Note: Descriptions are shown in the official language in which they were submitted.
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MARKER FOR MALE INFERTILITY AND/OR FERTILITY
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a marker for male
infertility and/or fertility and to a method for the
diagnosis of male infertility.
(b) Description of Prior Art
Infertility occurs in approximately 8% of North
American couples. In 50% of these cases, male factors
contribute to this pathological situation. Standard
semen analysis including sperm concentration, motility,
and morphology are widely used as principal indicators
of male fertility. However, these parameters fail to
explain all cases of male infertility and do not pro-
vide a precise prognostic value of human fertility in
vivo or in vitro. Due to the fact that standard semen
parameters often provide a poor prognostic value, many
other laboratory tests have been developed in order to
evaluate the fertilizing ability of human spermatozoa.
These include the determination of sperm membrane
integrity by hypoosmotic swelling test, nuclear matur-
ity, acrosomal status, motility assessment by video-
imaging as well as the ability of sperm to interact
with the oocyte. This sperm function has been evalu-
ated by the zona-free hamster test and by the ability
of human spermatozoa to bind to homologous zonae pellu-
cidae. Therapeutic in vitro fertilization represents a
direct evaluation of the fertilizing ability of semen.
The prognostic value of the various andrology tests can
thus be determined by correlating their conclusions
with in vitro fertilization results. The relationship
between the different tests and fertilization rates in
vitro have shown a very high correlation with the abil-
ity of sperm to bind to the zona pellucida, whereas
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normal morphological sperm and linearity of motility
correlated with a lower significance. Other sperm
parameters have been shown to be of no prognostic value
(Liu D.Y. et al., 1992, Fertil. Steril., 58:465-483).
While morphology and linearity are sperm parameters
that could easily be assessed, the sperm-zona pellucida
binding assay is difficult to perform mainly due to the
limited availability of human zonae pellucidae and the
technical difficulties associated with a large inter-
assay variability.
The binding of sperm to the zona pellucida is
an absolute prerequisite for fertilization in man as
well as in other mammalian species and appears to be
species-specific. The failure of sperm-zona pellucida
binding may be associated with certain cases of male
infertility. Therefore, laboratory tests that evaluate
the ability of sperm to undergo successfully this step
in gamete interaction could have clinical usefulness in
the diagnosis of male infertility.
Using different animal models, it has been
shown that during epididymal transit the male gamete
acquires its fertilizing ability. During this matura-
tional process, sperm surface proteins are added or
modified and these modifications are involved in the
acquisition of the ability to the spermatozoa to bind
to the zona pellucida.
A 34kDa human epididymal sperm protein (P34H)
was previously described and it was proposed to be
involved in the interaction of human spermatozoa and
the homologous zona pellucida (Boue F. et al., 1994,
Biol. Reprod., 51:577-587). P34H appears on the acro-
somal cap of the spermatozoa within the caput
epididymis. Furthermore, a polyclonal antibody against
P34H interferes with the ability of spermatozoa to bind
the human zona pellucida, without affecting motility,
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the acrosome reaction and the fusion with the plasma
membrane of the oocyte. Thus, P34H appears to be
involved in one of the absolute prerequisites of fer-
tilization, i.e. sperm-zona pellucida binding.
Boue F. et al. (1995, Biol. Reprod., 52:71,
Abstract No. 57) mentionned that 6/12 infertile men
showed a P34H of less than 20% of the normal value
whereas 6/12 of the infertile men were rated as
undetermined fertility or as normal.
It would be highly desirable to be provided
with a marker for male infertility and/or fertility.
It would be highly desirable to be provided
with a method for the diagnosis of male infertility.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
a marker for male infertility and/or fertility.
Another aim of the present invention is to pro-
vide for a method for the diagnosis of male infertil-
ity.
