Gerd Ludwig Julian Frick

Spermatology
Atlas and Manual

In Collaboration with Erwin Rovan

With a Contribution by
Wolf-Hartmut Weiske and Fred Maleika

With 101 Figures, Mostly in Color

in 215 Separate Illustrations, and 15 Tables

Springer-Verlag Berlin Heidelberg NewYork

London Paris Tokyo Hong Kong
Professor Dr. GERD LUDWIG
Urologische Klinik des
Stiidtischen Krankenhauses Frankfurt
Gotenstr. 6-8
D-6230 Frankfurt am Main-Hochst, FRG

Professor Dr. JULIAN FRICK

Urologische Abteilung
der Landeskrankenanstalten Salzburg
Miillner Hauptstr. 48
A-5020 Salzburg, Austria

Translated by:
PHILIP J. GIBSON
6 Sawtry Road
Glatton, Huntingdon
Cambridgeshire PE17 5RZ, UK

Title of the German Edition:

Praxis der Spermatologie
© Springer-Verlag Berlin Heidelberg 1987

ISBN-13: 978-3-642-73661-2 e-ISBN-13: 978-3-642-73659-9

DOl: 10.1007/978-3-642-73659-9

Library of Congress Cataloging-in-Publication Data. Ludwig, Gerd, 1942 - [Praxis der Spermatologie. English]
Spermatology: atlas and manual/ Gerd Ludwig, Julian Frick; in collaboration with Erwin Rovan: with a
contribution by Wolf-Hartmut Weiske and Fred Maleika; [translated by Philip J. Gibson]. Translation of:
Praxis der Spermatologie. Bibliography. Includes index. ISBN-13: 978-3-642-73661-21. Infertility, Male-Diagno-
sis-Atlases. 2. Infertility, Male-Diagnosis-Handbooks, manuals, etc. 3. Semen-Examination-Atlases. 4. Semen-
Examination-Handbooks, manuals, etc. I. Frick, Julian, 1933-. II. Rovan, Erwin. III. Title.
RC889.L7813 1990 616.6'92--dc20 89-11596 CIP

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Preface

The analysis of ejaculate, known as the spermiogram, is the crucial investigative

technique in the andrological check-up for childlessness. Althoug other impor-
tant factors such as medical history, physical examination, hormone analysis
and tests, biochemical, microbiological, immunological, and genetic tests, and
histological and histochemical investigations must all be considered before
a final assessment can be made of male fertility, the spermiogram is the guiding
factor in estimating the prospects of fertility. Furthermore, the improvement
in insemination techniques in recent years and the development of in vitro
fertilization have enhanced the importance of the accurate analysis of sperm
morphology and function.

In view of the multiplicity of andrological investigative techniques listed

above, urologists, gynecologists, physicians specializing in endocrinology and
immunology, general practitioners, pathologists, and dermatologists are routin-
ely involved in semen analysis today. We hope in this book to address people
working in all these different medical specialities.

Having regard for the requirements of daily practice, we will intentionally

restrict ourselves to the spermiogram, which we hope to present step by step
by means of a large number of illustrations. A substantial part of the book
is devoted to the morphological assessment of normal and pathological forms
of spermatozoa and other cellular elements, in the form of generously illustrat-
ed atlas. Dr. Rovan has given us outstanding assistance in preparing the majori-
ty of photomicrographs.

We have given the mixed antiglobulin reaction (MAR) test a section of its
own as it is, in our opinion, the only immunological test of relevance in daily
practice. In view of the tremendous growth in the importance of homologous
insemination and also in vitro fertilization, a separate chapter is included on
penetration and fertilization tests, which are central to intra- and extracorporeal
fertilization. Jeyendran's hypoosmotic swelling test, an important test of the
fertilizing ability of spermatozoa, is also discussed. Finally, in order to establish
the possibilities of optimizing pathological ejaculates for homologous insemina-
tion, details of the swim-up method are given. We are grateful to Dr. W.-H.
Weiske, a urologist, and to Dr. F. Maleika, a gynecologist, for their work
in producing this chapter. A book of this sort cannot be produced without
the help of a number of colleagues. As representatives of the many who cannot
all be named, we would like to express our sincere thanks to Mrs. Eva Potyka
for her help in preparing the spermiograms, Mr. Peter Wacht for the macros-
copic photography, Mr. Wolf-Dieter Korner for producing the drawings, Mrs.
Christiane Vinhage and Mrs. Anna Schmitz for typing the manuscript, and
Dr. Rolf Leissner for his help in proof-reading. We are particularly grateful
to the Springer-Verlag, especially Mr. Bergstedt and Mrs. Schuhmacher, for
their outstanding cooperation and the excellent presentation of the book.

V
 We hope that this book will live up to its claim to present a handy guide
to the preparation and assessment of a spermiogram, as well as to provide
an introduction to the preparatory work for insemination and to the assessment
of the prospects of in vitro fertilization.

Frankfurt-Hochst GERD LUDWIG

Salzburg JULIAN FRICK

VI
Contents

Introduction . . . . . . . . . . 1

1 Andrological Terminology 2
2 Ejaculate Analysis (Spermiograms) 3
2.1 The Ejaculate: Composition and Transport 3
2.2 Ejaculate Collection and Sexual Abstinence 5
2.3 Collecting the Ejaculate. . . . . . . . 5
2.4 Transport to the Laboratory for Analysis 6
2.5 Split Ejaculates . . . . . . . . . . 6
2.6 Macroscopic Examination of Ejaculates 7
2.6.1 Color . . 8
2.6.2 Odor . . . . 8
2.6.3 Viscosity . . 9
2.6.4 Liquefaction. 9
2.6.5 Volume . . . 10
2.6.6 pH . . . . . 10
2.7 Microscopic Examination of Ejaculates 11
2.7.1 Spermatozoal Motility . . . . . . . 11
2.7.1.1 Determining Motility by the Estimation Method 13
2.7.1.2 Objective Methods of Determining Motility . . 17
2.7.1.2.1 Multiple-Exposure Photography (MEP) in a Makler Chamber 17
2.7.1.2.2 Laser-Doppler Spectroscopy (Lazymot) . . . . . . . 20
2.7.2 Number and Density of Spermatozoa . . . . . . . . 20
2.7.2.1 Determining Spermatozoal Density in Hemocytometers 21
2.7.2.1.1 Neubauer Counting Chamber . . . . . . . . . . . 23
2.7.2.1.2 Thoma-ZeiB Counting Chamber . . . . . . . . . . 24
2.7.2.2 Determining Spermatozoal Density in a Makler Chamber 25
2.7.2.3 Determining Spermatozoal Density Using Electronic Counters 27
2.7.2.4 Evaluation of Spermatozoal Density 27
2.7.3 Morphology. . . . 28
2.7.3.1 Staining . . . . . . . . . . 29
2.7.3.1.1 Papanicolaou's Stain . . . . . 30
2.7.3.1.2 May-Griinwald-Giemsa Staining 32
2.7.3.1.3 Peroxidase Reaction . . . . . 32
2.7.3.1.4 Rapid Differentiation Using Testsimplets 33
2.7.3.1.5 Staining Using Hemafix. . . . . . . . 33
2.7.3.2 Specific Morphology . . . . . . . . . 34
2.7.3.2.1 Morphological Differentiation of Ejaculate Smears 38
2.7.3.2.2 Technique for the Morphological Differentiation of Ejaculate
Smears . . . . . . . . . . . . . . . . . . . . . . . . 39

VII
Atlas of the Cellular Elements in the Ejaculate

A The Spermatozoa (Fig. 43a-53t) . . 44

A1 The Hormonally Mature Spermatozoon (Fig. 43) . . . . . . 44
A2 Immature Spermatozoa = Late Spermatid Stages (Fig. 44a-e). 44
A3 Immature, Pathological Forms of Spermatozoa
= Late Spermatid Stages (Fig. 45 a-g). . . . . . . . . 47
A4 Pathological Forms of Spermatozoa with Abnormalities
of the Head and Head/Neck Junction ( = Bent Flagellum)
(Fig. 46 a-e). . . . . . . . . . . . . . . . . . . 50
A5 Megalocephalic Spermatozoa with Amorphous Heads
(Megalocephalic Abnormality 1) (Fig. 47a-t). . . . . 53
A6 Megalocephalic, Nonamorphous Forms of Spermatozoa
(Megalocephalic Abnormality 2) (Fig. 48 a-t). . . . . 56
A7 Pseudomegalocephalic Forms of Spermatozoa
(Megalocephalic Abnormality 3 = Pseudomegalocephaly)
(Fig. 49 a-e). . . . . . . . . . . . . . . . . . . . 59
A8 Microcephalic Spermatozoa (Microcephalic Abnormality)
(Fig. 50 a-e). . . . . . . . . . . . . . . . . . . . 62
A9 Atypical Head Shapes (Fig. 51 a-k) . . . . . . . . . . 65
A10 Abnormalities of the Nucleus and Acrosomal Malformations
(Fig. 52 a-d). . . . . . . . . . . . . . . . . . 70
A 11 Spermatozoa with Deformities of the Tail (Flagellum)
(Fig. 53 a-t) . . . . . . . . . . . . . . . . . . 73

B Cells from the Germinal Epithelium of the Seminiferous Tubules 76

B1 Spermatogonia (Fig. 54a, b) . 76
B2 Spermatocytes (Fig. 55a-l) 77
B3 Spermatids (Fig. 56a-c) . . . 83
B4 Pathologically Altered Forms of Germinal Epithelial Cells
(Fig. 57 a-t) . . . . . . . . . . . . . . . . . . . . 85
B5 Degenerating Giant Cell Forms with Advancing Expulsion
of the Nucleus (Fig. 58 a-g) . . . . . . 88

C Leukocytes and Macrophages (Fig. 59 a-I) 92

D Phagocytosis of Spermatozoa by Macrophages

(Spermatophagia) (Fig. 60a-t) . . . . . . . 98

E Peroxidase Reaction of Peroxidase-Positive Polymorphocellular

Leukocytes (Fig. 61 a-e). . . . . . . . . . . 102

F Various Types of Urinary Tract Cells (Fig. 62a-g) 105

G Smear Staining Using Testsimplets (Fig. 63a, b) . 109

H Smear Staining Using Hemafix (Fig. 64) . . . . 111

I Scanning Electron Micrographs of Various Forms of Spermatozoa

(Figs. 65-76) . . . . . . . . . . . . . . . . . . . . . 112

VIII
2.7.4 Vitality. 121
2.7.5 MAR Test (Mixed Antiglobulin Reaction Test) . 121
2.7.5.1 Principle of the MAR Test. 123
2.7.5.2 Performing the MAR Test. 123
2.8 Fructose 125
2.8.1 Determining Fructose in Seminal Plasma 125
2.8.2 Procedure. 126

3 Penetration and Fertilization Tests In Vivo and In Vitro

(WOLF-HARTMUT WEISKE and FRED MALEIKA) 129
3.1 "Dynamic" Parameters in the Spermiogram 130
3.1.1 Sperm Washing and Swim-up Method. 130
3.2 Penetration Tests. 131
3.2.1 The Postcoital Test (Sims-Huhner Test) 133
3.2.1.1 Evaluating the Postcoital Test 134
3.2.2 The Slide Test (Kurzrock-Miller Test) . 134
3.2.2.1 Evaluating the Slide Test (Kurzrock-Miller Test) 135
3.2.3 The Sperm-Cervical Mucus Contact Test (SCMC Test)
of Kremer and Jager 136
3.2.3.1 Evaluating the SCMC Test 137
3.2.4 The Kremer Test . 137
3.2.4.1 Evaluating the Kremer Test 138
3.2.5 The Bovine Mucus Penetration Test (BMP Test) (Penetrak Test) 140
3.2.5.1 Performing the Penetrak Test 140
3.2.5.2 Evaluating the Penetrak Test. 142
3.2.6 The Peritoneal Sperm Migration Test (PSM Test) 144
3.3 Tests of Membrane Stability . 144
3.3.1 The Hypoosmotic Swelling Test (HOS Test) 144
3.3.2 Freezability of Spermatozoa . 146
3.4 Hamster Oocyte Penetration Test (HOP Test = Heterologous
Ovum Penetration Test) . 146
3.5 Human In Vitro Fertilization (IV F) . 147
4 The Steps in Ejaculate Analysis in Chronological Order . 148
5 Conclusion 149
References . 150
Subject Index 159

IX
Introduction
Approximately 15% of marriages, or one in
six, remains childless against the wishes of
the partners (SCHILL 1980; LIPSHULTZ and
HOWARDS 1983a; SWERDLOFF et al. 1985). If
this situation persists for more than a year
despite attempts to conceive, the condition
is described as primary infertility (SIMMONS
1956). In such cases, a thorough andrological
examination should be undertaken, after no
more than 2 years, because it has been shown
that the success of treating a case of infertility
diminishes the longer it has been in existence
(LAMB 1972).
As husband and wife are almost equally
likely to be the cause of the infertility, the
man should be tested first due to the less
invasive nature of the necessary andrological
tests in his case.
Accurate history-taking, which must in-
clude finding out the individual's social back-
ground and establishing his sexual habits and
practices in addition to the customary medi-
cal history, is the first stage in the andrologi-
cal investigation.
This is followed by a thorough physical
examination, which means not only inspect-
ing and palpating the external genitalia and
rectal palpation of the prostate and seminal
vesicles, but also paying particular attention
to assessing the subject's constitution as a
whole, the relative lengths of the upper and
lower body, body hair, and the male breast.
History-taking and the general physical ex-
amination are thus the two most important
initial steps in establishing the individual's
fertility status (details can be found in the
following textbooks, which we can recom-
mend: KRAUSE et al. 1981; SCHIRREN 1982a;
HARGREAVE 1983; LIPSHULTZ and HOWARDS
1983).
Although male fertility depends on several
different factors, some of which interact
(anatomy, genetics, hormones, the biochem-
istry of the seminal plasma, accompanying
illnesses, dysfunction of the ejaculate, etc.),
the macroscopic and microscopic analysis of
the ejaculate - the simple spermiogram -, re-
mains the principal technique of andrological
examination (MACLEOD 1964, 1974; ELIAS-
SON 1971; AMELAR and DUBIN 1977; ZUKER-
MAN et al. 1977; HOFMANN 1979; ELIASSON
1981; SCHIRREN 1982a; HOFMANN et al.
1982; SCHILL and DASILVA 1983; KIESSLING
1984).
We want to deal in this book with sperma-
tology in practice, to give a step-by-step
guide to ejaculate analysis and to provide a
clear differentiation of spermatozoal pathol-
ogy, using a large number of exemplary illus-
trations.
As practical spermatology has gained in
importance in new areas due to the possibili-
ties of improved insemination technique and
in vitro fertilization, we are also concerned
to introduce the physician who is active in
the andrological field to the appropriate pre-
liminary tests through the wider application
of practical spermatology.
1
1 Andrological Terminology

ejaculate semen

spermatozoon = sperm cell

plural: spermatozoa = sperm cells
- spermia (suffix) = relating to semen
aspermia = absence of semen
hypo spermia = insufficient semen
«2 ml)
hyperspermia = too much semen
(>6 ml)
hemospermia = blood present in the
(or hematospermia) semen

pyospermia = pus present in the

semen
azoospermia = absence of sperma-
tozoa in the semen
oligospermia = < 20 million sperma-
tozoa/ml
polyspermia = > 250 million
spermatozoa/ml
asthenospermia = reduced motility
of spermatozoa
( < 50% with normal
morphology)
teratospermia = > 50% abnormally
formed spermatozoa
necrospermia = spermatozoa present
are dead
(confirmed by an
eosin test)
cryptospermia = very few spermatozoa
( < 1 million/ml),
only detectable
after sedimentation
globospermia = only round-headed
spermatozoa
OA T syndrome = oligoasthenotera-
tospermia syndrome

2
2 Ejaculate Analysis (Spermiograms)
2.1 The Ejaculate:
Composition and Transport
The totality of all the constituents of semen
expelled from the urethra at the climax of
sexual stimulation, usually combined with
orgasm, is called the ejaculate. This ejaculate
consists of a mixture of secretions entering
the urethra from Cowper's and Littre's
glands, the prostate gland, the seminal vesi-
cles, the vas deferens, and the epididymis and
the spermatozoa which are formed in the
testes. The primary function of this complex
mixture would appear to be to transport the
spermatozoa to the neck of the uterus (HEITE
and WOKALEK 1980; URRY 1985).
Transport of Spermatozoa. Sertoli cells push
the as yet immotile spermatozoa, imbedded
in a secretion, into the lumina of the semi-
niferous tubules. From there the spermato-
zoa are moved into the efferent ductules and
into the canal of the epididymis. While they
are being transported through the long,
meandering, coiled canal of the epididymis,
the spermatozoa undergo a process of matu-
ration. They become motile and become ca-
pable of fertilization (MANN and LUTWAK-
MANN 1981).
After leaving the tail of the epididymis, the
spermatozoa pass through the 40 cm long vas
deferens - with the assistance during ejacula-
tion of peristalsis-like contractions of the epi-
didymis and the seminal duct. The seminal
vesicles opening just behind the ampullae of
the vas deferens add a secretion high in fruc-
tose, which accounts for 50%-80% of the
total volume of the ejaculate. The secretions
of the prostate gland constitute 15%-30%
of the seminal fluid and contain mainly citric
acid and acid phosphatase (MANN 1974).
Ejaculation. Before ejaculation begins, the ef-
ferent ductules first contract between the tes-
tis and the epididymis. These contractions
spread via the epididymis to the vas deferens.
At the same time the seminal vesicles and
the glandular ducts of the prostate contract.
The mixture of all these secretions is thus
assembled in the prostatic segment of the
urethra. This process is termed emission.
When the actual ejaculation starts, the in-
ner sphincter of the urinary bladder contracts
and thereby prevents retrograde ejaculation.
At the moment when the seminal fluid is ex-
pelled, the external sphincter relaxes and
there is a synchronous contraction of the per-
ineal muscles (especially the bulbocavernosus
muscle and the ischiocavernosus muscle)
whilst the inner sphincter is closed and the
outer sphincter is open, and this causes the
semen to be expelled to the outside through
the urethra. The pressure created in this pro-
cess is so great that the ejaculate is expelled
in pulses in its different fractions.
One usually distinguishes between four
ejaculate fractions (HEITE and WOKALEK
1980; SCHIRREN 1982a):
1. the preejaculatory fraction
2. the preliminary fraction
3. the main fraction
4. the terminal fraction
The preejaculatory fraction occurs as sex-
ual excitement increases. Secretions are pro-
duced by Cowper's and Littre's urethral
glands. This is a clear secretion containing
protein, with a mucilaginous, moderately
viscous consistency, which may possibly
serve to neutralize residues of urine in the
urethra, and also to lubricate the urethral ca-
nal to facilitate the subsequent evacuation of
the following seminal plasma fractions.
The preliminary fraction originates in the
prostate gland. It gives the semen as a whole
its characteristic "chestnut-blossom odor."
3
It contains a number of prostatic enzymes
whose function is to liquefy the spermatozoal
coagulum emerging from the epididymis and
vas deferens.
The main fraction consists of a mixture of
gelatinous and liquid constituents. They orig-
inate from the prostate gland on the one
hand, and are therefore similar in some re-
spects to the preliminary fraction, and on the
other they come from the seminal vesicles
and secretions of the testis and epididymis.
The preliminary fraction and the main
fraction contain the majority of the sperma-
tozoa.
The terminal fraction is formed exclusively
from secretions of the seminal vesicles. It is
entirely gelatinous in consistency, with large
numbers of immotile spermatozoa enclosed
in the firm masses.
Table 1. Information that the spermiogram ( = ejacu-
late analysis) provides
- Color, odor (milky-cloudy,
chestnut-blossom aroma)
- Liquefaction time (10-30 min)
- Viscosity
- Volume (2-6 ml)
- pH (7.2-7.8)
IUnprepared sample
- Agglutination (preferably none)
- Motility (50% overall motility,
at least 30% progressive Motility)
I Counting chamber
- Number of spermatozoa
(40-800 x 106 spermatozoa)
- Spermatozoal density
(20-250 x 106 spermatozoa/ml)
IStained smear
- Morphology of spermatozoa
(50% normal forms)
- Differentiation of "round cells"
I Eosin test
- Vitality (80% live spermatozoa)
I Fructose
- (> 1200 I1g/ml or 1.2 gil)
4
The entire ejaculate is analyzed when pre-
paring what is described as the spermiogram,
and thus it would be more accurate to talk
of ejaculate analysis. Normally it is sufficient
to determine the parameters listed in Table 1
for the purposes of a general examination
for fertility. To assess the prospects of fertil-
ization, at least two ejaculate analyses must
be performed with an interval of 14 days be-
tween each. It cannot do more than provide
information on the prospects of fertilization,
and then can only be evaluated in conjunc-
tion with the andrological examination as a
whole. If one of the two ejaculates is patho-
logically altered, a third ejaculate or even
more should be examined after a further peri-
od of 2--4 weeks.
Variability. There are fluctuations in the
sperm count and spermatozoal motility from
day to day even in the same individual. The
variability in spermatozoal density in a nor-
mal man is between 35 and 79 million/ml
(TITMAR 1978).
It is certain that the number of spermato-
zoa decreases as the frequency of sexual ac-
tivity increases (LAMPE and MASTERS 1956;
FREUND 1963; CONFINO et al. 1985; LEVIN
et al. 1986).
Opinions differ concerning seasonal varia-
tions: TJOA et al. (1982) deny that they exist
in man, whereas BAKER et al. (1981) in Aus-
tralia found a lower motility rate in winter
and a higher motility rate in summer.
Age-related variations are slight. In the re-
productive years, the results of the ejaculate
analysis remain fairly constant, although
there is said to be a tendency to deteriorating
motility from the age of 40 onwards (MAC-
LEOD 1951).
In studies of long-term variations in semen
parameters, no significant difference was
found with increasing age, but in individual
patients - disregarding the largely constant
mean values - there were marked fluctua-
tions from one examination to the next
(KRAUSE 1984). This underlines the impor-
tance of performing at least two ejaCUlate
analyses for a basic examination. In contrast,
there are considerable fluctuations caused by
accompanying illnesses, particularly viral in-
fections, and these fluctuations may be so
marked that the prospects of fertilization
may be apparently nil weeks after this infec-
tion has passed. This phenomenon has been
observed primarily with measles, hepatitis, or
infectious mononucleosis (MACLEOD 1964;
SCHILL 1985b). However, other viral diseases
may in principle cause transient spermato-
zoal diminution of this sort (SCHNEIDER and
SCHEUERLEIN 1946; CALLOMON and WILSON
1956; NIERMANN 1960; NIERMANN and NOLT-
ING 1971). The effect is reversible provided
both testes are not affected by a local inflam-
matory process, as in the well-known condi-
tion of mumps orchitis, which can result in
fibrous testicular atrophy (KIESSLING 1960;
NIERMANN 1960; SCOTT 1960; SCHIRREN and
THIESENHAUSEN 1972).
2.2 Ejaculate Collection
and Sexual Abstinence
The ejaculate is collected by means of mas-
turbation following a period of 3-5 days of
sexual abstinence. A shorter period of absti-
nence reduces semen quality to an extent
which varies from individual to individual,
whilst extending the period of abstinence
beyond 5 days brings no improvement (MAc-
LEOD and GOLD 1954 ; SCHWARTZ et al. 1979;
KRAusE and ROTHAUGE 1981; SCHIRREN
1982a; HARGREAVE and NILSSON 1983; URRY
1985; POLAND et al. 1985).
If masturbation is impossible in the exami-
nation room for any particular reason, the
patient is given the collecting vessel (see Sect.
2.3) to take home with him. It is important
that the analysis be performed within 2 h at
the most after ejaculation, and the optimal
time of 0.5 h after ejaculation.
In a very small number of cases, it is not
possible to collect the semen sample be means
of masturbation even at the subject's home.
In such cases there is no alternative but to
obtain the ejaculate either by means of coitus
interruptus or by collecting it in a special con-
dom containing none of the usual spermi-
cides found in commercial sheaths (SCHIRREN
1982a; URRY 1985).
Table 2. Equipment required for a spermiogram
1. Microscope (preferably with phase-contrast)
2. Collecting beaker for ejaculate (a Petri dish may
be used)
3. Glass rod (pipette)
4. Measuring cylinder or 5 ml syringe with a no. 1
needle (if there is no scale on the collecting
beaker)
5. Special indicator paper for pH 6.4-8.0 (Merck,
item no. 9557)
6. Microscope slides (polished, 76 x 26 mm)
7. Cover slips (18 x 18 mm)
8. Counting chamber (Neubauer, Thoma-ZeiB,
Biirker-Tiirk)
9. Hemocytometer cover slip (20 x 26 mm, 0.4 mm
thick)
10. Leukocytepipette
11. Suction tube
12. 3% NaCl solution or distilled water
13. Electric vibrator (optional)
14. Filter paper or cellulose wadding
15. Utensils and reagents for Papanicolaou's stain-
ing (see Sect. 2.7.3.1.1)
16. Testsimplets (Boehringer, Mannheim, FRG)
17. 0.5% yellowish eosin solution or 1% bluish eo-
sin solution
18. Immersion oil
19. Utensils and reagents for fructose determination
(hexokinase method; Boehringer, Mannheim,
FRG; see Sect. 2.8)
20. Forms on which to record findings (see Table 3)
Preparationfor the Ejaculate Analysis. Before
starting the ejaculate analysis, all the utensils
and reagents should be assembled on a suit-
able working surface (e.g., wipable Resopal
sheet). Table 2 lists the equipment required.
2.3 Collecting the Ejaculate
The seminal fluid should be collected in a
clean glass vessel with a wide neck (Fig. 1)
(HEITE and WOKALEK 1980; KRAUSE and
ROTHAUGE 1981; HARGREAVE and NILSSON
1983), in a glass beaker with a pourer lip
(Fig. 2), or in a plastic beaker (CALAMERA
1978) with a screw cap (Fig. 3). The plastic
beaker or a Petri dish, also made of plastic,
have the advantage of being cheap and of
being supplied in sterile packs. They enable
5

Fig. 1. Cylinder with ba e, coar e cale, and a funnel-
haped neck for collecting the ejaculate
Fig. 2. Collecting beaker with pourer lip (right) and
mea uring tube (Ie/t)
Fig. 3. Collecting beaker made of plastic with a
crew cap
6
a semen culture to be prepared at the same
time for microbiological examination. It is
essential that the collecting vessel be free of
chemical solutions, washing agents, or other
residues. To remove any residues which may
be present, glass containers should be rinsed
several times with double-distilled water after
they have been cleaned (SCHIRREN 1973).
2.4 Transport to the Laboratory
for Analysis
If the semen is not collected in the examina-
tion room, care should be taken to ensure
that the collecting vessel with its cap on is
not exposed to extreme variations in temper-
ature. The motility of spermatozoa can be
easily disturbed if exposed to temperature
variations, especially cold. The optimum is
transport at body temperature, e.g., in a
trouser pocket (HARGREAVE and NILSSON
1983).
2.5 Split Ejaculates
The term split ejaculate is used to describe
the fractionated collection of seminal fluid
in separate portions (usually two), i.e., split-
ting the total ejaculate sample. The physio-
logical justification for this is the fact that
the ejaculate is expelled in four to six portions
(ELIASSON and LINDHOLMER 1976) in a rapid
succession of pulses. In 95% of patients, the
first half of the ejaculate contains about two-
thirds of the total number of spermatozoa
(AMELAR and HOTCHKISS 1965; ELIASSON and
LINDHOLMER 1972; TAUBER et al. 1975;
ADONI and PAL TI 1979; MAR MAR et al. 1979;
COHEN et al. 1980; COHEN et al. 1981; Schill
1985a). As their progressive motility, density,
vitality, and morphology are significantly
better in this fraction (SCHILL 1980; SINGER
et al. 1982), splitting the sample provides a
better quality of ejaculate for insemination
purposes in cases of oligo- and asthenosper-
mia (see Sects. 2.7.1, 2.7.2) (SCHILL 1979).
 In addition, in cases of abnormal viscosity 2.6 Macroscopic Examination
(see Sect. 2.6.3) it may also be necessary to of Ejaculates
examine the individual ejaculate fractions for
differing coagulation or liquefaction behav-
IOr. When the ejaculate analysis begins, the fol-
lowing tests are performed in the sequence
shown, and the results are entered on the re-
cord form illustrated in Table 3:
Color and odor
Volume
pH

Table 3. Spermiogram record form (normal values in parentheses)

Surname ................................... . First name

Date of birth ............................................................................... .
Address .................................................................................... .
Insurance ................................... Referred by ................................. .
Date of examination ......................................................................... .

