1 University of Bristol, Clinical Science at South Bristol (Obstetrics & Gynaecology), St Michaels Hospital, Southwell Street, Bristol and 2 Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessop Wing, Royal Hallamshire Hospital, Sheffield, S10 2SF, UK
3 To whom correspondence should be addressed. E-mail: K.Whittington{at}bris.ac.uk
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Abstract |
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Key words: compliance/morphology/semen analysis/sperm/World Health Organization
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Introduction |
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Although a number of previously conducted studies have shown that the variables of sperm concentration and motility are correlated with the probability of conception following IVF (Kruger et al., 1986; Grow et al., 1994
) as well as in unassisted conception in cohorts of pregnancy planners (Bonde et al., 1998
; Guzick et al., 2001
), it is still controversial as to whether there exists an obvious threshold between fertile and infertile men in terms of their semen quality. This is disappointing because the information would be of immense value to clinicians when advising patients as to the probability of achieving a pregnancy, by whatever mode of conception was considered appropriate.
Interestingly, in their study of 696 fertile and 765 infertile men Guzick et al. (2001) found that although there was significant overlap between the semen variables of the two groups, sperm morphology was the greatest discriminator between them. This is perhaps of no surprise since it has been known for some time that only morphologically normal sperm can pass though mid-cycle cervical mucus (Katz et al., 1990
) and the human zona pellucida selectively binds sperm with normal morphology (Liu and Baker, 1992
) such that men with severe teratozoospermia frequently have defective spermzona pellucida interaction (Liu and Baker, 2003
). However, since the pioneering work of MacLeod (1956)
, it is the techniques of assessment of sperm morphology that have undergone the most change with successive versions of the WHO Laboratory Manual (WHO, 1980
, 1987
, 1992
, 1999
) providing increasingly stringent methodological approaches for the identification and classification of sperm with normal morphology. Therefore, with this knowledge it might be assumed that the techniques for sperm morphology assessment were becoming easier to implement and as such providing more robust data for the development of thresholds for use in the clinic.
Interestingly, however, a global survey performed by Ombelet et al. (1997) illustrated the relatively poor compliance of laboratories in performing sperm morphology according to the then relevant WHO (1992)
guidelines for sperm morphology. More recently Keel et al. (2002)
illustrated the general lack of standardization in laboratories across the USA, with only 85% of laboratories performing sperm morphology assessment as part of semen analysis and of these only 23% using the WHO (1999)
criteria for identifying normal sperm. Clearly, therefore, there is some way to go before the techniques of sperm morphology assessment are implemented consistently around the world.
It was in the knowledge of these observations, in addition to the fact that, anecdotally, laboratory personnel often report the assessment of sperm morphology as one of the hardest parts of semen analysis to perform, that a survey of current laboratory practice within the UK was conducted. The focus of this survey was to determine if the implementation of the methods described in the 4th edition of the WHO Laboratory Manual (WHO, 1999) has indeed reduced the high degree of variability in the techniques used to assess sperm morphology and thereby potentially increased the clinical usefulness of morphology as a predictive indicator of male fertility.
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Materials and methods |
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Results |
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Staining methods
When asked whether samples were stained prior to morphology assessment, 14 (74%) of the specialist laboratories and two (11%) of the DGH laboratories reported that they observed unstained preparations. However, two of the laboratories in each group reported that they observed both unstained and stained preparations. Therefore, 25 out of 37 of all laboratories (68%) assessed sperm morphology on stained preparations: seven (37%) of the specialist laboratories and 18 (100%) of the DGH laboratories (Table II).
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When these 25 laboratories were asked about the staining methods they used (Table II), only five specialist laboratories and 12 DGH laboratories used either Papanicolaou or Diff Quick as recommended by WHO (1999). Therefore 65% of all laboratories, and 22% of laboratories that did stain samples prior to analysis, were following procedures that were not compliant with current WHO guidelines for the staining of semen smears.
