Sperm motility assay and devices

The present invention provides devices, methods and kits for assessing fertility potential of semen samples. Semen samples may be assayed by the present invention to identify those samples having more than 20.times.10.sup.6 motile sperm per milliliter, a concentration associated with fertile samples.

BACKGROUND OF THE INVENTION 
The present invention provides devices, methods, and kits for convenient 
determination of male fertility parameters. More specifically, the present 
invention is able to detect semen samples having a sufficient 
concentration of motile sperm to support fertility. The devices, methods 
and kits of the present invention provide easy, one-step assays for such 
determinations. 
Fertility testing is becoming more widespread as increasing numbers of 
apparently infertile couples seek medical assistance in conception. 
Because reproductive abnormalities of both sexes may affect fertility, 
assessing male fertility is common in fertility evaluations. 
Male infertility may be due to a number of factors, including hormonal 
anomalies, work place exposures, and sequelae of infectious diseases. Even 
common childhood illnesses such as mumps may cause male infertility. Thus, 
most males are at some risk for infertility and should be evaluated in any 
fertility work-up. 
The most common starting point for evaluation of male fertility is an 
assessment of the sperm count in semen. The most commonly accepted figure 
as the lower limit of normal is 20.times.10.sup.6 motile sperm per 
milliliter. Even more important to fertility than absolute sperm count, 
however, is sperm motility. Fertility Study, 31:305 (1979). Therefore, in 
male fertility analyses sperm motility must also be determined. 
Currently available techniques for measuring sperm count and sperm mobility 
are microscopic in nature. Sperm morphology and motility is visually 
assessed by laboratory technicians. Because semen evaluation is 
essentially qualitative, substantial experience is required for accurate 
evaluation by the technicians. The high level of experience required by 
laboratory technicians precludes general office evaluation of semen 
samples and generally requires referral to a reference laboratory. 
Further, debris in semen samples can cause erroneous or inconsistent 
results. 
Accurate assessment of sperm motility also requires that the semen sample 
be fresh. Since the sample must be analyzed by a reference laboratory, the 
sample must usually be obtained at the laboratory. Often this requires 
additional appointments at medical facilities and time away from work. 
This can be inconvenient for many patients and even a hardship for 
patients living in rural areas far away from reference laboratories. 
Attempts to develop biochemical assays of semen have not resulted in simple 
procedures which may be performed in the physician's office. Most 
biochemical markers have failed to demonstrate correlations with either 
sperm number or motility. Fumarase activity, an enzyme present in semen, 
has been found to correlate to both sperm count and percentage motility. 
Crabbe, J. Reprod. Fert., 51:73-76 (1977). Crabbe measured fumarase 
activity by spectrophotometric measurements. Unfortunately, 
spectrophotometric assays are not generally suitable for office assays 
because of the cost of these specialized devices as well as the training 
required for accurate and reproducible operation. 
What is needed in the art is a simple assay for assessing sperm count and 
motility that does not require expensive specialized instrumentation or 
extensive training. One step assays that could be performed according to 
easy to follow instructions without specialized training would be optimal. 
Surprisingly, the present invention satisfies these and other needs. 
SUMMARY OF THE INVENTION 
The present invention provides devices for assessing sperm number and 
motility in semen samples. The devices comprise a solid support having a 
carrier matrix containing a fumarase substrate and malate dehydrogenase. 
Generally, the fumarase substrate will be fumaric acid, a fumarate salt, 
or a fumarate ester. Conveniently, the device is provided in a dipstick 
form. 
Also provided are methods for identifying a semen sample having more than 
about 20.times.10.sup.6 mobile sperm per milliliter. The methods generally 
comprise applying the sample to a carrier matrix on a solid support, which 
carrier matrix contains a fumarase substrate and malate dehydrogenase; and 
detecting a visual signal from the solid support resulting from metabolism 
of the fumarase substrate by fumarase present in the sample. Similar to 
the devices above, the fumarase substrate will generally be fumaric acid, 
a fumarate salt, or a fumarate ester. Often, the visual signal will be a 
color change that may be detected visually or by an instrument. Typically, 
the visual signal will be detected within 10 minutes of applying the 
sample to the carrier matrix. 