Ejaculation at home 0 ...... min ago In the clinic ................................. .

Color ....................................... Odor ...................................... .
Liquefaction time ............ (10-30 min) Viscosity ................................... .
Volume ............ (2-6ml) pH ............ (7.2-7.8)
Motility: · ......... % actively motile (30%) )
· ......... % moderately motile (20%) spermatozoa
· ......... % immotile (SO%)
Number: · •••••.••. X 10 6 sp.jml (20-2S0 x 10 6 sp.jml)
· ......... "round cells" ( < 2 x 10 6 /m!)
Morphology: · ......... % normally formed spermatozoa (SO%)
· ......... % abnormally formed spermatozoa
% malformations of the head
% malformations of the middle piece
% malformations of the tail
· ......... germinal cells ( < 1 OOOOOO/ml)
· ......... leukocytes, lymphocytes ( < SOOOOO/ml)
Vitality: · ......... % live spermatozoa (80%)

Fructose: · ......... Jlg/ml (> 1200 Jlg/ml or > 1.2 gil)

Assessment:

7
2.6.1 Color ly be attributed to injury (e.g., following
catheterization or cystoscopy), or to inflam-
matory processes in the posterior urethra
Normal, freshly expelled semen has a milky-
(BAuER 1963). In the case of true hemosper-
whitish, cloudy to gray-yellowish color. The
mia, the blood originates from the accessory
degree of cloudiness depends on the sperm
sex organs as a consequence of unspecific
count (KRAusE and ROTHAUGE 1981). The
prostatovesiculitis, and results in a dark, uni-
color changes as the period of abstinence in-
form mixture with the semen. The cause is
creases: the shorter the period of abstinence,
believed to be an increase in local fibrinolytic
the more transparent it is; as the length of
activity due to the inflammatory process
abstinence increases, the more yellowish the
(JECHT 1971).
semen becomes (SCHIRREN 1982a). In addi-
Tumors figure as the possible cause of he-
tion, a darker yellow color is observed in old-
mospermia only in extremely rare cases.
er subjects (KRAusE and ROTHAUGE 1981).
Figure 4 shows examples of different col-
A greater admixture of leukocytes, as is ob-
ors of ejaculates.
served in cases of inflammation of the male
accessory sex organs, also imparts a yellow
color to the ejaculate, which then becomes
2.6.2 Odor
rather dirty in appearance. This is then de-
scribed as pyospermia (LUDVIK 1976).
The admixture of blood gives the semen The odor is normally very characteristic and
a reddish to brownish color, depending on resembles the fragrance of chestnut tree blos-
when the bleeding occurred (hemospermia or som. The odor derives from the prostatic se-
hematospermia). cretions and is absent in cases of atrophy of
One can distinguish between false hemo- the prostate gland (SCHIRREN 1982a). Inflam-
spermia and true hemospermia (LUDVIK matory processes impart a fetid, foul smell
1976). In the case of false hemospermia, the to the semen (HARGREAVE and NILSSON
blood is mixed with the semen in the form 1983). Various other types of odor are of no
of small lumps or threads, and this can usual- significance (KRAUSE and ROTHAUGE 1981).

Fig. 4. Different colors of ejaculate. From left to of ejaculate after 5 days of abstinence ' brownish, true
right : red hemospermia with admixture of fresh hemospermia in a case of spermatocystitis ' greenish-
blood (fa I e hemo permia); yeUowi h, normal color yeUow, pyospermia in a case of prostatitis

8
2.6.3 Viscosity
Normally the semen coagulates immediately
after ejaculation, and this serves the function
of preventing it leaking too quickly from the
vagina. The coagulating enzymes originate
from the secretions of the seminal vesicles
(MACLEOD and HOTCHKISS 1942; AMELAR
1962; MANN and LUTWAK-MANN 1981). It
has a viscous, gelatinous, sometimes floccu-
lent-lumpy to sago-like consistency, and after
it has liquefied (see Sect. 2.6.4) it can usually
be poured out a drop at a time, or can be
drawn up into a pipette or syringe (SCHIRREN
1982a; HARGREAVE and NILSSON 1983; LIP-
SHULTZ and HOWARDS 1983a) (Fig. 5).
To measure the viscosity accurately, one
needs either a viscosimeter or a calibrated
pipette (LUDVIK 1976; SCHIRREN 1982a).
However, for routine testing in practice, the
following procedure is satisfactory: After
complete liquefaction (see Sect. 2.6.4), stir the
ejaculate with a glass rod (or pipette) to en-
sure that it is evenly mixed. Then estimate
the length of a thread of semen which adheres
to the glass rod when it is slowly raised. If
the viscosity is normal, a drop of semen
stretched like a thread of about 1 cm in
length will hang from the rod for 10- 15 s.
Fig. 5. After complete liquefaction , the emen can
normally be emptied a drop at a time from a syringe
fitted with a needle
Fig. 6. The" stringine "of the liquefied ejaculate
is a sign of normal viscosity. A drop of emen can
be pulled out like a thread of at least 1 cm in length
and remain tretched for 10- 15
This is described as "stringiness" (Fig. 6). If
the viscosity is low, the semen cannot be
pulled into a thread at all, and if it is high
the thread can be several centimeters in
length (V ASTERLING 1960; SCHIRREN 1976;
SCHIRREN 1982a).
However, no confirmed correlation has
been found so far between diminished or in-
creased viscosity and disorders of the seminal
vesicles or of the prostate gland (AAFJES et al.
1985; HUBNER et al. 1985; MANDAL and
BHATTACHARYYA 1985).
2.6.4 Liquefaction
Normally, semen which has coagulated im-
mediately after ejaculation will liquefy within
20 min (10- 30 min) (LUNENFELD and GLE-
ZERMANN 1981). The proteolytic enzymes
which induce liquefaction originate from the
prostate gland (MANN 1974; LILYA and
LAURELL 1984). Viscosity (see Sect. 2.6.3),
volume (see Sect. 2.6.5), and pH (see Sect.
2.6.6) cannot be checked or determined until
after liquefaction is complete and all frac-
tions of the ejaculate have been thoroughly
mixed .
9
2.6.5 Volume
The normal volume of the ejaculate after 3-5
days of sexual abstinence is 2-6 ml. The vol-
ume of the ejaculate increases as the duration
of sexual abstinence increases, or to put it
another way: there is an inverse correlation
between volume and sexual activity (MAC-
LEOD and GOLD 1954; SCHWARTZ et al.
1979).
The volume is measured after liquefaction
is complete (see Sect. 2.6.4) using a graduated
cylinder with a funnel-shaped neck, if the
ejaculate was collected in this vessel (see
Fig. 1). This has the advantage of avoiding
any loss of volume on transferring the semen.
If a Petri dish or a similar vessel without
graduations is used to collect the sample (see
Figs. 2, 3), the ejaculate must be poured into
a graduated cylinder after liquefaction is
complete, or drawn up into a syringe (Fig. 7),
although a minimal loss of volume is un-
avoidable. Drawing the semen into a syringe
has the advantage that drops of semen can
be placed with ease onto the various micro-
scope slides (see below) using the syringe. The
volume measured is entered straight away on
the spermiogram record form (see Table 3).
If the volume is less than 2 ml (hyposper-
mia) one should consider the following possi-
bilities: a disorder of the prostate gland and
Fig. 7. In order to measure the volume and to carry
out ub equent operation with the ejaculate (mount-
ing drop-wi e on sLides) the liquefied ejaculate can
be drawn up into a syringe. The minimal loss of vol-
ume which occurs in the process can be ignored
10
Table 4. Factors to be considered in the differential
diagnosis of hypo spermia « 2 ml of ejaculate)
- Extremely short period of sexual abstinence
- Partial loss during masturbation
- Retrograde ejaculation
- Occlusion of the ejaculatory duct
- Severe chronic prostatovesiculitis
(possibly tuberculosis as well)
- Hypoandrogenism
(e.g., due to gonadotropin deficiency)
- Congenital absence of
vas deferens
prostate gland
seminal vesicles
seminal vesicles (SCHIRREN 1982 b), retro-
grade ejaculation, (very rarely) the congenital
absence of the prostate and seminal vesicles,
and gonadotropin deficiency (HARGREA VE
and NILSSON 1983), or occlusion of the ejacu-
latory duct (LUDVIK 1976) (see Table 4).
There is no known explanation for a vol-
ume greater than 6 ml (up to a maximum
of 10 ml; hyperspermia) (KRAUSE and ROTH-
AUGE 1981).
However, with regard to the specific char-
acteristics of the spermatozoa such as motili-
ty (see Sect. 2.7.1), density (see Sect. 2.7.2)
and morphology (see Sect. 2.7.3), and with
regard to the fertilizing ability in a hamster-
oocyte penetration test (see Sect. 3.4), no dif-
ferences were found between cases of hypo-
spermia and hyperspermia (TANG and CHAN
1985).
2.6.6 pH
Normal pH is measured immediately after
liquefaction is complete, using a special indi-
cator paper (made by E. Merck, Darmstadt,
FRG; Fig. 8) which has a narrow range be-
tween pH 6.4 and 8.0 indicated by color
change.
The indicator strip is dipped half-way into
the semen, and the moistened end of the strip
will change color. By immediately comparing
Fig. 8. Indicator paper for pH determination (Merck, Fig. 9. Comparing the color of the indicator strip
item no. 9557) dipped in semen to determine pH

the color of the strip against a color scale below 7 (SCHIRREN 1976; SCHIRREN 1982a;
(Fig. 9), the pH of the semen can be very URRY 1985).
easily determined, and entered on the sper- Table 5 shows in summary form the differ-
miogram record form (Table 3). Using an ent values of pH and their possible connec-
electric pH meter to determine pH is time- tion with pathological conditions.
consuming and has no advantages over the
simple indicator paper test.
The normal pH of completely liquefied
and stirred ejaculate (see Sect. 2.6.4) varies 2.7 Microscopic Examination
between 7.2 and 7.8. of Ejaculates
As the time since ejaculation increases, pH
shifts towards the more strongly alkaline
In the microscopic examination of ejaculates,
range of 8 and above. In cases of acute in-
the classical semen parameters of motility,
flammation of the male accessory sex organs
number, and morphology of the spermatozoa
the pH is also frequently over 8, whereas in
are determined as the most important criteria
chronic diseases of the prostate gland and
to assess the fertilizing ability of the ejaculate.
seminal vesicles and when the ejaculatory
Spermatozoal motility and the number of
duct is occluded, it shifts into the acid range
spermatozoa, or the density or concentration
( = number/ml) of spermatozoa, are deter-
mined in unprepared, unstained samples of se-
men (Sects. 2.7.1, 2.7.2).
Table 5. Shifts in the pH of the ejaculate and their After this, various staining methods are
possible causes used for the morphological differentiation of
pH value
individual spermatozoa and other cellular el-
Possible cause
ements, and to investigate vitality (Sects.
7.2-7.8 Normal 2.7.3,2.7.4).
8.0 and above Acute prostatitis
Acute vesiculitis
Acute epididymitis (bilateral)
2.7.1 Spermatozoal Motility
6.6-7.0 Chronic prostatitis
Chronic vesiculitis
Chronic epididymitis The progressive forward motility of the sper-
(bilateral)
matozoon by means of beating movements
Occlusion of the ejaculatory duct
of its tail is essential to enable it to cover

11
the distance through the cervix, uterus, and
oviduct ampulla, in order finally to penetrate
the female ovum (OVERSTREET and KATZ
1981).
It does not matter how smart a car appears
if it does not go.
When related to the assessment of the indi-
vidual semen parameters, this truism ex-
presses in a few words the fact that spermato-
zoal motility is the most important criterion
for fertilizing ability (JANICK and MACLEOD
1970; SCHILL 1980; STEINBERGER et al. 1981;
ALBERT et al. 1986). It is therefore not sur-
prising that a high percentage of actively mo-
tile spermatozoa correlates with a high con-
ception rate and vice versa (EDVINSSON et al.
1983; BOSTOFTE et al. 1984). Even men with
severely reduced spermatozoal densities (se-
vere oligospermia - see Sect. 2.7.2 - of < 1
million/ml) have been able to induce preg-
nancies because 50%-60% of their spermato-
zoa exhibited good progressive motility
(SCHILL 1980).
It is not therefore unexpected that motility
also correlates with the ability of spermato-
zoa to achieve capacitation, which is crucially
important both for natural conception and
for in vitro fertilization.
WARTER et al. (1985) also found a reduced
number of spermatozoa capable of achieving
capacitation when there was a severe loss of
motility, and suggest that a loss of motility
is attributable to disturbances of the seminal
plasma.
A distinction is made between quantitative
and qualitative motility (AMELAR et al. 1973;
BELSEY et al. 1980).
In determining quantitative motility, one
distinguishes the percentage of motile sper-
matozoa from the percentage of immotile
spermatozoa in ten separate fields under the
microscope. This is also described as global
motility.
In determining qualitative motility, the na-
ture of the motility is additionally defined
as follows:
O=immotile
1 = poor progressive motility
2 = good progressive motility
3 = excellent progressive motility
12
As far as the practical demands of routine
examinations are concerned, however, it is
quite satisfactory to classify spermatozoal
motility as SCHIRREN (1982a) does:
a = very actively motile
b = moderately motile
c =immotile
The spermatozoa classified under a and b
should display progressive (directional) mo-
tility. Circular movements, oscillating on the
spot, or trembling movements (known as
"shaking") are pathological forms of move-
ment and must be distinguished from those
forms of progressive motility which are im-
portant for fertilization.
The duration of motility also has a part
to play. The loss of motility after 2 h should
not amount to more than 20% of the initial
motility (SCHIRREN 1982a). The spermatozoa
acquire their motility through various sub-
stances present in the seminal plasma which
have not as yet been completely identified,
and" the sperm motility stimulating principle
in human semen is very complex in nature
and of multifactorial origin" (MIZUTANI and
SCHILL 1985).
For a long time, an important role as an
energy source for spermatozoal motility was
attributed to the fructose in seminal plasma
(SCHIRREN 1971; LUDWIG et al. 1974), but
there were doubts about this early on (HAR-
TREE and MANN 1961; KINDLER and
MOLLMANN 1972).
Successsful conceptions with fructose-free
ejaculates in animal experiments (WAGEN-
KNECHT et al. 1974; KELtMI 1974; WAGEN-
KNECHT et al. 1977; KELAMI 1981), and most
recently in vitro fertilization with washed
spermatozoa free of seminal plasma have
made it even less likely.
In addition to maturing substances which
are added to the hitherto immotile spermato-
zoa during their passage through the epididy-
mis (MANN and LUTWAK-MANN 1981; Coo-
PER 1986), the greatest importance as intra-
cellular, and therefore primary, energy
sources for spermatozoal motility has since
been ascribed (CALAMERA et al. 1982; COM-
HAIRE et al. 1983) to a number of other sub-
stances (MIZUTANI and SCHILL 1985),
especially adenosine triphosphate (ATP).
In order to measure the speed of movement
of spermatozoa, quite complicated equipment
is required (comparison and evaluation of
specific methods and equipment in KAMI-
DONO et al. 1983; PUSCH 1985). Although
measuring the speed of spermatozoa move-
ment is of no importance in normal practice,
the mUltiple-exposure photography (MEP)
method described below (Sect. 2.7.1.2) used
in conjunction with a Makler chamber can
be employed by those interested.
A completely adequate method of deter-
mining spermatozoal motility for daily rou-
tine andrological examinations, which is also
simple and quick, is the estimation method
(EuAssoN 1975; SCHIRREN 1982 a; SCHIRREN
1982 c; MA TTHEUS and HEISE 1984; SCHILL
1985a). Fig. 10. Micro cope (preferably but not nece sarily,
with pha e-contra t)

2.7.1.1 Determining Motility

by the Estimation Method

Equipment Required (Figs. 10, 11)

- Microscope (preferably with phase-con-

trast)
- Slides (polished, 76 x 26 mm)
- Cover slips (18 x 18 mm)
- Pipette, glass rod or 5-ml syringe with nee-
dle

a
Procedure

Immediately after the ejaCUlate has complete-

ly liquefied (see Sect. 2.6.4), a drop of semen
is mounted on a slide using the pipette, glass
rod, or the 5-ml syringe and covered with
a cover slip. The drop should be sufficiently
large to just fill the area of the cover slip
(no empty edges and no floating). A drop
with a diameter of 3--4 mm is usually satisfac-
tory for this purpose. The slide is then exam-
ined under the microscope at a magnification
b
of 400 x (objective lens 40 x, eyepiece 10 x).
ig. 11 a, b. Micro cope slides. a Cover lip and 5-
In normal cases a number of rapidly ml yringe with needle. b Cover sli ps 18 x 18 mm ;
"swarming" spermatozoa will be seen. In ad- upper righI , cover lip 20 x 26 mm, 0.4 mm thick ,
dition there will be numerous weakly motile for covering the counting chamber (see Fig. 25)

13
Fig.12a .• Round cells" in an unprepared specimen. distinguj hed with certainty from leukocytes, phago-
Although differentiation is occasionally possible us- cytes or epithelial cells. Pha e-contrast, xenon lamp
ing pha e-contrast, germjnal cells cannot usually be blue filter x 2400

Fig. 12b.• Round cells" in an unprepared specimen. field without phase-contrast. Xenon lamp, blue filter
The same cell as in Fig. 12a, but in a simple bright x 2400

14
 Fig. 13. Spermatozoa in an unprepared specimen. Phase-contrast, halogen lamp, no filter
x 2400

Fig. 14. Spermatozoa in an unprepared specimen. Phase-contrast, xenon lamp, blue filter,
x 2400

spermatozoa, or spermatozoa merely trem- spermatogenesis, leukocytes, monocytes,

bling on the spot, together with cells which
cannot be differentiated. The latter, de-
scribed as "round cells," cannot be differen-
tiated in an unprepared specimen. They are
either cells at some stage in the process of
phagocytes, or epithelial cells (Figs. 12a and
b, 16). In an unprepared specimen it is also
impossible to distinguish the fine detail of
the morphological structure of spermatozoa
(Figs. 13- 16).

15
Fig. IS. Spermatozoa in an unprepared pecimen. Bright field blue filter, x 2400

Fig. 16. Spermatozoa and round cell in an unprepared pecimen. Bright field , blue filter,
x 2400

Simple light microscopy (bright-field mi-
croscopy) only enables one to distinguish be-
tween different types of motile and immotile
spermatozoa and round cells.
Phase-contrast microscopy may sometimes
provide a coarse differentiation of round cells
especially if filters and stronger light sources
(halogen or xenon lamps) are used as well,
but even then differentiation is not generally
reliable.
The same applies to interference phase-con-
trast microscopy in which differing color ef-

16
fects are achieved without the use of filters
by varying the diffraction of the light beam;
this results in a three-dimensional image.
Individual cells and the fine detail of their
morphological structure can only be positive-
ly differentiated by staining as described in
Sect. 2.7.3.1.
Head and tail agglutination of spermato-
zoa can be identified in an unprepared speci-
men, but this is achieved more easily in a
stained smear. The significance of these phe-
nomena is unclear. It may possible be an im-
munological process. In their agglutinated
formation they fail in the fertilization pro-
cess.
Estimating Motility
The crucial feature is progressive forward
motility, because this is the only way sperma-
tozoa can cover the distance to the ovum
which is to be fertilized.
One must therefore try to classify the sper-
matozoa in the field under the microscope
into three groups on a percentage basis:
- Those with very active progressive motility
- Those with moderate progressive motility
- Those which are immotile (which includes
those moving only weakly on the spot)
This is most easily done by examining the
field while focusing and defocusing the eye-
piece.
When the eyepiece is out of focus one can
see the immotile spermatozoa better and with
the eyepiece focused the motile spermatozoa
are clearer. With a little practice at constantly
changing the focus and assessing slides, one
soon achieves results which are comparable
with those obtained by objective methods of
measurement within an acceptable level of
error. At least ten fields should be examined
in this way at a magnification of 400 x (eye-
piece 10 x , objective lens 40 x).
However, one should bear in mind that
motility rates tend to be estimated on the
high side if numbers are high, and on the
low side if numbers are low (KRAUSE and
ROTHAUGE 1981).
2.7.1.2 Objective Methods
of Determining Motility
In the last 10 years a whole series of methods
for objectively determining motility has been
developed (PHILLIPS 1972; JECHT and Russo
1973; DUBOIS et al. 1975; KATZ and DOTT
1975; SOKOLOWSKI et al. 1977; MAKLER
1978a; OVERSTREET et al. 1979; KATZ and
OVERSTREET 1981; KAMIDONO et al. 1983;
HARTMANN et al. 1983). They all have advan-
tages and disadvantages: they are either time-
consuming, expensive, and accurate, or are
cheap and less accurate. Ultimately they offer
no advantages for routine daily andrological
examinations over the simple estimation
method, which although inaccurate is tolera-
bly so.
We will nevertheless describe two systems
because they cover the range of options from
empiricism to precise analysis, especially
when evaluating therapies or substances
which stimulate spermatozoa in vitro:
1. The multiple-exposure photography
(MEP) method using the Makler chamber.
2. Laser-Doppler spectroscopy using the La-
zymot.
We have chosen MEP because it ensures
objective measurement at justifiable cost, and
Lazymot because, although it is expensive,
it is extremely accurate, easy to operate, and
rapid in providing an assessment of motility
and speed of movement.
2.7.1.2.1 Multiple-Exposure
Photography (MEP)
in a Makler Chamber!
This is a microphotographic method using
the counting chamber described by Makler
(MAKLER 1978a; MAKLER 1978b). The stan-
dard counting chamber illustrated in Fig. 17
is only 10 11m deep. Progressive motility, de-
viant motility (circular movements, "shak-
1 Makler counting chamber, Sefi Medical Instru-
ments, P.O.Box 7295, Haifa 31070, Israel. Tel. 04-
251651, Telex 46400 Ext. 8796.
17
ing" on the spot), or immotility are recorded
on a photograph taken through a disk with
six slots, which is driven by an intermittent
electric motor, with an exposure time of 1 s.
The heads of motile spermatozoa are photo-
graphed several times during the relatively
long exposure time of 1 s, so that the motile
spermatozoa appear as chains composed of
six links, whilst the immotile spermatozoa are
clearly visible from head to tail as brighter
forms (Fig. 18).
Fig. 17. Makler chamber

Fig. 18. Multiple exposure photography method for
the objective measurement of moti lity ; immotile p r-
matozoa are brighter and appear a ingle oval rings,
surrounded by a " halo." Motile spermatozoa appear
as six-link chain , the hape and length of which indi-
cate their movement over 5/ 6 s. (Microphotograph,
after MAKLER 1978, from GLEZERMA N M (1982) Se-
men Analysis. In ; BANDHAUER K , FR I K J (ed ) Di -
turbance in male fertility . Springer Berlin eidel-
berg ew York, p 207)

18
Procedure

A drop of thoroughly mixed ejaculate is

• I

placed with a wooden stick into the lower

section of the 10 11m-deep Makler chamber,
which is immediately covered (Figs. 19, 20).
This automatically ensures that the thickness
of the drop is 10 11m.
The chamber with its drop of ejaculate (A,

1EI
Fig. 21) is then placed on the stage (B) of
a phase-contrast microscope under the 20 x

~
objective (C). A camera with bellows exten-
sion (D) and loaded with a 100 ASA (= 21
DIN) black-and-white film is attached to the
microscope. Extension rings can be used in-
stead of the bellows. The distance to the spec-
imen is adjusted so that the area photo-
graphed is approximately 0.5 x 0.35 mm. Fig. 20. After placing the drop of ejaculate in the
In the Makler chamber the immotile sper- bottom ection of the Makler chamber (upper draw-
matozoa will lie at the bottom, whereas the ing) , the chamber is immediately closed with the
cover slip (lower drawing). The thickne of the drop
motile spermatozoa will swim in the upper is then 10 j.1m. It is examined under the micro cope
levels in the chamber. Those swimming at at a magnification of 200 x (eyepiece lO x , objective
the top are brought into sharp focus . A stro- 20 x)
boscope, consisting of a black disk with six
slots in it and connected to a rotary electric
motor (E, Fig. 21) is positioned between the
condenser and the field diaphragm of the mi-
croscope. While the disk turns at a speed of
60 rotations/min ( = 1 rotation/s), the film is
exposed for 1 s.
By this means the film is exposed to six
pulses of light each of 1/ 200 S duration and
intervals between each of 1/ 6 S. As only 1/ 30
of the light emitted in 1 s reaches the film,
the immotile spermatozoa are brighter but

Fig. 21. Multiple-exposure photography method . A,

Fig. 19. A wooden tick i u ed to place a drop of
thoroughly mixed ejaculate into the Makler chamber
Makler chamber, loaded with a drop of ejaculate ;
B, micro cope tage' C, 20 x objective ' D, camera
with bellow exten ion; E, trobo cope, i.e., di k with
ix lot, driven by a rotary electric motor. (Modified
from MAKLER 1978 a, b ; ee text for explanation)

19
are not overexposed, despite being illumi-
nated for six times longer. In addition, the
six-link chains representing the progressively
motile spermatozoa and shown in Fig. 18 are
not underexposed.
As the distances covered by the spermato-
zoa in 1 s are captured at the same time, the
mean speed of the spermatozoa in microme-
ters per second can be calculated in addition
to the quality of their motility (for further
details see MAKLER 1978a).
ISHII et al. (1977) and OVERSTREET et al.
(1979) described similar methods to assess
motility objectively. However, as the evalua-
tion of all of these methods is a time-consum-
ing process, MAKLER added a rapid micro-
computer-controlled reading facility to MEP,
but this makes it considerably more expen-
sive, of course.
2.7.1.2.2 Laser-Doppler Spectroscopy
(Lazymot 1 )
The Lazymot equipment operates on the
Doppler principle and measures frequency
shifts of scattered laser light caused by the
movement of spermatozoa in the scattered
laser light.
The theoretical principles were developed
by NOSSAL (1971) and by STEINER et al.
(1981). Other authors have documented the
accuracy, rapidity, and reliability of the
equipment for the objective, quantitative
measurement of spermatozoal motility (Du-
BOIS etal. 1975; STEINER etal. 1977; HART-
MANN et al. 1983; PUSCH 1985).
The technical specification of the equip-
ment is described in detail in STEINER et al.
(1981) and HARTMANN et al. (1983). Despite
its complicated technology, this expensive
equipment is simple to use and its operation
presents no difficulties (PuSCH 1985).
Table 6 lists the possible factors which can
be investigated using the Lazymot.
1 Lazymot, BTG-Biotechnik GmbH, Zeppelinstr. 73,
D-8000 Munich, FRG, Tel. 089/4488896.
20
Table 6. Objective quantitative and qualitative mea-
surements of motility and speed of movement of sper-
matozoa which are possible using the Lazymot equip-
ment
- Global motility as a %
- Proportion of spermatozoa with progressive motil-
ity as a %
- Mean overall speed of spermatozoa in IJlll/s
- Speed of actively progressively motile spermatozoa
in Ilm/s
- Speed of poorly progressively motile spermatozoa
in Ilm/s
- Spermatozoal density in millions of spermatozoal
ml ejaculate
Evaluation of Motility
Diminished motility of spermatozoa is
termed asthenospermia. There is general
agreement that 60%, or a minimum of 50%,
of the spermatozoa as a whole should be mo-
tile, while 40%, or a minimum of 30%,
should exhibit progressive motility (BELSEY
et al. 1980; MANN and LUTWAK-MANN 1981;
HARGREAVE 1983; GUNTHER et al. 1983). As
far as routine operations are concerned, we
follow the recommendation made by SCHILL,
who specified a minimum of 50% motility
(SCHILL 1985b). However, this is only equiva-
lent to the statistical mean of normal values.
BOSTOFTE et al. (1984), on the basis of their
results in over 1000 men examined on ac-
count of childlessness and followed up 20
years later, therefore recommended defining
the boundary between normal and dimin-
ished fertility as 80% immotile spermatozoa.
The conclusion to be drawn from this is that
conception is quite possible even with lower
values, and incidentally this also applies to
all the other semen parameters.
2.7.2 Number and Density
of Spermatozoa
"Good motility together with high sperm
density is in practice usually the safest point-
er to satisfactory fertilizing potential of se-
men" (Thaddeus Mann, in MANN and Lu-
TWAK-MANN 1981). This statement by a man Equipment Required
who has spent his whole life researching and
teaching the physiology of reproduction, - Microscope with phase-contrast
highlights the second most important, classi- - Counting chamber (Neubauer, Thoma-
cal semen parameter after motility: namely, ZeiB, or Burker-Turk)
the number of spermatozoa or the number/ml - Hemocytometer cover slip (20 x 26 mm,
ejaculate, i.e., spermatozoal density. 0.4 mm thick)
In order to arrive at the total number, one - Leukocytepipette
needs only to multiply the spermatozoal den- - Suction tube
sity by the volume : - 3% Saline solution or distilled water
- Electric vibrator
Total number of spermatozoa
- Filter papers or cellulose wadding
=number/mlx volume.

