International Journal of Andrology, 1991, 14, pages 108-1 16

Male infertility due to asthenozoospermia and

flagellar anomaly: detection in routine semen
analysis
D. M A R M O R and F. G R O B - M E N E N D E Z Unitk de Medecine
de la Reproduction, Service du Pr Ch.Roux, Hopital Saint-Antoine,
184, rue du Faubourg Saint-Antoine, Paris, France

Summary
Major monomorphous teratozoospermia, due to sperm tail structural anomalies,
were detected in 42 out of 4231 infertile patients during routine semen analysis. The
flagella were very short or absent in 16 cases, shortened with thickness irregularity
in 18 cases, and of normal length with diameter anomalies in eight cases. These
syndromes were always associated with poor forward motility, while mobility and
penetration into human cervical mucus were sometimes impaired only partially.
With good sperm smears and an experienced observer, the diagnosis could be made
without the need for electron microscopic analysis. All the affected patients were
sterile and several syndromes could have been transmitted genetically.

Keywords: asthenozoospermia, flagellum, infertility, sperm, spermatozoa.

Introduction
Sperm tail anomalies have been recognized as responsible for asthenozoospermia
and infertility. The ultrastructural features of several syndromes are well
documented. For example, abnormalities of the axoneme have been described
extensively: they provoke major asthenozoospermia, generally associated with
broncho-pulmonary diseases - ‘the immotile cilia syndrome’ (Afzelius, 1981).
Other flagellar defects, related mainly to peri-axonemal anomalies have also been
reported (Dadoune, 1988).
As these anomalies affect the length and/or the thickness of the sperm tail, we
surmised that they could be recognized by light microscopic analysis. This was
particularly interesting as electron microscopy of spermatozoa is a long and com-
plex process and cannot be performed in every fertility centre.
We therefore paid special attention to flagellar defects that could be detected
during routine semen analysis, and we have recently proposed a new morphological
classification of sperm tail anomalies (Marmor & Grob-Menendez, 1990). The use
of this classification for routine semen analysis allowed us to recognize major
monomorphous teratozoospermia due to flagellar structural anomalies, associated
with asthenozoospermia and infertility. The aim of the present study was to report

Correspondence: Dr Dorninique Marrnor, Unite de Medecinc de la Reproduction. Service du Pr Ch

Roux. Hopital Saint-Antoine, 184, rue du Faubourg Saint-Antoine, 75012, Paris. France.

108
 Sperm tail anomaly and asthenozoospermia 109
the frequency of such syndromes among the patients of our fertility centre, and to
describe their seminal and clinical features.

Patients and methods

Patients
Between January 1981 and December 1987, 4231 infertile men who were referred
to our laboratory provided at least one semen sample for analysis. All men had a
complete history taken together with a physical examination.

Semen collection and analysis

Semen was collected by masturbation at the laboratory, into special sterile plastic
cups. Previous sexual abstinence of 3 or 4 days was recommended. Semen speci-
mens were kept in an incubator at 37°C during analysis.
Sperm mobility (percentage of mobile sperm) and forward motility (quality of
sperm progression) were evaluted at 1 and 4 h after ejaculation, on an aliquot of
semen deposited between a slide and a coverslip. Motility was scored as 0 (com-
plete immotility), 1 (low), 2 (mediocre), 3 (average), or 4 (good). Sperm counts
were performed using a Neubauer cell.
Thin semen smears were air-dryed, fixed with ethanol-ether ( M ; v h ) , stained
with Harris’ haematoxylin and Shorr’s stain, and mounted. A total of 100 sperm
were examined under high magnification ( X 1250) using interferential contrast,
and classified according to a system based broadly on that of David et a f . (1975),
which allows classification of a sperm with several anomalies according to each
abnormality. For sperm tails, the following anomalies were recorded: absent,
short, thick, irregular diameter, coiled, multiple. Their features have been de-
scribed previously (David er al., 1975; Marmor & Grob-Menendez, 1990).
All sperm analyses were performed by the same observer.

Cervical mucus penetration

In-vitro sperm penetration tests into human cervical mucus were performed in 50 p1
pipettes (length = 6 cm), according to a method described already (Simeon, 1986).
In each case, a simultaneous test was performed with normal sperm in order to
assess the quality of the mucus; this was regarded as optimal if at least 10 sperm
were observed under each microscope field ( x 125) at the tip of the pipette within
30 min.

Statistics
Chi-square tests were used for statistical analysis.