In accordance with the present invention there
is provided a method for the diagnosis of male
infertility which comprises the steps of:
a) determining the amount of P34H or a related
antigen thereof in a sperm sample; and
b) comparing the determined amount of step a) with
a positive control having an amount of P34H or a
related antigen thereof of the levels of fertile
samples, wherein a P34H or a related antigen value
lower than 30% relative to the positive control sample
is indicative of infertility.
Preferably, the amount of P34H or a related
antigen in step a) may be determined using an antibody
raised aqainst one member selected from the group
consisting of P34H, a related antigen thereof and
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fragments thereof. Most preferably, the antibody is
raised against P34H. The antibody may be a polyclonal
or a monoclonal antibody.
Preferably, with the method of the present
invention P34H is determined.
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In accordance with the present invention there
is provided A kit for the diagnosis of male infertility
which comprises:
a) an antibody raised against one member selected
from the group consisting of P34H, a related antigen
thereof and fragments thereof;
b) a reagent for detecting the antibody of a)
linked to P34H or a related antigen thereof; and
c) a positive control having an amount of P34H or a
related antigen thereof of the levels of fertile
samples.
In accordance with another embodiment of the
present invention, the antibody is enzyme-labeled and
the reagent is an enzyme substrate.
In accordance with another embodiment of the
present invention, the reagent is a radiolabeled P34H.
In accordance with another embodiment of the
present invention, the positive control is a fertile
sample.
For the purpose of the present invention the
following terms are defined below.
"P34H or a related antigen thereof" refers to
the protein P34H or a protein with an homology of about
70% with P34H which would have a biological function
equivalent to that of P34H. Also fragments of P34H or
of related antigens thereof may be used in accordance
with the present invention.
The method and the kit of the present invention
may be used to determine male infertility of man and
animals which express P34H or a related antigen
thereof. The common characteristic of all the male
tested in accordance with the present invention is the
present of P34H or a related antigen thereof in their
sperm sample.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1A and 1B illustrate silver stained
(Fig. 1A) and corresponding immunoblots (Fig. 1B)
probed with anti-P34H antiserum of SDS-PAGE of proteins
5 extracted from 5.106 human spermatozoa from fertile men
(C+) and infertile men (1-7);
Fig. 2 is an analysis of amounts of P34H as
determined by densitometric scan of immunoblots of pro-
teins extracted from 5.106 spermatozoa from fertile (o)
and infertile (=) men;
Figs. 3A and 3B illustrate silver stained
(Fig. 3A) and corresponding immunoblots (Fig. 3B)
probed with anti-P34H antiserum of SDS-PAGE of proteins
extracted from 5.106 human spermatozoa from fertile men
before (1) or after a discontinuous Percoll centrifuga-
tion (2,3); and
Figs. 4A-4C are Western blot analyses of the
number of spermatozoa from control fertile donor (X
axis) or from different men (Y axis) bound per human
zona pellucida.
DETAILED DESCRIPTION OF THE INVENTION
The interaction of spermatozoa with the zona
pellucida is a critical step of fertilization. Spe-
cific sperm surface proteins involved in this process
could be added or modified during epididymal transit.
A 34 kDa human epididymal sperm protein (P34H) was pro-
posed to be involved in sperm-zona pellucida interac-
tion. In this study, Western blot analyses were per-
formed to determine the level of P34H protein present
on the spermatozoa of 16 men with idiopathic infertil-
ity. These levels were compared with the amount found
in men of proven fertility. In addition, a sperm-zona
pellucida binding assay was performed with spermatozoa
from fertile and infertile men. Individual men have
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similar P34H levels on their spermatozoa obtained from
different semen samples. However, the amount of P34
varied from one man to another. Compared to a popula-
tion of fertile men, 9/16 infertile men had a P34H
level of less than 30% of the normal value, while the
remaining 7 males were in the normal range. Sperm from
infertile subjects with a normal P34H determination
bound to the zonae pellucidae as efficiently as fertile
controls. However, spermatozoa from subjects with a
low amount P34H exhibited a dramatic diminution in
their ability to interact with zonae pellucidae. The
results show that the quantity of the epididymal pro-
tein, P34H, varied from one male to another and that
low levels of this epididymal sperm protein are associ-
ated with certain cases of idiopathic infertility.
