Basic Laboratory Andrology

APracticalGuidetoBasic
LaboratoryAndrology

APracticalGuide
toBasicLaboratory
Andrology
Lars Björndahl
Karolinska Institutet
David Mortimer
Oozoa Biomedical Inc., Vancouver
Christopher L. R. Barratt
University of Dundee
Jose Antonio Castilla
Hospital Virgen de las Nieves
Roelof Menkveld
University of Stellenbosch
Ulrik Kvist
Karolinska Institutet
Juan G. Alvarez
Institut Marques
Trine B. Haugen
Oslo University College

CAMBRIDGE UNIVERSITY PRESS
Cambridge, New York, Melbourne, Madrid, Cape Town,
Singapore, São Paulo, Delhi
Cambridge University Press
The Edinburgh Building, Cambridge CB2 8RU, UK
Published in the United States of America by
Cambridge University Press, New York
www.cambridge.org
Information on this title: www.cambridge.org/9780521735902
© Cambridge University Press 2010
This publication is in copyright. Subject to statutory exception
and to the provisions of relevant collective licensing
agreements, no reproduction of any part may take place
without the written permission of Cambridge University Press.
First published 2010
Printed in the United Kingdom at the University Press,
Cambridge
A catalog record for this publication is available from the
British Library
ISBN 978-0-521-73590-2 Paperback
Cambridge University Press has no responsibility for the
persistence or accuracy of URLs for external or third-party
Internet websites referred to in this publication, and does not
guarantee that any content on such websites is, or will remain,
accurate or appropriate.
Every effort has been made in preparing this publication to
provide accurate and up-to-date information which is in
accord with accepted standards and practice at the time of
publication. Although case histories are drawn from actual
cases, every effort has been made to disguise the identities of
the individuals involved. Nevertheless, the authors, editors and
publishers can make no warranties that the information
contained herein is totally free from error, not least because
clinical standards are constantly changing through research
and regulation. The authors, editors and publishers therefore
disclaim all liability for direct or consequential damages
resulting from the use of material contained in this
publication. Readers are strongly advised to pay careful
attention to information provided by the manufacturer of any
drugs or equipment that they plan to use.

Contents
List of authors page vi
List of abbreviations vii
1Introduction 1
2Basic physiology 5
3Basic semen analysis 33
4Extended semen analysis 77
5Sperm function tests 113
6Tests of sperm–cervical mucus interaction 147
7Sperm preparation 167
8Sperm cryobanking 189
9Preparation of surgically retrieved spermatozoa 219
10Quality management and accreditation 227
11Risk management 249
12Reproductive toxicology 257
Appendices
1 Reference values 261
2 Equipment required for a basic andrology laboratory 277
3 Supply companies 287
4 Recommendations on the application of CASA technology in the analysis
of spermatozoa 291
5 Home testing 295
6 Example laboratory and report forms 297
7 Comparison of replicate counts 305
8 Safety 313
Index 323
The color plates are to be found between pages 148 and 149.

Authors
Lars Björndahl
Centre for Andrology and Sexual Medicine,
Department of Medicine, Karolinska
University Hospital, Huddinge, S-141 86
Stockholm, Sweden
David Mortimer
Oozoa Biomedical Inc., Box 93012,
Caufeild Village RPO,
West Vancouver BC V7W 3G4, Canada
Christopher L.R. Barratt
Reproductive and Developmental
Biology Group, Centre for Oncology
and Molecular Medicine, Division of
Medical Sciences, Ninewells Hospital,
University of Dundee, Dundee,
Scotland, DD1 9SY
Jose Antonio Castilla
Hospital Virgen de las Nieves, Ribera del
Beiro, Avda. de las Fuerzas Armadas,
Granada, Andalucia, 18005, Spain
Roelof Menkveld
Andrology Laboratory E3,
Tygerberg Academic Hospital and
University of Stellenbosch,
Tygerberg, 7505, South Africa
Ulrik Kvist
Centre for Andrology and Sexual Medicine,
Department of Medicine, Karolinska
University Hospital, Huddinge,
S-141 86 Stockholm, Sweden
Juan G. Alvarez
Instituto Marques, Manuel Girona,
33, 08034 Barcelona, Spain
Trine B. Haugen
Oslo University College,
Pb 4 St. Olavs plass, 0130 Oslo,
Norway

