ORIGINAL ARTICLE
Sperm morphology training in India D. R. Franken1 & C. Joshi2 1 Department of Obstetrics and Gynecology, Faculty of Health Science, University of the Free State, Bloemfontein, South Africa; 2 Mohak Hi Tech Test Tube Baby Center, Department of Reproductive Medicine, Sri Arobindo Institute of Medical Science, Indore, India
Keywords Sperm morphology training Correspondence Prof. Daniel R. Franken, Department of Obstetrics & Gynecology, Faculty of Health Science, University of the Free State, Bloemfontein 9300, South Africa. Tel.: 27 51 4053447; Fax: 27 51 4442660; E-mail: [email protected] Accepted: August 18, 2014 doi: 10.1111/and.12358
Summary Sperm morphology is regarded as possibly the most consistent sperm variable that is related to in vitro and in vivo fertilisation success or failure. There is also a re-emerging awareness of andrology, particularly in developing countries where healthcare resources are often limited. This study aimed to record the importance of hands-on training on the sperm morphology reading skills among 174 Indian infertility workers. During a series of five workshops presented by the Indian Academy of Clinical Embryologists, delegates were requested to record the percentage normal spermatozoa on pre-stained Diff Quick slides. A total of four slides each containing different percentages normal spermatozoa were used during the workshops. The delegates were requested to record the percentage normal spermatozoa prior and after training. The results were expressed as the percentage difference between the reference value and the pre-training and post-training values reported by the workshop delegates. The percentage difference indicated a significant improvement in the reading skills, for example from 253 244% to 20 93% for slide 1. Similar values were recorded for slide 2, 3 and 4. If we consider the present findings as representative of medical laboratory scientist’s sperm morphology reading skills, clinicians should be concerned about the diagnostic quality as far as male infertility is concerned.
Introduction The value of sperm morphology assessments as predictor of a man’s fertilising 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 specimen’s differences in proficiency among technicians and inherent differences in classification criteria and method. These differences in methodologies between laboratories are well known (Ombelet et al., 1998), but since the publication of the WHO manual, the between-laboratory variation can be negated. With proper hands-on training and preferably instalment of an external quality control program, intra-and inter-individual variability should reduce to nonsignificant levels of difference (Franken et al., 2000). An ever-remaining problem would be the comparison of predictive power of semen parameters of one setting with another and attempt to transfer the specific values to fit their patient population (Ombelet et al., 1998). Since the publication of the World Health Organization’s WHO manual for the examination of human 932
semen and sperm-cervical mucus interaction, concerns were raised regarding the interpretation of the new lower reference values (WHO, 2010). This is especially true for the lower limits reported for sperm morphology. The controversy that exists regarding the interpretation of the lower reference values for morphology divided clinicians into two groups, namely those who are willing to adopt the values and those who are not. Those who are adopting the values should be aware of the total lack in most cases among laboratory technicians regarding the interpretation of sperm morphology. Despite many laboratories claim to use the WHO manual as guidance to report sperm morphology, a survey concluded that only 5% of the laboratories in the United Kingdom adhered to the morphology rules set by the WHO (Riddell et al., 2005). This is also true for slide preparation and staining techniques (Ombelet et al., 1997). Therefore, it stands for a reason that earlier reports suggested that analysis of human semen has become a neglected test and should be regarded as a technique (Ombelet et al., 1997, McDonough, 1997; Chang et al., 1983). The time has therefore come for technological improvements that will bring cost-effective clinically use© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
Sperm morphology
D. R. Franken and C. Joshi
Table 1 Descriptive statistics of morphology scores during pre- and post-training sessions of 174 workshop participants
n Slide Slide Slide Slide Slide Slide Slide Slide
1 1 2 2 3 3 4 4
Pre-training Post-training Pre-training Post-training Pre-training Post-training Pre-training Post-training
88 88 48 48 16 16 22 22
Reference values % normal sperm 10 4 15 11
Mean
95% CI
Median
SD
Percentage difference from the reference value Mean SD
25.8 1.8 26.7 5.6 13.2 3.1 31.0 7.9
17–27 2–1 13–31 1–6 3 to 25 9 to 5 1–64 7 to 18
23.0 1.0 21 2 11,0 6.5 29.0 1.0
24.3 8.9 24.5 10.3 18.3 7.6 29.9 16.1
253 20 638 129 88 21 265 57