Spermatozoa collection procedure
Sixteen couples presenting with primary infer-
tility were included in this study. Infertility was
defined by a period of at least 30 months of unpro-
tected sexual intercourse. In all cases, infertility
was classified as idiopathic based on a full infertil-
ity work-up. In women, clinical investigation
included; negative hysterosalpingography and laparo-
scopy, normal ovulation as evaluated by basal body tem-
perature, plasma hormone concentrations, normal
endometrial biopsy and post-coital test. The men had
produced at least three normal spermiograms as defined
by the WHO criteria and were free from anti-sperm anti-
bodies as determined by a negative immunobead test.
These healthy men were between 23 and 39 years of age,
a group comparable to the group of fertile volunteers
used in this study. The latter had fathered a child
less than 3 years prior to this study and had shown
normal spermogram values in accordance with WHO stan-
dards. Between two and seven days of sexual abstinence
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was required before semen was collected by masturba-
tion. After a liquefaction period of 30-60 min. at
room temperature, the usual spermogram values were
determined before processing the semen samples. No
correlation could be established between these values
and the levels of P34H as determined in a constant
number of spermatozoa from each sample. Spermatozoa
were washed three times by centrifugation (800g) in D-
PBS (Dulbecco-phosphate buffered saline: Gibco) and
resuspended in SDS-PAGE sample buffer (50 mM Tris-HC1
buffer, pH 6.8, 2% sodium dodecyl sulfate and 5% b-mer-
captoethanol at a final concentration of 5.105 sperma-
tozoa/ l. These samples were heat denatured at 95 C
for three min., centrifuged at 14 000g and supernatants
were kept at -20 C until used.
To compare spermatozoa with different buoyant
densities, aliquots of semen samples were loaded onto a
discontinuous PercollT"" gradient. This gradient was
composed of two layers of PercollT"~ (90% and 45%) pre-
pared in D-PBS. After centrifugation at 1 300g, sper-
matozoa were collected at the 45-90% Percoll interface
and in the pellet below the 90% Percoll layer. Sper-
matozoa were washed in D-PBS and were resuspended in
sample buffer at the final concentration of 5.105 sper-
matozoa/ l and processed as described above.
Western blot analysis
Proteins from 5.106 spermatozoa from each semen
sample were submitted to electrophoresis in presence of
sodium dodecyl sulfate on a 12% acrylamide gel. The
gels were stained with silver nitrate or electro-trans-
ferred to nitrocellulose membranes. These Western
blots were saturated for two hours in PBS containing
10% defatted milk and 2% normal goat serum followed by
an overnight incubation with the anti-P34H antiserum
diluted 1:1000 in PBS supplemented with 1% goat serum.
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Three volumes of anti-P34H polyclonal antiserum was
first adsorbed overnight on one volume of powder of
human keratin. After three washes with 0.1% (V/V)
TweenT"" 20 in PBS, nitrocellulose membranes were incu-
bated for 1 hour with a peroxidase-conjugated anti-rab-
bit IgG (1/3000 in PBS) and washed three times with
PBS-TweenT"'. Immune complexes were detected with a
BioMaxT"' film (Kodak) following incubation with a
chemiluminescent substrate of peroxidase (ECL Kit;
Amersham, Buckinghamshire, UK) according to the sup-
plier's instructions.
Quantification of P34H
In order to quantitate the P34H level, three
independent Western blots of each sperm sample were
scanned using an LKB UltroScan XLTM Laser Densitometer.
The surface under the curve corresponding to the amount
of P34H in each sample of 5.106 spermatozoa was deter-
mined and expressed as a percentage of an internal
positive control. The latter consisted of a sperm sam-
ple from a healthy donor in which the level of P34H was
defined as 100%. In each Western blot, a sperm sample
from this donor was processed in parallel in order to
serve as an internal standard.
