Note: Descriptions are shown in the official language in which they were submitted.
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ASSAYS, DEVICES AND KITS FOR DETERMINING MALE FERTILITY
s Background of the Invention
Det~r",ini,.~ the Fertility of a Sperm Sample
According to recent studies, male infertility is responsible almost 40% of the
time that a couple is unable to conceive a child. In addition, use of male contraceptives is on
the rise. For example, according to current estimates, more than 500,000 vasectomies are
performed in the U.S. each year and about 2,000,000 jare performed worldwide. In addition
to vasectomies, a variety of oral col~llacep~ es for use by males are in development.
Although male contraceptives decrease the probability that a fertile male will initiate a
pregnancy, no male contraceptive is 100% effective. Further, most male contraceptives
require a certain period of time in which to take effect.
For a semen sample to be considered fertile according to standards set by the
World Health Org~ni7~tion, at least two 1.5-5.0 milliliter ejaculate volumes obtained from a
male must contain a sperm density of greater than 20 million spermatozoa/mL and/or a
percent motility of 60% with a forward progression greater than 2 (on a 1-4 scale). In
20 addition, the semen samples should show no evidence of sperm agglutination, pyospermia or
hyperviscosity (Sigman, M., et al., Evaluation of the subfertile male. In: Lipschultz, LI and
SS Howards eds. Infertility in the Male, 2nd ed. Chicago: Mosby-Year Book, 1991; p.l84).
By convention, male infertility is diagnosed based on low sperm motility
25 and/or count. However, motility analyses can produce false negatives, since viable sperm
may appear non-motile due to damage sustained during procecsing. With regard to sperm
count, 20 million spermatozoa/mL or greater is generally considered to be in the fertile range.
However, non-motile, non-viable sperm can be included in the count. Sperm count and
motility are typically ~se~ed using commercially available instruments, such as light
30 microscopes and computerized videoanalysis systems.
U.S. Patent No. 5,068,089 describes a home kit for testing fertility of human
sperm based on ability of the sperm to reduce a dye. The extent of reduction (displayed
colorimetrically), is said to be indicative of sperm fertilizing ability. However, this test is
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time consuming, requires incubation at a telllp~ldLule above room temperature and does not
distinguish between reduction due to sperm cells or other cells, which may be present in a
semen sample.
s U.S. Patent No. 5,219,729 describes a laboratory assay for ~let~rmining the
fertilizing ability of sperm based on the affinity of binding to an oocyte zona pellucida
fr~gment. The tighter the binding, the greater the fertilizing ability of the sperm sample.
However, this assay requires freshly prepared oocyte fragments and at least four hour's time
during which the sperm must be kept in contact with the oocyte fr~gm~nt.
U.S. Patent No. 5,434,057 describes assays and kits for determing male
fertility based on the detection of fumarase activity. However, since fumarase is ubiquitously
present in cells including cells that may be present in a sperm sample (epithelial, leukocyte
and bacterial or fungal), whether fumarase activity is an accurate indicator of sperm count
s and motility, as claimed in the patent, is dubious.
A simple, rapid and accurate assay for determining the fertility of a sperm
sample in a reference laboratory, doctor's office or at home is needed.
Increasing the E~ectiveness of an Assisted Reproduc~ive Technology
Assisted reproductive technologies (ARTs), such as in vitro fertilization (IVF),gamete intrafallopian transfer (GIFT), intrauterine insemination (IUI) and spermintracytoplasmic injection (ICSI) offer ways to initiate a pregnancy when natural approaches
have been unsuccessful. These techniques are also useful for breeding ~nim~l~ or producing
transgenic ~nim~
However, ARTs do not always result in a successful pregnancy. For example,
IVF has an estim~te~l success rate of about 25%, while GIFT is estimated as being successful
in about 31% of attempts. One factor, which may contribute to an unsuccessful ART attempt
is that not all sperm samples are capable of fertilization. According to recent studies, male
infertility is responsible almost 40% of the time that a couple is unable to conceive a child.
In addition, the chance that a sperm sample will be incapable of initiating a
pregnancy is increased when that sa~nple has been stored for any period of time or
3s cryopreserved. This finding has particular significance in view of the use of cryopreserved
sperm in ARTs. Cryopreservation can result in sublethal cryodamage, in which cell viability
post-thaw is lost more rapidly at later times than in fresh cells. Sublethal cryodamage has
been shown to be due in part to membrane embrittlement during the phase transitions
involved in freezing and thawing (Alvarez, J.G. and B.T. Storey (1993) J. Androl. 14 (3):
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199-209). To a lesser degree, sublethal cryodamage has been shown to be caused by
s~o~ eous lipid peroxidation (SLP) of sperm phospholipids (Alvarez, J.G. and B.T. Storey
(1993) J. Androl. 14 (3): 199-209 and J.G. Alvarez and B.T. Storey (1992) J: Androl 13(3):
232-241). Spontaneous lipid peroxidation appears to be the major factor limiting the motile
s lifetime of sperm that has not been cryopreserved (Alvarez, J.G. and B.T. Storey (1988)
Gamet Res. 23:77-90; and Alvarez, J.G. and B.T. Storey (1985) Biol. Reprod. 32: 342-351).
