1 Reproductive Biology Research Laboratory, Department of Obstetrics & Gynaecology, University of Stellenbosch, PO Box 19058, Tygerberg 7505, South Africa, 2 Kenyatta National Hospital, College of Health, Department of Obstetrics & Gynaecology, Nairobi, Kenya and 3 WHO Special Programme of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland
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Abstract |
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Key words: andrology/quality control/strict sperm-morphology/training
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Introduction |
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Therefore the value of sperm morphology assessments as a predictor of a man's fertilizing potential has often been challenged due to different classification systems. Several factors are responsible for this technical variation, including differences in the methods used to prepare and stain specimens (Katz et al., 1986; Davis and Gravance, 1993
), differences in proficiency among technicians (Zaini et al., 1985
; Baker and Clarke, 1987
; Dunphy et al., 1989
; Menkveld et al., 1990
) and inherent differences in classification criteria and methods (Davis and Gravance, 1994
; Ombelet et al., 1995
, 1998
). These differences in methodologies between laboratories are well known, but since the publication of the World Health Organization manual (WHO, 1999) the between-laboratory variation can be negated. Intra- and inter-individual variability should decrease to non-significant levels of difference. An ever-remaining problem would be the comparison of the predictive power of semen parameters of one setting with another and the attempt to transfer the specific values to fit their patient population. A recent comprehensive study of so-called normal couples attempting to achieve conception showed that the probability of conception increased with increasing sperm concentration up to 40x106/ml and that the proportion of spermatozoa with normal morphology was strongly related to the likelihood of pregnancy, independently of sperm concentration (Bonde et al., 1998
). These findings should be interpreted with caution, since these completely different populations of patients differ from those referred to assisted reproductive programmes. A meta-analysis has confirmed the importance of sperm morphology as a predictor of male fertility (Coetzee et al., 1998
). There was a pregnancy rate of 15.2% among the couples in the morphology group with <4% normal forms compared to 26% among the >4% group. Sperm morphology therefore is possibly the most consistent sperm variable which appears to be related to in-vitro fertilization (IVF) success (Enginsu et al., 1991
; Kobayashi et al., 1991
; Liu and Baker, 1992
; Ombelet et al., 1995
). A logistic regression model, including DNA status and morphology of spermatozoa revealed sperm morphology (strict criteria) and concentration of progressive motile spermatozoa to be the principle predictors for IVF (Duran et al., 1998
).
Based on the reported clinical importance of sperm morphology (Oehninger et al., 1991; Ombelet et al., 1994
, 1997a
; Coetzee et al., 1999), the Tygerberg Reproductive Biology Research Laboratory developed a strict criteria sperm morphology training course for sub-Saharan African scientists, which aimed to record and evaluate the efficiency of continuous sperm morphology training.
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Materials and methods |
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Laboratories from sub-Saharan African countries, i.e. Uganda, Zimbabwe, Zambia, Cameroon, Kenya, Nigeria, South Africa and Tanzania were requested during April 1997 (n = 10) and September 1998 (n = 10), to participate in semenology workshops at Tygerberg Hospital with the objective to establish a continuous quality control system for sperm morphology evaluation. In addition to basic semen analysis training, an intensive sperm morphology teaching course was presented during a 5 day hands-on workshop at the Reproductive Biology Research Laboratory at Tygerberg Hospital.
Prior to the training, participants performed a morphology evaluation (percentage normal cells) on pre-stained Papanicolaou slides. These results were compared with an evaluation by a highly experienced panel of morphology experts, and the data used as a reference set to evaluate improvement in participants' skills.
Training
Following the pre-training evaluation, delegates were provided with a take-home reference set of five pre-stained Papanicolaou slides which consisted of the following: slide 1 contained spermatozoa from a donor with >14% normal forms (this slide was also used during pre-training evaluation), slide 2 contained spermatozoa with <4% normal forms. Slides 1 and 2 were repeatedly used until each participant was familiar with the shape and microscopic appearance of the so-called normal spermatozoa according to strict criteria. Slides 3, 4 and 5, used during the teaching course, contained sperm samples with >14%, 514% and 4% normal forms, respectively. The latter slides served as a control to evaluate the participant's knowledge of normal sperm cells. For a spermatozoon to be considered normal, the sperm head, neck midpiece and the tail must be normal. The head should be oval in shape. Allowing for the slight shrinkage that fixation and staining induce, the length of the head should be 4.05.0 µm and the width 2.53.5 µm. The length-to-width ratio should be 1.501.75. These ranges are the 95% confidence intervals (CI) for Papanicolaou-stained sperm heads (Katz et al., 1986
). Estimation of the length and width of the spermatozoa were made with an ocular micrometer. There should be a well-defined acrosomal region comprising 4070% of the head area. The midpiece should be slender, <1 µm in width, about one-and-a-half times the length of the head, and attached axially to the head. The tail should be straight, uniform, thinner than the midpiece, uncoiled and ~45 µm long (WHO, 1999). This classification scheme requires that all `borderline' forms be considered abnormal (Kruger et al., 1986
; Menkveld et al., 1990
). It is also the method most widely used in andrology laboratories and is the method recommended by WHO (1999).