Human sperm-zona pellucida binding
Human sperm-zona pellucida binding assays were
performed with spermatozoa labeled with two different
fluorochromes. In this study, human oocytes obtained
in the context of therapeutic in vitro fertilization
and that had remained unfertilized after 48-60 h post
insemination were stored in liquid nitrogen. Immedi-
ately prior to use, oocytes were washed by transfer
into five successive baths of D-PBS containing 0.5% BSA
(bovine serum albumin, fraction V) followed by a final
wash in B2 medium (Bio Merieux, Marcy-L'Etoile,
France). Semen samples were submitted to a discon-
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tinuous Percoll gradient as described above. Motile
spermatozoa, recovered in the pellet under the 90% Per-
coll layer, were capacitated in B2 medium at 37 C in 5%
C02 for 3 hours. Spermatozoa prepared from semen of
different men were labeled with one of the two fluoro-
chromes FITC (fluorescein) or TRITC
(tetramethylrhodamine isothiocyanate). Spermatozoa
from a fertile donor that were used as a control were
labeled with the other fluorochrome. After labeling,
spermatozoa were washed and resuspended in B2 medium at
a final concentration of 2.105 motile spermatozoa/ml.
Equal volumes of FITC and TRITC labeled spermatozoa
were mixed and incubated at 37 C in 5% C02 with human
zonae pellucidae. Two hours later, the zonae pelluci-
dae were extensively washed by successive pipetting and
individually observed under an epifluorescence micro-
scope at the appropriate wavelength corresponding to
FITC and TRITC in order to determine the number of
spermatozoa from each category that bound to the zona
pellucida.
Statistical Analysis
The distribution-free test U of Mann and Whit-
ney was performed to evaluate the difference in the
level of P34H between the two populations of infertile
and fertile men. Human sperm-zona pellucida binding
assays were studied by linear regression analysis, and
compared to the theoretical line by the Student "t"
test.
Results
Silver stained electrophoretic patterns of
sperm proteins from different semen samples are illus-
trated in Fig. lA. Molecular weight standards (103
kDa) are indicated on the left. Minor differences in
the protein patterns were observed from one sample to
the other while the overall protein solubilized under
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reducing conditions were consistent in all samples.
Although the patterns of proteins were= similar, the
corresponding Western blots probed with the anti-P34H
revealed dramatic differences in the amount of this
particular sperm antigen (Fig. 1B). Western blotting
was necessary to detect these differences due to the
fact that P34H, which is a very minor sperm protein,
cannot be detected in the stained electrophoretic pro-
tein patterns, in spite of the use of the highly sen-
sitive silver staining procedure (Fig. lA).
Western blots probed with anti-P34H antiserum
exhibited a single band at 34 kDa corresponding to
P34H. Whereas the electrophoretic mobility of P34H was
similar from one sample to the other, the intensity of
this band was highly variable (Fig. 2). The results
are expressed as a percentage of an internal positive
control (C+ = 100%). Group A and Group B were similar,
whereas, Group C was significantly different from group
A(p<0.001). Western blotting was performed on a
constant number of spermatozoa obtained from 16 sub-
jects presenting with idiopathic infertility as well as
from 17 fertile donors. When quantified by densi-
tometric scanning, the levels of P34H in fertile donors
varied between 57% and 129% in comparison to the
internal positive control (C+) representing 100%. On
the other hand, the levels of P34H in those samples
obtained from infertile patients were between 3% and
117%. Two populations of infertile patients were
therefore defined according to the amount of P34H
detected. Those with a P34H value lower than 30% were
significantly different from the control fertile group
with a P< 0.001. Idiopathic infertile men with higher
values of P34H were comparable to the fertile group.
Thus, 9 out of 16 infertile men with normal spermogram
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values were characterized by values of P34H that were
significantly lower than the fertile group.