Procedure
2.7.2.1 Determining Spermatozoal Density
in Hemocytometers After complete liquefaction, the ejaculate
must first be thoroughly mixed (see Sect.
There are various types of counting chambers 2.6.4). To ensure the uniform distribution of
available, which are usually employed for spermatozoa and to simplify counting, the
counting blood cells and are therefore called ejaculate is diluted 1 : 20. As the spermatozoa
blood counting chambers or hemocy- must be immobilized to prevent them escap-
tometers.
The best known counting chambers are :
- Neubauer
- Thoma-ZeiB
- Burker-Turk
A counting chamber is a specially designed
microscope slide with engraved squares. The
different chambers are thus divided into var-
ious large and small squares forming a grid
pattern. The depth of the counting chambers Fig. 22. Mixing pipette (leukocytepipette)
is 0.1 mm or 0.2 mm depending on the type.
The principle of determining spermatozoal
density in a counting chamber is that the
ejaculate, in a standard dilution, is intro-
duced into the chamber, which is covered

L
with a cover slip, and the number of sperma-
tozoa is counted in a specified number of
squares. ;J
-....:.
........'.'
By taking account of the depth of the
chamber and the dilution factor , the number ~ ~~
, .. ',. .:. -,.'
,
.
of spermatozoa per milliliter of ejaculate can
be calculated.

Fig. 23. Mixing pipette fitted to a micropipeller. The

completely liquefied ejacu late i ucked up to the 0.5
mark , then 3% aline olution i ucked up to the
11 mark . Thi dilute the ejaculate to a dilu tion of
1 :20

21

Fig. 24. Electric vibrator
ing from the counting chamber square while
counting is in progress, solutions which im-
mobilize spermatozoa are used to dilute the
ejaculate, e.g. :
- Physiological saline solution containing a
drop of triphenyltetrazolium chloride solu-
tion (SCHIRREN 1982a)
- Distilled water (GLEZERMANN 1982)
- 3% saline solution (KRAUSE and ROTH-
AUGE 1981)
1. Suck up the thoroughly mixed ejaculate
into the 1: 20 mixing pipette (leukocytepi-
eubauer counting chamber with cover lip
22
pette) as far as the 0.5 mark, using a suc-
tion tube. Any excess can be corrected by
blotting on filter paper or cellulose wad-
ding.
2. Then suck up 3 % saline solution into the
leukocytometer as far as the 11 mark. The
sample is now diluted 1 : 20 (Figs. 22, 23).
3. In order to obtain a homogeneous mix-
ture, shake the mixer pipette for 3-5 min
either by hand (holding the two ends
closed with thumb and middle finger after
removing the suction tube), or using an
electric vibrator (Fig. 24).
4. Before introducing the sample, cover the
counting chamber (Fig. 25) with a special
cover slip (0.4 mm thick, area 20 x
26 mm). First moisten the shoulders of the
chamber and gently press down the cover
slip.
5. If any part of the capillary of the mixing
pipette contains sample which is poorly
mixed, this part is discarded by expelling
it or blotting it on filter paper or cellulose
wadding.
6. Fill the counting chamber by introducing
one drop from the mixing pipette on both
sides (Fig. 25 b). The spermatozoa are
counted under the microscoppe with a
magnification of 400 x .
uer
 Tiefu
O.JOO",m

Fig. 25b. Introducing the diluted ejaculate into the eubauer counting chamber

Counting Spermatozoa in the Chamber

and Calculating Density

2.7.2.1.1 Neubauer Counting Chamber

The Neubauer counting chamber (Fig. 26)

has a depth of 0.1 mm and is divided into 1 2
a grid of nine large squares, from which we
select those positioned like the dots of a 5
on a die to perform the count, although the r 5a ~
central large square is usually sufficient. The I
central square (no. 5) is divided in turn into 5< 5e
a grid of 25 small squares, from which we 1
can again select the squares shown as 5a - 5. l 5c Sd

corresponding to the dots of a 5 on a die.

Depending on the level of accuracy re-
quired, the time available, and the total
number of spermatozoa involved, three dif-
ferent methods of counting and calculating
can be employed. The density determinations
shown comply with the recommendations of
the WHO (BELSEY et al. 1980).
3 t.
Fig. 26. Subdivi ion of the eubauer counting
chamber: the sum of all the spermatozoa in the cen-
tral square 5 x 10000 x 20 gives the number of per-
matozoa per milliliter and thus the spermatozoal den-
sity (see text for further detail)

23
I Accurate Count
Count all the spermatozoa in the large square
no. 5 (the central square). Multiply the total
by 10000 (because the volume of the field
counted is 10 Ill) and by the dilution factor
(20 for a dilution of 1: 20) to obtain the
number of spermatozoa per milliliter. Any
spermatozoa touching the upper border and
the border on the right, including those pro-
jecting over the borderline, will be counted,
but not those touching the lower and left
borders (although, of course, this principle
can be applied in reverse).
II Quick Count
Count all the spermatozoa in the small
squares 5 a, b, c, d and e. Multiply the total
by 50000 (because the volume is only one-
fifth that of a large square) and by 20 (the
dilution factor) to obtain the number of sper-
matozoa per milliliter of ejaculate.
III Counts in Cases of Low Density
« 10 Million Spermatozoa/ml)
Count all the spermatozoa in the large
squares 1- 5. Multiply the total by 2000 (be-
cause they contain 5 times the volume of one
large square) and by 20 (the dilution factor)
Fig. 27 a. Thoma-Zei13 counting chamber
24
to obtain the number of spermatozoa per
milliliter of ejaculate.
Table 7 shows the three options for deter-
mining spermatozoal density as abbreviated
formulae.
2.7.2.1.2 Thoma-Zein Counting Chamber
(Fig. 27 a and 27 b)
The Thoma-ZeiB counting chamber has a
depth of 0.1 mm and is divided by parallel
lines into a grid of 16 large squares. Each
of these large squares is divided into 16 small
Table 7. Abbreviatied formulae for the three methods
of determining spermatozoal density using the Neu-
bauer counting chamber (assuming a dilution of
1 :20)
I. Accurate count
Total number of spermatozoa in large square 5
x 10000 x 20 = density
II. Quick count
Total number of spermatozoa in small squares
5a- 5.
x 50000 x 20 = density
III. Counts in cases of low density
Total number of spermatozoa in large squares
1- 5
x 2000 x 20 = density
Thom ·
e. IV
 I
V IVI/ V Abbreviated formula for determining, sper-
l(.j/ V matozoal density using the Thoma-ZeifJ
-
~ v~ V counting chamber
V V V 1/ (assuming a dilution of 1 : 20)
IV V V V Total number of spermatozoa m large
1/ v2 V squares 1-5
1/ :;/ 1/ x 1 million = density
1/1/ / /
VVV V
1/ /~; '/
1/ V Multiplying total number of spermatozoa
V V 1/ 1/ obtained from counting the contents of five
V 1/V V V V V 1/ large squares by 1 million gives the number
- VS/ V
~ v~ V
1/
1//0 ~ f- of spermatozoa per milliliter for a dilution
of 1 : 20 and therefore the spermatozoal den-
IV VV V 1/ IN 1/
I I sity.
Fig. 27b. Subdivisions of the Thoma-ZeiB counting
chamber: the sum of all the spermatozoa in the Principle to be Followed
squares numbered 1-5 gives the spermatozoal density
in millions per milliliter with Different Counting Chambers

Apart from the procedure described in detail

squares. One large square fits almost exactly
into the field of the microscope at a magnifi-
cation of 400 x .
The spermatozoa are counted in five large
squares, and, as described for the Neubauer
chamber, any spermatozoa lying on one of
the two horizontal lines or on one of the two
vertical lines are included in the count. The
most convenient approach is to count the
contents of four diagonal squares and a fifth
corner square (see Fig. 27b).
II
I!
above for calculating spermatozoal density
with the Neubauer and Thoma-ZeiB counting
chambers, the following formula can be ap-
plied in principle to the Burker-Turk
chamber (Fig. 28) and other counting
chambers:
c
c sp = A xHxD x 1000
Csp = number of cells/mm 3
C = number of cells counted
A = area counted (mm 2 )
H = height (depth) of the chamber
D = dilution.

(In the first part of the formula one obtains

the number/mm 3 . This must be multiplied by
1000 in order to obtain the number/cm 3 (=
ml)=density.)

2.7.2.2 Determining Spermatozoal Density

in a Makler Chamber

In the Makler chamber which was mentioned

in Sect. 2.7.1.2.1 and which was subsequently
improved still further (MAKLER 1980), it is
possible to determine the number of sperma-
Fig. 28. Biirker-Tiirk counting chamber tozoa with ease even in undiluted ejaculate.

25
 Fig. 29a. Maider chamber, Overview
through the microscope, x t 6

Fig. 29b. Makler chamber. Sectional

view, bright-lield, without lilter x 40

. .
'""
'" .. , ~

.... .., ..
I
'. . . . ..
'i~
-..,..... ..
~
~
l. ~~-
\:.,-" "
"
..
,

,"

,,' ,.
~
~'~ . '
•
~~

...~
\ "
.
c ,\ . " \
I>'t~~
.. , .~-'• . ~ ... ,
~

~
~"").<
'-
'
.,
I,. ~~ ~ .
, ... ;-:-.~ .
,- ~ '.. - -u-
....:.c·
.
-..... ,
...
.
'--'
~
t~) ... .
01 ...
.. ~
.;. ~
....
l ' ..,
..
r
"-'
~
. " ..
,.\ '

- , ~.
".
.L"\ .. \.1.:.
............ .,
,"\'"
,", ~ .
\
'-
.. ,~~~
r-
;'oj ,,,.,

~ .. } ..!... • i ,
•
Fig. 29c. Makler chamber. Sectional view interference pha e-contra t, x 60

26
The method of measurement is more accu-
rate than that in hemocytometers (MENK-
VELD et al. 1984).
Counting in Makler Chamber
A drop of liquefied, thoroughly mixed, un-
prepared ejaculate is placed on the base plate
(lower part) as illustrated in Fig. 19. The
cover slip (upper part) is pressed onto it in
such a way that colored rings (Newton's
rings) become visible on the shoulders. This
guarantees that the depth of the chamber is
uniform. The lower surface of the cover slip
is engraved with a grid covering a total area
of 1 mm 2 , subdivided into 100 small squares
each of 0.01 mm 2 . The thickness of the drop
of the sample is therefore exactly 10 Jlm.
Using a magnification of 200 x, the con-
tents of one strip of ten squares are counted
under the microscope. The volume equals
10 Jll. Therefore, if one wishes to determine
the number of spermatozoa or other cellular
elements per milliliter (and therefore their
density), the number counted is multiplied
by 1 million. However, in order to ensure
that the results are statistically reliable, one
should count the contents of three strips each
of ten squares, from at least two samples.
Abbreviated formula for determining
spermatozoal density using
the M akler chamber:
Total number of spermatozoa
in 10 squares x 1 million = density
Figures 29 a, 29 band 29 c shows different
views in a Makler chamber under the micro-
scope.
2.7.2.3 Determining Spermatozoal Density
Using Electronic Counters
1. Coulter Counter
The difficulties of counting spermatozoa us-
ing equipment which is extremely suitable for
counting the cellular elements of the blood
are due to the fact that it cannot differentiate
between spermatozoa and other cellular ele-
ments, clumps of cells, or detritus (BROTHER-
TON 1973; READ and SCHNIEDEN 1978;
KRAUSE and ROTHAUGE 1981; SCIllRREN
1982a). In addition, it is inaccurate at low
densities « 10 million/ml) according to HAR-
GREAVE and NILSSON (1983), and according
to KRAUSE and ROTHAUGE (1981) it is even
inaccurate at densities below 20 million sper-
matozoa/ml.
2. Cytophotometry
A good estimate of the number of spermato-
zoa can be obtained by means of cytopho-
tometry of spermatozoal DNA, although an
elevated number of abnormally formed sper-
matozoa (teratospermia, see Sect. 2.7.3.2.1)
will distort the results (LACROIX and WARTER
1982).
3. Laser-Doppler Spectroscopy
The method described in Sect. 2.7.1.2.2 is
also quick, simple, and accurate when used
for determining the total number of sperma-
tozoa and spermatozoal density, but it does
require the acquisition of very expensive
equipment.
2.7.2.4 Evaluation of Spermatozoal Density
A complete absence of spermatozoa in the
ejaculate, even after sedimentation, is called
azoospermia. A reduction in spermatozoal
density is called oligospermia. In its most se-
vere form it is termed cryptospermia, when
a few isolated spermatozoa are found only
after sedimentation. The normal values quot-
ed in the literature vary: the value of 60 mil-
lion spermatozoa/ml established on the basis
of studies of ejaculates in only four men by
the Viennese biology student ALOIS LOHDE
(1891) nevertheless stood for almost 80 years,
even though there was evidence some time
ago (MACLEOD 1951, 1965a) that 60 million
spermatozoa/ml was too high a figure for de-
termining prospects of fertility.
The Andrology Club took account of this
and in 1970 set the lower limit for normal
findings at 40 million spermatozoa/ml
(SCHIRREN 1972).
27
 Further studies in subsequent years found
no differences in fertilization rate between
volunteers with > 40 million and those with
20 million spermatozoa/ml or slightly above
(EUASSON 1971; EUASSON 1975; VAN ZYL
et al. 1975; FREUND and PETERSON 1976;
EUASSON 1981; GUNTHER et al. 1983), and
therefore, today, a normal lower limit for
spermatozoal density of 20 million spermato-
zoa/ml is generally accepted and is also speci-
fied as such in the WHO report (BELSEY et al.
1980), (see Table 8).
Indeed, the critical level for impaired fertil-
ity is actually considered to be 10 million
spermatozoa/ml (VAN ZYL et al. 1975; ZUK-
ERMAN et al. 1977; SCHILL 1980; GUNTHER
et al. 1983; URRY 1985; HOFMANN and
FREUNDL 1986).
However, it should be borne in mind that
the studies on which these proposals were
based are necessarily only of an empirical or
catamnestic nature. The other semen param-
eters, particularly motility and morphology
(see Sect. 2.7.3) but also acrosomal function,
penetration ability (see Sect. 3), and biochem-
ical parameters of seminal plasma, must be
taken into account as well when assessing the
prospects of fertility. A reliable prognosis
cannot be made even then in the individual
case.
Elevated spermatozoal density, polyzoosp-
ermia, exists if one finds more than 250 mil-
lion spermatozoa/ml or more than 800 mil-
lion spermatozoa in the total ejaculate. Its
significance has not yet been precisely estab-
lished. Its incidence is between 1.2% and 5%
(REHAN et al. 1975; SCHILL 1987). There have
been reports of increased frequency of sub-
fertility, particularly when disturbed motility
is greater at the same time (DOEPFMER 1962),
and a higher rate of miscarriages among the
Table 8. Diagnostic evaluation of different spermato-
zoal densities
Normospermia 20-250 million/ml
Oligospermia < 20 million/ml
Polyspermia > 250 million/ml
Cryptospermia spermatozoa not
discovered until
after sedimentation
Azoospermia no spermatozoa
28
wives of men with polyzoospermia (REHAN
et al. 1975; SCHILL 1987).
ZUKERMAN et al. (1986) found that poly-
zoo spermia increased as the duration of ab-
stinence increased, and concluded that the re-
duction in fertility in cases of polyzoospermia
could be caused by disturbed feedback con-
trol to a hypothetical spermatozoa-releasing
mechanism (ZUKERMAN et al. 1986).
As a marked loss of acrosin activity was
observed in a not inconsiderable proportion
of men with polyzoospermia (SCHILL and
FEIFEL 1984; TOPFER-PETERSEN et al. 1985),
possibly caused by a severe disorder of the
acrosin inhibitor system (SCHILL et al. 1986;
SCHILL 1987), which would explain infertility
due to a defective acrosome, Schill recom-
mends that acrosin be determined when in
vitro fertilization is planned (SCHILL 1985 a).
2.7.3 Morphology
The assessment of morphology comprises the
qualitative and quantitative differentiation of
normally formed from pathologically formed
and destructured spermatozoa, and the dif-
ferentiation of various "round cells" into
spermatogenic cells from the wall of the semi-
niferous tubules, granulocytes, lymphocytes,
monocytes, histiocytes, and epithelial cells.
After motility (Sect. 2.7.1) and the number
or density of spermatozoa (Sect. 2.7.2), mor-
phology is the third essential pillar support-
ing the diagnostic framework of ejaculate
analysis.
For most investigators, there is no doubt
about its importance in male fertility (JOEL
1953; DOEPFMER 1960; MACLEOD 1964;
EUASSON 1975; SCHIRREN et al. 1975; SCHIR-
REN et al. 1977; SHERINS et al. 1977; SCHILL
1980; MANN and LUTWAK-MANN 1981;
SWERDLOFF et al. 1985; Pinatel1985).
There is a significant correlation between
high percentages of pathologically deformed
spermatozoa and reduced pregnancy rates,
but there is no correlation with miscarriages
or pathological pregnancies (BOSTOFTE et al.
1982; GUNTHER et al. 1983). If abnormal
forms account for over 50% of spermatozoa
examined, one speaks of teratospermia. In
addition, in an ejaculate with an increased
incidence of morphological abnormalities,
one often finds simultaneously oligo- and as-
thenospermia (SINGER et al. 1980), and one
can therefore describe this as OAT syndrome
(= oligoasthenoteratospermia syndrome).
The reduced fertilizing ability of abnormal-
ly formed spermatozoa is also manifest in the
fact that in a postcoital test more normally
formed spermatozoa are found in the upper
section of the cervical canal than in the lower
section. As they move along the cervical ca-
nal, the spermatozoa apparently undergo a
process of selection in which those with poor
motility and abnormal morphology are
trapped in the "filter" of cervical secretions
(RAGNI et al. 1985).
Although spermatozoal morphology can
be assessed to some extent in an unprepared
sample in an improved Makler chamber (see
Sect. 2.7.2.2) with phase-contrast micropho-
tography or with interference phase-contrast
microscopy, the most successful method of dif-
ferentiating between the individual forms and
cells is to use a stained ejaculate smear.
2.7.3.1 Staining
Thefollowing staining methods may be used:
A "Classical," time-consuming staining
methods:
1. Hematoxylin and eosin (H&E)
2. Mayer-Stiasny
3. May-Grunwald-Giemsa (Pappenheim's
panoptic stain)
4. PAS (Periodic-Acid-Schiff)
5. Papanicolaou
6. Schorr
7. Couture
8. Bryan-Leishman
9. Peroxidase reaction (=benzidine-
cyanosine staining)
10. Eosin and nigrosin (see Sect. 2.7.4)
B Simplified, rapid staining procedures
1. Testsimplets (Boehringer, Mannheim,
FRG)
2. Hemafix (Biomed, Munich, FRG)
3. Sangodiff G (E. Merck, Darmstadt,
FRG)
The methods shown in italics are described
in greater detail below. Examples of ejaculate
smears stained using these methods can be
found in the section on specific morphology
(see Sect. 2.7.3.2).
Using Papanicolaou's stain, which is pre-
ferred by us and by other authors despite
being rather more complicated (GLEZERMAN
1982; HARGREAVE and NILSSON 1983; HOF-
MANN and FREUNDL 1986), one obtains good
differentiation of the heads of spermatozoa.
The round cells are stained panchromati-
cally, and therefore leukocytes can be easily
distinguished from germinal cells.
Pappenheim's method using May-
Griinwald-Giemsa stain gives excellent selec-
tive differentiation of round cells and the
staining of the karyomere is also strongly
contrasted.
The WHO (BELSEY et al. 1980) recom-
mends the Bryan-Leishman method to distin-
guish germinal cells. We prefer the peroxidase
reaction, particularly when it is difficult to
distinguish leukocytes and lymphocytes from
immature spermatogenic cells.
However, the requirements of routine prac-
tice can be adequately satisfied by the use of
prestained slides coated with a dye (Testsim-
plets, Boehringer) or alternative rapid staining
methods such as staining with Hemafix
(Biomed) or staining films coated with a dye
(SCHIRREN et al. 1977; CALAMERA and VILLAR
1979; WERNICKE and SCHIRREN 1982; LUD-
WIG 1986).
The clarity of differentiation is quite ade-
quate and economical. The maximum error
in comparison with Papanicolaou's stain is
4%-5%, and in addition the image quality
of Testsimplets improves after 24 h due to
the longer exposure to the dye (SCHIRREN
et al. 1977). However, permanent specimens
cannot be produced as the dye fades over
time.
"Classical", More Time-Consuming Staining
Methods
Equipment Required
- Microscope
- Slides (polished, 76 x 26 mm)
29
- Cover slips (24 x 50 mm)
- Pipette, glass rod, or record syringe
- Staining bench with appropriate reagents
(described under each staining method,
Sects. 2.7.3.1.2-2.7.3.1.4)
Procedure
a) For Densities < 10 Million Spermatozoa/
ml:
After the ejaculate has completely liquefied
and has been thoroughly mixed (see Sect.
2.6.4), place a drop of semen measuring
about 3 mm in diameter onto the slide using
a pipette, glass rod, or record syringe, and
smear but do not cover with a cover slip or
second slide.
Leave this smear to dry in the air for 1
or 2 h, then fix and stain in accordance with
the method chosen (see Sects. 2.7.3.1.2-
2.7.3.1.4).
b) For Densities > 10 Million Spermatozoa/
ml:
In such cases it is necessary first to concen-
trate the cellular components by centrifuging
the completely liquefied and thoroughly
mixed ejaculate in a test tube. It should be
centrifuged for 15 min at a speed of 2000 ro-
tations/min. Place a drop of the sediment
onto a slide as described above, smear, dry
in the air, and fix and stain in accordance
with the method chosen.
For assessing the individual cellular ele-
ments, see Sect. 2.7.3.2.
2.7.3.1.1 Papanicolaou's Stain
This staining technique was developed specif-
ically for the identification of cell types in
malignant tumors. It is also excellent for
smears of samples taken from the male and
female genital tract.
Equipment Required
- 13 glass cuvettes with lids (Fig. 30)
- 1 slide rack (Figs. 31-33)
- 1 diamond scriber or water-resistant pencil
30
Solutions Required
1. Diethyl ether DAB 6 (Merck)
2-5. Ethanol 96%, 80%, 70%, 50%
6. Distilled water
7. Harris' hematoxylin solution (Merck)
( = Papanicolaou I)
8. Ammoniacal alcohol 70%
9. 0.05% aqueous lithium carbonate solu-
tion
10. Orange G solution (OG 6) (Papanico-
laou II)
11. Absolute alcohol DAB 6 (Merck)
12. Polychrome solution EA 36 (Papanico-
laou III)
13. Xylene
- Canada balsam (or similar)
Procedure for Papanicolaou's Staining
Fix the smears in a mixture of ether and 96%
alcohol (1: 1),5-15 min.
Immerse in an alcohol series of decreasing
concentration from absolute alcohol to 50%
(96%, 80%, 70%, 50%), allowing about
2 min per concentration or dipping the slides
ten times. Immerse in distilled water for
2 min or dip ten times.
Staining with Harris' hematoxylin (=Pa-
panicolaou I), 3 min. Rinse the specimens for
3-5 min in three quantities of distilled water
or under running water.
Blue in ammoniacal alcohol (70%) for a
few seconds or up to 2 min.
Transfer to 70% alcohol then rinse with
tap water. Immerse in a 0.05% aqueous solu-
tion of lithium carbonate for 2-3 min. Then
rinse with tap water.
Immerse in an alcohol series of increasing
concentration from 50% to 96% (50%,70%,
80%, 96%), allowing about 2 min per con-
centration or dipping the slides ten times.
Staining in Orange G solution ( = Papanico-
laou II). Immerse for 2 min or dip ten times.
Wash twice in 96% alcohol for 2 min each
time or dip ten times.
Staining in polychrome solution EA 36 ( =
Papanicolaou III), 2 min.
Rinse off excess dye by transferring the
slides to three separate cuvettes containing
96% alcohol (dip five times in each).
Fig. 30. 13 glass cuvettes with lids for Papa nicolaou' taining series

Absolute alcohol, two quantities, 2 min in Basophilic cells: bluish-green

each. Leukocytes: pale red
Xylene, 20 min. Mucus: green
Mountant resin (Canada balsam). Karyomeres
Cover slip. Acrosome: pink
Label. Postacrosome: dark blue
Figure 30 shows the arrangement of glass Tail of spermatozoa: pink
cuvettes used in Papanicolaou's staining se- Papanicolaou's stain (PAPANICOLAOU
ries numbered in sequence of use. Figures 1942) seems to us to be the most important
31- 33 show how the slides are inserted into staining method and has been described in
a rack which is then dipped into the appro-
priate solution. The precise composition of
the dye solutions can be found in the Merck
catalogue.