Abbreviations
A list of common abbreviations and acronyms used in andrology and assisted conception
laboratories, which may be used in this book without further definition, is provided below.
Standard SI units and chemical formulae are not defined here, nor are many abbreviations
that are in general English usage.
A23187 a calcium ionophore
AATB American Association of Tissue Banks (USA)
AI arti ficial insemination (unspecified source of spermatozoa)
AID arti ficial insemination by donor (no longer used since the advent of AIDS,
see DI and TDI)
AIDS acquired immunode ficiency syndrome, caused by HIV infection
AIH arti ficial insemination homologousorartificial insemination by husband
ALH amplitude of lateral head displacement, a sperm kinematic measure (µm)
APAAP alkaline phosphatase:anti-alkaline phosphatase complex
AR acrosome reaction
ARIC acrosome reaction following ionophore challenge
ART assisted reproductive technology
ASAB(s) antisperm antibody(ies)
ASRM American Society for Reproductive Medicine (USA)
ATP adenosine triphosphate
AZF azoospermia factor, a region on the Y-chromosome
BBT basal body temperature
BCF beat cross frequency, a sperm kinematic measure (Hz)
BSA bovine serum albumin (Cohn fraction V)
CAP College of American Pathologists (USA)
CASA computer-aided sperm analysis (notcomputer-automated semen analysis)
CBS Cryo Bio System (Paris, France), the manufacturer of High Security Straws
for cryobanking spermatozoa
CD (-ROM) compact disk (read-only memory)
CD45 the “pan-leukocyte”antigen
C.I. Color Index number (for stains)
CJD Creutzfeldt –Jakob disease
CLIA Clinical Laboratory Improvement Amendments (USA)
CMV cytomegalovirus
COLA Commission on Laboratory Accreditation (USA)
CPA cryoprotective agent orClinical Pathology Accreditation (UK)

CPM cryoprotectant orcryopreservation medium
Cryo cryopreservation orcryopreserved
CSF cryosurvival factor, the proportion of motile spermatozoa post-thaw
expressed as a percentage of the motile spermatozoa pre-freeze
CV coe fficient of variation
CZBT competitive (sperm –)zona binding test
–D sometimes added to acronyms such as IVF to denote the use of donor
spermatozoa
DGC density gradient centrifugation (sperm preparation method)
DHA docosahexaenoic acid
DI donor insemination
DMSO dimethyl sulfoxide (a cryoprotectant and also a carrier molecule for
A23187)
DNA deoxyribonucleic acid
DPX a microscopy mountant
DSUS direct swim-up from semen (sperm preparation method)
DTT dithiothreitol
DVD digital versatile disk
EBV Epstein –Barr virus
ECA European Cooperation for Accreditation
EDTA ethylenediamine tetraacetic acid, a chelator of divalent cations
EFQM European Foundation for Quality Management
EGTA ethyleneglycol tetraacetic acid, a chelating agent with a high affinity for
calcium ions
EQA external quality assurance
EQAP external quality assessment program
EQAS external quality assessment scheme
EQC external quality control
ESHRE European Society of Human Reproduction and Embryology
EtOH ethyl alcohol or ethanol
EU European Union ( notenzyme unit)
FITC fluorescein isothiocyanate, afluorochrome
FMEA failure modes and e ffects analysis
FMLP formyl-methionyl-leucyl-phenylalanine, a purportedly leukocyte-specific
probe to induce the generation of ROS
FNA fine needle aspirate (a technique for performing TESA)
FSH follicle-stimulating hormone
GAT Kibrick ’s gelatin agglutination test for spermagglutinating antisperm
antibodies
GEYC glycerol-egg yolk-citrate, a modified Ackerman’s CPM for human
spermatozoa
GLP Good Laboratory Practice
GMP Good Manufacturing Practice
H33258 Hoechst dye 33258, a DNAfluorochome that can be used as a vital stain
HA hyaluronic acid orhyperactivation/hyperactivated
HBV hepatitis B virus
List of abbreviations
viii