ful sperm morphology results at least partially. This development will enhance the clinical value of the routine semen analysis (Ford, 2010; Esteves & Agarwal, 2011). This study reports on the results of multiple morphology training workshops in India. Materials and methods A total of 230 delegates were invited by the Academy of Clinical Embryologists (ACE) in India to attend a two semenology workshops series during 2013 and 2014. The 2013 workshops were presented in Mumbai, Delhi and Bengaluru, while the 2014 series were presented in Chennai, Bhopal, Ludhiana, Guwahati and Jaipur. The morphology training programme consisted of two sessions, namely pre- and post-training periods. During the pretraining session, each delegate received two pre-stained DiffQuik slides which they had to score for the percentage normal spermatozoa on each slide. Each slide contained different percentages of normal spermatozoa. The results of the slides were kept blind after the pre-training session. Following this session, delegates were lectured on the WHO 2010 criteria for sperm morphology. All aspects of sperm morphology were addressed, that is normal forms as well as abnormal forms. Special attention was given to the slightly abnormal spermatozoa, and delegates were encouraged to classify slightly abnormal forms (borderline forms) as abnormal. This is in agreement with the criteria set by the WHO (2010) and by Menkveld et al. (1990). During the training session, the author used computerprojected high-quality photomicrographs of individually numbered spermatozoa. Each numbered spermatozoa was individually discussed and aberrations, if any, were underlined and explained. During the post-training session, delegates were requested to re-evaluate the same two pre-stained slides that were provided during the pre-training session. Only at this point, the results of the percentage normal spermatozoa on the slides were provided. © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
244 93 610 258 122 51 278 149
Results The pre-training and post-training results are depicted in Table 1. The results in Table 1 and Fig. 1 underline the lack of knowledge regarding the configuration of a normal spermatozoon described by Menkveld et al., 1990;. The percentage difference for slide 3 of minus 20% (reference value 15%) is a typical adjustment that is often encountered. This is caused by the individual being too strict and subsequently reports mostly abnormalities. The data were used to calculate the percentage difference from the mean recorded for the pre- and post-training sessions. Discussion Our experience has indicated that the main problem in recording sperm morphology values correctly concerns the classification of those forms that the inexperienced
Fig. 1 Percentage difference between pre- and post-training sperm morphology values. (a–d) P ≤ 0.001 Student’s paired t-test.
933
Sperm morphology
workers are uncertain of, that is, whether these forms are normal, abnormal or borderline. In order to set up an internal control system and to ensure consistency during evaluations, laboratory technicians are encourage to prepare copies of those spermatozoa that they are uncertain of the correct classification. These forms are then collected and stored as a collage of forms that caused uncertainty as far as its classification is concerned. With the experience gained and with the clinical information (fertilisation rates) at hand, the technician can reclassify these forms as normal or abnormal. According to a survey, the percentage of laboratories using the WHO-recommended Papanicolaou and Shorr staining procedures decreased, and on the other hand, an increase is reported in the use of rapid staining methods, that is Testsimplets or DiffQuick (Mallidis et al., 2012). Classification methods vary from Tygerberg strict criteria, WHO, 1999; D€ usseldorf (Hoffman & Haider, 1985) and in-house handdown techniques. In certain cases, we received information from nonfertility laboratories recording sperm morphology data using unstained smears as well as eosin–nigrosin-stained slides. These practices are a total contrast with the procedures described by the World Health Organization, 2010 manual and are partly responsible for the confusion as the clinical value of sperm morphology is concerned. It is also true that many nonfertility laboratories are performing semen analysis and providing inferior results that are subsequently use to guide the management of infertility consulting patients. If standards are to be set forth, they need to be adhered to the guidelines set by the World Health Organization (2010). Although the present data illustrate a vast improvement in the technical knowledge of the workshops attendees, our previous work has highlighted the important role of a follow up of external quality control program (Franken et al., 2000). During that study, the reported data over a period of 21 months indicated the reading skills as excellent in 83% of the participating laboratories. Participants maintained their morphological reading skills, and their results agreed with the reference laboratory by not exceeding a SD limit of 0.2 (Franken et al., 2003, 2010). The findings of this study should be seen as a wake-up call for laboratories that provide semen analyses for referring clinicians. If we consider the present findings as representative of medical laboratory scientist’s sperm morphology reading skills, clinicians should be concerned about the diagnostic quality as far as male infertility is concerned. It is well known that the evaluation of the sperm morphology with light microscopy is subjective and therefore difficult to compare between laboratories in cases where different techniques are used. Standardising the slide preparation and staining procedure should be encouraged to improve the intra- and inter-technical variations. 934