Results of Staining

The individual cellular features will assume the

following colors:
Cell nuclei: blue to bluish-violet
Acidophilic cells: pink
Hornified cells: orange to yellowish-orange
Erythrocytes: orange to reddish-brown Fig. 31. Slide rack

31

32
33
Fig . 32 33. lmmer ing a slide rack into a gla
vette containing dye solution
detail for this reason, although May-
Grunwald-Giemsa's stain is also excellent,
especially for differentiating round cells, and
is the staining method we prefer for that pur-
pose. The other stains are described rather
more sketchily. The composition of the dye
solutions can be found either in the Merck
catalogue or in the manufacturer's cata-
logues, and can be readily obtained from sup-
pliers of chemical solutions.
2.7.3.1.2 May-Griinwald-Giemsa Staining
(Pappenheim's panoptic staining):
- Fix the smears by coating with May-
Grunwald solution (0.8-1 ml), 3 min.
- Add an equal quantity of distilled water,
1 min.
32
- Tip off the diluted dye solution, do not
rinse off.
- Coat with dilute Giemsa's solution (0.3 ml
Giemsa's stain in 10 ml distilled water),
about 20 min.
- Rinse vigorously with distilled water.
- If the blue color is too dark, differentiate
with acetic acid solution.
- Dry and mount.
Results of Staining
Nuclei: reddish-violet
Plasma of lymphoid cells: light blue
Lymphoid azure granules: purplish-red
Myeloid azure granules : violet to violet-
brown
Neutrophil granules: brownish to bluish pink
Eosinophil granules: orange to brick red
Basophil granules: ultramarine to bluish vio-
let
Erythrocytes : pink
Polychrome forms of erythrocytes: predomi-
nantly bluish
Basophilic stippling of erythrocytes: cobalt
cu· blue
Karyomeres: pale blue to dark blue
Acrosome: pink
2.7.3.1.3 Peroxidase Reaction
(Benzidine-cyanosine staining to differentiate
round cells in the ejaculate)
oj Stock Solution
Completely dissolve 125 mg benzidine and
150 mg cyanosine (Floxin) in 50 ml alcohol
(96%).
Dilute the solution with 50 ml distilled water.
The stock solution should be stored in a dark
bottle.
b) Working Solution
Shortly before use, add 2 drops of a 3% hy-
drogen peroxide solution to 4 ml of the stock
solution. The prepared reaction mixture can
be kept for, about 12 h.
Procedure

Thoroughly mix together 1 drop of complete-

ly liquefied, weB mixed ejaculate with one
drop of the reaction mixture on a slide.
After 2 min the results can be examined
under a bright-field microscope with a mag-
nification of 400 x or 1000 x .

Results of Staining

- Neutrophil granulocytes: brown

- Granules of basophil and eosinophil gran-
ulocytes: reddish brown to violet
- Lymphocytes: merely a tinge of pink, as
peroxidase negative

2.7.3.1.4 Rapid Differentiation Fig. 35. lmmer ion oil

Using Testsimplets

Equipment Required must first be concentrated by centrifugation,

- Microscope
- Testsimplets (Boehringer, Mannheim, FRG)
(Fig. 34)
- Immersion oil (Fig. 35).
Procedure
Place a drop of liquefied ejaculate with a pi-
pette or glass rod or from the record syringe
onto the dye-coated area of the prestained
Testsimplets slide and cover with a cover slip.
If the spermatozoal density is less than 10
mi11ion spermatozoa/ml, the ceBular elements
as described above for the classical staining
methods. The results of the staining reaction
are similar to those of panchromatic staining
(see Atlas section for examples, pp. 109- 11 0).
2.7.3.1.5 Staining Using Hemafix 1
The rapid staining set consists of
- a fixing solution,
- an acidophilic dye solution,
- and a basophilic dye solution.
Depending on the type and density of the
smear specimen, immerse the slide for be-
tween 4 and 20 s, first in the fixing solution
and then in the two dye solutions. When
staining is complete, the ceBs can be differen-
tiated. The stained smears can be mounted
wet or after drying.

Results

These are similar to those with May-

Griinwald-Giemsa staining and are com-
pletely satisfactory (see Sect. 2.7.3.1.3).

Fig. 34. Te t implets (Boehringer, Mannheim, FRG): 1 Biomed, Bruckmannring 28, D-8042 Ober-
prestained slides for rapid staining schleiBheim (Munich), FRO, Tel. 089/3151619.

