Human Reproduction vol.11 no.6 pp.1240-1243, 1996
Relationships between motility parameters, morphology
and acrosomal status of human spermatozoa
Jean Parinaud 1 , Gerard Vieitez, Helene Moutaffian,
Gerard Richoilley and Pierrette Milhet
Laboratoirc de F6condation In Vitro, CHU La Grave, 31052
Toulouse Cedex, France
'To whom correspondence should be addressed
A total of 130 semen samples were examined for motility
(by computer-assisted sperm analysis), morphology and
acrosomal status. A high positive correlation was found
between percentages of normal forms and progressive
motility in the whole semen (r = 0.539, P < 0.0001) as well
as in the Percoll fraction (r = 0.702, P < 0.0001). Among
the specific abnormalities, acrosome defects were most
highly correlated with progressive motility (r = -0.492,
P < 0.0001, in the Percoll fraction). The percentage of
total spontaneously acrosome-reacted spermatozoa in the
Percoll fraction was negatively correlated with the progressive motility (r = -0.499, P < 0.0001) and with the percentage
of normal forms (r = -0.430, P < 0.0001). Surprisingly,
the percentage of total spontaneously acrosome-reacted
spermatozoa was poorly linked with head abnormalities
but displayed significant positive correlations with the
percentages of bent tails (r = 0359, P < 0.0001) and of
coiled tails (r = 0371, P < 0.0001). These data suggest
that sperm defects are often linked together, reflecting
spermiogenesis and/or epididymal dysfunctions.
Key words: acrosome/human/morphology/motility/spermatozoa
Introduction
The fertilizing ability of spermatozoa involves numerous
functions such as motility, normal morphology and acrosome
function, and impairments of these functions have been reported
individually as causes of in-vitro fertilization (IVF) failures.
Indeed, Kruger et al. (1986) and Enginsu et al (1991) have
shown that high percentages of abnormal spermatozoa are
associated with impaired fertilization rates and we have demonstrated (Parinaud et al, 1993) that sperm morphology influences embryo quality and pregnancy rate in IVF. Motility,
especially computerized motion parameters, has been also
reported to be predictive of IVF results (Check et al, 1990;
Fetterolf et al., 1990; Kaskar et al, 1994). Acrosome dysfunctions, assessed through either the spontaneous acrosome reaction (Parinaud et al, 1995a) or the response to inducers such
as calcium ionophore A23187, progesterone or phorbol ester
(Fenichel et al, 1991; Tesarik and Mendoza, 1992; Parinaud
et al, 1995b), have been shown to discriminate between IVF
successes and failures. However, Heywinkel et al (1993) and
1240
Carrell et al. (1994) have shown close correlations between
morphological defects and decreased responses to acrosome
reaction inducers. Concerning motility, Calvo et al. (1994) and
Aitken etal (1994) have reported correlations between motility
and acrosome response to follicular fluid and calcium ionophore
respectively. Moreover, using an immunological technique to
select and characterize acrosome-reacted spermatozoa, our
group (Moutaffian and Parinaud, 1995) has reported that an
induced acrosome reaction occurs in the most morphologically
normal spermatozoa and is followed by a loss in longevity.
Therefore these data suggest the existence of a strong correlation between the parameters of motility, morphology and
acrosome function, and the present study aimed both to verify
this hypothesis using a large number of normal and abnormal
semen samples and to determine if the assessment of these
parameters has an additional predictive value for IVF results.
Materials and methods
Semen samples
A total of 130 semen samples were obtained from 130 patients
undergoing IVF. The causes of infertility were as follows: tubal (n =
34, 43%), endometriosis (n = 4, 5%), ovarian dysfunction (n = 6,
8%), male factors (n = 22, 28%), immunological factors (n = 2,
3%) and unexplained (n = 11, 14%). The mean abstinence period
prior to sample production was 5 ± 0.4 days. According to World
Health Organization (1992) criteria, 69 samples (53%) were classified
as normal. The means ± SEM for semen parameters were as follows:
112 ± HXlOfym] (median: 78, range: 1.8-815) for sperm count, 76
± 1% (median: 78, range: 8-95) for viability, 44 ± 2% (median: 44,
range: 2-85) for motility and 49 ± 1% (median: 50, range: 5-79)
for morphology. Fertilization was subsequently achieved in 109
cases (84%).