Kits for the detection of semen samples containing more than about 
20.times.10.sup.6 motile sperm cells per milliliter are also provided by 
the present invention. The kits include devices of the present invention 
and semen collection vials. Instruments to detect the visual signal or 
reference color charts may also be included in the kits.

DESCRIPTION OF SPECIFIC EMBODIMENTS 
The present invention provides convenient assays for detection of motile 
sperm in a semen sample. As noted above, fumarase enzyme activity has been 
correlated with the concentration of viable, motile sperm in a semen 
sample. The assays quantitatively detect fumarase enzyme in the sample. 
The assays of the present invention may be constructed on a dipstick which 
provides a one-step method of analysis. Generally, fumarase activity is 
assessed by a color change on the dipstick. The assay is rapid, easy to 
use with little training, and can be performed in the clinician's office. 
Fumarase enzyme converts fumaric acid to malic acid. Fumarase also converts 
fumarate salts and esters to malic acid. Hereinafter, fumaric acid will be 
understood to include fumarate salts and fumarate esters. The assay 
provides fumaric acid in a carrier matrix. The fumaric acid is typically 
supplied in excess in the carrier matrix so that formula fumarase activity 
will be rate limiting. The conversion of fumaric acid to malic acid is 
coupled to a system that will generate a visual signal. The visual signal 
will provide a quantitative assessment of the fumarase activity in the 
sample. The fumarate substrate coupling of the generation of the visual 
signal to reduction to malic acids generally occurs as outlined below: 
##STR1## 
Generally, the color generating system will be a nicotinamide adenine 
dinucleotide (NADH) dehydrogenase system capable of reducing a dye to 
create a color change. Preferably, malic dehydrogenase is present in the 
carrier matrix. The malic dehydrogenase converts malic acid to 
oxaloacetate and reduces NAD.sup.+ to NADH. The NADH couples the reaction 
to reduction of a tetrazolium salt. The tetrazolium salt is reduced using 
phenazine methosulfate as an electron acceptor. Reduction of the 
tetrazolium salt produces formazan which has a blue color. Appearance of 
the blue color in the assay indicates that sufficient fumarase was present 
in the sample to correlate with a motile sperm concentration associated 
with fertility. Persons of skill will appreciate that other reducing 
systems and dyes may be employed in the present invention. 
One aspect of the present invention are devices for assessing sperm 
motility in semen samples. Typically the devices comprise a solid support 
having a carrier matrix containing a fumarase substrate and malic 
dehydrogenase. The devices are capable of differentiating semen samples 
that have more than about 20.times.10.sup.6 motile sperm per milliliter 
from semen samples having fewer than about 20.times.10.sup.6 motile sperm 
per milliliter. Because this concentration of motile sperm is generally 
indicative of fertile semen, the devices of the present application 
provide a means for identifying both adequate absolute sperm count and 
motile sperm count in semen samples. 
The devices of the present invention include a solid support. The solid 
support provides a convenient platform for performance of the assay. The 
composition and shape of the solid support are not critical and may vary. 
Typically, the solid support is a plastic or nylon dipstick. The dipstick 
will usually be semi-rigid. In embodiments configured as a dipstick, the 
carrier matrix will generally be located on one end of the dipstick. 
Alternatively, the solid support may be shaped so that semen samples may 
be applied to the device by means other than dipping, e.g., application of 
controlled amounts of semen by pipettes or the like. 
The solid support has a carrier matrix. The carrier matrix may be the whole 
solid support, a part of the solid support, or a structure attached to the 
solid support. The carrier matrix will typically be an insoluble bibulous 
or non-bibulous material that maintains structural integrity when exposed 
to physiological fluids, such as semen. Suitable bibulous materials 
include paper, cellulose, synthetic resin fleeces, glass fibers, woven and 
non-woven fabrics and the like. Suitable non-bibulous matrices include 
organo-plastic materials such as polystyrene, polypropylene, and the like. 
Often, the carrier matrix will be a portion of a commercially available 
filter or a porous membrane. The matrix is generally attached to the solid 
support by suitable adhesive means, such as double-faced adhesive tape, 
epoxy resin adhesives, and the like. 