33
 '"
.~
Spermatozoa-- ~~~
2.7.3.2 Specific Morphology c

~~~I~i~t~hSrom~. ~ ~Q
(l)
CJl
o
.~

In order to understand the morphological

~
~
en
number)
features which can be found in a stained ejac-
2nd maturation division
ulate smear, one must first examine the var- (mitosis)
I
ious stages of the complicated, individually Secondary spermatocyte
(diploid chromosome number)
variable development process known as sper- I I
matogenesis, which has not yet been fully elu- TelophaseC?f _ _
tst maturallOn~~'
~~.;~£t\."
cidated even today. division . I· ,.'
In its widest sense, this process describes Metaphase
I
the development of the spermatozoon from
the embryonal spermatogonium to the fully Pachytene
mature spermatozoon which is capable of fer-
tilization (BUSTOS-OBREGON et al. 1975). In
the narrower sense, it is the process, which
first occurs at puberty, by which cells of the Zygotene
(crossing-over)
germinal epithelium in the seminiferous tu-
bules of the testis mature from spermatogo-
nium to spermatozoon. For teaching pur-
Leptotene
poses, one can distinguish between three suc-
cessive stages (CLERMONT 1963; HOLSTEIN
and ROOSEN-RUNGE 1981; DE KRETSER et al.
1982):
1. Spermatocytogenesis Ad spermato- Ap spermatogonium B spermatogonium
gonium (diploid chromosome
(the spermatogonium stage) number)

2. Meiosis Fig. 37. Illustration of the different cell forms in the

(the spermatocyte stage) process of spermatogenesis from the spermatogonia
residing in the basement membrane via spermato-
3. Spermiogenesis
cytes and spermatids, up to the spermatozoa which
(the spermatid stage) project into the seminiferous tubules. Ad spermato-
Spermatogenesis gonium, type A dark spermatogonium; Ap spermato-
Maturation of the germinal epithelium
to spermatozoon in the seminiferous tubules
gonium, type A pale spermatogonium; B spermato-
gonium, type B spermatogonium. (Modified after
CLERMONT 1963)
Lumen of the seminiferous tubule

Figures 36 and 37 are simplified diagrammat-

i
Spermatozoa Spermiogenesis
} = spermatid
stage ic representations of the various stages. With
Spermatids (haploid number
of chromosomes) regard to the details of spermatogenesis and
+-____ 2~~ ~aturation
diVISion
meiosis, we would draw your attention to the
(reduction relevant morphological publications and

r 1
division)
Secondary spermatocyte (diploid number
of chromosomes)
MeioSiS
= spermatocyte
textbooks (STIEVE 1930; ROOSEN-RUNGE and
... ____ ~~i~~~uration
stage
BARLOW 1963; CLERMONT 1963; HELLER and
CLERMONT 1964; DE KRETSER 1969; CLER-

1
(equational

l
division)
Primary spermatocyte
MONT 1970; ROSEMBERG and PAULSEN 1970;
Spermatocytogenesis
HOLSTEIN and WARTENBERG 1970; NISTAL
Pairingo!
chromosomes
(crossing-over)
---.-----+ = spermatogonium
stage and PANIAGUA 1984; HOLSTEIN and ROOSEN-
RUNGE 1981; DE KRETSER et al. 1982: COOPER
Spermatogonia
1986).
Basement membrane of the seminiferous tubule
The final stage, spermiogenesis, is impor-
tant for a clear understanding of the origin
Fig. 36. Greatly simplified diagrammatic representa- of aberrant forms, and it will therefore be
tion of spermatogenesis dealt with in greater detail below.

34
Spermatid Differentiation (Spermiogenesis) acrosome, becomes flattened and the postac-
rosomal part becomes distended (Fig. 39 b,
The complex cytological conversion of sper- and c). The resulting form displays the char-
matids into spermatozoa takes place in the acteristic shape of the head of a spermato-
final stage of spermatogenesis. This stage is zoon, which is oval when viewed from above,
also known as spermiogenesis. The develop- and pear-shaped flattening towards the tip
ment process is illustrated stage by stage in when viewed from the side (Fig. 41).
Figs. 38-40, similar to a time-lapse photo- The maturing spermatozoa are suspended
graphic technique. from long plasma processes of the Sertoli
The sequence in which the individual cells, projecting into the lumen of the semi-
stages appear is as follows: the acrosomal niferous tubules (Figs. 38h, 40b, 42).
cap develops first and spreads over the acro- When the mature spermatozoon is ejected,
somal vesicle and part of the surface of the the plasma processes of the Sertoli cells with-
nucleus (Figs. 38a- d, 39a--c). draw into the margin of the tubule again
The acrosomal vesicle and the acrosomal (Fig. 40c).
cap together form the acrosome. This is an In man the differentiation of spermatids
organelle enclosed in a membrane and is es- proceeds in definite, morphologically recog-
sential for the penetration of the ovum. The nizable stages, which do not form synchro-
Golgi apparatus separates from the acrosome nously as in animals, but successively, ap-
and floats free into the cytoplasm which is pearing topographically in column-like spi-
tending to move to the opposite pole rals, crossing over each other (CLERMONT
(Fig. 38e). 1963; HOFMANN and FREUNDL 1986).
The nucleus with the acrosome covering These maturation processes are controlled
it moves closer and closer to the margin and by a complicated interplay of hormones in
pushes out of the cell until the plasma is left which hormones of the hypothalamus (go-
behind it like a cylindrical bulge (Figs. 38 f, nadotropin-releasing harmone, GnRH), pi-
40b). As the acrosome continues to develop, tuitary gland (follicle-stimulating hormone,
the spermatid becomes increasingly elongat- FSH; luteinizing hormone, LH), the intersti-
ed, while the tip, i.e., the part bearing the tial cells of Leydig (testosterone), and

e f

9
c

b h

Fig. 38 a-h. Differentiation of a spermatid into a sper- skopische Spermaanalyse. Fertilitiit 2: 135). Figures
matozoon: partially diagrammatic photomontage of 38-40 were kindly made available by Professor
semithin sections (Prepared by Mrs. B. HILSCHER; Dr. N . HOFMANN, University Dermatology Clinic,
from HOFMANN N , FREUNDL G (1986). Die mikro- Dusseldorf, FRG

35
 Fig. 39a--c. Differentiation of the
acrosome and chromatin condensation:
partially diagrammatic representation of
semi thin sections. (Prepared by Mrs. B.
HILSCHER; from HOFMANN N, FREUNDL
G (1986). Die mikroskopische Sperma-
analyse. Fertilitiit 2: 135)

Fig. 4Oa--c. Final phase of

differentiation of the spermatozoon
from the spermatid (spermatid
elongation): partially diagrammatic
semithin sections. (Prepared by Mrs.
B. HILSCHER; from HOFMANN N,
FREUNDL G (1986). Die
mikroskopische Spermaanalyse.
Fertilitiit 2 : 135)

Frontal view Side VIew

(anleroposl erlor)
the Sertoli cells (androgen-binding protein,

}} Acrosome

Head
ABP; cyclic adenosine - 3' , 5' - monophos-
phate, cAMP) mutually interact and comple-
ment each other (STEINBERGER 1971; DE

}
KRETSER 1979; NIESCHLAG et al. 1979; DOR-
Neck and
middle piece RINGTON 1980; LUNENFELD and GLEZERMAN
1982; VIGERSKY 1983 ; HOFMANN et al. 1985;
HOFMANN and FREUNDL 1986).
Figure 42 illustrates in diagrammatic form
the interplay of hormones in testicular tissue,
Tao! and in particular shows the transport of tes-
(flagellum)
tosterone from the interstitial cells of Leydig
via the cells of the wall of the tubule into
the lumen of the tubule.

The Endocrine Regulation

a b
of Testicular Function
Fig. 41. Diagram of a spermatozoon viewed from
a above and b from the side. Seen from above the
head displays its characteristic oval shape. From the The gonadotropic hormones LH and FSH
side, the narrower acrosomal section becomes appar- reach the testis via the vascular system. As
ent, giving the head a pear-like shape macromolecular peptide hormones, they do

36
Fig. 42. The upper two-thirds of the diagram shows o In inhibin
a section of the germinal epithelium, with the lumen <=:) LH luteinizing hormone
of the seminiferous tubule at the top, and the base- o aP activated protein
ment membrane forms the boundary above the bot- C::> R receptor for FSH
tom third of the diagram. The bottom third shows the o RP regulator protein
interstice with the cells of Leydig and blood vessels go Spre steroid precursors
o T testosterone
cAMP cyclic adenosine-3' ,5' -monophosphate
Key to Symbols
ATP adenosine triphosphate
G R receptor for LH Sc spermatocyte
D ABP androgen-binding protein Sd spermatid
C> Adc adenylate cyclase Sg spermatogonium
G FSH follicle-stimulating hormone TJ tight junctions (blood-testis barrier)

37
not reach their target cells directly from the
capillaries, but bind to specific receptors on
the cell membrane and thereby liberate the
enzyme system of adenylate cyclase. This re-
leases the energy-rich cAMP from ATP.
cAMP activates protein kinase and this
causes phosphorylation and this activation of
a regulator protein, thereby stimulating the
specific cell response.
The gonadotropic hormones therefore act,
albeit indirectly, as stimulators of cell metab-
olism, membrane transport, and various se-
cretory processes.
The receptors for LH are located in the
cell membrane of the Leydig cells of the test-
icular interstice.
By binding to the receptors, LH stimulates
the synthesis of steroids, primarily the forma-
tion of testosterone.
The precursors necessary for steroid syn-
thesis are either delivered by the vascular sys-
tem and reach the receptors on the Leydig
cells, or they are synthesized in the Leydig
cell itself.
The receptors for FSH are located in the
cell membranes of Sertoli cells and spermato-
gonia. When FSH binds to the receptor, en-
zyme systems are liberated which cause cer-
tain processes of synthesis such as the pro-
duction of the testosterone receptor ABP (an-
drogen-binding protein) or the synthesis of
the FSH antagonist inhibin. These processes
take place in the Sertoli cells. In addition,
cAMP regulates membrane transport and the
permeability of the cell membrane. It thereby
also indirectly controls the transport of tes-
tosterone in and out of the Sertoli cells.
Testosterone diffuses from the peri tubular
matrix into the Sertoli cell and binds to a
receptor (ABP) in the cytoplasm of the cell.
The complex thus activated causes transcrip-
tion in the nucleus of the Sertoli cell and sub-
sequently coded protein synthesis either on
free polyribosomes (e.g., regulator proteins,
receptor proteins) or on ribosomes of the
granular endoplasmic reticulum (e.g., ABP).
The products of synthesis move from the en-
doplasmic reticulum into the cisternae of the
Golgi apparatus to complete their formation.
Terminal vesicles of the Golgi apparatus be-
come sealed off and are transported to the
38
cell membrane, where they are released either
into the intracellular space or into the lumen
of the tubule by membrane fusion. This is
how ABP gets into the lumen of the tubule
and from there via the rete testis into the
epididymis.
The testosterone-ABP complex is redis-
solved in the nucleus. Inactive ABP passes
through the nuclear pores back into the cyto-
plasm, where it can again form an activated
complex with testosterone.
Testosterone similarly diffused through the
nuclear pores into the cytoplasm and from
there into the intercellular space and into the
lumen of the tubule, or it may be metabolized
in the Sertoli cell (e.g., to DHT=dihydrotes-
tosterone).
Testosterone thus enters the adluminal ger-
minal cells (spermatocytes and spermatids),
in which it similarly induces processes of syn-
thesis which are responsible for the specific
course of spermatogenesis and spermiogene-
sis.
2.7.3.2.1 Morphological Differentiation
of Ejaculate Smears
The morphological differentiation of sperma-
tozoa into normal and various pathological
forms is subject to less individual variability
than their number and motility (HOFMANN
and FREUNDL 1986). The categorization of
deviations from the "normal" forms, i.e.,
forms mostly encountered among progres-
sively motile spermatozoa, is ultimately a
matter of subjective judgement (FREDRICSSON
1979), despite all attempts at classification
(MACLEOD and GOLD 1951; MACLEOD
1965a, b; FREUND 1966; ELIASSON 1971; DA-
VID et al. 1975; SCIllRREN et al. 1975; RIEDEL
and SCIllRREN 1978; MANN and LUTWAK-
MANN 1981; HOFMANN et al. 1982;
SCHWARTZ et al. 1984).
Nevertheless, the means of the various dis-
tribution profiles from large centers were
close to one another and were therefore com-
parable with one another (MACLEOD and
GoLD 1951; SCIllRREN et al. 1975; FREDRICS-
SON 1979; SCHWARTZ et al. 1984).
As the various changes to which the head,
middle piece, and tail of spermatozoa are
subject often occur in all possible combina-
tions, it is questionable whether the classifica-
tion often used ~ even by ourselves ~ into
malformations of the head, middle piece, and
tail is of any value other than for teaching
purposes. However, it is to be recommended
from the practical point of view and is of
benefit, especially for prospective and precise
retrospective studies aimed at verifying the
effectiveness of treatment given and to pro-
vide the possibility of prognosis.
Many investigators actually rate the mor-
phological examination highly in relation to
spermatozoal function and attribute patho-
genetic mechanisms to certain malformations
(MACLEOD 1964; FREUND 1966; HOFMANN
1979; MANN and LUTWAK-MANN 1981; HOF-
MANN et al. 1982).
The best known example is varicocele,
which we mention as the controversial dis-
cussion concerning specific morphology in
the stained differential spermiogram is clear-
est in this connection. Whereas MACLEOD
(1965) described typical immature forms,
especially those known as "tapered heads,"
which were also found by a number of inves-
tigators (GELEZERMANN et al. 1976; BUTLER
1979; ANNIBALO 1979; COCKETT et al. 1984),
other authors deny any morphological ab-
normality specific to varicocele in the sper-
miogram (RODRIGUEZ-RIGAU et al. 1978;
PANIDIS et al. 1984). A recently published,
very precise prospective study (AYODEJI and
BAKER 1985) found no spermatozoal mor-
phology specific to varicocele.
However, as insufficient is known at pres-
ent about the morphologically identifiable
and sometimes measurable malformations of
spermatozoa, both with regard to their
pathogenesis and their fertilizing ability, we
must welcome the scientific effort being made
to elucidate this problem further, such as the
studies by the Diisseldorf Morphology Study
Team under Hofmann (HOFMANN 1979; HOF-
MANN et al. 1982; HOFMANN and FREUNDL
1986). We also welcome light and electron
microscopic and histochemical studies ofma-
ture and immature germinal cells in the ejacu-
late and in the testicular tissue itself (DE
KRETSER 1969; RIEDEL 1980; HOLSTEIN and
ROOSEN-RUNGE 1981; SIGG and HEDINGER
1982; DE KRETSER et al. 1982; MORTIMER
et al. 1982; BUSTOS-OBREGON and LEIVA
1983; HOLSTEIN 1983; HILSCHER B. 1983;
HILSCHER W. 1983; SCHUTTE 1985; ZAINI
et al. 1985).
As our understanding of uncertain rela-
tionships generally begins with descriptive
observation, a large amount of space is allo-
cated in the Atlas section which follows to
illustrations and descriptions of spermato-
zoal morphology and other cells in the ejacu-
late (see pp. 44-119).
2.7.3.2.2 Technique for the Morphological
Differentiation of Ejaculate Smears
What are the cellular structures we should
expect to see? In a stained smear, we find
predominantly normally and abnormally
formed spermatozoa and a few free immature
germinal cells. If there is disturbed matura-
tion or transport, there is a greater number
of malformed, incompletely mature sperma-
tozoa, primarily spermatids and spermato-
cytes, but rarely spermatogonia as well. In
addition, in cases of inflammatory or other
pathological conditions of the testes and ac-
cessory sex organs, leukocytes, lymphocytes,
phagocytes, epithelial cells, erythrocytes, and
bacteria may be found.
Equipment
~ Microscope
~ Stained smear (see Sect. 2.7.3.1)
~ Immersion oil
Procedure
Examine the stained smear under the micro-
scope at a magnification of 1000 x (objective
lens 100 x, immersion oil, eyepiece 10 x).
Count off 100 spermatozoa and classify and
list them according to abnormalities of the
head, middle piece, and tail. A spermatozoon
may exhibit more than one deformity or ab-
normality. Express the total number of
pathological forms as a percentage.
39
 At the same time, classify into immature
germinal cells (spermatids, spermatocytes,
spermatogonia), leukocytes, epithelial cells,
and phagocytes, all round cells present in the
same field as the 100 spermatozoa counted.
These are recorded as the number per 100
spermatozoa.
If one wishes to express the concentration
of the various round cells in millions/ml of
ejaculate, the following formula can be used:
C=nxS
100
where
C = concentration
n = number of cells of that type found in
the same field as the 100 spermatozoa
S = spermatozoal density
Examples:
A. Immature germinal cells (spermatids,
spermatocytes, etc.):
n = 25, S = 52 million/ml
then
C = 25 x 52 x 10 6 13 million immature
100
germinal cells/ml ejaculate
B. Leukocytes:
n=12, S=18 million/ml
then
18 x 10 6 2 16 '11'
C = 12 x 100
. m! 10n
leukocytes/ml ejaculate
To satisfy the requirements of everyday
practice, it is sufficient to differentiate into
normal and pathological forms of spermato-
zoa, immature germinal cells, and leukocytes,
and to enter the values found on the record
form.
However, as the significance of the various
malformations has not yet been fully eluci-
dated with regard to their effect on fertilizing
ability, it is generally advisable to make the
following classification:
1. Spermatozoa
- Changes to the head
- Changes to the middle piece
- Changes to the tail (flagellum)
40
One frequently finds several of these
changes in one spermatozoon, and these
must then be listed in the appropriate col-
umn. It follows from this that this classifica-
tion is worthwhile for teaching purposes, but
is not necessarily logical in pathomorpholog-
ical terms.
2. Round cells
- Mature cells in the process of spermato-
genesis
- Degenerative forms and phagocytes
- Granulocytes
- Lymphocytes
- Epithelial cells
Certain malformations call for more de-
tailed functional andrological diagnostic
measures, especially if homologous insemina-
tion is planned or if in vitro fertilization is
being considered (SCHILL 1985a). For this
reason it is quite reasonable to differentiate
cell types in the ejaculate smear on purely
morphological grounds following the morpho-
logical criteria listed in the Atlas section, pro-
vided this is possible using light microscopy.
In consequence, if the "round-headed
spermatozoa" first described by SCHIRREN
et al. (1971) are found - a condition de-
scribed as globozoospermia if they occur ex-
clusively -, more detailed functional andro-
logical tests must be carried out. These
round-headed spermatozoa lack an acrosome
and the postacrosomal sheath. The diagnosis
is confirmed by determining acrosin activity
(SCHILL 1985a).
Although the lack of physical impairment
of round-headed spermatozoa was proved by
the vitality test (see Sect. 2.7.4) and the hy-
poosmotic swelling test (see Sect. 3.3.1), they
could not penetrate zona pellucida-free ham-
ster oocytes in the hamster oocyte penetra-
tion (HOP) test (see Sect. 3.4) and were there-
fore infertile (JEYENDRAN et al. 1985).
However, as no acrosin was detectable in
some of the morphologically intact sperma-
tozoa when there were only 20% round-
headed spermatozoa present (FU>RKE-GER-
LOFF et al. 1984), an acrosin test and a test
for acrosomal reaction by means of TALBOT
and CHACON'S triple stain technique (TALBOT
and CHACON 1981) should be carried out.
 Deformities of the middle piece or cyto-
plasmic appendages may also be an indica-
tion for more detailed functional andrologi-
cal tests. The functional integrity of the mito-
chondria of the middle piece can be demon-
strated by determining spermatozoa-specific
lactate dehydrogenase (LDH-X) in the semi-
nal plasma (ELIASSON et al. 1980). This all
points to the importance of evaluating mor-
phological changes in stained ejaculate
smears, although assessment requires prac-
tice and experience (SINGER et al. 1981).
The following Atlas section therefore pre-
sents a large number of examples of the dif-
ferent morphological variations of the cellu-
lar elements in the ejaculate.
41
Atlas of the Cellular Elements in the Ejaculate
The obvious way to subdivide this Atlas sec-
tion for the sake of clarity would be to work,
so to speak, from top to bottom, i.e., to di-
vide this section into the abnormalities of the
head, middle piece, and tail of spermatozoa.
Although a subdivision of this sort might
be logical in a listing of specific abnormal
forms, and is indeed followed in the record
form for reasons of clarity and simplicity, the
Atlas has been structured differently.
As you can see from the extensive illustra-
tions, there are very many more "mixed"
morphological abnormalities than there are
isolated departures from the norm affecting
just one of the parts of a spermatozoon. In
consequence, all the pathological changes
present in each instance are described, al-
though they are divided into two major
groups: (1) abnormalities of the head and (2)
abnormalities of the tail (flagellum abnor-
malities). The abnormalities of the neck and
of the middle piece, which only rarely occur
alone, are described under the abnormalities
of the head or tail.
In order to simplify differentiation, the ob-
viously pathological forms follow the normal
forms which are placed first.
These are then followed by less obvious
abnormalities, which are not apparent until
examined more closely and compared with
other forms.
The illustrations of the large-headed and
small-headed forms are followed by atypical
head shapes, and, finally, some obvious and
some less obvious changes in the form of nu-
clear abnormalities and acrosomal malfor-
mations.
Abnormalities of the tailor flagellum come
next, together with descriptions of the asso-
ciated secondary abnormalities.
Subsequent sections illustrate the various
cells of the germinal epithelium, leukocytes,
macrophages, phagocytes, and various cells
of the urinary tract, which are readily distin-
guishable structures when stained, but in un-
prepared specimens are described only in
general terms as "round cells. "
The peroxidase reaction is a reliable method
of distinguishing between leukocytes and ger-
minal cells, which is important.
Testsimplets and Hemafix are quick, prac-
ticable methods of staining, using in part
ready-prepared components, which are quite
satisfactory for everyday use.
Nevertheless, for instructional reasons, Pa-
panicolaou's stain and May-Griinwald-
Giemsa staining were used predominantly in-
stead to provide more distinct illustrations
of structures.
Scanning electron micrographs at the end
of the Atlas section will give the reader a
three-dimensional picture of the individual
cellular elements.
43
A The Spermatozoa

A 1 The HormonaUy Mature Spermatozoon

Figure 43

Fig. 43. Normal. mature spermatozoon with an oval head, neck, middJe piece and extended nagellum. Papani-
colaou, x 2400

A2 Immature Spermatozoa = Late Spermatid Stages

Figures 44 a-e

Fig.44a
~ Immature spermatozoon with

t
an oval, vacuolated head and
distinct cytoplasm in the region of
the neck and middle piece
-+ Normally formed spermatozoon
with a spherical head. Papanicolaou,
x 2400

44
A The Spennatozoa

Fig. 44b. Immature spermatozoon with an oval head and atypically shaped cytoplasm in the region of
the neck and middle piece. Papanicolaou, x 2400

Fig.44c. Immature form of

spermatozoon with patchy
condensed chromatin in the region
of the head and markedly
vacuolated cytoplasm in the region
of the neck and middle piece.
Papanicolaou, x 2400

45
A The Spermatozoa

Fig. 44d. Immature spermatozoon

with an o(1al head and slightly thick-
ened middle piece (~ cytoplasmic
remnant). Papanicolaou x 2400

Fig. 44e. Almost completely mature

spermatozoon with a normal
oval head and a small cytoplasmic
remnant at the neck, i.e.,
a .. thjckened " neck ( ~ ).
Papanicolaou, x 2400

46
A The Spermatozoa

A3 Immature, Pathological Forms of Spermatozoa = Late Spermatid Stages

Figures 45 a- g

Fig. 45a. Spermatozoon with acorn-

,
shaped head and club-shaped tail,
i.e., flagellum coiled up in the
cytoplasm. Papanicolaou, x 2400

Fig.45b
~ Immature spermatozoon with
pathological shape of head (acorn-
shaped) and large cytoplasm in the
region of the neck and middle piece
•
--. Spermatozoon with normal,
oval-shaped head. Papanicolaou,
x 2400

47
A The Spermatozoa

Fig. 45c. Immature spermatozoon

with asymmetrical, megalocephalic
head shape, and distinct cytoplasm
in the region of the neck and middle
piece. Papanicolaou, x 2400

Fig. 45d. Two immature

spermatozoa with pathological shapes
of head (pear-shaped) and
a common cytoplasm. Papanicolaou,
x 2400

48
A The Spermatozoa

Fig. 45e. Immature spermatozoa

with a common cytoplasm and only
one free flagellum. Papanicolaou,
x 2400

Fig.45f
~ Several immature spermatozoa
(late spermatids) with a common
cytoplasm in which the flagella are
coiled up
- . Spermatozoon with
a pathological round head
--t> Mature spermatozoon with
a normal oval head. Papanicolaou,
x 2400

49
A The Spermatozoa

Fig.4S g
-i> Spermatozoon with a spherical (round) head. Papanicolaou, x 2400
I> Spermatozoon with an atypically haped head and thickening in the region of the middle piece
~ Immature permatozoon with cytoplasm around the head

.... Normally formed spermatozoa. Papanicolaou, x 2400

A4 Pathological Forms of Spermatozoa with Abnormalities of the Head

and Head/Neck Junction ( = Bent FlageDorn) Figures 46a--e

Fig.46a
~ Immature permatozoon with
cross-wise oval head and thickened
middle piece
.... Mature spermatozoon
with an oval, vacuolated head .
Papanicolaou, x 2400

50
A The Spermatozoa

I>

Fig.46b
~ Spermatozoon with a normal oval head, bent head/neck junction, and a cytoplasmic remnant at the
neck
I> Degenerate, early spermatid
-+ Amorphou megalocephalic permatozoon. Papanicolaou, x 2400

Fig.46c
~ Spermatozoon with distinct
thickening in the region of the
middle piece and bent head/neck
junction
-+ Normally formed, mature
spermatozoon. Papanicolaou, x 2400

51
A The Spermatozoa

Fig. 46d. Immature spermatozoon with a mushroom-shaped head and a cytopl,smic remnant at the neck.
Papanicolaou, x 2400

Fig.46e
~ Immature permatozoon with a pear-shaped head , bent neck and cytoplasmic remnant at the neck
and on tbe nagellum
-+ Spermatozoa with normally formed beaded and recognizable cytopla mic remnant at the neck. Papani-
colaou, x 2400

52
A The Spermatozoa

AS Megalocephalic Spermatozoa with Amorphous Heads

(Megalocephalic Abnormality 1)
These are pathological developments in the process of spermatid differentiation.
Figures 47a- f

,
Fig. 47 a. Typical amorphous,
megalocephalic form of
spermatozoon. Papanicolaou, x 2400

Fig. 47b. Amorphous,

megalocephalic spermatozoon with
bent head/neck junction and
a cytoplasmic remnant at the neck.
Papanicolaou, x 2400

53
A The Spermatozoa

..

Fig. 47 c. Immature, megalocephalic

amorphous form of spermatozoon
with distinct cytoplasmic remnant at
the neck. Papanicolaou, x 2400

..
• .Fig. 47 d. Fusion of several
megalocephalic spermatozoa into
a single amorphous "giant form. "
Papanicolaou, x 2400

54
A The Spermatozoa

Fig. 47e. Spermatozoa with

megalocephalic, amorphous head
shape and fusion in the head region.
Papanicolaou, x 2400

Fig.47f
~ Extremely amorphous form of
spermatozoon with an asymmetrical,
vacuolated acrosome and distinct
nuclear abnormality
--. Microcephalic spermatozoon
with a pointed head. Papanicolaou,
x 2400

55
A The Spermatozoa

A6 Megalocephalic, Nonamorphous Forms of Spermatozoa

(Megalocephalic Abnormality 2)
Figures 48 a- f

Fig. 488
~ Spermatozoon with a mega-
locephalic, spherical Oarge round)
head, and pent head/neck junction
- . Spermatozoon with trapezoid,
hypochromatic head. Papanicolaou,
x 2400

Fig.48b. Typical megalocephalic,

spherical form of spermatozoon.
Papanicolaou, x 2400

56
A The Spermatozoa

Fig.48c
.... Spennatozoon with an acorn-
shaped head
~ Extremely megalocephalic
spennatozoon <l

I> Microcephalic, spherical

spennatozoon. Papanicolaou, x 2400

Fig. 48d. Immature, megalocephalic,

spherical spennatozoon with
a cytoplasmic remnant at the neck
and an asymmetrical head/neck
junction. Papanicolaou, x 2400

57
A The Spermatozoa

Fig.48e
Slightly megalocephalic, spherical
spermatozoon with twin flagella.
Papanicolaou, x 2400

Fig.48f

, ~ Megalocephalic spermatozoon
with bulges on the acrosome
--. Megalocephalic, spherical

I i
spermatozoon
[> Megalocephalic spermatozoon
with asymmetrical acrosome
-t> Normally formed, mature
spermatozoa. Papanicolaou, x 2400

58
A The Spermatozoa

A7 Pseudomegalocephalic Forms of Spermatozoa

(Megalocephalic Abnormality 3 = Pseudomegalocephaly)
These are malformations in the form of twins which give the impression of megaloce-
phaly due to fusion at the head. Figures 49 a-e

Fig.49a
~ Megalocephalic twins fused at
the head
- . Normally formed spermato-
zoon. Papanicolaou, x 2400

59
A The Spermatozoa

Fig. 49b. Fusion of two mega-

locephalic, spherical spermatozoa.
Papanicolaou, x 2400

Fig. 49c. Twinning due to fusion

of two megalocephalic, oval
spermatozoa at the head and neck.
Papanicolaou, x 2400

60
A The Spermatozoa

Fig.49d t
~ Fusion of two
immature spermatozoa with
oval heads
f
---. Normally formed,
mature spermatozoa.
Papanicolaou, x 2400

Fig. 4ge. Pseudomegalocephalic

spermatozoon due to fusion
(adhesion?) of two normocephalic,
spherical spermatozoa.
Papanicolaou, x 2400

61
A The Spermatozoa

AS Microcephalic Spermatozoa (Microcephalic Abnormality)

Figures 50a--e

Fig.50a. Microcephalic
spermatozoon with a pointed head.
Papanicolaou, x 2400

v Fig.50b
~ Microcephalic spermatozoon
with pointed head and cytoplasmic
remnants in the region of the middle
piece
- . Megalocephalic spermatozoon
with bulges on the acrosome
[> Normally formed spermatozoa.
Papanicolaou, x 2400

62
A The Spermatozoa

Fig. SOc
~ Microcephalic spermatozoon
with an acorn-shaped head
I> Spermatozoon with a spherical
head and thickened neck.
Papanicolaou, x 2400
<l
--{> Megalocephalic, immature
spermatozoon with an acorn-shaped
head and a large cytoplasm with
flagellum coiled up in it

Fig. SOd. Microcephalic spherical spermatozoa in the form of twin (-+) and mUltiple malformation (I»
due to fusions at the head. Papanicolaou, x 2400

63
A The Spermatozoa

Fig. SOe
~ Microcephalic spermatozoon witb an acorn-shaped abnormality of the head
.... Normally formed , mature permatozoa with oval head . Papanicolaou, x 2400

64
A The Spermatozoa

A9 Atypical Head Shapes

Figures 51 a- k

Fig.5la
~ Spennatozoon with tapered
postacrosomal section (pear-shaped)
and bent flagellum
--. Two nonnally fonned
spennatozoa with oval heads
I> Degenerate spermatozoon.
Papanicolaou, x 2400

Fig. SIb
~ Spennatozoon with an acorn-
shaped head
,
--. Spennatozoon with a nonnal,
oval head and supranuclear
vacuoles. Papanicolaou, x 2400

65
A The Spermatozoa

Fig. SIc. Spermatozoon with

bulging abnormalities
of the acrosome (acorn shape).
Papanicolaou, x 2400

Fig. SId. Spermatozoon with bulges

on the head in the postacrosomal
section and clearly recognizable
vacuoles. Papanicolaou, x 2400

66
A The Spermatozoa

Fig. 51 e. Characteristic acorn shape

with only one postacrosomal bulge.
Papanicolaou, x 2400

Fig. 51 r. Spermatozoon with pear-

shaped head and a large supranuclear
vacuole. Papanicolaou, x 2400

67
A The Spermatozoa

Fig. 51 g. Spermatozoon with

a typically pear-shaped head.
Papanicolaou, x 2400

Fig. 51 h. Spermatozoon with

a drop-shaped head. Papanicolaou,
x 2400

68
A The Spermatozoa

Fig. S1i. Spermatozoon with a drop-

shaped head and flagellum attached
asymmetrically (golf-club shape).
Papanicolaou, x 2400

Fig. Slk
I> Spermatozoon with bulges on
the acrosome and thickening at the
neck
~ Spermatozoon with an
oval head and flagellum attached
asymmetrically
--. Spermatozoon with a drop-
shaped head. Papanicolaou, x 2400

69
A The Spermatozoa

A 10 Abnormalities of the Nucleus and Acrosomal Malformations

These malformations arise from disorders of spermatid differentiation, affecting either

the dark-colored portion of the nucleus close to the neck of the spermatozoon, or
the lighter-colored acrosome at the tip, which is tapered in lateral view.
Spermatozoa of this sort are definitely incapable of penetration. Figures 52 a- d