Classification of sperm morphology
The WHO (1999) criteria for sperm classification (size parameters) were the most popular, being used by 32 of the 37 laboratories (86%) (i.e. 16 laboratories from each group). The now outdated WHO (1992)
criteria were being used by two laboratories: one from each group. Two specialist laboratories used the Kruger criteria and one DGH laboratory used the Mortimer (1994)
classification.
Magnification and sample size
When asked about the microscope magnification used for carrying out a morphology assessment and whether a graticule (or equivalent) was used to determine sperm dimensions, 32 out of 37 (86%) of the participants completed this section of the questionnaire. Of the 17 specialist laboratories that replied, only three (18%) were using WHO (1999)-recommended optics (x1000 magnification with oil immersion and graticule) compared to two (13%) of the 15 DGH laboratories that responded to the question. Furthermore, only 30% of laboratories (11 out of 37) reported that they classify
200 sperm during morphology assessment with the majority (69%) choosing to classify only 100 (or fewer) sperm.
Reporting morphology
Unfortunately, the WHO (1999) Laboratory Manual fails to provide a reference value for sperm morphology assessment to assist clinicians in discriminating between fertile and subfertile men. Therefore, laboratories performing semen analysis must provide their own. A total of 32 laboratories (19 specialist laboratories and 13 DGH laboratories) responded to this section of the questionnaire (Figure 1) and reported what reference value they provided to clinicians on their semen analysis report form. In brief, a total of 19 (51%) laboratories (10 specialist laboratories and nine DGH laboratories) use a reference value of 15% normal forms. This is in line with the footnote to Table 1b of the WHO (1999)
Laboratory Manual, which suggests that data from assisted reproductive technology programmes indicates that fertilization rates in vitro will decline when sperm morphology falls to <15% normal forms. By comparison, four (11%) laboratories have taken a stricter view with a reference value of <14% or <10% with a further eight (22%) laboratories taking a more lenient view with threshold values of between 20 and 30% normal forms. Finally, one of the specialist laboratories included in this survey had identified their own reference range based on their local fertile population. This group reports % oval forms and had previously found that <5% oval forms is consistent with fertility impairment as determined from dimensional parameters recorded via scanning electron micrographs (SEM) on donor sperm known to have produced pregnancies within six treatment cycles.
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Quality control
A greater proportion of DGH laboratories (16 out of 18) participated in an external quality control scheme in comparison to the specialist laboratories (13 out of 19). Participation in the scheme organized by UK NEQAS (St Marys Hospital, Manchester, UK) was the option all laboratories subscribed to, with one specialist laboratory also participating in the scheme organized by ESHRE. Surprisingly, the levels of compliance with internal quality control were notably lower with under half of all laboratories reporting that they were undertaking internal quality control: eight out of 18 (44%) of DGH laboratories and eight out of 19 (42%) specialist laboratories.
Cumulative compliance with WHO 1999 Guidelines for Morphology
Table III illustrates the number of laboratories that are compliant with each of the components of the guidelines for sperm morphology assessment as published by WHO (1999). In summary, when all aspects of staining methods, classification criteria, magnification and objectives and quality control are taken into account, only two of the 37 laboratories surveyed (5%) were compliant with all aspects of the current WHO (1999)
guidelines for sperm morphology assessment.