The carrier matrices will contain chemical compositions that participate in 
the chemical assay reactions. Generally, the chemical compositions will be 
applied to the carrier matrices in solution and dried. In some instances, 
the chemical compositions may be lyophilized into the carrier matrices. A 
fumarase substrate will be present in the carrier matrix. The fumarase 
substrate may be fumaric acid, a fumarate salt, a fumarate ester or the 
like. Generally, the sodium salt of fumaric acid is employed in the 
devices of the present invention. Generally the carrier matrices of the 
present invention have about 10 .mu.M to about 100 .mu.M of fumaric acid. 
Also, because it is desirable that the fumarase concentration in the semen 
sample be the rate limiting step in the assays of the present invention, 
the fumaric acid will generally be present in excess amounts. 
Also present in the carrier matrix is malic dehydrogenase. Typically, about 
0.1 units to 100 units of malic dehydrogenase is in the carrier matrices 
of the present invention. The malic dehydrogenase will usually be present 
in excess in the carrier matrices. 
In the devices of the present invention, fumarase concentration in the 
sample will be assessed by a visual signal resulting from conversion of a 
fumarase substrate to malic acid which is then further metabolized by 
malic dehydrogenase. The visual signal will generally be produced by 
chemical reactions coupled to the reduction of malic acid. Compounds 
participating in these reactions are contained in the carrier matrices. 
A preferred system for producing the visual signal is an NADH dehydrogenase 
system. NAD.sup.+ present in the carrier matrix can transfer electrons 
from the conversion of malic acid to reduce dye compounds. The reduced 
dyes will have a different optical characteristic than the non-reduced 
dyes. For example, a colorless tetrazolium salt dye may accept electrons 
and acquire a blue color in the reduced state. Additional chemical 
compounds may be required for reduction of the dye. In the above example, 
phenazone methosulfate and magnesium chloride are required for the 
reduction of the tetrazolium salt. All of the compounds required for 
reduction of the dye will be contained in the carrier matrices of the 
present invention. 
Adjuvants which do not participate in any of the chemical test reactions 
may be also be contained in the carrier matrices. These adjuvants may 
include thickening agents and enzyme stabilizers. In some instances, the 
thickening agents will also function as enzyme stabilizers. Suitable 
thickeners and stabilizers include polyvinylpyrrolidone, sodium alginate, 
gelatin, bovine serum albumin, polyvinyl alcohol, methylvinyl ether-maleic 
anhydride polymers, and the like. 
Malic acid may be contained in the carrier matrices of some embodiments of 
the present invention. The malic acid functions to increase the 
sensitivity of the device by lowering the threshold of malic acid that 
must be produced by the fumarase to produce detectable color changes. 
In devices in which the visual signal is a color change, the solid supports 
of the devices of the present invention may also have a color indicator 
panel. The color indicator panel is a reference standard for comparison to 
the color of the carrier matrix following the assay. The color panel 
indicator will generally be comprised of one segment which is the color of 
a positive assay on the carrier matrix. The color panel indicator may also 
have a segment that is the baseline color of the carrier matrix. Providing 
color reference standards for comparison will assist the operator in 
reading the results of the assay. 
Some embodiments of the present invention may have two carrier matrices on 
the solid support. The carrier matrices are not in contact. The matrices 
contain identical chemical compounds. One matrix is the sample matrix as 
described above. The sample is applied to the matrix for assay purposes. 
The other matrix is the control matrix. A control fluid is applied to the 
control matrix. The control fluid contains fumarase activity. The level of 
fumarase activity in the control fluid may vary. A positive control fluid 
will contain a level of fumarase activity equivalent to the fumarase 
activity in semen having more than about 20.times.10.sup.6 motile sperm 
per milliliter. A negative control fluid will contain a level of fumarase 
activity equivalent to the fumarase activity in semen having less than 
about 20.times.10.sup.6 motile sperm per milliliter. Application of a 
control fluid may provide internal positive and negative controls for the 
assay, depending on the control fluid employed. 