Results
Semen analysis
Of the 4231 patients, 69 men were detected with a major monomorphous terato-
zoospermia (1.63%). In four cases, all of the sperm were macrocephalic and/or
multi-headed; in 23 cases the acrosome was malformed or absent, and in 42 cases
the sperm tails were abnormal. The latter patients had the following semen
110 D. Marmor and F. Grob-Menendez
features: their sperm count varied from 2 to 174 x 10' ml-' (mean = 55.2 x 10'
ml-' k 50.6). In 3 men, the sperm count was below 5 x lo6 ml-', and in 10 men it
was between 5 and 20 x lo6 ml-'. The other 29 men had normal sperm counts.
Sperm mobility varied from 0 to 50% (mean = 11.7 k 14%): it was zero or
below 10% in 30 cases, between 10 and 30% in four cases and above 30% in eight
cases.
In all cases, forward progressive motility was zero or very low for the majority
of sperm.

Sperm morphology
Sperm analysis revealed flagellar anomalies: sperm tails were abnormal in at least
80% of the sperm examined, with a distinct predominant anomaly. In 34 cases, the
flagella were shortened, and mobility was below 10% in all but six cases. In 16 men,
the flagella were very short ( 510 vm) and thick, or were absent (Fig. 1). In 18 men
the flagella were more or less shortened, with diameter anomalies (thick and/or
irregular) (Fig. 2).
In eight cases, the diameter of the sperm tail was irregular. In two men, the first
third of the flagellum was thickened, while the other two-thirds were abnormally
thin, as in a terminal piece (Fig. 3). In six cases, normal regions alternated with
abnormally thin zones of variable length (Fig. 4). Only two of these men had sperm
mobility below 30%.

Fig. 1. Very short tailed sperm (interference

contrast X 1250).
Sperm tail anomaly and asthenozoospermia 111

Fig. 2. Sperm exhibiting shortened flagella

with diameter anomalies (interference con-
trast x 687.5).

Fig. 3. Sperm tail diameter anomaly: long

midpiece syndrome (interference contrast
x 875).
112 D . Marmor and F. Grob-Menendez

Fig. 4. Flagellar diameter anomaly: altern-

ance of normal and thin zones (interference
contrast x 1187.5).

Fig. 5. A normal spermatozoan (interfer-

ence contrast x 750).
 Sperm tail anomaly and asthenozoospermia 113
Cervical mucus penetration
An in-vitro sperm penetration test into human cervical mucus was performed in 10
cases. Sperm penetration was nil or very poor in seven men (six cases of short
flagella, one case of irregular flagella). In three men with sperm exhibiting irregular
flagella, the sperm penetration was mediocre, a few sperm reaching the tip of the
micropipette filled with optimal cervical mucus within 30 min.

Clinical features
The mean age of the 42 men was 33 k 6.3 years (range 26-45 years). All were
referred for primary infertility, lasting from 1 to 17 years (mean = 5.4 k 4.5 years).
For 19 men, the duration of infertility was 2.5 years.
Twenty five of these patients (59.5%) were of Mediterranean origin, in particu-
lar 22 of the 34 patients with short sperm tails. For the other men attending our
laboratory for semen analysis during the course of the study (control population),
Mediterranean origin was noticed in 21.2% of cases. This difference was highly
significant (P<O.OOl).
Parental consanguinity and/or a notion of primary infertility in close family
(brother, uncle, first cousin) was reported by 10 patients (23.8%) versus 1.19% in
the control group (P<O.OOl).
Respiratory problems were reported by 11 men (26.2%) versus 8.9% in the
control population ( P < 0.001). These problems were generally minor: no patient
suffered serious bronchiectasis or necessitated repeated hospitalization. The trou-
bles consisted mainly of chronic sinusitis and frequent bronchitis.