D. R. Franken and C. Joshi
References Chong AR, Waiters CA, Weinrelb SA (1983) The neglected laboratory test. The semen analysis. J Androl 4:280–282. Esteves SC, Agarwal A (2011) Impact of the new WHO guidelines on diagnosis and practice of male infertility. Open Reprod Sci J 3:7–15. Ford WCL (2010) Comments on the release of the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen. Asian J Androl 12:59–63. Franken DR, Smith M, Menkveld R, Kruger TF, SekaddeKigondu C, Mbizvo M, Akande EO (2000) The development of a continuous quality control (CQS) program for strict sperm morphology among sub-Sahara African laboratories. Hum Reprod 5:667–671. Franken DR, Menkveld R, Kruger TF, Sekadde-Kigondu C, Lombard C (2003) Monitoring technologist reading skills in a sperm morphology quality control program. Fertil Steril 79:1637–1643. Franken DR, Aneck-Hahn N, Lombaard C, Kruger TF (2010) Semenology training programs: 8 years’ experience. Fertil Steril 94:2615–2619. Hoffman N, Haider SG (1985) Neue Ergebnisse morphologisher Diagnostik der Spermatogenesest€ orungen. Gyn€akologe 18:79–80. Mallidis C, Cooper TG, Hellenkemper B, Lablans M, Uckert F, Nieschlag E (2012) Ten years of semen analysis external quality control. Fertil Steril 98:611–616. McDonough P (1997) Editorial comment: has traditional sperm analysis lost its clinical relevance? Fertil Steril 67:585–587. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA (1990) The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 5:586–592. Ombelet W, Pollet H, Bosmans E, Vereecken A (1997) Results of a questionnaire on sperm morphology assessment. Hum Reprod 12:1015–1020. Ombelet W, Bosmans E, Janssen M, Cox A, Maes M, Punjabi U, Blaton V, Gunst J, Haidl G, Wouters E, Spiessens C, Bornman MS, Pienaar E, Menkveld R, Lombard CJ (1998) Multicenter study on reproducibility of sperm morphology assessments. Arch Androl 4:103–114. Riddell D, Pacey A, Whittington K (2005) Lack of compliance by UK andrology laboratories with World Health Organization recommendations for sperm morphology assessment. Hum Reprod 20:3441–3445. World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Interaction, 4th edn. Cambridge University Press, Cambridge, UK. World Health Organization (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization, Geneva.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
Sperm morphology training in India D. R. Franken1 & C. Joshi2 1 Department of Obstetrics and Gynecology, Faculty of Health Science, University of the Free State, Bloemfontein, South Africa; 2 Mohak Hi Tech Test Tube Baby Center, Department of Reproductive Medicine, Sri Arobindo Institute of Medical Science, Indore, India
Keywords Sperm morphology training Correspondence Prof. Daniel R. Franken, Department of Obstetrics & Gynecology, Faculty of Health Science, University of the Free State, Bloemfontein 9300, South Africa. Tel.: 27 51 4053447; Fax: 27 51 4442660; E-mail: [email protected] Accepted: August 18, 2014 doi: 10.1111/and.12358
Summary Sperm morphology is regarded as possibly the most consistent sperm variable that is related to in vitro and in vivo fertilisation success or failure. There is also a re-emerging awareness of andrology, particularly in developing countries where healthcare resources are often limited. This study aimed to record the importance of hands-on training on the sperm morphology reading skills among 174 Indian infertility workers. During a series of five workshops presented by the Indian Academy of Clinical Embryologists, delegates were requested to record the percentage normal spermatozoa on pre-stained Diff Quick slides. A total of four slides each containing different percentages normal spermatozoa were used during the workshops. The delegates were requested to record the percentage normal spermatozoa prior and after training. The results were expressed as the percentage difference between the reference value and the pre-training and post-training values reported by the workshop delegates. The percentage difference indicated a significant improvement in the reading skills, for example from 253 244% to 20 93% for slide 1. Similar values were recorded for slide 2, 3 and 4. If we consider the present findings as representative of medical laboratory scientist’s sperm morphology reading skills, clinicians should be concerned about the diagnostic quality as far as male infertility is concerned.