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Discussion |
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In addition to selecting the correct staining methods, WHO (1999) also recommends that x100 oil immersion brightfield objective (with at least a x10 ocular) should be used to observe the stained smear. Yet only 14% of all laboratories in this survey reported using such optics. Similarly, 69% of laboratories reported that they were observing
100 sperm in order to derive a value for sperm morphology. The need for compliance with WHO (1999)
recommendations for these parameters is more than a matter of simply following the rules as both have the potential to introduce significant sampling error into sperm morphology assessments, although the impact of sample size is arguably more profound. The influence of sample size on the estimation of sperm morphology has recently been the subject of a more detailed analysis by Kuster et al. (2004)
and although this paper is primarily aimed at the veterinary field where animal species have higher percentages of morphologically normal sperm in their ejaculates, the theory is equally applicable to human samples as is illustrated in the chapter on quality control (QC) in the WHO (1999)
manual. Briefly, the fewer sperm that are counted during a morphology assessment, the wider the corresponding 95% confidence interval of the result obtained. Ironically, more sperm need to be evaluated in order to obtain the same confidence with regard to sperm morphology in a specimen with a low percentage of normal forms, such as exists in human males in comparison to many animal species. WHO (1999)
have determined that the counting of 200 sperm is the absolute minimum requirement on which to base a sperm morphology assessment. If an assessment is made on fewer than this number, it is entirely plausible that teratozoospermia warranting the use of ICSI may be diagnosed, whereas in reality the sample is quite normal. How well laboratory scientists understand the effect on the reported result of classifying too few sperm is unclear, but needs to be addressed in training programmes and in future editions of the WHO Laboratory Manual.
A new topic introduced into the WHO (1999) Laboratory Manual was the importance of QC within the diagnostic laboratory. Although both internal and external quality control are now integral to laboratory accreditation (Burnett, 1996
), it was surprising to find that although 78% of laboratories were taking part in a external quality assurance scheme, only 43% undertook internal quality control. Although internal quality control in Andrology can be considered cumbersome and difficult to implement (Clements et al., 1995
) its benefits are well known and WHO (1999)
recommends that between 1 and 5% of samples should be used for internal QC. The success of external quality control schemes is well recognized (Cooper, 1996
; Jorgensen et al., 1997
; Auger et al., 2000
).
The omission of a normal reference range for morphology in Table 1a of WHO (1999) has certainly added to the ambiguity surrounding morphology assessment and further increased confusion as to its clinical relevance. Although the Manual did include a footnote to indicate that as sperm morphology falls to <15% normal forms the fertilization rate decreases, presumably based on the work of Kruger et al. (1986)
, the results of this survey (Figure 1) suggest that many UK laboratories have taken a pragmatic decision to cite this value as the reference range for fertile men. Although this decision has now been given some validity following the study of pregnancy planners by Guzick et al. (2001)
, the usefulness of this decision in the many laboratories that are poorly implementing sperm morphology assessment should be questioned.
Finally, it is interesting to consider whether the differences in the approach of sperm morphology assessment, and therefore compliance with WHO (1999) guidelines, differs between laboratories where andrology is carried out in a mixed discipline environment DHG laboratories as opposed to that performed in specialist laboratories usually associated with assisted conception units. The possibility for different approaches may come from the fact that the staff mix in these two types of laboratories is quite different (Table I) with typically more embryologists and PhD scientists working in specialist laboratories as opposed to staff in DGH laboratories being almost entirely biomedical scientists, with quite different training routes. Whilst it could be argued that DGH laboratories are arguably closer to compliance with WHO (1999)
recommendations than specialist laboratories linked to assisted conception units (by virtue of more DGH laboratories observing stained smears rather than unstained wet preparations), in reality only one laboratory from each group was fully compliant with all of the WHO (1999)
recommendations (Table III). Although it is perhaps understandable that andrology laboratories linked to embryology laboratories (specialist laboratories) would want to avoid the use of unnecessary volatile compounds, such as solvents, contaminating the atmosphere in which embryos are cultured (Elder and Dale, 2000
) it is of concern that this may mean that diagnostic semen analysis is being performed inappropriately or attempts are being made to correlate sperm morphology results with treatment outcomes such as fertilization rate or pregnancy.
In conclusion, these results illustrate that there remains a great need for improvements in the standardization of methodology in the UK with regard to sperm morphology assessment. Improvements in andrology training and education are urgently needed to address these issues.
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References |
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Submitted on May 26, 2005; resubmitted on June 15, 2005; accepted on June 24, 2005.