Methods for detection of semen samples having a sufficient concentration of 
motile sperm for fertility are also provided. The methods generally 
comprise applying the sample to a carrier matrix on a solid support, which 
carrier matrix contains a fumarase substrate and malate dehydrogenase; and 
detecting a visual signal from the solid support resulting from metabolism 
of the fumarase substrate by fumarase present in the sample. 
The semen assayed in the present invention will generally be fresh. 
Following collection, the semen is usually incubated at room temperature 
for 15-30 minutes. The semen liquifies during the incubation providing 
easier and more uniform application of the sample to the carrier matrix. 
When the carrier matrix is mounted on a dipstick, the dipstick is dipped 
into the sample until the carrier matrix is wetted. If the carrier matrix 
is not mounted on a dipstick, the sample may be applied to the carrier 
matrix by a swab, pipette, or the like. 
The sample is incubated on the carrier matrix at room temperature. The 
color of the carrier matrix is assessed at 1, 3, 5, 7, and 10 minutes. A 
bluish purple color on the pad indicates a positive result. No color 
change or a very faint color change indicates a negative result. The color 
of the carrier matrix may be compared to the color reference to assist the 
operator when making a visual comparison. The color reference may be on a 
color indicator panel on the solid support or a separate color key. 
Alternatively, the color of the carrier matrix may be analyzed by an 
instrument such as a reflectance analyzer or a video image analyzer. 
When a control matrix is present on the solid support, the control fluid is 
applied to the control matrix immediately after application of the sample 
to the sample matrix. The color of the control matrix is assessed with the 
color of the sample matrix. When used as a positive control, the control 
matrix should be a bluish purple color within 10 minutes of application of 
the control fluid if the assay is performing properly. When used as a 
negative control, the control matrix should be colorless or only a very 
faint bluish color when the device is performing properly. 
Kits for detection of semen samples having more than about 
20.times.10.sup.6 motile sperm per milliliter are also provided. The kits 
include a device of the present invention and a semen collection vial. The 
shape and composition of the vial is not critical and may vary. A pipette 
may also be included as appropriate. The kits may also contain vials of 
control fluids. Both positive and negative control fluids may be included 
in the kits. 
A color key may also be provided in the kits of the present invention. The 
color key will generally have segments of different colors. Generally, the 
color key will have bluish purple segments typical of a positive assay and 
faint bluish segments typical of a negative assay. The kits may also 
contain a reflectance analyzer or video image analyzer. 
The following examples are offered by way of illustration and not of 
limitation. 
EXAMPLE 1 
This example demonstrates the construction of a device of the present 
invention and use of the device for assessing sperm count and sperm 
motility in patients attending an infertility clinic. 
A solution containing sodium fumarate, malic acid, malic dehydrogenase and 
a dye system coupled to NAD+ was prepared with the following constituents: 
______________________________________ 
Volume 
______________________________________ 
1 mM Fumarate 200 .mu.l 
1 mM Malic acid 50 .mu.l 
2.5 mM magnesium chloride 
100 .mu.l 
MTT (10 mg/ml) 50 .mu.l 
NAD (10 mg/ml) 100 .mu.l 
Ethoxy PMS (2.5 mg/ml) 10 .mu.l 
Malic dehydrogenase 5 U/10 .mu.l 
Total Volume 520 .mu.l 
______________________________________ 
The solution was diluted 1:5 with deionized water to a final volume of 2.5 
ml and mixed with 2.5 ml of 10% gelatin in phosphate buffered saline. 
Filter paper strips were dipped in the solution and dried at 45.degree. C. 
in a forced air oven for 15 minutes. The dried filter paper containing the 
reagents was attached to a piece of plastic using double-faced adhesive 
tape. The plastic was cut into dipsticks. 
The dipsticks were taken to an infertility clinic for office testing of 
fresh semen samples. Patients supplied samples in the clinic which were 
allowed to liquify at room temperature for 15-30 minutes. A dipstick was 
placed in each sample until the reagent pad was wetted. The dipsticks were 
examined five minutes following application of the sample. Positive 
reactions were determined by a non-de minimis blue coloration of the 
reagent pad after five minutes. Negative responses were determined by no 
color change or a very faint blue tint of the reagent pad after five 
minutes. 