Introduction The value of sperm morphology assessments as predictor of a man’s fertilising 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 specimen’s differences in proficiency among technicians and inherent differences in classification criteria and method. These differences in methodologies between laboratories are well known (Ombelet et al., 1998), but since the publication of the WHO manual, the between-laboratory variation can be negated. With proper hands-on training and preferably instalment of an external quality control program, intra-and inter-individual variability should reduce to nonsignificant levels of difference (Franken et al., 2000). An ever-remaining problem would be the comparison of predictive power of semen parameters of one setting with another and attempt to transfer the specific values to fit their patient population (Ombelet et al., 1998). Since the publication of the World Health Organization’s WHO manual for the examination of human 932
semen and sperm-cervical mucus interaction, concerns were raised regarding the interpretation of the new lower reference values (WHO, 2010). This is especially true for the lower limits reported for sperm morphology. The controversy that exists regarding the interpretation of the lower reference values for morphology divided clinicians into two groups, namely those who are willing to adopt the values and those who are not. Those who are adopting the values should be aware of the total lack in most cases among laboratory technicians regarding the interpretation of sperm morphology. Despite many laboratories claim to use the WHO manual as guidance to report sperm morphology, a survey concluded that only 5% of the laboratories in the United Kingdom adhered to the morphology rules set by the WHO (Riddell et al., 2005). This is also true for slide preparation and staining techniques (Ombelet et al., 1997). Therefore, it stands for a reason that earlier reports suggested that analysis of human semen has become a neglected test and should be regarded as a technique (Ombelet et al., 1997, McDonough, 1997; Chang et al., 1983). The time has therefore come for technological improvements that will bring cost-effective clinically use© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
Sperm morphology
D. R. Franken and C. Joshi
Table 1 Descriptive statistics of morphology scores during pre- and post-training sessions of 174 workshop participants
n Slide Slide Slide Slide Slide Slide Slide Slide
1 1 2 2 3 3 4 4
Pre-training Post-training Pre-training Post-training Pre-training Post-training Pre-training Post-training
88 88 48 48 16 16 22 22
Reference values % normal sperm 10 4 15 11
Mean
95% CI
Median
SD
Percentage difference from the reference value Mean SD
25.8 1.8 26.7 5.6 13.2 3.1 31.0 7.9
17–27 2–1 13–31 1–6 3 to 25 9 to 5 1–64 7 to 18
23.0 1.0 21 2 11,0 6.5 29.0 1.0
24.3 8.9 24.5 10.3 18.3 7.6 29.9 16.1
253 20 638 129 88 21 265 57
ful sperm morphology results at least partially. This development will enhance the clinical value of the routine semen analysis (Ford, 2010; Esteves & Agarwal, 2011). This study reports on the results of multiple morphology training workshops in India. Materials and methods A total of 230 delegates were invited by the Academy of Clinical Embryologists (ACE) in India to attend a two semenology workshops series during 2013 and 2014. The 2013 workshops were presented in Mumbai, Delhi and Bengaluru, while the 2014 series were presented in Chennai, Bhopal, Ludhiana, Guwahati and Jaipur. The morphology training programme consisted of two sessions, namely pre- and post-training periods. During the pretraining session, each delegate received two pre-stained DiffQuik slides which they had to score for the percentage normal spermatozoa on each slide. Each slide contained different percentages of normal spermatozoa. The results of the slides were kept blind after the pre-training session. Following this session, delegates were lectured on the WHO 2010 criteria for sperm morphology. All aspects of sperm morphology were addressed, that is normal forms as well as abnormal forms. Special attention was given to the slightly abnormal spermatozoa, and delegates were encouraged to classify slightly abnormal forms (borderline forms) as abnormal. This is in agreement with the criteria set by the WHO (2010) and by Menkveld et al. (1990). During the training session, the author used computerprojected high-quality photomicrographs of individually numbered spermatozoa. Each numbered spermatozoa was individually discussed and aberrations, if any, were underlined and explained. During the post-training session, delegates were requested to re-evaluate the same two pre-stained slides that were provided during the pre-training session. Only at this point, the results of the percentage normal spermatozoa on the slides were provided. © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
244 93 610 258 122 51 278 149
Results The pre-training and post-training results are depicted in Table 1. The results in Table 1 and Fig. 1 underline the lack of knowledge regarding the configuration of a normal spermatozoon described by Menkveld et al., 1990;. The percentage difference for slide 3 of minus 20% (reference value 15%) is a typical adjustment that is often encountered. This is caused by the individual being too strict and subsequently reports mostly abnormalities. The data were used to calculate the percentage difference from the mean recorded for the pre- and post-training sessions. Discussion Our experience has indicated that the main problem in recording sperm morphology values correctly concerns the classification of those forms that the inexperienced
Fig. 1 Percentage difference between pre- and post-training sperm morphology values. (a–d) P ≤ 0.001 Student’s paired t-test.