In order to ascertain whether variations in the
P34H levels could be associated with certain cases of
infertility rather than with an intersample variabil-
ity, we have determined this parameter on a constant
number of sperm from more than one semen sample col-
lected from different men. This data demonstrated that
the expression of P34H was constant from one semen sam-
ple to the other in any one individual (Table 1). This
was true for fertile men as well as for infertile men
that expressed or did not express P34H on their sperma-
tozoa. Even though the case history of PGO and JLA
were similar, the amount of P34H that characterized
these two infertile men was quite different. Sperma-
tozoa from semen samples separated by Percoll gradient
centrifugation also exhibited comparable P34H values
even though they were characterized by different buoy-
ant densities (Fig. 3).
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Table 1
P34H determination in different semen samples from
fertile and infertile men
Semen sample P34H Mean
(% of control +)
Fertile
#1 2 125 % 108 %
91 %
#2 2 100 % 107.5 %
115 %
Infertile
PGO 2 92 % 102.5 %
113 %
JLA 2 8 0 9%
0
In order to correlate the presence of P34H with
fertilizing ability, we have performed in vitro zona
pellucida binding assays using spermatozoa with various
10 levels of P34H. Each zona pellucida was inseminated
with sperm from a fertile donor along with the same
number of sperm from a second fertile donor (Fig. 4A),
as well as with sperm obtained from infertile men with
a normal level of P34H (Fig. 4B) or infertile men with
low levels of P34H (Fig. 4C). The Y axis corresponds
to fertile (A), infertile men with (B) or without (C)
P34H. Each value corresponds to one zona pellucida.
The regression line corresponding to each panel is
illustrated. The number of sperm from each experimen-
tal group (Y axis in Fig. 4) was compared with the
internal fertile control (X axis in Fig. 4). Simple
regression analysis of each experiment indicated that
the evaluation of the sperm binding ability by the
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double staining technique used was consistent from one
zona pellucida to another with a significance of
p<0.002 (Table 2).
Table 2
Statistical analysis of zona pellucida binding ability
of spermatozoa from fertile and infertile men
Linear r.squared simple linear regression
regression regression vs. theoretical line
equation probability significant differ-
ence
theoretical Y=1X 1
(median)
Fertile/Fertile Y=1.1X - 0.75 0.85 P< 0.001 NS
Fertile/infertile with Y=0.97X + 1.3 0.75 P< 0.001 NS
P34H
Fertile/infertile Y=0.14X + 1.44 0.52 P < 0.002 P< 0.001
without P34H
This allowed the comparison of the regression
line calculated from each experiment with a theoretical
line where all zonae pellucidae would be on the median
e.g. an ideal situation where each spermatozoon on the
Y axis shows the same binding ability as those from a
positive fertile control on the X axis. In this
situation, only the comparison of the sperm binding
ability between fertile positives and infertile sub-
jects without P34H (Fig. 4C) showed a highly signifi-
cant difference at P<0.001. Thus, spermatozoa with
normal levels of P34H had the same ability to bind to
zonae pellucidae regardless of the fertility status of
the donor. Compared to healthy donors, spermatozoa
obtained from males without P34H showed a highly sig-
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nificant decrease in their ability to bind to the zona
pellucida with a P<0.001 (Table 2).
Discussion
During the past 15 years the increasing inves-
tigation of the causes of infertility has shown that
male reproductive dysfunctions are primary factors in
up to 40% of those couples consulting for infertility.
Standard semen analysis including sperm concentration,
motility and morphology have provided relevant clinical
information mainly in those extreme cases of male
infertility such as azoospermia, severe oligo-astheno-
spermia and total terato or necrospermia. However, in
the majority of cases, the usual spermiogram parameters
have low prognostic values for the in vivo and in
vitro fertilizing capacity of spermatozoa. In vitro
fertilization is useful to evaluate sperm function but
cannot be used for diagnostic purposes. Many new func-
tional tests have thus been developed and used to
evaluate the fertilizing capacity of human sperm.