hCG human chorionic gonadotrophin
HCV hepatitis C virus
HEPES N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic) acid, a zwitterionic
pH buffer, pKa at 25°C = 7.5, useful pH range = 6.8–8.2
HEPT (zona-free) hamster egg penetration test
HFEA Human Fertilisation and Embryology Authority (UK)
hFF human follicular fluid
H-H head-to-head spermagglutination
HIV human immunode ficiency virus, types-1 and -2 (causes AIDS)
H-MP head-to-midpiece spermagglutination
HMT hyaluronate migration test
HOS test hypo-osmotic swelling test
HPF high power field (40× objective)
HPM high power magni fication (40× objective)
HPV human papillomavirus
HSA human serum albumin (Cohn fraction V)
HSPM human sperm preservation medium, a particular CPM formulation for
human spermatozoa
HSV herpes simplex virus
H-T head-to-tail spermagglutination
HTF human tubal fluid culture medium
HTLV human T-cell lymphotropic virus, types-I and -II (HTLV-III was renamed
HIV-1)
HZA hemizona assay
IAAC InterAmerican Accreditation Cooperation (USA)
IBT immunobead test
ICI intracervical insemination
ICSI intracytoplasmic sperm injection
ID identi fication
Ig immunoglobulin
ILAC International Laboratory Accreditation Cooperation
IMV Instruments de Medecine Veterinaire (L’Aigle, France), the original
manufacturer of semen straws
IPA isopropyl alcohol
IQA internal quality assurance (scheme)
IQC internal quality control (scheme)
ISO International Organization for Standardization (Geneva, Switzerland)
IU International Unit (IU for enzymes refers to the conversion of 1 µmol
of substrate/minute at 37°C)
IUI intrauterine insemination
IVF (–ET) in-vitro fertilization (and embryo transfer)
JCAHO Joint Commission on Accreditation of Healthcare Organizations (USA)
LCR ligase chain reaction
LH luteinizing hormone
LIN linearity, a sperm kinematic measure calculated as VSL/VCL × 100 (%)
LN2 or as LN
2, liquid nitrogen
List of abbreviations
ix

LPF low power field (10× objective)
LPM low power magni fication (10× objective)
MAR mixed antiglobulin reaction
MESA micro-(surgical) epididymal sperm aspiration
MI (sperm) motility index
MOPS 3-( N-morpholino)propanesulfonic acid, a zwitterionic pH buffer, pKa
at 25°C = 7.2, useful pH range = 6.5–7.9
MSDS material safety data sheet
mtDNA mitochondrial DNA
NA numerical aperture (of a microscope objective)
NADH β-nicotinamide adenine dinucleotide, reduced form
NAFA Nordic Association for Andrology
NATA National Association of Testing Authorities (Australia)
NCD nuclear chromatin decondensation
NH negative high phase contrast microscope optics
NPM non-progressively motile (spermatozoa)
OI oil immersion
PBS phosphate bu ffered saline
PCR polymerase chain reaction
PCT post-coital test
PDCA the Plan-Do-Check-Act quality cycle or Deming wheel
PESA percutaneous epididymal sperm aspiration
PETG polyethylene terephthalate glycol
PL positive low phase contrast microscope optics
PMA phorbol 12-myristate 13-acetate, an agent that provokes the generation
of ROS by leukocytes and spermatozoa
PNA peanut agglutinin, a lectin fromArachis hypogeathat binds to the outer
acrosomal membrane
PNP p-nitrophenol
PNPG p-nitrophenylα-d-glucopyranoside
PR (sperm motility) progression rating
PSA prostate-speci fic antigenor Pisum sativum(pea) agglutinin, a lectin that
binds to the acrosomal contents
PT pro ficiency testing
PVA polyvinyl alcohol
PVC polyvinyl chloride
PVP polyvinylpyrrolidone
QA quality assurance
QC quality control
QI quality improvement
QMS quality management system
RBC red blood cell (erythrocyte)
RCA root cause analysis
RFID radiofrequency identification (device)
ROS reactive oxygen species (a type of free radical)
RPLND retroperitoneal lymph node dissection
List of abbreviations
x

SCD test sperm chromatin dispersion test
SCMC test sperm–cervical mucus contact test
SCSA sperm chromatin structure assay
SD standard deviation
SDS sodium dodecyl (lauryl) sulfate
SEM standard error of the mean orscanning electron microscopy
SIT Isojima ’s sperm immobilization test for complement-dependent cytotoxic
antisper