WHO laboratory manual for the Examination and processing of human semen

FIFTH EDITION

Chapter 1 Background 1

1.1 Introduction 1

1.2 The fi fth edition 1 1.3 Scope of the manual 3

PART I. SEMEN ANALYSIS

Chapter 2 Standard procedures 7

2.1 Introduction 7

2.2 Sample collection 10

2.2.1 Preparation 10 2.2.2 Collection of semen for diagnostic or research purposes 11 2.2.3 Sterile collection of semen for assisted reproduction 11 2.2.4 Sterile collection of semen for microbiological analysis 11 2.2.5 Collection of semen at home 12 2.2.6 Collection of semen by condom 12 2.2.7 Safe handling of specimens 13

2.3 Initial macroscopic examination 13

2.3.1 Liquefaction 13 2.3.2 Semen viscosity 14 2.3.3 Appearance of the ejaculate 15 2.3.4 Semen volume 15 2.3.5 Semen pH 16

2.4 Initial microscopic investigation 17

2.4.1 Thorough mixing and representative sampling of semen 17 2.4.2 Making a wet preparation 18 2.4.3 Aggregation of spermatozoa 19 2.4.4 Agglutination of spermatozoa 19 2.4.5 Cellular elements other than spermatozoa 21

2.5 Sperm motility 21

2.5.1 Categories of sperm movement 22 2.5.2 Preparing and assessing a sample for motility 22 2.5.3 Worked examples 25 2.5.4 Lower reference limit 26

2.6 Sperm vitality 26

2.6.1 Vitality test using eosin–nigrosin 27 2.6.2 Vitality test using eosin alone 29 2.6.3 Vitality test using hypo-osmotic swelling 30

2.7 Sperm numbers 32

2.7.1 Types of counting chamber 34 2.7.2 The improved Neubauer haemocytometer 34 2.7.3 Using the haemocytometer grid 35 2.7.4 Care of the counting chamber 35 2.7.5 Fixative for diluting semen 36 2.7.6 Importance of counting suffi cient spermatozoa 36

2.8 Routine counting procedure 37

2.8.1 Determining the required dilution 38 2.8.2 Preparing the dilutions and loading the haemocytometer chambers 39 2.8.3 Assessing sperm numbers in the counting chambers 41 2.8.4 Calculation of the concentration of spermatozoa in semen 43 2.8.5 Worked examples 43 2.8.6 Lower reference limit for sperm concentration 44 2.8.7 Calculation of the total number of spermatozoa in the ejaculate 44 2.8.8 Lower reference limit for total sperm number 44

2.9 Low sperm numbers: cryptozoospermia and suspected azoospermia 45

2.10 When an accurate assessment of low sperm numbers is not required 45

2.10.1 Taking no further action 45 2.10.2 Examination of centrifuged samples to detect spermatozoa 45 2.10.3 Examination of non-centrifuged samples to detect motile spermatozoa 46

2.11 When an accurate assessment of low sperm numbers is required 48

2.11.1 Assessing low sperm numbers in the entire improved Neubauer chamber (phase-contrast microscopy) 48 2.11.2 Assessing low sperm numbers in large-volume disposable chambers (fl uorescence microscopy) 52

2.12 Counting of cells other than spermatozoa 55

2.12.1 Calculation of the concentration of round cells in semen 55 2.12.2 Sensitivity of the method 56 2.12.3 Worked examples 56

2.13 Sperm morphology 56

2.13.1 The concept of normal spermatozoa 57 2.13.2 Preparation of semen smears 58

2.14 Staining methods 62

2.14.1 Traditional fixation and sequential staining 62 2.14.2 Papanicolaou staining procedure for sperm morphology 63 2.14.3 Shorr staining procedure for sperm morphology 65 2.14.4 Rapid staining procedure for sperm morphology 66