Discussion
Among our infertile population of 4231 men, flagellar anomaly syndromes repre-
sented the most common monomorphous morphological abnormality of sperm
(60.9% of the major teratozoospermia). However, syndromes of flagellar anomaly
are rarely recognized during a routine semen analysis, and are even reputed to be
undetectable by conventional methods of semen analysis (Zamboni, 1987). In
general, diagnosis is established by electron microscopy, performed for low sperm
mobility or poor motility. This can be explained in several ways: first, many
morphological classifications of sperm do not include any sperm tail abnormality,
or only absent, coiled and multiple forms (MacLeod, 1968; Schoysman, 1988). In
particular, thick and irregular sperm tails are almost never taken into account.
Second, the diagnosis of subtle flagellar anomalies, such as irregular diameter,
requires thin sperm smears with no background, very good fixation and coloration
and an experienced observer. The system of David’s classification, which notes all
the anomalies of each spermatozoon, and the use of interferential contrast, greatly
help the diagnosis.
It is interesting to note that sperm mobility was not always very low, while
forward motility was constantly poor. Several authors have reported the import-
ance of a good sperm progression for in-vitro or in-vivo fertilization (Bostofte et a[.,
1983; Pusch, 1987; Talbert et al., 1987).
Penetration of human cervical mucus by sperm with irregular flagella was
possible in some cases, when mobility exceeded 30%. For these patients, several
114 D. Marmor and F. Grob-Menendez
attempts of intra-cervical insemination, intra-uterine insemination with washed
sperm and finally in-vitro fertilization have been performed, without any success.
Every patient affected by a syndrome of monomorphous sperm tail anomaly
had primary infertility, and no pregnancy has occurred since the diagnosis. It is
therefore probable that these syndromes are responsible for the sterility, as has
been described already for several mammals: rabbit (Maqsood. 195l ) , mouse
(Hollander er al., 1960), bull (Blom, 1966; Arriola et al., 1985), stallion (Che-
noweth er al., 1970) and boar (Holt, 1982).
The majority of the patients, especially those with short sperm tails were of
Mediterranean origin. The most frequent ethnic origin was North Africa (Algeria
and Tunisia). This has already been reported by Bisson et al. (1979). The same
authors also noted the high frequency of parental consanguinity and of infertility in
close male family: they hypothesed that the short tail anomaly could be a recessive
autosomic syndrome, as has been demonstrated for flagellar anomalies in several
mammals (MacLeod & McGee, 1950; Johnson & Hunt, 1971; Arriola et al., 1985;
Vierula et al., 1987). Our data on a larger population strengthen this interpretation.
The ultrastructural features of the sperm tail anomalies that we have detected
by light microscopic analysis have been described already. The very short flagella
were first reported by Baccetti er al. (1975) and Bisson & David (1975): the sperm
tail was replaced by a large cytoplasmic droplet, the axoneme and peri-axonemal
structures being absent or completely disorganized (McClure ef al., 1983).
The short and irregular sperm tails correspond to the fibrous sheath dystrophy
described by Ross et al. (1973) and de Chemes et al. (1987): the fibrous sheath
appeared hyperplastic and disorganized, and the axoneme was missing or grossly
distorted. The flagella of subnormal length, thickened in the proximal third and
then abnormally thin correspond to ‘the long middle piece syndrome’, the first
monomorphous sperm tail anomaly reported (Williams, 1950), characterized by an
unusually long mid-piece and an absence of fibrous sheath along the principal piece
(Pedersen et al., 1971).
For the flagella with thin regions alternating with areas of normal diameter, the
ultrastructural anomaly was described by Bisson et al. (1979), as localized interrup-
tions of the fibrous sheath.
Peri-axonemal structures were almost always involved in the flagellar anomalies
detectable by light microscopy. The role of these structures is not well known. They
are only present in species with internal fertilization and, therefore, have not been
much studied, as most of the fundamental experiments on flagella and on fertiliza-
tion have been performed in animals with external fertilization.
Several hypotheses have been raised: peri-axonemal structures could confer
stiffness to the sperm tail, thus helping propagation of the motility wave. O r they
could have some contractile properties due to actin-like and myosin-like substances
which enter into composition of the fibrous sheath (Fawcett. 1975; Gibbons, 1981).
It is interesting to note that peri-axonemal anomalies are often associated with
various axoneme abnormalities (Escalier & David, 1984). This could explain the
high frequency of respiratory problems in our patients, also noted by Bisson et al.
(1979). In fact, as respiratory cilia have no peri-axonemal structures they should
not be altered in men affected by a fibrous sheath anomaly. It is however probable
 Sperm tail anomaly and asthenozoospermia 115
that the axoneme anomalies frequently associated with the peri-axonemal anomaly
within the sperm tail are also present in the respiratory epithelium. On the other
hand, axoneme anomalies described in the immotile cilia syndrome are often
associated with various anomalies of the peri-axonemal structures of the sperm tail
(Afzelius & Eliasson, 1979; Escalier & David, 1984).
It can therefore be assumed that axoneme and periaxonemal structures are
encoded by very close genes or that their differentiation during spermiogenesis is
interdependent or is under the control of the same structures (Escalier & Serres,
1985).
No treatment can be proposed today for the patients affected by a sperm tail
structural anomaly. It has, however, been proved that sperm with abnormal tails
can undergo the acrosome reaction and penetrate zona-free hamster ova (Moryan
et at., 1986). Therefore, these men could benefit from the new techniques of
in-vitro fertilization with sperm micro-injection under the zona pellucida.

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Received 20 March 1990; accepted 7 September 1990