The samples were also assessed microscopically. Experienced laboratory 
technicians measured both the absolute sperm count and sperm motility by 
standard methods. The results were compared and are set forth in Table 1 
below: 
TABLE 1 
______________________________________ 
Data From an Infertility Clinic 
Individual Results 
Absolute Motile 
Con- Con- Micro- 
Sample 
centration 
Motility centration 
scopic 
Dipstick 
No. (millions) 
(%) (.times. 10.sup.6) 
Result 
Result 
______________________________________ 
1 142.5 68 97 + + 
2 129.5 67 87 + + 
3 30.6 65 20 + + 
4 40.0 66 26 + + 
5 261.5 45 118 + + 
6 148.8 43 64 + + 
7 95.8 67 64 + + 
8 54.6 43 23 + + 
9 30.1 61 18 + + 
10 4.1 0 0 - - 
11 144.0 67 97 + + 
12 0.15 1 0 - - 
13 22.0 53 12 + +/- 
14 41.3 74 31 + + 
15 21.9 28 6 - - 
16 134.0 51 68 + + 
17 82.3 66 54 + + 
18 26.3 66 17 + +/- 
19 46.5 70 33 + + 
20 104.1 66 69 + + 
21 55.0 75 41 + + 
22 240.0 67 160 + + 
23 312.9 65 203 + + 
24 161.5 67 108 + + 
25 78.3 44 34 + + 
26 106.0 75 79 + + 
27 15.5 16 2.5 - - 
28 60.3 62 37 + + 
29 145.5 58 84 + + 
30 114.8 63 72 + + 
31 64.0 71 54 + + 
32 118.3 57 67 + + 
33 38.3 41 16 + + 
34 43.0 64 28 + + 
35 8.7 22 2 - - 
36 21.9 15 3 - - 
37 230.0 66 151 + + 
38 108.0 71 77 + + 
39 0.0 0 0 - - 
40 48.0 78 37 + + 
41 364.0 64 232 + + 
42 23.1 6 1 - - 
43 25.1 62 15 + + 
44 91.3 60 54 + + 
45 2.0 58 1 - - 
46 176.0 68 119 + + 
47 157.0 56 87 + + 
48 394.5 64 252 + + 
49 36.5 63 22 + + 
50 0.99 2 0 - - 
51 79.5 73 58 + + 
52 165.0 65 107 + + 
53 189.5 68 128 + + 
54 23.5 27 6 - +/- 
55 97.0 58 56 + + 
56 4.1 69 3 - - 
57 153.0 63 96 + + 
58 0.0 0 0 - - 
59 49.0 60 29 + + 
60 64.8 35 23 + + 
61 67.5 50 34 + + 
62 34.9 2 0 - - 
63 7.4 48 36 - - 
64 57.5 71 41 + + 
65 252.5 62 156 + + 
66 48.6 83 40 + + 
67 32.3 65 21 + + 
68 203.0 65 132 + + 
69 160.3 48 77 + + 
70 11.6 29 3 - - 
71 1.1 0 0 - - 
72 17.3 47 8 - - 
73 105.5 73 77 + + 
74 0.6 0 0 - - 
75 50.0 45 28 + + 
76 65.6 50 33 + + 
77 55.0 60 33 + + 
78 30.0 70 21 + + 
79 188.0 82 154 + + 
80 111.5 45 50 + + 
81 2.5 62 1.5 - - 
82 45.3 65 29 + + 
83 3.9 36 1.4 - - 
84 35.3 42 15 + + 
85 0.0 0 0 - - 
86 0.25 0 0 - - 
87 82.5 63 52 + + 
88 123.0 72 89 + + 
89 1.3 41 0.5 - - 
90 2.5 62 1.6 - - 
91 19.0 42 8 - +/- 
92 0.79 1 0 - - 
93 68.3 0 0 - - 
94 15.9 36 6 - - 
95 142.5 55 78 + + 
96 14.0 41 6 - - 
97 40.0 66 26 + + 
98 261.5 68 179 + + 
99 40.0 66 26 + + 
100 0.0 0 0 - - 
101 1.2 29 0 - - 
102 46.7 67 31 + + 
103 72.3 35 25 + + 
104 213.0 68 145 + + 
105 97.5 67 65 + + 
106 10.9 43 5 - - 
107 33.3 66 22 + + 
108 43.9 65 29 + + 
______________________________________ 
Total Positive = 
73 
Total Negative = 
31 
Borderline = 
4 
Total # = 108 
______________________________________ 
This example demonstrates that the dipstick was highly sensitive and 
specific with a correlation of 96%. 