2.15 Examining the stained preparation 67

2.15.1 Classification of normal sperm morphology 67 2.15.2 Classifi cation of abnormal sperm morphology 69

2.16 Morphology plates 70

2.17 Analysing a sperm morphology smear 99

2.17.1 Assessment of normal sperm morphology 99 2.17.2 Worked examples 100 2.17.3 Lower reference limit 100 2.17.4 Assessment of abnormal sperm morphology 101 2.17.5 Worked example 101 2.17.6 Assessment of specifi c sperm defects 102

2.18 Assessment of leukocytes in semen 102

2.18.1 Staining cellular peroxidase using ortho-toluidine 103

2.19 Assessment of immature germ cells in semen 107

2.20 Testing for antibody coating of spermatozoa 108 2.20.1 The mixed antiglobulin reaction test 109 2.20.2 The direct immunobead test 111 2.20.3 The indirect immunobead test 113

Chapter 3 Optional procedures 115

3.1 Indices of multiple sperm defects 115

3.1.1 Calculation of indices of multiple morphological defects 115 3.1.2 Worked example 116

3.2 Panleukocyte (CD45) immunocytochemical staining 117

3.2.1 Principle 117 3.2.2 Reagents 118 3.2.3 Procedure 118

3.3 Interaction between spermatozoa and cervical mucus 122

3.3.1 In-vivo (postcoital) test 122 3.3.2 In-vitro tests 125 3.3.3 In-vitro simplifi ed slide test 126

3.3.4 Capillary tube test 127

3.4 Biochemical assays for accessory sex organ function 130 3.4.1 Measurement of zinc in seminal plasma 130 3.4.2 Measurement of fructose in seminal plasma 132 3.4.3 Measurement of neutral -glucosidase in seminal plasma 134

3.5 Computer-aided sperm analysis 136

3.5.1 Introduction 136 3.5.2 Use of CASA to assess sperm motility 137 3.5.3 Use of CASA to estimate sperm concentration 140

3.5.4 Computer-aided sperm morphometric assessment 140

Chapter 4 Research procedures 142

4.1 Reactive oxygen species 142

4.1.1 Introduction 142 4.1.2 Measurement of reactive oxygen species generated by sperm suspensions 143

4.2 Human sperm–oocyte interaction tests 146

4.3 Human zona pellucida binding tests 146

4.4 Assessment of the acrosome reaction 147

4.4.1 Procedure for the fluorescence assessment of acrosomal status 147 4.4.2 Induced acrosome reaction assay 150 4.5 Zona-free hamster oocyte penetration test 152

4.5.1 Protocol 152

4.6 Assessment of sperm chromatin 157

PART II. SPERM PREPARATION

Chapter 5 Sperm preparation techniques 161

5.1 Introduction 161

5.1.1 When spermatozoa may need to be separated from seminal plasma 161 5.1.2 Choice of method 161 5.1.3 Effi ciency of sperm separation from seminal plasma and infectious organisms 162

5.2 General principles 162

5.3 Simple washing 163

5.3.1 Reagents 163 5.3.2 Procedure 163

5.4 Direct swim-up 164

5.4.1 Reagents 164 5.4.2 Procedure 164

5.5 Discontinuous density gradients 165

5.5.1 Reagents 165 5.5.2 Procedure 166

5.6 Preparing HIV-infected semen samples 166

5.7 Preparing testicular and epididymal spermatozoa 167

5.7.1 Enzymatic method 167 5.7.2 Mechanical method 167 5.7.3 Processing sperm suspensions for intracytoplasmic sperm injection 167

5.8 Preparing retrograde ejaculation samples 168

5.9 Preparing assisted ejaculation samples 168

Chapter 6 Cryopreservation of spermatozoa 169

6.1 Introduction 169

6.2 Semen cryopreservation protocols 172

6.2.1 Standard procedure 172 6.2.2 Modifi ed freezing protocols for oligozoospermia and surgically retrieved spermatozoa 175 6.2.3 Labelling of straws and records 176