Fig. 52a. Microcephalic

spermatozoon with a rudimentary
nucleus and an atypical shape
of acrosome. Papanicolaou, x 2400

70
A The Spermatozoa

Fig. 52 b. Spermatozoon with an

asymmetrical acrosome and
a thickened neck . Papanicolaou,
x 2400

Fig. 52c. Spermatozoon with an

atypical, oval head, abnormalities in
the nucleus and acrosome,
and a flagellum coiled at the end.
Papanicolaou, x 2400

71
A The Spermatozoa

Fig. 52d. Spermatozoon with a triangular bead and rudimentary columnar nucltus. Papanicolaou, x 2400

72
A The Spermatozoa

A 11 Spermatozoa with Deformities of the Tail (Flagellum)

Figures 53 a- f

Fig. 53a. Spermatozoon with a

pointed, vacuolated head and two
flagella with nodular lumps.
Papanicolaou, x 2400

..

..

Fig. 53b. Spermatozoon with a

normally formed head, cytoplasmic
remnants in the region of the middle
piece, and twin flagella.
Papanicolaou, x 2400

73
A The Spermatozoa

Fig. 53c. Spermatozoon with a

drop-shaped head and several
. flagella, some of which are coiled at
the middle. Papanicolaou, x 2400

Fig.53d
~ Spermatozoon with an oval
head and twin flagella
---. Spermatozoon with a normally
formed head and triple flagella
(" thick flagella")
I> Megalocephalic, spherical
spermatozoon
-I> Spermatozoon with trapezoid
malformation of the head.
Papanicolaou, x 2400

74
A The Spermatozoa

Fig.53e
~ Spermatozoon with nuclear
chromatin of varying density (spot-
like condensed chromatin or
" nuclear patches") and several
fused rudimentary flagella
- . Spermatozoon with a
hypochromatic, trapezoid head and
with two flagella which are partially
fused . Papanicolaou, x 2400

Fig.53f
[> Normally formed spermatozoa
~ Spermatozoon with a drop-
shaped head and a flagellum coiled
at the end
- . Spermatozoon with bulging
malformations of the head, bent
head/neck junction, and a flagellum
coiled in the middle
--c> Spermatozoon with just a
suggestion of a bulge on the
acrosome, thickening of the middle
piece, and a bent tail. Papanicolaou,
x 2400

75
B Cells from the Germinal Epithelium of the Seminiferous Tubules

B 1 Spermatogonia
Figures 54a, b

Fig. 54a. Spermatogonium of the

Ad type (type A dark) with
a characteristic nuclear vacuole.
Papanicolaou, x 2400

Fig. 54b. Spermatogonium of the

Ap type (type A pale). Papanicolaou,
x 2400

76
B Cells from the Germinal Epithelium of the Seminiferous Tubules

B2 Spermatocytes
Figures 55a- 1

- t

Fig. 558
~ Early prophase stage of a pennatocyte (primary spennatocyte)
- . Normal spermatozoon
[> Suggestion of, and -i> fully developed spherical head (probable acro omal defect). May-Grunwald-
Giem a, x 2400

t
-
Fig.55b
~ Prophase stage of a spermatocyte
(primary spermatocyte)
- . Normal spennatozoa. May-
Grunwald-Giemsa, x 2400

77
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. 55e. Pachytene spermatocyte

with a round nucleus.
May-Griinwald-Giemsa, x 2400

Fig. 55d. Pachytene spermatocyte

(--.) with an atypical, crescent-
shaped nucleus. May-Griinwald-
Giemsa, x 2400

78
B Cells from the Germinal Epithelium of the Seminiferous Tubules

-
Fig. SSe
~ Degenerating pachytene
spermatocyte <l
- . Polymorphonuclear leukocyte
I> Trinucleate spermatid. May-
Griinwald-Giemsa, x 2400

-
Fig. SSf
~ Secondary spermatocytes. May-
Griinwald-Giemsa, x 2400

79
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. SSg. Degenerating spennatocyte.

May-Griinwald-Giemsa,
x 2400

Fig. SSh. Degenerating spermatocyte

with incipient nuclear pyknosis
and vacuolation of the cytoplasm.
May-Griinwald-Giemsa, x 2400

80
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig.55i
~ Secondary spermatocyte in the
process of division (telophase J)
-
- . Normal spermatozoon
[> Normal spermatozoon and I>
nuclear vacuoles
-!> Spermatozoon with "parachute
head." May-Griinwald-Giemsa,
x 2400

Fig.55k
~ Advanced telophase of a
secondary spermatocyte (upper left)
- . Secondary spermatocyte,
incomplete division (lower right).
May-Griinwald-Giemsa, x 2400

81
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. 551. Almost completely

concluded division of a secondary
spermatocyte. May-Grunwald-
Giemsa, x 2400

82
B Cells from the Germinal Epithelium of the Seminiferous Tubules

B 3 Spermatids
Figures 56a~

-
Fig.56a
!> Megalocephalic spermatozoon
with a thickened neck

t
--. Spermatozoon with a spherical
head
~ Early spermatid stage
-t> Normal spermatozoon.
Papanicolaou, x 2400

Fig. 56b. Degenerating, early

spermatid with pyknotic nucleus.
May-Griinwald-Giemsa, x 2400

83
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig.56c
~ Early spermatid stage
- . Spermatozoon with an elongated
oval head
<l l> Spermatozoon with a rounded
head. Papanicolaou, x 2400

84
B Cells from the Germinal Epithelium of the Seminiferous Tubules

B4 Pathologically Altered Forms of Germinal Epithelial Cells

Figures 57 a- f

Fig. 57 a. Pathological giant cell

fonn (~) of a spermatid.
May-Griinwald-Giemsa, x 2400

Fig. 57b. Binucleate giant cell fonn

of a spermatid with numerous
vacuoles. May-Griinwald-Giemsa,
x 2400

85
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. 57c. Trinucleate giant eel\.

spermatid. May-Griinwald-Giemsa,
x 2400

Fig. 57 d. Trinucleate spermatid stage

with typical polar positioning of
the nuclei (giant cell). May-Griinwald-
Giemsa, x 2400

86
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. 57 e. Tetranucleate spermatid

stage with polar positioning of the
nuclei (giant cell). May-Griinwald-
Giemsa, x 2400

Fig. 57f. Multinucleate giant cell

(spermatid stage). May-Griinwald-
Giemsa, x 2400

87
B Cells from the Germinal Epithelium of the Seminiferous Tubules

B5 Degenerating Giant Cell Forms with Advancing Expulsion of the Nucleus

Figures 58 a-g

-
Fig.58a
~ Mononucleate giant cell with a
pyknotic nucleus positioned at the
pole and with a severely vacuolated
cytoplasm
---. Polymorphonculear leukocyte
(granulocyte) May-Griinwald-
Giemsa, x 2400

Fig. 58b. Trinucleate spermatid with

extreme nuclear pyknosis (very small
nuclei) and granular cytoplasm'.
May-Griinwald-Giemsa, x 2400

88
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. SSe. Binucleate spermatid with

pyknotic nuclei and nuclear
vacuoles; expulsion of the nucleus is
incipient. May-Griinwald-Giemsa,
x 2400

Fig. SSd. Advanced expulsion of the

nucleus. May-Griinwald-Giemsa,
x 2400

89
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. S8e
~ Expulsion of the nucleus almost
complete. The nucleus is only
connected to the parent cell by a thin
extension of the cytoplasm
---. Degenerate giant cell of
uncertain origin with nucleolus and
numerous vacuoles (residual
bodies ?). May-Griinwald-Giemsa,
x 2400

, ,

Fig. S8f
~ Cytoplasmic remnant from
spermatids after expulsion of the
nucleus
---. Cell remnants after cytolysis.
May-Griinwald-Giemsa, x 2400

90
B Cells from the Germinal Epithelium of the Seminiferous Tubules

Fig. SSg
~ Advanced degeneration of a
-
giant form of spermatid with
severely pyknotic nuclei of different
sizes
- . Normal spermatozoon with a
large vacuole in the head. May-
Griinwald-Giemsa, x 2400

91
C Leukocytes and Macrophages

Figures 59 a-I

Fig. 59a-g. Various types of

polymorphonuclear leukocytes. May-
Griinwald-Giemsa, x 2400

Fig.59a

Fig.59b

92
C Leukocytes and Macrophages

Fig.59c

Fig.59d

93
C Leukocytes and Macrophages

Fig.5ge

Fig.59f

94
C Leukocytes and Macrophages

Fig. 59g

Fig. 59h-k. Degenerate

polymorphonuclear leukocytes. May-
Griinwald-Giemsa, x 2400

Fig. 59h

95
C Leukocytes and Macrophages

Fig.59i

Fig.59k

96
C Leukocytes and Macrophages

Fig. 59 I. Macrophage (monocyte).

May-Griinwald-Giemsa, x 2400

97
D Phagocytosis of Spermatozoa by Macrophages
(Spermatophagia)

For the sake of consistency with the change in terminology from" sperm" to "spermatozoa, "
we also recommend changing the term " spermiophagia, " which is still in common use,
to "spermatophagia." The term spermatophagia describes the phagocytosis of spermatozoa.
Cells which can phagocytose spermatozoa (enclose them in their cell membrane, "ingest"
them) are called spermatophages. They are usually large cells (macro phages) such as mono-
cytes. Figures 60a- f

Fig.60a. Macrophage with the head

of a spermatozoon ingested.
Papanicolaou, x 2400

98
D Phagocytosis of Spermatozoa by Macrophages (Spermatophagia)

Fig.60b
- . Spermatozoon with a triangular
-
head
~

[>
Macrophage with spermatozoa
Immature spermatozoon with t
cytoplasmic remnants in the region
of the middle piece
~ Spermatozoon with an oval
head and slight cytoplasmic remnants
at the neck. May-Griinwald-Giemsa,
x 2400

Fig. 60c
~ Macrophage with phagocytosed
center section of the flagellum
- . Spherical, megalocephalic
spermatozoon. May-Griinwald-
Giemsa, x 2400

99
D Phagocytosis of Spermatozoa by Macrophages (Spermatophagia)

Fig.60d
~ Spermatophagia of a largely
denatured megalocephalic
spermatozoon
---. Spermatozoon with an elongated
oval head. May-Griinwald-Giemsa,
x 2400

Fig.60e
---. Expulsion of a trumpet-shaped,
deformed nucleus from a spermatid
~ Phagocytosis of a spermatozoon
with bulging mal-formations
of the acrosome
<1
I> Cytoplasmic remnants
after expulsion of the nucleus.
Papanicolaou, x 2400

100
D Phagocytosis of Spermatozoa by Macrophages (Spermatophagia)

Fig. 60f. Phagocytosis of several

spermatozoa by a macrophage.
Papanicolaou, x 2400

101
E Peroxidase Reaction of Peroxidase-Positive
Polymorphocellular Leukocytes

In contrast to germinal cells, leukocytes exhibit a positive peroxidase reaction, i.e., they
stain a deep brown. This enables leukocytes to be distinguished from other "round cells, "
which cannot be positively differentiated in unprepared specimens or even sometimes by
the usual staining methods. Figures 61 a--e

<l Fig. 613. Positive peroxidase

reaction (brown color) of
polymorphonuclear leukocytes:
normal number in a nonpathological
ejaculate. Secondary finding :
numerous crystals. Papanicolaou,
x680

Fig. 61 b, c. Deep brown color [>

indicating a positive reaction
of polymorphonuclear leukocytes.
Germinal cells exhibit a negative
peroxidase reaction and only display
the background stain with cyanosin.
x 1700

102
 E Peroxidase Reaction of Peroxidase-Positive Polymorphocellular Leukocytes

c ~________~. .__~______________________~_

103
E Peroxidase Reaction of Peroxidase-Positive Polymorphocellular Leukocytes

Fig.6Id. Higher number of peroxida e-positive leukocyte

cells remain un tained . x 1700

Fig. 61 e. Aggregation of peroxidase-

positive leukocytes in a case of
pyospermia. x 1700

104
F Various Types of Urinary Tract Cells

Cover cells from the transitional epithelium of the kidney, ureter, bladder, and upper urinary
tract, plus squamous epithelial cells from the lower urinary tract are occasionally found
in the ejaculate smear. Figures 62 a-g

Fig. 62a. Degenerate cover cell from

the transitional epithelium
of the urinary tract. May-Grunwald-
Giemsa, x 2400

105
F Various Types of Urinary Tract Cells

Fig. 62b. Multinucleate cover cell

from the transitional epithelium.
May-Griinwald-Giemsa, x 2400

Fig. 62c. Trinucleate cover cell from

the transitional epithelium.
May-Griinwald-Giemsa, x 2400

106
F Various Types of Urinary Tract Cells

Fig. 62d. Cell cluster made up

partly of multinucleate cover cells
from the transitional epithelium.
May-Griinwald-Giemsa, x 2400

Fig. 62e. Cluster of cover cells from

the transitional epithelium.
May-Griinwald-Giemsa, x 2400

107
F Various Types of Urinary Tract Cells

Fig.62f. Isolated, binucleate cover cell

of the transitional epithelium.
May-Griinwald-Giemsa, x 2400

Fig. 62g. Squamous epithelial cell

with massive bacterial colonization.
May-Griinwald-Giemsa, x 2400

108
G Smear Staining Using Testsimplets

Although the results are not as striking or as permanent as with specimens prepared using
Papanicolaou's stain or May-Griinwald-Giemsa stain, prestained slides coated with dyes,
such as Testsimplets from Boehringer, Mannheim, FRG, are quick and quite satisfactory
for everyday use, and are suitable for the morphological differentiation of the various cellular
structures in the ejaculate. Figure 63 a, b

,
.....

Fig.63a
~ Normally formed spermatozoa with oval heads

-+ Spermatozoa with drop-shaped heads

[> Spermatozoon with a spherical head
-(> Agglutination of spermatozoa. x 2400

109
G Smear Staining Using Testsimplets

Fig.63b
~ Early spermatid stage with
vacuolated cytoplasm
~ Elongated spermatozoa head
(described as the" tapering form" by
MACLEOD 1965 b) with the tail
coiled up in the large cytoplasmic
remnant of the middle piece, x 2400

110
H Smear Staining Using Hemafix

The Hemafix rapid staining system from Biomed, Munich, FRG, is also a quick and practic-
able method of assessing the morphology of components of the ejaculate (Figure 64). It
provides approximately the same amount of information as Testsimplets (Sect. G).

Fig. 64. Immature spermatozoon with

a cytoplasmic remnant in the region
of the neck and middle piece.
Hemafix, x 2400

111
 I Scanning Electron Micrographs of Various Forms
of Spermatozoa

Only scanning electron microscopy provides a truly three-dimensional image of spermatozoal

morphology with its striking clarity in shades of grey. These images give us a genuine
insight into the dimensional relationships of the individual cellular structures. The clarity
of the pictures speaks for itself. Figures 65- 76

Fig. 65. Overview showing various forms of sperma- Fig. 66. Detailed view of a nonpathological ejaculate I>
tozoa and round cells
\l Fig. 67. a Spermatozoon seen from the side with cy- I>
toplasm (=-) at the neck. b Spermatozoon with
round head, thickened neck (=-) and bent flagellum
(-{». c Spermatozoon with pointed head and slight
cytoplasmic remnant at the neck

65

112
113
 I Scanning Electron Micrographs of Various Forms of Spermatozoa

Fig. 68. a Spermatozoon with amorphous head and Fig. 69. Spermatozoon with a pointed head I>
flagellum attached asymmetrically (pipe shape).
b Normally formed spermatozoa seen from different Fig. 70. Spermatozoon with a round head I>
views. c Spermatozoon with an oval head and a cyto-
plasmic remnant at the neck
'V

68

114
115
 I Scanning Electron Micrographs of Various Forms of Spermatozoa

Fig. 71. Spermatozoon with an extremely abnormal- Fig. 72. Spermatozoon with a mushroom-shaped [>
ly formed head and flagellum attached asymmetri- head. The tip is spherical and there is an equally
cally distinct spherical cytoplasmic remnant at the neck.
'V Head and neck together thus make a dumbbell shape

Fig. 73. a Spermatozoon with an oval head, thick- [>

ened neck and tail coiled at the end. b Drops of
cytoplasm on the head and at places along the flagel-
lum (-.)

7J

116
117
 I Scanning Electron Micrographs of Various Forms of Spermatozoa

Fig. 74. Cytoplasmic remnants Fig. 75. Spermatozoon with twin flagella [>
\l
Fig. 76. Various superficial structures on round cells [>

74

118
119
2.7.4 Vitality

As immotile spermatozoa may easily be alive,

one can only distinguish between dead sper-
matozoa and immotile but live spermatozoa
by using the eosin test (ELIASSON and
TREICHL 1971).
The determination of vitality is therefore
an important part of the spermiogram, and
is one of the additional diagnostic tests which
may be performed (SCHILL 1980; PINATEL Fig. 77. Equipment for the eo in Ie t : eo in solution
1985). nigro in olution for counter taining when u ing a
SINGER et al. (1980) found an increase in light micro cope, !ide , yringe with needle, watch
glas with gla rod for mixing
nonvital spermatozoa in association with
worsening oligospermia.
Vitality is also of importance in assessing
the fatal and nonfatal malformations in tera-
tospermia. Many malformations of the head, red spermatozoa =
especially enlargement, were found among dead spermatozoa
the fatal malformations, whereas tapering
forms, cytoplasmic appendages, and abnor-
mal tail formations were of less fatal signifi-
Although this method, which is described by
cance (FREDRICSSON 1978).
ELIASSON and TREICHL (1971), benefits from
the use of a phase-contrast microscope to ex-
amine the slides, it is possible to distinguish
Equipment the red (dead) spermatozoal heads from the
unstained (live) spermatozoa using a simple
- Microscope slides light microscope by counterstaining with
- Cover slips 10% nigrosin in distilled water (DOTT and
- 0.5% or 1 % eosin solution FOSTER 1972).
- 0.5% yellowish eosin solution or 1 % blu-
ish eosin solution in 100 ml physiological
saline (see Fig. 77) or Evaluation
- 10% fuchsin solution :
fuchsin 10 ml, glacial acetic acid 10 ml, Ten fields should be evaluated under the mi-
physiological saline 80 ml croscope (BELSEY et al. 1980; HARGREA VE
and NILSSON 1983). At least 80% (KIESSLING
1960; LUDVIK 1976), or between 65% and
Method 85% (HEITE and WOKALEK 1980) should be
alive, i.e., unstained.
Place a drop of semen on a slide, add 1 drop
of the 0.5% aqueous yellowish eosin solution
and cover with a cover slip. After 1- 2 min,
2.7.5 MAR Test
the spermatozoa stained red can be distin-
(Mixed Antiglobulin Reaction Test)
guished from the unstained spermatozoa.
The difference is due to the fact that live sper-
matozoa repel eosin, whereas the dye can Spermatozoa contain antigenic components
penetrate dead spermatozoa through the de- and this factor may be of significance in the
fective membrane of the head and stain it infertility of the male partner (UPADHYAYA
red. et al. 1984; SCHILL 1985a).

121
 One must distinguish between autoantibo-
dies to his own spermatozoa in the serum
of the man and isoantibodies to the male's
spermatozoa in the genital secretions of the
woman.
Either may display agglutinating or immo-
bilizing properties, which at high titers may
impede penetration of the cervical mucus
(ALEXANDER 1984; ALEXANDER and BEAR-
WOOD 1984).
In about 10% of couples unable to fulfil
their desire for children, the man has specific
autoantibodies to his spermatozoa which
lead to agglutination or immobilization
(HENDRY et al. 1977; JONES 1982; ROGERS-
NEAME et al. 1986). Using radioimmunoassay
(RIA), METTLER and CZUPPON (1985) found
antibodies to spermatozoa in 15% of women
and in 12% of men who were unable to fulfil
their desire for children. This is a significantly
higher level than in the negative control
group.
The possibility of an immunological cause
for infertility in the man is therefore of
greater importance, and consequently a diag-
nostic test for antibodies should be per-
formed, at least if the spermiogram proves
typical and normal.
Tests for antibodies can be applied to:
- serum,
- seminal plasma, and
- cervical mucus at ovulation.
Unspecific antibody tests (Sims-Huhner
test, slide test = Kurzrock-Miller test,
Kremer-Jager test = sperm-cervical mucus
contact test, Kremer test and Penetrak test)
are described in detail under penetration tests
(Sect. 3).
The following tests are available for specif-
ic antibodies to spermatozoa:
- The macrosperm agglutination test (gelatin
agglutination test, GAT) after KIBRICK
et al. (1952)
- The sperm immobilization test after ISOJI-
MA et al. (1968)
- The microscope slide test after Friberg
(tray agglutination test, TAT) (FRIBERG
1974)
122
- Detection of antibodies by ENZYME-
LINKED IMMUNOSORBENT ASSAY
(ELISA) (ACKERMANN et al. 1981; WOLFF
and SCHILL 1985).
The evaluation of immunologically unspe-
cific penetration tests, which may acquire
great significance in relation to insemination
and in vitro fertilization, is described in detail
in Sect. 3.
Kibrick's test, Isojima's test, and Friberg's
test have the advantage of being specific and
quantitative, but their disadvantage is that
they require a normal test ejaculate and can-
not therefore be used in cases of OAT syn-
drome. In addition, they are insufficiently
sensitive and their reproducibility depends on
a large number of variables.
Modern ELISA tests have the advantage
of being simple and quick. They are therefore
of practical value as well as being sensitive
and reproducible. As such they are very suit-
able as screening tests. The disadvantage with
them is the possibility of a false-positive reac-
tion.
The mixed antiglobulin reaction test
(MAR) is an unspecific, simple, and reliable
diagnostic test for antibodies to spermatozoa
(HENDRY et al. 1982; STEDRONSKA and
HENDRY 1983).
CERASARO et al. (1985) also found a con-
firmed correlation between positive IgG-
MAR results and spontaneous spermatozoal
agglutination. The MAR test is particularly
suitable in cases of low motility to screen for
possible immunological factors in infertility
(CIMINO and BARBA 1985).
JENSEN and HJORT (1985) confirmed that
it is quite feasible to detect antibodies to IgG
and IgA on spermatozoal membranes using
the MAR test. They also found a close corre-
lation between sperm agglutinins in the se-
rum and in the seminal plasma. According
to SCARSELLI et al. (1985) an IgA-MAR test
is necessary in all cases when an IgG test
is MAR positive, in order positively to ex-
clude or confirm immunological factors. In
some cases it therefore appears important to
identify the class of immunoglobulin (LEWIS
and OVERSTREET 1986).
FRANCAVILLA et al. (1984) considered the
MAR test to be so effective and easy that
they stated that more complex and more ex-
pensive tests should not be performed in ev-
eryday practice for the detection of anti-
bodies to spermatozoa.
2.7.5.1 Principle of the MAR Test
The test is intended to couple IgO antibodies
bound to spermatozoa with the aid of biva-
Ll
A.ftIIM'UftI~OiCO!'! ....
M.... o· I,G
t·:·"lI' /C~II. ,.ui ••
Fig. 78. Materials for the mixed antiglobulin reac-
tion (MAR) test: antiserum to human IgG (y-chain ;
Behring-Werke, Marburg, FRG ; te t erythrocyte,
sen itized with anti-Rh antibodie (Coomb Control
erum, Dr. Molter, 0-6903 Neckargemiind, F RG)
Fig. 79a. MAR te t u ing interference phase-contrast microscopy. Po itive MA R test. Aggregation of eryth-
rocyte on a permatozoon. Examination under the microscope reveals free- wimming spermatozoa in the
u pension of erythrocytes
lent anti-IgO antibodies to test erythrocytes
laden with IgO antibodies. If the test is posi-
tive, aggregations of erythrocytes are formed
like a ruff, preferentially around the neck and
middle piece of the spermatozoa. However,
they may be mixed up together or occur on
other parts of the spermatozoon.
If the test is negative, there is no aggrega-
tion of test erythrocytes on the spermatozoa.
2.7.5.2 Performing the MAR Test
Equipment and R eagents Required
· 'I
- Microscope slides
- Cover slips (18 x 18 mm)
- Tuberculin syringe with a slender needle
- Antiserum to human IgO (y-chain; Behr-
ing-Werke, Marburg, FRO)
- Test erythrocytes, sensitized with anti-Rh
antibodies (Coombs Control Serum, Dr.
Molter, 0-6903 Neckargemiind, FRO).
The reagents are illustrated in Fig. 78
One drop of the fresh ejaculate to be tested
is placed on a microscope slide and mixed
with a drop of the erythrocyte suspension.
123
Fig. 79 b. Negative MAR te t using interference pha e-contra t microscopy: permatozoa and round cell
in an unstained pecimen : no aggregation

ig.80. Po itive MAR te t using interference phase-contra t micro copy: ruff-like aggregation of erythro-
cytes at the neck of a spermatozoon

124

Fig. 81. Positive MAR te t : erythrocyte aggregation
using dark-field, interference pha e-contra t micro -
copy
The test erythrocytes do not clump togeth-
er and on the spermatozoa until a drop of
the anti-IgG antibody solution is added
(Figs. 79a, 80, 81).
2.8 Fructose
The sugar found in relatively high concentra-
tions in seminal plasma was identified as
fructose by MANN (1945, 1946). Ninety per-
cent of it is formed in the seminal vesicles
and 10% in the vas deferens (BANDHAUER
and KOVESDl 1970). The production of the
fructose in seminal plasma is regulated by
testosterone. A normal level of fructose in
seminal plasma is therefore dependent on:
1. intact seminal vesicles and 2. a normal level
of testosterone.
It was logical to consider the fructose as
the energy source - in a way, the fuel for
the engine - of spermatozoal motility,
especially as motility decreased in propor-
tion to increasing fructolysis (MANN 1946;
SCHIRREN 1971; LUDWIG et al. 1974; KRAUSE
and ROTHAUGE 1981). This was, however,
disputed (KINDLER and MOLLMANN 1972;
LISCHKA 1975; THIEL et al. 1983; GUNTHER
et al. 1983) and was refuted, at least as a spe-
cific phenomenon, by fertilizations using
fructose-free semen (KELAMI 1981). The high
concentration of fructose in seminal plasma
nevertheless permits one to conclude that the
sugar could play an important role as an en-
ergy source for spermatozoal motility, even
though this role could be assumed by other
substances and is probably not essential.
In any event, the fructose in seminal plas-
ma is an indicator of the functional status
of the seminal vesicles (KRAUSE and ROTH-
AUGE 1981; HASELBERGER et al. 1983; HAR-
GREAVE et al. 1983; MAWHINNEY 1983; LUD-
WIG 1986). Although it may fall below 1.2 gi l
even when the spermiogram is completely
normal (LUDVIK 1976), such a level does rep-
resent a disturbance of the biochemistry of
the seminal plasma (SCHILL 1980).
Until its significance in seminal plasma is
conclusively established, it will remain an es-
sential part of ejaculate analysis to measure
the level of fructose.
2.8.1 Determining Fructose
in Seminal Plasma
The simplest method of determining fructose
in seminal plasma is enzymatically by the
hexokinase method from Boehringer, Mann-
heim, FRG (LUDWIG et al. 1973).
Principle of the Enzymatic Determination
of Fructose in Seminal Plasma
The enzymatic determination of fructose in
seminal plasma uses the hexokinase method
for glucose, with the sole exception that an
additional enzyme, phosphoglucoisomerase
(PGI), is used which converts the fructose
into glucose by enzymatic action (SCHMIDT
1961). In consequence the very low concen-
tration of glucose present in the ejaculate is
measured at the same time. This may be ig-
nored because it accounts for such a minimal
percentage. Glucose and fructose, with aden-
osine triphosphate (A TP), are phosphorylat-
ed by the enzyme hexokinase (HK) to glu-
cose-6-phosphate (G-6-P) and fructose-6-
phosphate (F-6-P):
125
 glucose + ATP---.G-6-P + ADP
fructose + ATP---.F-6-P + ADP
In the presence of the enzyme glucose-6-
phosphate-dehydrogenase (G6P-DH), glu-
cose-6-phosphate (G-6-P) will be oxidized by
nicotinamide-adenine-dinuc1eotide phosphate
(NADP) to gluconate-6-phosphate, while
NADP will be reduced to NADPH:
G-6-P + NADP+ ---. gluconate-6-
phosphate + NADPH + H+.
NADPH is the substance measured. The
quantity of NADPH + H+ formed by this
reaction can be determined photometrically
using an appropriate wavelength, and serves
as a measure of the glucose concentration.
The added enzyme PGI converts F-6-P to
G-6-P, and the quantity of NADPH + H+
measured is now equivalent to the concentra-
tion of fructose:
PGI
F-6-P -----+1 G-6-P.
Equipment Required
- Centrifuge
- Centrifuge tubes
- Refrigerator
- Photometer
- Cuvettes (glass or plastic)
- Micropipettes (e.g., Eppendorf), 20-100 III
and 200-1000 III
- Glass or plastic spatula.
Reagents Required
- Potassium perchlorate 10%
- Potassium bicarbonate (crystalline form)
- Solutions I-IV of the Glucose Test Kit,
Boehringer, Mannheim, FRG,
- Phosphoglucoisomerase, Boehringer,
Mannheim, FRG, no. 15433.
2.8.2 Procedure
Prepare the test solution by pipetting 0.2 ml
of completely liquefied ejaculate into 1.0 ml
10% potassium perchlorate solution in a cen-
trifuge tube. Place the tube in a refrigerator
for 60 min at a temperature of + 4° C. After
126
centrifuging for 5 min at high speed, decant
the supernatant liquid and add a knife-tip
(about 100 mg) of potassium bicarbonate.
Decant the supernatant. This is the test solu-
tion containing the protein-free, neutralized
seminal plasma. Take a glass or plastic cu-
vette with a depth of 1 cm, and pipette into
it 1.0 ml solution I (triethanolamine buffer
pH 7.6, NADP 64 mg, ATP 160 mg, magne-
sium sulfate), 0.1 ml seminal plasma, and
1.9 ml double-distilled water from the Glu-
cose/Fructose VV Test Kit (Boehringer,
Mannheim, FRG). Mix the solution thor-
oughly with a plastic spatula, and after 3 min
measure the extinction (E 1 ) through a filter
with a wavelength of Hg 334 nm. Prepare an
equivalent reference solution (Rs) without
seminal plasma and measure the extinction
(E1). When measurement is complete, add
0.02 ml solution II (enzyme suspension con-
taining 100 IV G6P-DH, and 200 IV hexoki-
nase) to both solutions. After 15 min measure
the extinctions (E2). Calculate the extinction
difference (LlE= E2 - E 1 ) for the test solution
(Ts) and also for the reference solution (Rs).
The extinction difference for glucose (LlE-
glucose) can be calculated by subtracting LIE
of the reference solution (Rs) from LIE of the
test solution (Ts) :
Add to both samples 0.02 ml solution III
(PGI490 IV) from the Glucose/Fructose Test
Kit (Boehringer/Mannheim), and measure
the extinctions (E3) after 15 min. Calculate
the extinction differences (E3 - E 2) for both
samples. To get the extinction difference for
fructose (LiEfructose), subtract LIE of the refer-
ence solution (Rs) from LIE of the test solu-
tion (Ts):
LlEfructose = LlETs - LlERs .
The concentration of fructose (c) can be cal-
culated using the following equation:
c ( /1)= FV x MW x LlEx DF,
g exdxvx1000
where
LIE = difference E3 - E2
FV = final volume in the test
 MW = molecular weight (for fructose
180.16)
e = coefficient of molar extinction for
NADPH at Hg 334 nm:
6.18 (l mmol- I cm- I )
d = depth of layer in the cuvette (cm)
v = volume of seminal plasma used
(ml)
DF = so-called dilution factor obtained
by perchlorate treatment of native
semen sample.
To make this clearer, the entire procedure
for determining fructose in seminal plasma
is shown step by step in Table 9.
Figures 82 and 83 show the materials used
for protein removal and to perform the actu-
al test.
Figure 84a and b shows the traditional Ep-
pendorf photometer. Figure 85 shows the
quite reasonably priced modern photometer
made by Shimadzu for the determination of
various biochemical factors in seminal plas-
ma.

Table 9. Fructose determination

i. Preparing the test solution

Pipette
1.0 ml 10% potassium perchlorate solution into
0.2 ml semen (completely liquefied, thoroughly
mixed),
leave to stand for 60 min at 4° C in the refrigera-
tor,
then centrifuge for 5 min at 3500 rpm.
Decant the supernatant and neutralize with about
100 mg (equivalent to 1 knife-tip) potassium bi-
carbonate (crystalline form).
Stand in the refrigerator for 10 min, then centri-
fuge at high speed for 10 min. Decant the super- Fig. 82. Fructose determination by the hexokina e
natant liquid into a centrifuge tube. method (Boehringer Mannheim, FRG); material
for protein removal
The result is a protein-free, neutralized seminal
plasma for use as the test solution.

2. Test
Place in a cuvette (test solution T,) 1.0 ml solu-
tion I and 0.1 ml protein-free seminal plasma,
and add 1.9 ml double-distilled water.
Place in a cuvette (reference solution Rs) 1.0 ml
solution I and add 1.9 ml double-distilled water.
After 3 min measure the extinctions (E.) at Hg
334 nm.
Add 0.02 ml solution II (enzyme mixture) to T,
and also to R,.
After 15 min measure the extinctions (E2)'
Add 0.02 ml solution III (PGI) to T, and also to
R,.
After 15 min measure the extinctions (E3)' Fig. 83. Fructo e determination by the hexokina e
Calculate L1E for T, and for R, = E3 - E2 . method : te t equipment
Calculate L1Efruclo,e by subtracting L1ER from
L1~ ,
,
. FVxMWxL1ExDF
Use the equatIOn c= ex dx v x 1000
to get the concentration of fructose in grams per
liter

127
Fig.84a. Fructose determination by the hexokina e Fig.84b. Detail from Fig. 84a
method: the Eppendorf photometer

Fig. 85. Double-beam pectrophotometer with a

graphic display of the ub lance mea ured vi ible on
the creen. Made by Shimadzu, ii eldorf, FRG

128
3 Penetration and Fertilization Tests In Vivo and In Vitro
WOLF-HARTMUT WEISKE and FRED MALEIKA
"The only test of fertility is pregnancy." This
pithy statement by ELIASSON (1971) high-
lights the dilemma in which we fid ourselves
when we resort to laboratory tests which are
intended to assess a man's fertility. On the
other hand, history-taking can establish