EXAMPLE 2 
This example demonstrates an assessment of the time required for detection 
of positive reactions in the assays of the present invention. Most 
positive reactions were apparent within 5 minutes of applying the samples 
to the dipsticks. 
Fresh semen samples were provided to be assayed. The samples contained 
varying numbers of sperm and motile sperm. The semen was analyzed by 
traditional microscopic techniques as described above to determine 
absolute sperm counts and motility. Dipsticks were constructed as 
described in Example 1. Following liquefaction of the semen by incubation 
at room temperature, the dipstick reagent pads were wetted in the samples. 
The color of the pads was assessed at 1, 3, 5, 7, and 10 minutes following 
application. The color was scored 0-4+. Zero was no color change and 4+ 
was a marked color change. A 1+ reading was considered a positive result. 
The results are set forth in Table 2 below. 
TABLE 2 
__________________________________________________________________________ 
MALE FERTILITY TEST 
Sperm Count No. of Motile 
Color Results in Minutes 
Sample .times. 10.sup.6 
Motility 
Sperm .times. 10.sup.6 
1 3 5 7 10 
__________________________________________________________________________ 
SID9230 
0 0% 0 -- 0 0 0 0-1 
D241 267 65% 173.6 -- 0 0-1 1 2 
D283 39 83,85% 
32.8 -- 0-1 1-2 3 4 
D257 94 77,78% 
77,78 1 2 2-3 3 3 
SID9233 
2.4 51% 1.2 0 0 0 0 0-1 
SID9234 
.22 8% .018 0 0 0 0-1 0-1 
SID9240 
5.5 47% 2.6 0 0 0-1 1-2 2-3 
SID9244 
4.4 25% 1.1 0 0 1 1-2 2-3 
SID9247 
4.4 40% 1.8 0 0 0 0 0 
D283 6.5 79% 51.4 0 0-1 1 2-3 3 
D275 18.2 74% 134.7 0 1 2 3 3-4 
D257 43.5 77,79% 
33.9 0 0-1 1-2 3 3 
SID9256 
81 59% 47.8 0 0-1 1 2 2-3 
SID9257 
4.0 46% 1.84 0 0 0 0 0 
NTW 15.3 64% 9.8 0 0 0 0-1 1 
SID9259 
29.8 48% 14.3 0 0 0-1 1 1-2 
SID9263 
29 36% 10.4 0 1 1-2 2 2-3 
SID9265 
1.8 20% .36 0 0 0-1 1 2 
SID9266 
25.8 51% 13.2 0 0 0 0 0-1 
SID9267 
9.8 43% 4.2 0 0 0 0 0-1 
SID9269 
36 0% 0 0 0 0 0 0-1 
SID9270 
17 26% 4.42 0 0 0 0 1 
SID9271 
0.22 11% 0.02 0 0 0 0 0 
SID9275 
0 0% 0 0 0 0-1 0-1 1 
SID9284 
13.7 51% 6.49 0 0-1 1 1-2 2 
SID9283 
0.5 21% 0.105 0 0 0 0 0 
SID9288 
5.0 39% 1.95 0 0 0-1 1-2 2 
SP 0248 
0 0% 0 0 0 0 0 0 
SID9286 
40 56% 23 0 0 0-1 1 2 
SID9292 
39.8 48% 19 0 0 0-1 1-2 2 
SP0197629 
0 0% 0 0 0 0 0 0-1 
SID929 40 48% 19 0 0 1 2 2-3 
__________________________________________________________________________ 
All publications, patents and patent applications mentioned in this 
specification are herein incorporated by reference into the specification 
to the same extent as if each individual publication, patent or patent 
application was specifically and individually indicated to be incorporated 
herein by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.