly an impact on the results of the sperm morphology assessment (Baker and Clarke, 1987). However, the level of experience, when taken into consideration alone, is not a guarantee of low inter-individual variability in the assessment of human sperm morphology, even if trained technicians do show lower inter-individual variability than among less trained technicians in some EQA [Jequier and Ukombe, (1983), see Table IV; Baker and Clarke, 1987]. In the present EQA, it was interesting to see that participants who do not routinely use the recommended procedures were less strict and gave significantly different results in comparison with participants who follow the guidelines (irrespective of their level of experience) and to the biologists who set reference values.
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In conclusion, the present study shows that the practical experience as well as the compliance with recommended methodologies have a serious impact on the results of the sperm morphology assessment. It demonstrates the imperative need for standardization, training and quality control for human sperm morphology assessment.
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Acknowledgements |
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We would like to thank B.Foliguet and A.Clavert for the organization of the meeting, all the participants in this EQA and R.Dolan for reviewing the English revision of the manuscript.
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Submitted on February 25, 2002; resubmitted on December 6, 2002; accepted on January 14, 2003.
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