Continuous quality control (CQC) system
Participants were requested to enroll in the CQC system that entailed the following: (i) A communication line via a fax/e-mail facility, through which each enrolled laboratory obtained direct access to advice and slide information, in order to ensure the continuous educational value of the CQC system. (ii) On a quarterly basis participants received a single Papanicolaou pre-stained sperm slide of e.g. spermatozoa from normo-, terato- or severe teratozoospermic sperm samples, evaluated for percentage normal cells by a panel of highly experienced morphology experts prior to shipment. Participants had to record the percentage normal cells for this slide and forward this information via the communication line to Tygerberg Hospital, where all the information were recorded in a data base. The `correct' results according to the reference laboratory, i.e. the percentage normal forms present on each of the slides, were subsequently supplied to the participating laboratory.
Evaluation of the system
The results of the participants' morphology evaluation at each stage of the training programme were compared with the evaluation of the reference laboratory, and the difference in score was calculated. This change in agreement with the reference laboratory, measured over time, was the outcome parameter of interest, which would be indicative of the participants' change in skill in evaluating sperm morphology on a slide. The ability of participants to correctly classify spermatozoa was further assessed by the group classification described for strict criteria at each stage of the programme, namely, p-pattern (4% normal forms), g-pattern (514% normal forms) and normal pattern (>14% normal forms). A group classification was regarded as incorrect when the mean percentage normal forms reported surpassed the 95% CI (>2xSD) of the mean score assessed by the reference laboratory.
Statistical analysis
Statistical evaluation was performed with Statistica for Windows Release 5.1 (Statsoft Southern Africa Ltd, Private Bag X131, South Africa; 1997) and consisted of evaluation of individual scatterplots for each participant's data and pairwise comparisons of differences at and between evaluation times with the Wilcoxon matched pairs test.
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Results |
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Pairwise comparison of the differences at each evaluation time revealed the following: Baseline differences (pre-training) differed significantly from the differences at 3 months (P = 0.0002) as well as at 6 months after training (P = 0.007). The differences at 6 months did not differ significantly from those at 3 months (P = 0.27).
Individual progress for each of the 20 participants were analysed using categorized scatterplots (Figure 1AT). The learning curve for each participant is shown in the scatterplots by the curve between individual points. The initial difference recorded between participant and the reference laboratory shown in Figure 1A
for example, was 51% and further decreased to 2%. The dramatic improvement is illustrated by the steep slope of the regression line. Similar findings were recorded for all but Figure 1C, D, Q and R
. The slope of the regression line indicates that these individuals had a better understanding of the morphological configurations of a normal sperm cell. The participant represented in Figure 1R
did not enter the CQC programme, since this individual gained morphology experience at Tygerberg Hospital prior to the semenology workshop.
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Discussion |
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Individual scatterplots of the participants (Figure 1) demonstrate the difference in the initial knowledge between the trainee and trainer, which represents the trainee's inability to identify the normal sperm cell. The scatterplots may also be very useful in presenting such a technician training project on a national basis, because the referring laboratory will be able to monitor the technical quality and performance of each participating laboratory. Changes in individual standard can immediately be identified from such a scatterplot and steps taken to rectify the problem. The correct group classification may be a stronger indicator of the success of training in andrology; the initial classification revealed only one laboratory (5%) that had a correct morphology classification, while 3 months later 42% (8/19) of the laboratories were correct. After 6 months of continuous quality control, 83% (10/12) of the participating laboratories classified spermatozoa correctly according to the groups outlined by strict criteria. Equally important is the identification of laboratories that do not improve during the 6 months period. These laboratories should be alerted about the technical quality of the evaluation and brought back to the reference laboratory for advanced training sessions.
The results of the present study are encouraging since the 20 participants, once trained, maintained for a period of at least 6 months a high level of accuracy in their morphology assessments. Training of technicians as well as regular proficiency testing will ensure continuous communication with the referring laboratory. Proficiency testing of technicians' skills is of the utmost importance if andrology laboratories want to secure a professional code of conduct. The authors firmly believe that global quality control measurements in andrology laboratories will eventually become mandatory. A high quality semen analysis still represents the cornerstone in the investigation of the infertile couple. In order to maintain low intra- and inter-technician variation and high quality proficiency testing among laboratory technicians, continuous teaching programmes should be available to all.
The authors feel satisfied with the results of this ongoing study, since the initial training and continuous quality control of the andrology laboratories in Africa provides a standard of sperm morphology evaluation that will eventually be beneficial to the clinician and patient. Sperm morphology training programmes are of the utmost importance in all andrology laboratories, especially since the literature clearly indicates that improper technical skills can leave clinicians without a proper diagnosis.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 2, 1999; accepted on November 19, 1999.