Sperm preparation
After 30 min liquefaction at 37°C, motile spermatozoa were isolated
using a discontinuous (60, 80, 90%) Percoll (Sigma Chemical Co,
St Louis, MO, USA) gradient Following centrifugation for 30 min
at 200 g, the 90% Percoll fraction was recovered and the sperm cells
were then washed once with Tyrode's solution (GIBCO BRL, Cergy
Pontoise, France). The final pellet was resuspended in INRA Me'ne'zo
B2 medium (Bio-Merieux, Marcy l'Etoile, France).
Sperm analysis
Sperm viability was evaluated after eosine-nigrosine staining.
Morphology was evaluated, in whole semen and after Percoll preparation, using Shorr staining (Merck, Darmstadt, Germany) according
to the criteria described by Jouannet et aL (1988). Acrosome morphology was classified according to Adelman and Cahill (1989).
Motility parameters were assessed in 20 (ll of semen for each
sample in a 2-well microcell (BICEF, L'Aigle, France), both for
© European Society for Human Reproduction and Embryology
Motility, morphology and acrosome
Table I. Correlations between motility parameters and sperm morphology in the whole semen
Motility
Normal forms (%)
Tapered heads (%)
Thin heads (%)
Microcephalous (%)
Acrosomal abnormalities (%)
Bent tails (%)
Coiled tails (%)
Multiple anomalies index
0.471 1
-0.096
-0.105
-0.287 b
-0.356"
-0214
-0197
-0.067
Progressive
motility (%)
VAP
(jim/s)
Rapid
motility (%)
ALH
0.539"
-0.185
-0.24 l c
-0.299*"
-0.405'
-O.235c
-0.242°
-0.178
0.4801
-0234 c
-0348 1
-O^SK)5
-0.394'
-0.201
-0.139
-0.3O2b
0.531'
-0.201
-0.229 c
-0.269*
-0.396'
-0.209
-0.262 c
-0.162
0.286b
-0.224 c
-0.214
-0.235 c
-0.22 T
-0.121
dun)
-0.045
VCL
(um/s)
VSL
Linearity
0.442"
-0.254 c
-0.326°
0.447"
-0.197
-0.324'
-O.3O7b
-0.370"
-0.194
-0.128
-O.SOO6
0.154
0.031
-0.124
-0.247 c
-0.143
-0.078
-0.108
-0.128
-o^g*
•
-0.174
-0.359"
-0.183
-0.119
-0.28 l b
"P < 0.0001; bP < 0.001; CP < 0.01.
whole semen and immediately after Percoll preparation, at 37°C using
a Hamilton-Thom Motility Analyzer (version 7.2; Hamilton-Thorn
Research, Beverly, MA, USA). The machine parameters were: frame
rate of 20 at 25/s, minimum contrast 8, minimum size 6, low/high
size gates 0.5/1.7, low/high intensity gates 0.4/1.7, non-motile head
size 9, non-motile intensity 200, a path velocity > 5 (irn/s to be
counted as motile. The variables measured included the percentage
of motile spermatozoa, straight line velocity (VSL), mean path
velocity (VAP), curvilinear velocity (VCL), mean linearity, mean
amplitude of lateral head displacement (ALH) and percentage of
motile spermatozoa with a path velocity >25 um/s expressed as the
rapid motility. Hyperactivated motility was defined as follows: VCL
>100 um/s, linearity <65% and ALH >7.5 urn.
Acrosomal status was assessed after Percoll preparation and resuspension of the pellet in B2 medium, using FTTC-GB24 monoclonal
antibody (Th6ramex, Monaco), which is specific for the inner acrosomal membrane (Ffnichel et aL, 1989). Viability was simultaneously
measured by staining with an ethidium homodimer (Interchim,
Montlucon, France). Following Percoll preparation, spermatozoa were
incubated for 30 min at 37°C in 100 |il of phosphate-buffered saline
(PBS), 0.15 mol/1 pH 7.4, containing 0.1% bovine serum albumin
(Sigma) (PBS-BSA), 50 mg/1 FTTC-GB24 (50ug/ml) and 10 umol/1
ethidium homodimer. After one wash with PBS-BSA, spermatozoa
were resuspended in 250 |il of PBS containing 0.3% formaldehyde.