PART III. QUALITY ASSURANCE

Chapter 7 Quality assurance and quality control 179

7.1 Controlling for quality in the andrology laboratory 179

7.2 The nature of errors in semen analysis 179

7.3 Minimizing statistical sampling error 180

7.4 The quality assurance programme 182

7.5 Laboratory procedures manual 182

7.6 Internal quality control 182

7.6.1 Purchased QC samples 183 7.6.2 Laboratory-made QC samples 183 7.6.3 Stored samples (purchased or laboratory-made) 183 7.6.4 Fresh QC samples (laboratory-made) 184

7.7 Statistical procedures for analysing and reporting within- and among-technician systematic errors 185

7.7.1 The Xbar chart 185 7.7.2 The S chart 188

7.8 QC for percentages 189

7.9 Assessing Xbar and S charts 189

7.9.1 How to recognize out-of-control values 189 7.9.2 Causes of out-of-control values 190 7.9.3 Responses to out-of-control values 191

7.10 Statistical procedures for analysing and reporting among-technician variability 191

7.10.1 Comparing results from two or more technicians 191 7.10.2 Monitoring monthly means 194

7.11 External quality control and quality assurance 194

7.11.1 Assessment of EQC results 196 7.11.2 Responses to out-of-control results 197

7.12 Frequency and priority of quality control 197

7.13 Training 198

7.13.1 Practical hints when experiencing difficulty assessing sperm concentration 198 7.13.2 Practical hints when experiencing diffi culty assessing sperm morphology 200 7.13.3 Practical hints when experiencing diffi culty assessing sperm motility 200 7.13.4 Practical hints when experiencing diffi culty assessing sperm vitality 202

REFERENCES 205

APPENDICES

Appendix 1 Reference values and semen nomenclature 223

A1.1 Reference values 223 A1.2 Nomenclature 225

Appendix 2 Equipment and safety 227

A2.1 Basic supplies needed in an andrology laboratory 227 A2.2 Potential biohazards in an andrology laboratory 230 A2.3 Safety procedures for laboratory personnel 230 A2.4 Safety procedures for laboratory equipment 232 A2.5 Safety precautions when handling liquid nitrogen 233

Appendix 3 Microscopy 234

A3.1 Loading the sample 234 A3.2 Adjusting the oculars 236 A3.3 Focusing the image 236 A3.4 Focusing the oculars 236 A3.5 Focusing the light condenser 236 A3.6 Centring the condenser 237 A3.7 Adjusting the phase rings 237 A3.8 Fluorescence microscopy 237

Appendix 4 Stock solutions 238

A4.1 Biggers, Whitten and Whittingham 238 A4.2 Dulbecco’s phosphate-buffered saline 238 A4.3 Earle’s medium 239 A4.4 Ham’s F-10 medium 239 A4.5 Hanks’ balanced salt solution 240 A4.6 Human tubal fl uid 240 A4.7 Krebs–Ringer medium 240 A4.8 Tris-buffered saline 241 A4.9 Tyrode’s solution 241 A4.10 Papanicolaou stain 241

Appendix 5 Cervical mucus 245

A5.1 Introduction 245 A5.2 Collection and preservation of cervical mucus 246 A5.3 Evaluation of cervical mucus 247

Appendix 6 Record forms for semen and cervical mucus analyses 251

A6.1 Template for a semen analysis recording form 251 A6.2 Template for a cervical mucus recording form 253

Appendix 7 Sampling errors and quality control 254

A7.1 Errors in measurement of sperm concentration 254 A7.2 The importance of understanding sampling errors 256 A7.3 Errors in measurement of percentages 257 A7.4 Production of semen samples for quality control 260 A7.5 Preparation of a video-recording for internal quality control of analysis of sperm motility 261 A7.6 Preparation of diluted semen for internal quality control of determination of sperm concentration 265 A7.7 Preparation of slides for internal quality control of assessment of sperm morphology 268 A7.8 Calibration of equipment 269

WHO laboratory manual for the Examination and processing of human semen (Fifth Edition)