whether the patient was fertile in the past
and provide a basis for our subsequent prog-
nosis. AITKEN (1983) states: "It is an unfortu-
nate fact that the techniques currently em-
ployed by andrologists for diagnosing male
fertility are so insensitive that the conditions
they describe are generally untreatable. There
is therefore an urgent need to develop more
sensitive diagnostic techniques capable of
identifying subfertile states which are ame-
nable to the few therapeutic options open to
us." This statement by AITKEN is an appear
to complement the classical analytical meth-
ods of spermatology with dynamic biological
test methods.
Consideration of the characteristics of the
ejaculate alone is often not enough, particu-
larly if only the seminal plasma is examined,
as it is not the destiny of spermatozoa to
remain in the seminal plasma. On the con-
trary, after ejaculation the spermatozoon
struggles to escape from the environment of
its surrounding seminal plasma in order to
work its way through the fluids of the female
genital tract to finally penetrate the ovum.
ROGERS (1985) showed that if spermatozoa
are delayed for more than half an hour in
the seminal plasma, their penetration ability
is reduced in a hamster oocyte test.
The test methods required to extend the diag-
nosis of spermatozoal function can be di-
vided into four groups:
1. Tests of "dynamic" parameters in the
spermiogram
2. Penetration tests
3. Tests for membrane stability
4. Fertilization tests
In more detail, this means:
1. Tests of dynamic parameters in the sper-
miogram:
a) counting the normally formed sperma-
tozoa with unimpaired progressive mo-
tility in the total ejaculate
b) examination of the spermatozoa iso-
lated by the" swim-up" method
2. Penetration tests:
a) the postcoital test (Sims-Huhner test)
b) the slide test (Kurzrock-Miller test)
c) the sperm cervical mucus contact test
(Kremer-Jager test)
d) the Kremer test
e) the Penetrak test
f) the peritoneal sperm migration test
3. Tests for membrane stability:
a) the hypoosmotic swelling test (HOS
test)
b) examination of motility after freezing
4. Fertilization tests:
a) the hamster oocyte penetration test
(HOP test, or heterologous ovum pene-
tration test)
b) human in vitro fertilization (from the
diagnostic angle)
By selecting the appropriate tests or a com-
bination of a few of them, one will be able
to predict with about 80% certainty whether
the semen of a particular man is or is not
capable of fertilization (IRVINE and AITKEN
1986).
129
3.1 "Dynamic" Parameters
in the Spermiogram
Before proceeding with actual penetration
tests, one should first establish whether an
alternative way of approaching ejaculate
analysis might improve the predictability of
an individual's prospects of fertility. The ha-
bit of considering the three major aspects of
the spermiogram - spermatozoal density,
motility, and morphology - often distorts our
assessment, because by doing so we overlook
the numerical situation entirely. Assuming an
ejaculate of 20 million spermatozoa/ml, a to-
tal motility of 60%,50% normal forms, and
a volume of 1 ml, there is a total of 7 million
normally formed, actively motile spermato-
zoa in the total ejaculate. Assuming identical
data from the spermiogram but with a vol-
ume of 4 ml, this makes 28 million spermato-
zoa. Despite a large degree of numerical
equality between the basic parameters, the
two ejaculates have quite different prospects
of fertility.
ALBERT et al. (1986) highlighted the fact
that there is a direct correlation between the
number of motile spermatozoa and the pene-
tration rates in the hamster oocyte penetra-
tion (HOP) test. Both pure laboratory data
and epidemiological data therefore show that
there is a direct relationship between the
number of actively motile, normally formed
spermatozoa in the total ejaculate and the
prospects of fertility.
Using the fashionable objective analyses of
motility with computer-assisted image analy-
sis techniques, it was demonstrated in both
the HOP test and in human in vitro fertiliza-
tion that fertile ejaculates have faster moving
spermatozoa in them than infertile ejaculates
(HOLT et al. 1985).
3.1.1 Sperm Washing
and Swim-up Method
It has been demonstrated by many authors
that there is a positive correlation between
fertility and both the total number of motile
130
spermatozoa and their penetration of the
fluids in the female genital tract. That is to
say: the higher the number of actively motile
spermatozoa which can escape from the
seminal plasma, the higher are the prospects
of fertility. These observations were also of
use for in vitro operations.
The original demand for a small number
of highly motile, normal spermatozoa arose
from the requirements of in vitro fertiliza-
tion. COHEN et al. (1985) accordingly de-
scribed three methods for the preparation of
spermatozoa:
1. Standard method:
The standard method, as it is known, con-
sists of centrifuging twice at 200 g, and
diluting 1 part of semen in about 10 parts
of medium.
2. Sedimentation method:
Another preparation method was the sedi-
mentation method in which the washed
spermatozoa are allowed to fall as a sedi-
ment to the bottom of a Petri dish and
the supernatant liquid containing the
highly motile spermatozoa is pipetted off
after incubation.
3. Overlay method:
The third technique is known as the over-
lay method, in which the ejaculate is over-
laid with a medium through which the
spermatozoa swim up.
The disadvantage with all three methods
is that a considerable number of spermatozoa
are lost in the preparation process. The sedi-
mentation method and the overlay method with
swim-up therefore only have the advantage of
selecting the highly motile, debris-free sperma-
tozoal populations.
As these techniques were developed for in
vitro fertilization, low concentrations of ac-
tively motile spermatozoa were adequate. On
the other hand, to obtain a practical method
for insemination we introduced a modifica-
tion, as further losses of spermatozoa in the
course of preparation are not acceptable
when semen parameters are frequently ab-
normal anyway.
Procedure (Sperm Washing)
- Centrifuge the entire ejaculate (10 min at
200 g).
- Pipette off the seminal plasma immediately
and discard it.
- Shake the pellet in 2 ml Ham's F 10 medi-
um.1
- Centrifuge for 10 min at about 200 g.
- Discard the supernatant immediately.
- Carefully overlay the pellet with 0.3 ml
Ham's F 10 medium. 1
(In order to make it easier for the sperma-
tozoa to get free, the pellet can be broken
up a little by gently tapping the tube.)
Procedure (Swim-up)
- Incubate the overlaid pellet for about
60 min at 37° C (may be done in an atmo-
sphere of 5% CO 2 ).
- Draw up about 0.2 ml of the supernatant
into a tuberculin Syringe.
- Shake well.
- Assess motility.
- Perform intrauterine insemination.
Variations
- Instead of Ham's F 10 medium, serum
from the female partner may be used.
- If swim-up is poor, shape up the pellet in
0.1 m1 medium and also inseminate within
the cervix.
- In cases of severe OAT syndrome without
bacteria, detritus, or leukocytes, the pellet
may be shaken in 0.2 ml medium after
washing and inseminated complete within
the uterus.
With this method of washing the entire
ejaculate, the losses of spermatozoa are natu-
rally less.
1 The following solutions may be used in place of
Ham's F 10 medium: (1) the patient's serum; (2)
Ringer's solution; (3) Gelifundol.
In cases of asthenospermia, ANDOLz et al.
(1986) found that the swim-up method re-
sulted in an improvement in motility from
28.8% to 65.6%. In normozoospermia, we
observed that the proportion of motile sper-
matozoa in the swim-up method was regular-
ly between 90% and 100%. We noticed that
in cases of oligoasthenozoospermia there
were often marked fluctuations in the swim-
up test.
Another aspect of the swim-up technique
which is worthy of note was demonstrated
by PENG et al. (1986) in studies using electron
microscopy. After sperm washing in media
containing antibiotics, no microorganisms at
all were found on the spermatozoa. Washing
also frees the spermatozoa of surface anti-
bodies, although only antibodies of the IgG
class and not those of the IgA class, which
bind to the spermatozoa before liquefaction
(ADEGHE 1986).
3.2 Penetration Tests
Following the logic imposed by the physio-
logical process, we were interested first in
tests determining spermatozoal penetration
of cervical mucus, to quantify what is known
as the cervical factor.
The cervical mucus is a hydrogel contain-
ing a highly viscous component (the gel
phase), and a component of low viscosity
consisting of electrolytes, organic substances,
and soluble proteins. The component of high
viscosity consists of a macromolecular net-
work of mucin which is the main determinant
of the rheological properties of the mucus.
Depending on the production of estrogen,
the daily production of mucus varies from
60 mg in midcycle to 20-60 mg during other
phases of the menstrual cycle. To perform
these tests, one should use the mucus in its
optimal condition. The cervical mucus per-
forms firstly the function of a massive filter
to spermatozoa, and secondly has the func-
tion of storing spermatozoa (MOGHISSI 1977).
The mucus is collected after establishing
the level of mucus in the vagina, and if neces-
131
 0 1 2 3

Width of os uteri closed pinhole slightly open gaping

Fig. 86. The cervical score of
Quantity of mucus 0 just 1 thick cascade Insler assesses the quality of the
apparent drop mucus by summing points for
Stringiness (cm) (H 1-4 4-8 >8 width of the os uteri, quantity
of mucus, "stringiness," and the

*- ~
Fern test

/ ~
fern test. The physical properties
of the cervical mucus are
normal if the cervical score
No. of exceeds 8. (After INSLER et al.
branches 0 2 3
1972)
Compulsory of the evaluation of the postCOital test:
pH : > 6.4-8.5
Insler's cervical score: > 8
Sum of scores = cervical index (0-12). Evaluation of cervical function: 0-3, inadequate; 4-6, restricted;
7-9, good; 10-12. very good

sary after wiping the cervix with a dry swab. et al. 1972). Mucus which is to be used in
If there is insufficient mucus present, mucus a penetration test must attain a cervical score
can be expressed from the os uteri by means of 8 or more. With lower scores, the physical
of pressing on the cervix with the posterior properties of the mucus are so impaired that
and anterior blades of a speculum. The mu- penetration is unlikely to take place irrespec-
cus is drawn up into a tuberculin syringe tive of the quality of the semen.
placed at the os uteri. The mucus should be
used immediately if at all possible. If a delay
is unavoidable, the sample must be prevented
from drying out by sealing the tuberculin sy- Score
ringe with paraffin paper. It may be accept- 12
able to store the mucus for 5 days in a refrig-
erator at 4° C. Spermatozoal penetration 11

tests should not be performed with mucus 10

samples which have been frozen and thawed,
9
as this process destroys the fine structure of
the mucus. 8
When the mucus is poor, we try to achieve
7
some improvement by administering gonado-
tropins, as the resulting increase in the level 6
of estradiol leads in most cases to better cer- 5
vical mucus scores.
If the cervical score is low, intrauterine in- 4

semination should be the method adopted. 3

2
Evaluating the Cervical Mucus

Penetration studies of cervical mucus can 9-18 19-28 29-48 49-60 80-160
only be evaluated if the mucus is of a certain Estrogen in urine
quality. This can be established on the basis Fig. 87. Correlation between urinary estrogen and
of Insler's cervical score (Fig. 86; INSLER the cervical score. (After INSLER et al. 1979)

132
pH of the Cervical Mucus
The pH of a sample of mucus to be tested
should be between 6.4 and 8.5. The pH test
is performed using the special indicator paper
made by Merck, item no. 9557. Ifthe mucus
is acid, the spermatozoa will generally be im-
mobilized (CAMPANA et al. 1981).
A mucus penetration test performed on
spermatozoa should, therefore, only be eval-
uated if the cervical score for the mucus is
> 8 and the pH is between 6.4 and 8.5.
3.2.1 The Postcoital Test
(Sims-Huhner Test)
The postcoital test was first described in 1886
by Sims. He observed spermatozoa in the cer-
vical mucus a few minutes after coitus and
was able to show that live spermatozoa were
present in the cervical mucus up to 48 h after-
wards. Huhner is credited with having popu-
larized the test, and that is why we call it
the Sims-Huhner test.
It has undoubtedly been the most widely
practiced fertility test to date, together with
the cervical score. Despite this, it is also un-
doubtedly the most misunderstood and most
overused test. It has never been precisely
standardized. It has come to be overused be-
cause it was correlated with pregnancy rates
and is regarded in general practice as a fertili-
ty test in its own right. In consequence, doc-
tors were unable to understand why no preg-
nancy occurred despite a normal menstrual
cycle, normal fallopian tubes, and a normal
postcoital test.
However, if we come down to what the
test actually tells us, we see that the postcoital
test examines the interaction between the sper-
matozoa and the cervical mucus. The test
shows whether the physicochemical proper-
ties of the mucus are adequate, and there is
a direct correlation with the number of motile
spermatozoa. In addition, there is a distinct
correlation with the cervical antisperm anti-
bodies.
It provides no more than a description of
the cervical environment for penetrating sper-
matozoa.
Considerable numbers of spermatozoa can
be found in the cervical mucus only 90 s after
ejaculation during coitus. After 15 min, the
number of spermatozoa in the mucus reaches
its peak. After 20 min, 95% of the spermato-
zoa have already migrated from the vagina
into the cervical canal (OVERSTREET et al.
1980).
Some of the spermatozoa are held in the
cervical canal. It is clear that spermatozoa
stored in the cervical canal for 80 h are still
capable of penetrating the human zona pellu-
cida (LAMBERT et al. 1985).
This storage function of the cervix makes
it possible for coitus several days before ovula-
tion to lead to pregnancy. SCHWARTZ et al.
(1980) demonstrated in 1132 cycles, during
which patients were inseminated once with
frozen donor semen due to their husband's
azoospermia, that the frequency of concep-
tion on the 3 days prior to ovulation was
identical to that on the day of ovulation,
whilst the maximum was recorded on the day
before ovulation. HANSON and OVERSTREET
(1981) also proved that the percentage motili-
ty, swimming speed, and the morphology of
spermatozoa in the mucus after and 48 hare
the same, i.e., over the entire period.
This epidemiological finding has practical
consequences: firstly, it shows that when we
are advising patients about the best way of
conceiving we should not play the part of
the pedantic, petty "sex counsellor," but
should take a more liberal attitude like
Luther, who is well known to have recom-
mended coitus "twice a week. "
Secondly, the long period during which the
spermatozoa remain in the mucus after coitus
means that a postcoital test should only be
used to check what it is designed to check:
- rapid penetration and
- the storage function of the cervix.
MORTIMER et al. (1982) reported that sper-
matozoa recovered from the cervix displayed
a level of normal morphology one-third high-
er than the original ejaculate. On the other
hand, they found that this filtering action is
not a property of the female genital tract,
but that it occurs to the same degree with
amorphous filter systems such as nickel
133
gauze. It is therefore the varying speed of One criticism which must be made is that
movement of the spermatozoa which is re- there is no standardization in this widely used
sponsible for their selection, rather than their test. Even the WHO classification completely
environment. omits to state what thickness of layer should
In postcoital tests in which the results are be used to assess the spermatozoal density
good, antibodies to the spermatozoa are per field. Basically, the test could only be re-
found in a maximum of 10% of cases. If the garded as standard if a suitable counting
results are moderate or negative, on the other chamber with a defined depth is used.
hand, antibodies to the spermatozoa will
probably be found in almost 50% of cases
(HAAS 1986).
3.2.2 The Slide Test
(Kurzrock-Miller Test)
3.2.1.1 Evaluating the Postcoital Test
The slide test described in 1928 by KURZ-
Basically, the function of the postcoital test ROCK and MILLER is the simplest method of
can be defined quite simply: If it has been simulating the penetration of spermatozoa
performed sufficiently frequently with similar into the mucus in vitro, and also of arriving
results for a particular couple, intrauterine at a certain general quantification of this pa-
insemination should be adopted if the results rameter.
are negative or moderate. Constantly good The test is indicated if the Sims-Huhner
results in the postcoital test practically ex- postcoital test has produced negative or incon-
clude the possibility of cervical hostility. clusive results on several occasions. However,
We follow the guidelines of the WHO (BEL- the test may be used in the initial assessment
SEY et al. 1980) for assessing the postcoital of fertility, as it reveals the presence of anti-
test. sperm antibodies on the spermatozoa or in
The results are classified as "normal," the cervical mucus. It can also be used in
"uncertain," or "abnormal" (see Table 10). a cross-over manner, i.e., with donor mucus
The assessment is carried out at a magnifi- or donor spermatozoa, in order to discover
cation of 400 x (40 x objective). In order to which partner has the hostile factor. How-
scan the sample quickly to get a general im- ever, we consider a cross-over slide test to
pression, the 100 x magnification is selected be of little relevance in clinical practice, be-
first; this also identifies variable density of cause therapeutic measures need only be tak-
spermatozoa from field to field in the speci- en to treat pathological phenomena found
men. in both partners.

Table 10. Assessment of the postcoital test following the WHO classification (1976)

Normal: More than 7 spermatozoa with progressive motility

Uncertain: 1-7 spermatozoa with good motility
Abnormal: No spermatozoa or only immotile or agglutinated spermatozoa
counted per field at 400 x magnification (40 x objective)

e>= ~------

However, the WHO classification does not state the thickness of layer.

134
Performing the Slide Test
The preconditions are that the mucus should
have a cervical score after Insler > 8 and a
pH between 6.4 and 8.5, as described above.
Smear the mucus onto a slide and press
a cover slip firmly onto it so as to leave only
a narrow, air-filled capillary gap between the
cover slip and slide. Apply the liquefied, thor-
oughly mixed semen onto the edge between
the cover slip and the slide. Capillary action
will draw it up to the margin of the mucus.
The margin is wavy.
Phalanges of semen move deep into the
mucus. This is simply the phenomenon which
occurs when two fluids of differing viscosities
meet. The spermatozoa first colonize these
phalanges, and this is also where the first
penetration of the mucus generally takes
place, to be followed after an initial hesita-
tion by the rapid and complete colonization
of the mucus (Fig. 88).
3.2.2.1 Evaluating the Slide Test
(Kurzrock-Miller Test)
The test is evaluated 5 and 15 min after the
drop of semen has been applied to the slide.
Count the number of spermatozoa in the first
field beginning at the boundary between the
semen and mucus, and in the second field
above it and directly adjoining it (fields F 1
and F 2; see Fig. 89). The examination can
be performed under 200 x or 400 x magnifi-
cation. Record the magnification used on
each occasion. To interpret the tests in accor-
dance with the WHO guidelines, use the
400 x magnification

Tuberculin syringe Evaluation of the Slide Test

excellent = in F 1 25 spermatozoa under

400 x magnification
Coverslip in F 2 25 spermatozoa under
400 x magnification
a good = in F 1 15 spermatozoa under
400 x magnification
in F 2 10 spermatozoa under
400 x magnification
Mucus
moderate = in F 1 5 spermatozoa under
400 x magnification
b
in F 2 0-1 spermatozoon
under 400 x
magnification
negative = no penetration in F 1 or in F 2
Tuberculin syringe

The relevant category is determined after

Semen 5 and 15 min.
Although the WHO guidelines give no in-
Mucus
formation about the percentage of progres-
sively motile spermatozoa, we consider it
c helpful to determine the number as a percent-
age per field at the same time.
Fig. 88a-c. Performing the slide test (Kurzrock-Mil- Certain specific examinations conducted
ler test). See text for description during this test provide information concern-

135

C.M.
I
\ ,, ,
Fig. 89. Evaluating the slide test. C.M., = cervical
mucus; Sp, spermatozoon; Ph, phalanx ofspermato-
zoa; F I , first field; F 2 , second field. (After MOOIDSSI
1966)
ing the presence of agglutinins both on the
spermatozoa and in the cervical mucus. Only
antibodies of the IgA class lead to agglutina-
tion. IgG class antibodies do not lead to ag-
glutination in either medium (KREMER et al.
1977).
Agglutinations should not be confused
with spermatozoa found at the margin of the
mucus or in the phalanges which are moving
from place to place. True agglutination is
when the head is stuck fast in the cervical
mucus, which indicates the presence of anti-
bodies to the spermatozoa in the mucus. The
rarer case, when the tails of the spermatozoa
are stuck at the edge of the mucus, indicates
agglutinins attached to the tail of the sperma-
tozoon, as described by KREMER et al. (1977).
Spermatozoa of this sort are able to penetrate
the mucus initially, but after a short while
they are only able to move on the spot, dis-
playing the characteristic "shaking" phe-
nomenon. This comes about when agglutina-
tion due to antibodies occurs between the
spermatozoon and the glycoprotein myce-
lium of the mucus. It allows the spermato-
zoon only limited mobility before complete
immobilization comes about.
Instead of mucus, serum from the female
partner may, of course, be used in the slide
test in order to detect or exclude antibodies
to the spermatozoa.
136
The slide test is a simple aid for everyday
use. However, its interpretation is very sub-
jective and depends entirely on the experience
of the observer. More precise quantification
of penetration ability can only be gained
from the Kremer Test (see Sect. 3.2.4).
3.2.3 The Sperm - Cervical Mucus
Contact Test (SCMC Test)
of Kremer and Jager
With the slide test we have already seen that
IgA antibodies, both on the spermatozoon
and in the mucus, can lead to agglutination,
which is associated with the characteristic
shaking phenomenon (Fig. 90).
The sperm--cervical mucus contact
(SCMC) test, which is simple to perform,
provides a quantitative assessment of this
shaking phenomenon. Spread a drop of mu-
/,======,7A Press down cover slip firmly
" +
Slide
Mucus Semen
Observing the "shaking" phenomenon
Evaluation
Shaking present in 0%- 25% =-
25%- 50% =+
50%- 75% = ++
75%-100% = +++
Fig. 90. Positive "shaking" phenomenon in the
SCMC test: due to aggluination, the head of the sper-
matozoon (right) or, less commonly, the tail of the
spermatozoon (left) becomes stuck in the cervical
mucus. In either case the spermatozoon has only lim-
ited mobility on the spot (= shaking), before com-
plete immobilization comes about. The percentages
correspond to the presence of different levels of ag-
glutinins in the mucus or on the spermatozoa

cus on a slide, and place a small drop of
liquefied semen in the center of the spread
drop of mucus. By applying a constant, but
gentle, pressureon the two media, they
should mingle. After about 10 min, examine
a number of different fields at a magnifica-
tion of 200 x to estimate the percentage of Fig. 91 a. Sperm penetration meter after Kremer
spermatozoa displaying the shaking phenom- (modified by FREUNDL 1985). I , Capillary tube -
enon. from lOp to bo(/om : capillary tube containing the
3.2.3.1 Evaluating the SCMC Test
0%- 25% = negative
25%- 50% = single positive (+)
50%- 75% = double positive (+ +)
75%-100% = triple positive (+ + +)
A triple-positive shaking phenomenon is clin-
ically significant and is associated with a high
local antibody titer (KREMER et al. 1977) (see
Fig. 90).
The SCMC test is a simple microscopic
screening test which provides an indication
of clinically significant IgA antibodies in the
mucus and on the spermatozoa.
3.2.4 The Kremer Test
The Sperm Penetration Meter test, originally
described by KREMER in 1965, quantifies the
penetration of spermatozoa in glass capillary
tubes filled with cervical mucus.
Later a second capillary tube was added,
containing serum from the man's wife. Thus
an elegantly simple design of functional test
was conceived giving information of great
clinical relevance, in that it enabled penetra-
tion to be analyzed semiquantitatively in vi-
tro in the two principal fluids - cervical mu-
cus and serum - which correspond to the en-
vironment of the uterine tubes.
AB serum from a fertile woman is used
as a reference.
All possible combinations of cross-over
tests can also be carried out using donor mu-
cus, donor semen, artificial mucus, and bo-
vine mucus. However, to assist in making the
2
4
patient's mucus, capillary tube containing the pa-
tient' erum, capillary tube containing AB serum
from a rerti le woman as reference. 2, Slide with en-
graved cale. 3, Container ror emen. 4, Putty or
paraffin wax plug to render capillary tube airtight
routine decision whether to bypass the cervi-
cal environment by intrauterine insemina-
tion, the following arrangement is sufficient:
- Capillary tube containing the patient's mu-
cus
- Capillary tube containing the patient's se-
rum
- Capillary tube containing AB serum from
a fertile woman as reference.
Performing the Kremer Test
The appropriate chamber l is required with
three small containers at the lower end to
receive the semen, and with centimeter gradu-
ations (Fig. 91 a).
The mucus is drawn up into the microcapil-
lary tubes (3 mm wide, 0.3 mm deep) using
a tuberculin syringe fitted to the end.
Once the mucus has completely filled the
capillary tube, with no air bubbles present,
its end is sealed with putty or paraffin wax
to make it airtight.
The serum passes into the narrower capil-
lary tube 2 (2 mm wide, 0.1 mm deep) by cap-
illary pressure.
I Made by Jan de Groot, Kastanjelaan 1, 3481 XD
Harmelen, Netherlands.
2 Obtained from Ahrin bv Instrumenten, Postbus 80,
2280 AB Rijswijk, Netherlands; manufactured by Vi-
tro Dynamics Inc., New Jersey, USA.
137
 However, an incubation time of 2 hours has
proved expedient. Reading the results under
15
a 10 x objective requires some practice.
___ c
3.2.4.1 Evaluating the Kremer Test

10 Using a simplified version of the WHO scor-

5
o nation of the scoring system are presented
Fig. 91 b. Sperm penetration meter after Kremer De-
tail from Fig. 91 a: a ingle capillary tube. A Capil-
lary tube ; 8 semen reservoir ; measuring cale (in
accordance withWHO 7957 1; BEL EY et al. 1980)
ing system, the depth of penetration is read
off, and the density of spermatozoa penetrat-
ing to the 5 cm mark and the degree of mot il-
ity in the upper third of the capillary tube
are determined. A diagram showing the eval-
uation of the test results and a detailed expla-
in Tables 11 and 12.
If the penetration of the mucus is negative
or moderate, intrauterine insemination is indi-
cated.
If there is no difference between the degree
of penetration in the patient's serum and that
in the AB serum of the fertile control, one
can exclude the possibility of a humoral im-
munological factor of any clinical signifi-
cance.

Table II. Evaluation of results of the Kremer test

Maximum
penetration (em)
n=99 n= 17

Fig. 91 c. Kremer te t equipment assembled: the te t

chamber with the three eparate capi llary tube on
6

5
I.......
.....
......
•

the slide i placed on the moi tened filter paper on

the bottom of a Petri dish , thus providing a humidity
chamber
4
.........
•• •

3- ......
......
••
....
The capillary tubes are then fixed onto the
chamber with small pieces of putty so that
their open ends just dip into the semen con- 2- .......
•
....
tainers. The entire test assembly is placed in
a humidity chamber, consisting of a Petri
dish lined on the bottom with moistened filter - ... ••
paper (Fig. 91 c).
This is then incubated at 37° C. In princi- o
.......
ple, the test results can be obtained within 7 -1 2 1- 6
incubation times of up to 24 h. Cervical score

138
Table 12. Scoring system for the Kremer test

Kremer Test (test time 2 h) Date:

Patient: Born:

Partner: Born:
-
Score

m
Patient's mucus Depth of penetration (cm)

Density penetrated to 5 cm
Os width
Quantity Motility
Stringiness
Fern test
total
Insler's cervical score: -
pH:
-
Score

Patient's serum Depth of penetration (cm)

Density penetrated to 5 cm

Motility

total
'---

r---
Score

AB serum, fertile control Depth of penetration (cm)

Density penetrated to 5 cm

Motility

total

The simplified WHO classification covers:

Score 0 1 2 3
Depth of penetration in centimeters
Depth of penetration 0 0-2 2-5 5
Density penetrated number of spermatozoa per field
(approx.)
t05cm (lOx objective) at the 5 cm mark
Motility classified as in the upper third of the capillary tube Density penetrated 0 1-10 11-50 >50
O=no progressive motility (5cm)
1 =25% of spermatozoa progressivelymotile
Motility 0 1 2 3
2=25o/~50% progressively motile
3=over 50% progressively motile
Score: O=neg ative; 1-3=moderate; 4-6= g ood; 6-9=excelient

The Kremer test is a microscopic screening
test that gives us general information about
a possible immunoreaction which correlates
to a high degree with the clinical picture and,
of course, with the results of the spermio-
gram.
In our own studies, we found that after
2 h there was a direct correlation between
penetration to the 2 cm mark and progressive
motility, and there was a direct correlation
between penetration to the 5 cm mark and
the number of spermatozoa with unimpaired

139
progressive motility (MALEIKA et al. 1983). of the studies mentioned above, a standard-
KOLODZIEJ et al. (1986) confirmed that motil- ized bovine mucus penetration test (BMP)
ity is the factor of greatest prognostic value test was developed, similar to the Kremer
for the spermatozoal penetration of cervical test, and it can now be obtained commercial-
mucus, and they were able to show that re- ly as the Penetrak® Test! (Fig. 92a and b).
sults read after 2 h incubation have a signifi- The special feature of this test is that -
cant, although relatively minor, correlation apart from time and temperature - penetra-
with pregnancy rates. PANDYA et al. (1986) tion ability is the only variable.
showed that one can predict the outcome of It frees one from dependence on human
the Kremer test with 70% accuracy on the mucus. The advantage of this is that the fe-
basis of the calculated motility index. male partner of an infertile couple is not re-
MORTIMER et al. (1986) found that the per- quired for examination at the start and the
centage of morphologically normal sperma- collection of a mucus sample at the time of
tozoa correlates with the results of the ovulation, which is administratively time-
Kremer test. If the test result was "moder- consuming, is superfluous. It also, of course,
ate," there were significantly more abnormal- eliminates all the other variables associated
ities of the tail. with human mucus, such as the time of sam-
The use of donor semen and mucus enables pling, viscoelasticity, pH, and any possible
one to detect incompatible partners, if the contamination with bacteria and cellular de-
partners have excellent test results with either tritus.
the donor mucus or donor semen as appli- The involved and time-consuming process
cable (JONSSON et al. 1986). of filling the capillary tubes homogeneously
in the Kremer test, which is necessary to
achieve reproducibility, also ceases to be nec-
essary.