933
Sperm morphology
workers are uncertain of, that is, whether these forms are normal, abnormal or borderline. In order to set up an internal control system and to ensure consistency during evaluations, laboratory technicians are encourage to prepare copies of those spermatozoa that they are uncertain of the correct classification. These forms are then collected and stored as a collage of forms that caused uncertainty as far as its classification is concerned. With the experience gained and with the clinical information (fertilisation rates) at hand, the technician can reclassify these forms as normal or abnormal. According to a survey, the percentage of laboratories using the WHO-recommended Papanicolaou and Shorr staining procedures decreased, and on the other hand, an increase is reported in the use of rapid staining methods, that is Testsimplets or DiffQuick (Mallidis et al., 2012). Classification methods vary from Tygerberg strict criteria, WHO, 1999; D€ usseldorf (Hoffman & Haider, 1985) and in-house handdown techniques. In certain cases, we received information from nonfertility laboratories recording sperm morphology data using unstained smears as well as eosin–nigrosin-stained slides. These practices are a total contrast with the procedures described by the World Health Organization, 2010 manual and are partly responsible for the confusion as the clinical value of sperm morphology is concerned. It is also true that many nonfertility laboratories are performing semen analysis and providing inferior results that are subsequently use to guide the management of infertility consulting patients. If standards are to be set forth, they need to be adhered to the guidelines set by the World Health Organization (2010). Although the present data illustrate a vast improvement in the technical knowledge of the workshops attendees, our previous work has highlighted the important role of a follow up of external quality control program (Franken et al., 2000). During that study, the reported data over a period of 21 months indicated the reading skills as excellent in 83% of the participating laboratories. Participants maintained their morphological reading skills, and their results agreed with the reference laboratory by not exceeding a SD limit of 0.2 (Franken et al., 2003, 2010). The findings of this study should be seen as a wake-up call for laboratories that provide semen analyses for referring clinicians. If we consider the present findings as representative of medical laboratory scientist’s sperm morphology reading skills, clinicians should be concerned about the diagnostic quality as far as male infertility is concerned. It is well known that the evaluation of the sperm morphology with light microscopy is subjective and therefore difficult to compare between laboratories in cases where different techniques are used. Standardising the slide preparation and staining procedure should be encouraged to improve the intra- and inter-technical variations. 934
D. R. Franken and C. Joshi
References Chong AR, Waiters CA, Weinrelb SA (1983) The neglected laboratory test. The semen analysis. J Androl 4:280–282. Esteves SC, Agarwal A (2011) Impact of the new WHO guidelines on diagnosis and practice of male infertility. Open Reprod Sci J 3:7–15. Ford WCL (2010) Comments on the release of the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen. Asian J Androl 12:59–63. Franken DR, Smith M, Menkveld R, Kruger TF, SekaddeKigondu C, Mbizvo M, Akande EO (2000) The development of a continuous quality control (CQS) program for strict sperm morphology among sub-Sahara African laboratories. Hum Reprod 5:667–671. Franken DR, Menkveld R, Kruger TF, Sekadde-Kigondu C, Lombard C (2003) Monitoring technologist reading skills in a sperm morphology quality control program. Fertil Steril 79:1637–1643. Franken DR, Aneck-Hahn N, Lombaard C, Kruger TF (2010) Semenology training programs: 8 years’ experience. Fertil Steril 94:2615–2619. Hoffman N, Haider SG (1985) Neue Ergebnisse morphologisher Diagnostik der Spermatogenesest€ orungen. Gyn€akologe 18:79–80. Mallidis C, Cooper TG, Hellenkemper B, Lablans M, Uckert F, Nieschlag E (2012) Ten years of semen analysis external quality control. Fertil Steril 98:611–616. McDonough P (1997) Editorial comment: has traditional sperm analysis lost its clinical relevance? Fertil Steril 67:585–587. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA (1990) The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 5:586–592. Ombelet W, Pollet H, Bosmans E, Vereecken A (1997) Results of a questionnaire on sperm morphology assessment. Hum Reprod 12:1015–1020. Ombelet W, Bosmans E, Janssen M, Cox A, Maes M, Punjabi U, Blaton V, Gunst J, Haidl G, Wouters E, Spiessens C, Bornman MS, Pienaar E, Menkveld R, Lombard CJ (1998) Multicenter study on reproducibility of sperm morphology assessments. Arch Androl 4:103–114. Riddell D, Pacey A, Whittington K (2005) Lack of compliance by UK andrology laboratories with World Health Organization recommendations for sperm morphology assessment. Hum Reprod 20:3441–3445. World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Interaction, 4th edn. Cambridge University Press, Cambridge, UK. World Health Organization (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization, Geneva.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 932–934