The percentage of labelled spermatozoa was measured by an Epics
Profile cytofluorometer (Coultronics, Margency, France) (Parinaud
et al., 1995a). The percentages of live reacted spermatozoa and total
reacted spermatozoa were 8.3 ± 0.7% (median: 5.9, range: 0.6-47.1)
and 13.0 ± 0.9% (median: 9.7, range: 1.4-53) respectively.
Statistical analysis
All parameters were studied in all semen samples. Data are means
± SEM. Correlations were studied using the Pearson test and
were considered as statistically significant when P < 0.01. Partial
correlations were performed with the Programme Conversationnel de
Statistiques pour les Sciences et le Marketing software (Deltasoft,
Meylan, France).
Results
Correlations between motility parameters and morphology of
spermatozoa
As reported in Table L, there were high positive correlations
between the motility parameters, except for the ALH and the
linearity, and the percentage of normal forms, when studied
in whole semen. Among the specific abnormalities, acrosomal
defects had the greatest negative correlation with the motility
&
20
40
60
Progressive rootffltr (%)
Figure 1. Correlation between the percentages of normal forms and
of progressive motility after Percoll preparation (r = 0.702,
P < 0.0001) [% progressive motility = -0.0845 + (0.7531 X
% normal forms)].
parameters, and in particular with the percentage of progressive
motility. Conversely, tail abnormalities were only poorly correlated with motility.
These correlations were higher in the Percoll fraction than
in the whole semen. The percentage of progressive motility
was directly correlated with the percentages of normal forms
(r = 0.702; P < 0001) (Figure 1) and inversely with acrosomal
abnormalities (r = -0.492; P < 0.0001). However, the latter
correlation was due to the correlation between progressive
motility and percentage of normal forms since no significant
correlation (r = 0.083) was found between acrosome morphology and progressive motility for a constant percentage of
normal forms. The percentage of hyperactivated spermatozoa
was not significantly correlated with any of the morphological
parameters. On the other hand, the percentage of motile
spermatozoa and the viability were significantly correlated
(r = 0.398, P < 0.0001 in whole semen; r = 0.608, P <
0.0001 in the Percoll fraction).
1241
J.Parinaud et aL
#
2*
«
t»
M
Prognnin motillty (%)
Figure 2. Correlation between the percentages of total
spontaneously acrosome-reacted spermatozoa and of progressive
motility after Percoll preparation (r = -0.499, P < 0.0001)
[% progressive motility = 57.5643 - (0.9866 x % reacted
spermatozoa)].
Correlations between motility parameters and acrosomal
status of spermatozoa
The percentage of total spontaneously acrosome-reacted spermatozoa was significantly correlated with the motility parameters
and particularly with the percentage of progressive motility
(r = -0.499, P < 0.0001), as shown in Figure 2. This
correlation was independent of the morphology, since for a
constant percentage of normal forms, there was a significant
correlation between the percentages of total spontaneously
acrosome-reacted spermatozoa and of progressive motility
(r = -0.315, P < 0.0001).
The percentage of total spontaneously acrosome-reacted
spermatozoa showed no significant correlation with the percentage of hyperactivated spermatozoa (r = -0.195, NS).
Correlations between morphology and acrosomal status of
spermatozoa
Figure 3 shows a negative correlation between the percentages
of total spontaneously acrosome-reacted spermatozoa and of
normal forms (r = -0.430, P < 0.0001). Concerning the
specific abnormalities, the percentage of total spontaneously
acrosome-reacted spermatozoa was significantly linked to the
percentages of tapered heads (r = 0.247, P < 0.01), thin
heads (r = 0.267, P < 0.01), bent tails (r = 0.359, P <
0.0001), short tails (r = 0.273, P < 0.01) and coiled tails
(r = 0.371, P < 0.0001) and to the multiple abnormalities
index (r = 0.304, P < 0.001).