Although some of these are recommended by the WHO, they
provide limited information with regard to fertiliza-
tion and are of questionable prognostic value. It
would appear that those experimental procedures that
evaluate the ability of spermatozoa to bind to homolo-
gous zona pellucida have a high correlation with fer-
tilization potential (Liu D.Y. et al., 1992, Fertil.
Steril., 58:465-483).
The binding of spermatozoa to the homologous
zona pellucida involves a receptor on the male gamete
with a high affinity for its ligand. In the hamster,
we have previously identified a sperm protein, P26h,
that meets many criteria that are essential in order to
be considered as a zona pellucida receptor (B6rube B.
et al., 1994, Bio. Reprod., 51:1255-1263). Using anti-
bodies raised against P26h we have recently identified
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a human sperm protein, P34H, that shows antigenic and
functional homologies with the hamster P26h (Boue F. et
al., 1994, Biol. Reprod., 51:577-587). We have thus
proposed that P34H is involved in the processes medi-
ating sperm-zona pellucida interaction in humans.
P34H is localized on the surface covering the
acrosomal cap of ejaculated human spermatozoa (Boue F.
et al., 1994, Biol. Reprod., 51:577-587); a localiza-
tion that is in agreement with the proposed function.
In fact, it is via this subcellular region that the
male gamete interacts with the zona pellucida. The
amount of P34H found on a constant number of spermato-
zoa was highly variable from one individual to the
other (Figs. 1 and 2). Interestingly, the protein is
present on spermatozoa of all these fertile men that
were investigated while lower amounts of this sperm
antigen were encountered only in normospermic infertile
men (Fig. 2). This observation is in agreement with
the proposed function of P34H and the fact that the
sperm zona binding ability is of high prognostic value
for male fertility (Liu D.Y. et al., 1992, Fertil.
Steril., 58:465-483). This is directly demonstrated by
the fact that spermatozoa from fertile as well as from
infertile men with normal P34H values are able to bind
to homologous zona pellucida whereas spermatozoa from
infertile men with low levels of P34H are unable to
interact with the egg's extracellular coat (Fig. 4 and
Table 2). Considering that the presence of sufficient
amounts of P34H can be correlated with human sperm
ability to bind to the zona pellucida, this sperm
antigen could thus be considered as a biochemical
marker of male fertility. In this regard, it is
important to note that the variability of P34H is due
to an inter-individual variability and that this
parameter is consistent from one semen sample to the
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other from the same individual (Table 1). The
variation of P34H on spermatozoa of different buoyant
densities could not explain the important variation of
P34H level found in our group of infertile men
(Fig. 3). Two populations of idiopathic infertile men
can be defined on the basis of P34H levels. Over 50%
(9/16 in this study) of men presenting with idiopathic
infertility are characterized by low levels of P34H
(group C in Fig. 2) and spermatozoa from the latter
group are unable to bind to zona pellucida (Fig. 4C and
Table 2). It is obvious that infertility is a
multifactorial disease and the absence of one sperm
antigen cannot account for all of those cases of unex-
plained male infertility. Nevertheless, a high pro-
portion of these men from infertile couples investi-
gated in this study show an abnormally low level of
P34H.
P34H is a biochemical marker that could be
readily used for the diagnosis of male infertility. In
these cases, infertility is associated with the inabil-
ity of the spermatozoa to interact efficiently with
the zona pellucida. The rather high occurrence of this
defect in our normospermic infertile man population
suggests that dysfunction may be a common cause of
infertility. In vitro fertilization is presently
applied to overcome certain cases of male infertility.
Compared to the results obtained when these technolo-
gies are applied to overcome tubal diseases, the
chances of successful in vitro fertilization applied to
male infertility is much lower. Thus, the determina-
tion of P34H levels could be relevant by avoiding
clinical procedures that cannot overcome the inability
of the spermatozoa to penetrate the egg investments in
vivo as well as in vitro.
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While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