3.2.5 The Bovine Mucus
It is independent of the pathological
Penetration Test (BMP Test) changes which may be present in some cir-
(Penetrak® Test)
cumstances (Insler's cervical score below 8,
IgA class antibodies), and it tests exclusively
Apart from the quality of the semen, the ca- the penetration ability of the spermatozoa
pability of spermatozoa firstly to migrate through cervical mucus.
through the cervical canal to reach the ovum, The Penetrak ® test is a standardized test.
and secondly to penetrate the ovum, is of Specially prepared, purified bovine mucus is
crucial importance to their fertilizing ability. filled in flat capillary tubes which are sealed
Tests have been developed to investigate and airtight. A weak point at one end of the
both processes and will be described below. capillary tube enables it to be opened. The
We will also comment on the quality of infor- flat capillary tubes must be stored at minus
mation they provide and on their practica- 20° C.
bility.
Bovine mucus is very similar to human cer-
vical mucus in its properties, particularly its 3.2.5.1 Performing the Penetrak® Test
viscoelastic characteristics (LEE et al. 1977;
GADDUM-RosSE et al. 1980; ALEXANDER The test should be carried out within 2--4 h
1981; FREUNDL 1985). Human spermatozoa of thawing the tubes. The individal steps are
can penetrate bovine mucus without diffi- as follows:
culty. Their behavior in this medium is identi-
1. Remove from the pack the actual number
cal to that when penetrating human mucus
of test tubes needed for the proposed test
(LEE et al. 1977; KATZ et al. 1980; ALEX-
ANDER 1981; BERGMANN et al. 1981; LEE
et al. 1981; MOGHISSI et al. 1982; Blasco 1 Serono Diagnostica GmbH, Merzhauser Str.134,
1984; Stumpf and LLOYD 1985). On the basis . D-7800 Freiburg, Tel. 0761/40928, FRG.

140
Fig. 92a. Penetrak test (penetration test with bovine
mucus, Serono Diagnostica). The materials are laid
out from left to right in the order in which they are
used. In the center are the sample beakers filled with
semen in which the flat capillary tubes containing
Fig. 92 b. Enlarged picture of the" work bench" used in the Penetrak test containing the sample beakers
(two tubes per sample). Tubes cannot be
refrozen once thawed. Before use, bring
the test tubes up to room temperature
(20°- 25° C). The thawing process will take
about 12-15 min. The tubes must be stood
upright to thaw, with the graduated end
of the tube uppermost. If there are any
small air bubbles in the tube, tap the tubes
the bovine mucus stand. Below, to the right of center,
are the graduated microscope slides on which the
capillary tubes are placed to determine the distance
covered by the spermatozoon which has migrated
furthest (see text for details)
until the bubbles are above the mark.
Break open one tube at the weak point
holding the tube so that the graduations
are facing you. Snap the end off the tube
carefully to avoid causing air bubbles or
allowing the cervical mucus to escape from
the tube (Fig. 93).
141
2. Pipette 4 drops (about 0.2 ml) of the lique-
fied and thoroughly mixed ejaculate into
a sample beaker (Fig. 94).
3. Place the open end of the tube into the
sample beaker ensuring that the open end
is completely covered (Fig. 95).
4. Repeat the entire procedure with a second
tube. Both tubes should then be left to
stand in the sample for 90 min at room
temperature (Fig. 96).
5. After 90 min, remove one tube from the
sample and carefully wipe off the semen
adhering to it with a soft paper tissue.
Take care not to pull any cervical mucus
out of the tube when wiping it (Fig. 97).
6. Determine the depth of penetration of the
spermatozoa under a microscope, by plac-
ing the tube with its opening on the scale
marked on the microscope slide (Fig. 98).
7. Examine the tube, refocusing constantly
to identify the spermatozoon which has
penetrated furthest into the cervical mu-
cus. The distance covered is measured
against the scale on the slide (Fig. 99).
8. Repeat steps 5-7 with the second tube and
calculate the mean of the two samples
(Fig. 100).
3.2.5.2 Evaluating the Penetrak ® Test
Penetration is normal if the 30 mm mark is
passed. If the spermatozoa have penetrated
less than 20 mm the test result is considered
pathological. The distance between 20 and
30 mm is inconclusive. It could be described
as a gray area. On the basis of clinical trials,
the manufacturer claims the sensitivity of the
test to be 83% and its specificity to be 94%
(comparison with human cervical mucus).
Critical Comments on the Penetrak ® Test
j. Advantages:
a) Availability, independent of cervical mu-
cus from the woman
b) Standardization: variables are minimized
in comparison with human mucus
2. Disadvantage: Relatively high price
142
It is possible to reduce the cost a little by
not using two flat capillary tubes per sample
as the manufacturer recommends, but only
one. If the result is pathological (less than
20 mm) or uncertain (between 20 and
30 mm), the test can be repeated with two
capillary tubes, if necessary with a new sam-
ple of semen at a different time, if the semen
sample in question already exhibits an abnor-
mal loss of motility.
Studies by MOSLEIN et al. (1987) suggest
that the Penetrak® test does not only mea-
sure the ability of spermatozoa to penetrate
mucus. The test was performed on 30 ejacu-
lates before and after a selection procedure
had been carried out (Separon) to increase
the density of motile spermatozoa. The re-
sulting mean values were 28.76+7.7 mm for
the untreated sample and 38.86+10.51 mm
for the treated samples. These differing
values can be interpreted as an indication of
abnormal motility.
In any event, the Penetrak® test is a simple
method for checking the effectiveness of var-
ious selection techniques for increasing the
density of progressively motile spermatozoa.
Another fact worth mentioning is that the
results of the test can be pathological in
about 10%-30% of cases when the spermio-
gram is nominally normal (ALEXANDER 1981;
SCHUTTE and SCHIRREN 1987). Conversely, it
was demonstrated that penetration tests can
be normal when the spermiogram is patho-
logical (BLASCO 1984; SCHUTTE and SCHlR-
REN 1987).
The correlation between the results of the
Penetrak ® test and the motility characteris-
tics of human spermatozoa was studied using
a computerized image analysis system, with
the following results:
The comparison with important parame-
ters in the spermiogram (density, percentage
of oval heads, percentage of motile spermato-
zoa, mean speed of movement, lateral move-
ment of head, beating frequency and progres-
sive motility) shows that 52% of the clinical
results in the Penetrak ® test can be predicted.
As in studies using human mucus (ULSTEIN
and FJALLBRAND 1973; BLASCO 1984), the
Penetrak test correlates best with progressive
motility and total motility (r=0.7,p<0.001).
 Thaw 2 capillary tubes and bring them to
Pipette 0.2 m lof the completely liquefied
1 room temperature (15 min); snap open at the 2 semen into the sample beaker
weak point provided

93 ~ __________________________ ~94

3 Stand both capillary tubes in the sample 4 Incubate for 90 min at room temperature

95 96
Take one capillary tube out of the sample and
carefully wipe off traces of the sample (with- Place the capillary tube on the graduated
5 out drawing the cervical mucus out of the tube) 6 slide under the microscope (40 x - 100 x)

97 98

Determine the distance covered by the Repeat steps 5-7 with the second tube ;
7 spermatozoon which has travelled furthest 8 Calculate the mean of the two

99 100

Figs. 93-100. The eight steps in performing the Penetrak test (see text for details)

143
 In addition, a high degree of correlation
was demonstrated with the HOS test (hy-
poosmotic swelling test of Jeyendran et al.
1984; R=O.77, p<O.OOl) (BLASCO 1984;
GERING 1987; SCHUTTE and SCHIRREN 1987).
These findings are a clear indication that
the Penetrak ® test reveals spermatozoal
characteristics which are undetectable in the
classical spermiogram. Our recommendation
is, therefore, that the test be performed as
a screening test prior to planned in vitro fer-
tilization.
The Penetrak ® test thus complements the
spermiogram and provides a more compre-
hensive assessment of male fertility than the
spermiogram alone.
3.2.6 The Peritoneal Sperm Migration
Test (PSM Test)
The peritoneal sperm migration (PSM) test, Table 13. Swelling test (method of VAN DER VEN et al.
which involves performing insemination 1986)
2-8 h before diagnostic pelviscopy, is an in-
teresting attempt to realize the in vitro bene- 1. Swelling medium
fits of the Kremer test under in vivo condi-
tions.
In diagnostic pelviscopy, fluid is tapped
from Douglas' pouch and the spermatozoa
which have migrated there are detected in
that fluid. Detection is easier if the cellular
constituents of this fluid are first sedimented
out by centrifugation and resuspended in a
smaller quantity of fluid. As a large number 2. Test
of erythrocytes are present, substances which
lyse erythrocytes can be used. The test proves
whether the spermatozoa can actually reach
the site of fertilization. In practice, however,
the test has not been widely adopted, because
spermatozoa are detected in the fluid in
Douglas' pouch in only about half the cases,
even if the fallopian tubes are clear. This is
due primarily to the enormously high dilu-
tion factor in the fluid in Douglas' pouch
and to the small number of spermatozoa
which reach the fimbria of the uterine tube.
Using the PSM test, STONE and HIMSEL
(1986) showed that mild endometriosis does
not affect the transport of spermatozoa in
the tubes and their survival in the pouch of
Douglas.
144
3.3 Tests of Membrane Stability
3.3.1 The Hypoosmotic Swelling Test
(HOS Test)
The hypoosmotic swelling (HOS) test identi-
fies in percentage terms the ability of the
spermatozoal membrane to tolerate hypoos-
motic stresses. It is postulated that membrane
stability is an additional parameter of the vi-
tality of spermatozoa and that membrane
stability correlates with fertility.
When spermatozoa are incubated in a so-
lution of low osmolality, water enters and
the tails swell. The percentage of spermato-
zoa swollen in this way is counted (Fig. 101).
The preparation of the hypoosmotic solu-
tion is described in Table 13.
a) Sodium citrate 1.47 g in 100 ml water
b) Fructose 2.75 gin 100 ml water
Mix a and b to produce 200 ml swelling medium.
Sterilize by filtration into 250 ml Erlenmeyer
flasks.
Adjust osmolality to 150 mosmol/l using an os-
mometer.
The solution can be filled into 1-ml ampoules and
stored deep-frozen.
Bring 1 ml of the swelling medium up to room
temperature, transfer to a sterile test tube, and
mix with 0.1 ml semen (0.5 min). Incubate in a
water-bath (37 C for about 30 min).
0
Examine for swollen cells (see Fig. 101): (100, or
preferably 200, spermatozoa on the slide. Magnifi-
cation 400 x under a phase-contrast microscope).
The percentage of coiled-up tails in the unpre-
pared specimen must be subtracted because their
shape can be confused with swollen tails. The den-
sity of spermatozoa in the swelling medium can
be increased by centrifuging briefly before placing
them on the slide for evaluation.
 1 2 3 5
Fig. 101. HOS test. Diagrammatic representation of
the typical morphological changes to the tail of sper-
matozoa under hypoosmotic conditions. 1, No
change; 2, "chain-like swelling," minimal, segmented
Evaluating the "OS Test
> 60% swollen spermatozoa = normal
< 60% swollen spermatozoa = abnormal
The test initially aroused great hope, as
JEYENDRAN et al. (1984) were able to demon-
strate that the percentage of swollen sperma-
tozoa correlates with the percentage of ham-
ster oocytes penetrated in vitro in a HOP
test (see Sect. 3.4) with a correlation coeffi-
cient of 0.9.
Furthermore, VAN DER YEN et al. (1986)
showed that ejaculates which fertilized 00-
cytes in vitro always displayed more than
60% swelling, whereas nonfertilizing ejacu-
lates had less than 60% swelling. At first it
was believed that one simple test would en-
able one to distinguish clearly between a fer-
tile and an infertile man. Unfortunately,
other research teams produced contradictory
observations. CHAN et al. (1985) showed that
the correlation between spermatozoal pene-
tration in a HOP test and the percentage
6 7
swelling of the entire tail; 3- 7, different forms of
swelling of the tail of increasing magnitude (swollen
section of tail shaded). (Modified after JEYENDRAN
et al. t 984)
swelling was insignificant (r = 0.05). In our
own study (MALEIKA et al. 1985) we found
that, although significantly different penetra-
tion rates were indeed found in the HOP test
between ejaculates that were and were not
successful at human in vitro fertilization,
there was absolutely no difference in the re-
sults of the swelling test. The result was
71 .8% in the swelling test with successful fer-
tilization and 72.9% with no fertilization (see
Table 14). Our study involved a population
of men with normozoospermia, whose fertili-
ty had been proven by the earlier extrauterine
pregnancies of their wives, and therefore the
two groups should not have differed for this
reason alone.
In agreement with this, various authors
found correlations between swelling rate and
the classical spermiogram parameters (Ta-
ble 15). The fact that the correlation between
the swelling test and motility is most marked
and with the eosin test is least marked (see
Sect. 2.7.4) shows that swelling rate can be
regarded as a new parameter of vitality. As
145
Table 14. Results of the HOP test and swelling test solution, and additionally to a double hot-
in relation to success of in vitro fertilization cold/cold-hot shock. The better the motility
of spermatozoa is after thawing, the greater
Fertilization No p
(n=23) Fertilization is their fertilizing ability. For example, in
(n= 18) 6327 insemination cycles, SPIRA (1986)
showed that if motility after thawing was at
Mean SEM Mean SEM least 40%, the pregnancy rate per cycle was
10%, and with motility after thawing of more
HOP test (%) 36.6 3.77 27 3.67 0.05
Swelling test (%) 71.8 2.10 72.9 2.63
than 60% the pregnancy rate was 20% per
n.s.
cycle (in patients with good cervical scores).
Significantly different penetration rates were found
in the HOP test between spermatozoa producing pos-
itive results and those producing negative results in
human in vitro fertilization. In contrast, the swelling 3.4 Hamster Oocyte Penetration Test
test revealed no differences between successful and (HOP Test = Heterologous Ovum
unsuccessful attempts at fertilizations (MALEIKA et al.
1985)
Penetration Test)

In the hamster oocyte penetration (HOP)

Table 15. Swelling test correlation with "classical"
spermiogram parameters
test, superovulation is induced in female
hamsters by human menopausal gonadotro-
r pin (HMG) and human chorionic gonadotro-
pin (HeG). The follicle cell layer and the
JEYEN- MALEIKA VAN DER zona pellucida are removed biochemically
DRAN et al. YEN et al.
(1984) (1985) (1986)
from the ova obtained. The naked oocytes
are then incubated with capacitated human
Motility 0.61 0.56 0.51 n=40 spermatozoa, which first bind with the mem-
Eosin 0.52 0.49 n=38 brane (known as sticking), and then pene-
Morphology 0.30 0.27 0.29 n=40 trate the ooplasm, where they swell into the
Spermatozoal 0.26 0.20
male pronucleus.
density
The number of oocytes penetrated is
n 38-40 218 80-83 counted. This technique is very difficult and
the experimental aspect is more involved
The correlation with motility is greatest for the swell- than that of human in vitro fertilization. A
ing test and least with the eosin test detailed description would be beyond the
scope of this book. We would refer anyone
a measure of the functional integrity of sper- interested to the article by ROGERS (1985).
matozoa, it measures something fundamen- The author cites 97 references in the litera-
tally different from the other well-known pa- ture.
rameters. For this reason alone, it is worth The HOP test is the only test in which the
carrying out the swelling test. However, in potential fertility of the man can be demon-
view of the controversial data so far ob- strated in vitro. It may be used to establish
tained, we are not yet in a position to judge the success of drug therapy. It could be help-
its importance. ful in diagnosing the need for surgery for var-
icoceles. It can also be used to test the fertility
of specific groups (old men) who are no lon-
ger personally considering reproduction. Un-
3.3.2 Freezability of Spermatozoa
fortunately the test entails a high cost in
keeping the animals needed, a very skilled
In contrast to the hypoosmotic swelling test, experimenter, and a lot of time, and for these
the preservation of spermatozoa by freezing reasons has not been widely accepted to date
exposes them to an extremely hyperosmotic in the Federal Republic of Germany.

146
3.5 Human In Vitro Fertilization
(IVF)
In principle, human in vitro fertilization
(IVF) would be the ideal test to prove the
actual fertility of a couple. The clinical in-
volvement required is becoming acceptable
with the recently introduced technique of
transvaginal follicle puncture, and the suc-
cess rate is rising constantly. IVF also reveals
functional disorders of the spermatozoa,
such as an insufficient acrosome, in ejaculates
which are otherwise completely normal.
However, the serious ethical considerations
as well as the mental stress on couples make
this technique currently only suitable for
therapeutic application.
147
4 The Steps in Ejaculate Analysis in Chronological Order

Basic Ejaculate Analysis (The Spermiogram)

1. Prepare equipment (see p. 5)

2. "Complete the top part of the record form (see p. 7)
3. Color (see p. 8)
4. Odor (see p. 8)
5. Liquefaction time (see p. 9)
6. Stringiness (viscosity) (see p. 9)
7. Volume (see p. 10)
8. pH (see p. 10)
9. Estimate motility in the unprepared specimen (see p. 13)
10. Fill into a leukocytometer (up to 0.5 mark) (see p. 21)
11. Dilute with 3% NaCI solution (up to 11 mark) (see p. 21)
12. Agitate (vibrator) (see p. 22)
13. Fill the counting chamber (see p. 23)
14. Count (determination of density) (see p. 24)
15. Stain: Testsimplets (see p. 33); Papanicolaou or similar (see p. 30)
16. Assess the stained smear (Atlas; see pp. 43-111)
17. Test vitality by means of the eosin test (see p. 121)
18. Determine fructose (see p. 125)

Extended Ejaculate Analysis in Practice

1. Test for IgG antibodies using the mixed antibody reaction test (see p. 121)
2. Penetration tests:
Penetrak test (see p. 140)
Sims-Huhner postcoital test (see p. 133)
Kremer-Jager sperm--cervical mucus contact test (see p. 136)
Hamster oocyte penetration test (see p. 146)
3. Hypoosmotic swelling test (see p. 144)
4. Semen separation (swim-up test; see p. 130)

148
5 Conclusion

The spermiogram only takes account of some of the possible aspects

of disturbed male fertility. Nevertheless, it is a simple and therefore
practicable method of investigation to provide a basic assessment of
a man's ability to reproduce. Although the ejaculate analysis provides
no conclusive evidence of a man's prospects of fertility, as the most
important parameter of semen quality it will nevertheless retain its cen-
tral role in the search for the reasons for involuntary childlessness.
Tests of spermatozoal function, some of which take account of factors
on the woman's side, are an extension to the diagnosis of fertility and
open up new prognostic perspectives.
When combined with and assessed simultaneously with the classical
semen parameters, they permit the prospects of fertility to the estimated
in a far higher percentage of cases. By means of simplified and improved
standardized tests it is possible today for any physician interested to
make a comprehensive diagnostic evaluation of a sterile couple in his
own practice and to draw his own conclusions with regard to their
treatment.

149
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157
Subject Index
The page numbers printed in italics are the pages on which the topic in question
is dealt with in greater detail

Abnormalities 39jJ., 43, 44-75 Cytology 90

ABP, see Antibody binding protein Cytophotometry 27
Abstinence 5,8, 10 Cytoplasmic remnant 90, 100
Acrosome 351[, 40, 147 Cytoplasmic appendages 41
Adenosine triphosphate 36jJ. Cytoplasmic receptors 38
Agglutination 17, 109, 122, 136
Aggregation 123, 124, 125 Degenerate spermatogenesis cells 79, 80, 88jJ.
Androgen binding protein 35ff. Density 21jJ.
Antigens 121 - determination in a Makler chamber 25
Antibodies 122, 131, 134, 135 - determination in counting chambers 23ff.
Antibodies to spermatozoa in cervical mucus - determination using electronic equipment 27
136 - evaluation 27
Aspermia 2 Differentiation
Astheno spermia 2, 7, 20, 28, 131 - of the acrosome 35ff.
Autoantibodies, see Antibodies - of leukocytes 29, 32jJ., 40, 92jJ., 102-104
Azoospermia 2, 27, 28 - of lymphocytes 29, 321[, 40, 102-104
- of macrophages 92ff.
Benzidine-cyanosin stain 9, 32jJ., 102-104 - morphological, of an ejaculate smear
Blood-testis barrier 37 38ff.
BMP test, see Penetrak test - of "round cells" 29, 32, 40, 921[, 102-104
Bovine mucus penetration test, see Penetrak test - of spermatogenesis cells 29, 34jJ., 40
Burker-Turk chamber 25 - of spermatozoa heads 29
Dihydrotestosterone 38
cAMP, see Cyclic adenosine-3' ,5' -monophosphate Dynamic spermiogram parameters 129,
Capacitation 12, 147 130jJ.
Cellulose wadding 21, 22
Cervical factor 131 Ejaculate 3jJ.
Cervical mucus 131-134, 137, 138 - analysis 4,148
- pH 133 - collection 5ff.
Cervix - color 8
- function 132 - composition 3
- index 132 - fractions 3, 4
- storage function 131 - liquefaction 9
- score 132 - macroscopic examination 7ff.
Chromatin condensation 36 - microscopic examination 11 ff.
Chromosome number 34 - morphological differentiation of the smear
Color 4 38ff.
Coulter counter 27 - odor 8
Counting chambers 21 ff. - obtaining 5
- Burker-Turk 25 - pH 10ff.
- Neubauer 21, 23jJ. - sexual abstinence 5, 8, 10
- Thoma-ZeiB 21, 24jJ. - split ejaculates 6
Counting the cellular elements in the ejaculate - variability 4ff.
21ff. - transport 3
- accurate count 23 - viscosity 9
- quick count 24 - volume 10
- when density is low 24 Ejaculation 3
Cover slips 13, 21, 22 ELISA, see Enzyme-linked immunosorbent assay
Cowper's glands 3 Emission 3
Crossing over 34 Endocrine regulation of testicular function 36ff.
Cryptorchidism 2, 27, 28 Enzymatic determination of fructose in seminal
Cyclic adenosine-3' ,5'-monophosphate 35 ff. plasma 125ff.

159
Enzyme-linked immunosorbent assay
Eosin test 4, 29, 121, 146
Epithelial cells 28, 40, 105-108
Estimation of motility 17
Expulsion of the nucleus 88-90
Extended ejaculate analysis 148
Fatal malformations 121
Fern test 132
Fertilization tests 129, 147
Fertilization prospects 130, 149
Fertilizing ability 12
Fertility, impaired 27, 28
Filter paper 21, 22
Flagellum abnormalities, see Tail abnormalities
Follicle-stimulating hormone 35 ff.
Freezability of spermatozoa 129, 146
Friberg's test 122
Fructose 4, 12, 125jJ.
Fructose in seminal plasma 125
FSH, see Follicle-stimulating hormone
Fuchsin solution 121
GAT, see Macrosperm agglutination test
Gelatin agglutination test, see Macrosperm, agglu-
tination test
Germinal cells 14, 34jJ., 40, 44-50, 76-91
Giant cells 85-91
Global motility 20
Globospermia 2, 40
Gonadotropin-releasing hormone 35ff.
Granulocytes 28, 40, 92-97, 102-104
Hamster oocyte penetration test 129, 145, 146,
147
Head agglutination 17
Head abnormalities 39, 43
Head cap 35
Hemafix staining 29, 33, 111
Hematospermia 2, 8
Hemospermia 2, 8
Hemocytometer 21
Heterologous ovum penetration test, see Hamster
oocyte penetration test
Hexokinase method 125ff.
HOP test, see Hamster oocyte penetration test
Hormonal interplay 35ff.
HOS test, see Hypoosmotic swelling test
Human in vitro fertilization 129, 147
Hyperspermia 2, 10
Hypoosmotic swelling test 129, 144jJ.
Hypospermia 2, 10
Hypothalamus 35
IgA antibodies 122, 131, 136, 140
IgG antibodies 122, 131, 136
Immobilization 122
Infertility, primary 1
Inhibin 37, 38
Insler's cervical score 132, 133, 140
In vitro fertilization 130, 147
160
122 Isoantibodies, see Antibodies
Isojima's test 122
Jeyendran's test, see Hypoosmotic swelling test
Kibrick's test, see Gelatin agglutination test
Kremer-Jager test, see Sperm-cervical mucus con-
tact test
Kremer test 129, 137jJ.
Kurzrock-Miller test, see Slide test
Lactate dehydrogenase 4
Laser-Doppler spectroscopy 17,20,27
Lazymot 17,20,27
Leptotene 34
Leukocytes 14, 40, 92-97, 102-104
Leukocytometer, see Mixing pipette
Leydig, cells 35ff.
LH, see Luteinizing hormone
Liquefaction of the ejaculate 9, 10
Littre's glands 3
Luteinizing hormone 35ff.
Lymphocytes 28, 40, 102-104
Main fraction (ejaculate) 3, 4
Makler chamber 17ff., 25, 26
Macrophages 97, 98-101
Macrosperm agglutination test 122
MAR test, see Mixed antiglobulin reaction test
Maturation division 34
May-Griinwald-Giemsa stain 29, 32, 33
Meiosis 34
Membrane stability test 144ff.
Metaphase 34
Micropipetter 21
Microscopy 13, 16
- bright-field 16
- interference phase-contrast 16, 123, 124, 125
- light 16, 121
- phase-contrast 13, 14, 15, 19, 121
Middle piece abnormalities 39, 43
Mitosis 13
Mixed antiglobulin reaction test 121 ff.
Mixing pipette 12
Monocytes 28, 97, 98-101
Morphology 28 ff.
- abnormalities 39jJ., 43, 44-75
- differentiation in the ejaculate smear 38 ff.
- differentiation of leukocytes 29, 92-97,
102-104
- differentiation of lymphocytes 29, 92-97,
102-104
- differentiation of "round cells" 29, 32
- differentiation of spermatogenesis cells 29,
34jJ., 44-50, 76-91
- specific 34 ff.
- in cases of varicocele 39
- in cervical mucus 133
Motility lljJ.
- after thawing 146
- and capacitation 12
- in cervical mucus 133 Receptor for luteinizing hormone 37, 38
- determination 13 Residual bodies, see Cytoplasmic remnant
- duration 12 "Round cells" 14, 15, 16,28,29,32,40, 124
- energy source 12, 13 Round-headed spermatozoa 40
- estimation method 13
- evaluation 20 SCMC test, see Penetrak test
- global 20 Sedimentation method of preparing spermatozoa
- loss of 12 130
- objective methods of determination 17ff. Seminal vesicles
- progressive motility 11, 12, 17, 20 - function 125
- qualitative 12, 20 - secretions 38
- quantitative 12, 20 Sertoli cells 3, 35/f
- speed 13, 20, 130, 133 Shaking 12, 18, 136, 137
- and the Swelling test 140 Sims-Huhner test, see Postcoital test
Movement, see Motility Slide test 129, 134/f
Multiple-exposure photography 17 ff. Slide racks 31, 32
Specific diagnostic tests for antibodies 122
Necrospermia 2 Sperm 2
Neubauer counting chamber, see Counting Sperm-cervical mucus contact test 129, 136jJ.
chambers Sperm washing 130 ff.
Nigrosin 121 Spermatids 34jJ., 40
Normospermia 28, 131 - early stages 83, 84
Number of spermatozoa 21 ff. - elongation 36
- late stages 44, 51
Oil immersion 33 - stage 34
Oligoasthenoteratospermia syndrome 2, 29, 122 Spermatocytes 34, 40, 77-82
Oligospermia 2, 7, 12, 27, 28, 121, 131 - stage 34
Overlay method of preparing spermatozoa 130, Spermatocytogenesis 34
131 Spermatogenesis 34ff.
Spermatogenesis cells 28, 34jJ., 40, 44-50, 76-91
Pachytene 34 Spermatogonia 34, 40, 76
Papanicolaou's stain 29, 30jJ. - stage 34
Pappenheim's stain 29, 32jJ. Spermatophagia 98-101
Penetrak test 129, 140jJ. Spermatozoa 2, 36
Penetration of cervical mucus 122, 134 - acrosomal malformation 70-72
Penetration ability 140 - atypical head shapes 65-69
Penetration tests 129, 131jJ., 140jJ. - bent tail 50-52
Peritoneal sperm migration test 129 - concentration 11, 21/f
Peritubular matrix 38 - cytoplasm 45, 51, 101
Peroxidase reaction 29, 32jJ., 102-104 - density 11, 21jJ.
pH 10ff. - freezability 129, 146
Phagocytes 28, 40 - head 35
Phagocytosis 98-101 - hormonally mature 44
Photometer - immature 44--46
- Eppendorf 127, 128 - immature, pathological 44, 47-50
- Shimadzu 127, 128 - immobilization 22
Plasma processes 35 - macrocephalic, see megalocephalic abnormality
Poly spermia 2, 28 - megalocephalic abnormality, amorphous 53-55
Postacrosomal part (sheath) 35, 40 - megalocephalic abnormality, nonamorphous
Postcoital test 29, 129, 133jJ. 56-88
Preejaculatory fraction 3 - megalocephalic abnormality, pseudomegaloce-
Preliminary fraction of the ejaculate 3, 4 phaly 59-61
Primary infertility 1 - microcephalic abnormality 57, 62-64
Progressive forward motility 11, 17, 140 - morphology 11
Prophase 34 - motility 11 ff.
Prostate gland, secretions of 3, 8 - number 11, 21jJ.
PSM test, see Peritoneal sperm migration test - preparation 130ff.
Pyospermia 2, 8, 104 - scanning electron micrographs 112-119
- speed of movement 13, 20, 130, 133
Regulator protein 37, 38 - tail abnormalities 71, 73-75
Receptor for follicle-stimulating hormone 37, 38 - transport 3

161
Spermatozoa, twinning 60 Tail agglutination 17
- unprepared specimen 16 Tail abnormalities 39, 43
Spermiogenesis 34, 35 ff. Tapering form 39, 110
Spermiogram 4 TAT see Tray agglutination test
- record form 7 Telophase 34
Spermiophagia 98 Teratospermia 2, 28, 12
Split ejaculate 6 Terminal fraction 3, 4
Staining 29 ff. Testosterone 35 ff.
--.: eosin and nigrosin 29, 121 Testsimplets 29, 33
- Hemafix 29, 111 Tight junctions 37
- May-Griinwald-Giemsa 29, 32, 33 Thoma-ZeiB, see Counting chambers
- Papanicolaou 29, 30jJ. Tray agglutination test 122
- peroxidase reaction 29, 32jJ.
- Testsimplets 29, 109jJ. Unprepared specimen 14, 15
Standard method of spermatozoal preparation
130
Steps in ejaculate analysis 148 Varicocele and specific morphology 39
Steroid synthesis 8, 38 Vibrator 21, 22
Sticking 147 Viscosimeter 9
Stringiness 9, 132 Viscosity 7, 9
Stroboscope 19 Vitality 121, 146
Surface antibodies 131, 134 Volume 10
Swelling test, see Hypoosmotic swelling test
Swim-up 129, 130f! Zygotene 34

162
 V.Jonas, University of Hannover; N.F.Dabhoiwala,
University of Amsterdam; F.M.J.Debmyne, Univer-
sity ofNijmegen (Eds.)

Endourology
New and Approved Techniques
1988. XI, 162 pp. 99 figs. 22 tabs. Hardcover
ISBN 3-540-18415-5
Contents: Internal Urethrotomy in Male Urethral Stric-
tures. - Bladder Neck Incision in the Male. - Endo-
urethral Teflon. - Antegrade Resection of Posterior
Urethral Valves. - Transperineal 1251 Seed Implantation
in Prostatic Cancer Guided by Transrectal Ultrasonog-
raphy. - Endoscanning of the Bladder and Prostate. -
Surgical Aspects of Transurethral Resection of Superfi-
cial Bladder Tumors. - Electrohydraulic Lithotripsy of
Bladder Stones. - TUR Using the Spoonloop Resec-
toscope. - Flexible Endoscopy of the Upper and Lower
Urinary Tract. - Endoscopic Correction ofVesicoure-
teric Reflux Using Subureteric Teflon Injection - the
Sting. - Ureterorenoscopy. - Role of the Ureteroscope
in Urological Surgery. - Percutaneous Treatment of
Staghorn Calculi. - Stones in Caliceal Diverticula:
Removal by Percutaneous Nephrolithotomy. - Percu-
taneous Coagulum Nephrolithotripsy: Clinical Experi-
ence. - Extraperitoneal Pelvioscopy.
Endourology provides a summary of the different
endourological modalities, especially the more
advanced and controversial techniques, such as the
antegrade resection of urethral valves, the transperineal
1251 seed implantation, the spoonloop resectoscope,
flexible endoscopy, teflon injection to correct vesico-
ureteral reflux, stone manipulation in calyceal divertic-
Springer-Verlag
Berlin Heidelberg New York ula, as well as the extraperitoneal pelvioscopy. These
London Paris Tokyo techniques are supported by descriptions of the stan-
Hong Kong dard urologic and endoscopic procedures.
 K.-H. Bichler, W. L. Strohmaier,
University ofTlibingen (Eds.)

Nephrocalcinosis,
Calcium Antagonists
and Kidney
1988. X, 190 pp. 107 figs.
Hardcover
ISBN 3-540-18119-9

The effect of calcium antagonists on the

kidney is to a large extent unknown. Pre-
liminary results of physiological investiga-
tions indicate that calcium antagonists
influence the canals in the distal part of
the tubule and the influx of calcium.
Experimental investigations on different
models in nephrocalcinosis point to the
effect of this group of substances as protec-
Springer-Verlag tives in acute renal failure, and also to a
Berlin Heidelberg New York possible application in renal transplantation
London Paris Tokyo
Hong Kong and urolithiasis.