Predictive value of sperm parameters on FVF results
A multiple logistic regression analysis showed that the parameter most predictive of the percentage of fertilized oocytes
was the percentage of progressive motility, then the viability,
and then the percentages of acrosomal abnormalities, of total
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Normal forms (%)
Figure 3. Correlation between the percentages of normal forms and
of total spontaneously acTOsome-reacted spermatozoa after Percoll
preparation (r = -0.430, P < 0.0001) [% normal forms =
69.2090 - (0.5835 X % reacted spermatozoa)].
spontaneously reacted spermatozoa and of normal morphology.
However, when studied alone none of these parameters was
able to explain more than 14% of variance, while when taken
together, they could explain up to 21% of variance.
Discussion
The results of this study show strong correlations between the
parameters of motility, morphology and acrosomal status.
Indeed, we found a significant association between morphology
and motility. This is in good agreement with the data from
Kaskar et aL (1994), who, using a different classification
of morphology (Kruger et aL, 1986), reported significant
differences in some motility parameters (VSL and VCL)
among the different patterns of morphology (poor, good and
normal). However, this is the first report of correlations between
the motility parameters and specific abnormalities. A surprising
result concerns the absence of any linkage between tail
morphology and motility. Two hypotheses could explain this
finding. First, the immotility of sperm cells may be due in
part not to a defect in gross morphology but to a defect in
metabolism, and thus not be related to morphology. Furthermore, most immotile cells were non-viable cells and high
correlations occurred between the percentage of motile spermatozoa and viability. Second, most tail abnormalities leading to
impaired motility concern the microtubular and mitochondrial
ultrastructure and are not detectable using conventional assessment of morphology (Wilton et aL, 1992).
The percentage of spontaneously acrosome-reacted spermatozoa has been previously reported to be increased in cases
of male infertility (Fenichel et aL, 1991; Parinaud et aL,
1995a). It was negatively correlated to the percentage of
progressive motility. This is in good agreement with our
previous report concerning acrosome-reacted sperm selection
Motility, morphology and acrosome
(Moutaffian and Parinaud, 1995). Indeed, we have shown that
spermatozoa progressively lose their motility and have a
decreased longevity after acrosome reaction. The calcium flux
within the cells, an essential step in acrosome reaction (Fraser,
1993), may have a detrimental effect on motility and viability
(Fraser, 1987; Byrd et al, 1989) and explain the close
correlations between acrosomal status, motility and viability.
Moreover, since maturation of spermatozoa, and especially the
acquisition of motility and ability to undergo the acrosome
reaction, are known to occur in the epididymis (Haidl et al,
1994), impairments of both motility and acrosome function
could reflect an epididymal dysfunction (Haidl et al, 1993).
The absence of any correlation between the acrosomal status
and the percentage of hyperactivated spermatozoa is not
surprising since hyperactivation is known to reflect capacitation, which occurs before the acrosome reaction and thus,
even though these two events are associated, acrosome loss
occurs subsequent to hyperactivation (Robertson et al., 1988).
The percentage of total spontaneously acrosome-reacted
spermatozoa was negatively correlated with the percentage of
normal forms and positively with numerous abnormalities of
the head and the tail. This is in good agreement with data
from Heywinkel et al (1992) and Carrell et al. (1994), who
reported acrosome dysfunctions in spermatozoa with head
abnormalities. The correlation between acrosomal status and
tail abnormalities is more surprising. It could be explained by
a global spermiogenesis defect which may simultaneously
impair different parts of the spermatozoa, as suggested by the
linkage between acrosomal status and the multiple abnormalities index.
Despite high correlations between these parameters, their
predictive values are additive, highlighting the involvement of
all functions in the fertilizing ability of spermatozoa.
In conclusion, it appears that defects in motility, morphology
and acrosome function are closely linked, suggesting a global
impairment of spermiogenesis and/or epididymal functions.
Acknowledgements
This work was supported in part by grants from Theramex Laboratory,
Monaco and from Coultronics, Margency, France. The authors wish
to thank Roger Mieusset, MD, PhD (Centre de Sterilite Masculine,
Centre Hospitalier Universitaire La Grave, Toulouse, France) for his
help in the preparation of the manuscript
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Received on January 2, 1996; accepted on March 18. 1996
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