The selective oxidation of phospholipid-bound polyunsaturated fatty acid moieties resulting
from sl,olll~leous lipid peroxidation leads to extensive oxidative damage to the sperm plasma
and acrosomal membranes and DNA.
However, cryopreservation of sperm samples is routinely p~lr~,lllled in ART
procedures and in fact is required in order to test the donor for the presence of tr~n~mi~.~ible
infectious agents (e.g., HIV) prior to insemination. For example, a donor is typically tested
six months after producing a particular sample and only if the test is negative will the stored
5 sample be used for insemination.
More and more couples are turning to ARTs to conceive a child. In addition,
in vitro fertilization technologies are increasingly being used by breeders of livestock and in
generating transgenic anim~l~ A method for increasing the success rate of ARTs is needed.
Summary of the Invention
In general, the invention features devices, procedures and kits for isolating and
quan~ hlg motile sperm from a sperm-co"l~i"il-g sample (e.g. semen) and/or for
determining the pregnancy potential of a sperm sample. In general, the devices comprise a
2s container for isolating a sperm cont~ining sample, which is preferably adapted to separate
sperm based on motility. The container can optionally be conformed to facilitate collection
(e.g. funnel-shaped). In a preferred embodiment, a retainer for accomodating entry and
migration of motile sperm, but not non-motile cells in the sperm colll;.i~ g sample, is
positioned inside of the container. Preferably at an a~ iate site within the retainer is
30 positioned a means to determine the pregnancy potential of sperm that migrate into the
retainer. Alternatively, isolated motile sperm can be obtained from the retainer and
separately tested.
In one embodiment, the retainer is a colllp~llllent (e.g. a tube), which is in
35 fluid communication with the sperm Col-t~il-il-g sample. In another embodiment, the retainer
is a fluid, which is less dense than the sperm cont~ining sample and thereby facilitates
passage of motile sperm, but not non-motile cells. In a further embodiment, the retainer
includes a porous membrane, which separates the sperm cont~ining sample from an isolation
area and which thereby prevents passage of non-motile sperm or other cells that may be
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present in the sperm co"~ g sample, but allows passage of motile sperm into the isolation
area.
In a second aspect, the invention features assays for identifying sperm samples
s with high pregnancy potential. The assays can be used to provide an indication of the fertility
status of the male donor or can be selected for use in an Artificial Reproductive Technology
(ART). In a preferred embodiment, the process comprises the steps of: i) obtaining multiple
sperm samples from a donor over time, ii) obtaining an aliquot from each sperm sample, iii)
testing each aliquot to determine pregnancy potential, and iv) using at least one sperm sample
0 having high pregnancy potential in an ART to initiate a pregnancy. In a ple~llcd
embodiment, the testing is based on qua~ g an indicator of lipid peroxidation or a
change in an indicator in~lced by a stress. A particularly l~lcr~llcd indicator of sperm lipid
peroxidation is sperm superoxide dismutase (SOD) activity, which can be quantitated, for
example, using an anti-SOD antibody.
In a third aspect, the invention features screening assays for detecting the
pregnancy potential of sperm samples and thereby identifying potentially infertile males or
evaluating males following vasectomy or once a particular contraceptive has been~lmini~tered. In a preferred embodiment, the screening assay is based on determining motile
20 sperm count. In one embodiment, motile sperm are first isolated and counted. Quantitation
of less than about 50,000 motile spermatozoa/mL indicates that the contraceptive has been
effective and that the sperm sample has low pregnancy potential. In another embodiment,
ua~ a~ion of sperm is performed directly on the sperm cont~ining sample (withoutseparation of motile and immotile sperm) and quantitation of less than about 20 million
2s spermatozoa/mL indicates that the sample has low pregnancy potential; greater than about 20
million spermatozoa/mL and less than about 40 million spermatozoa/mL indicates that the
sample has borderline pregnancy potential; and greater than about 40 million
spermatozoa/mL indicates that the sample has high pregnancy potential. The cutoff value of
20 million spermatozoa/mL includes both motile and immotile spermatozoa. Although this
30 value is not diagnostic of infertility, it is intended to be used as part of a screening test. In
this way, males that use the kit to test their fertility potential and have less than about 20
million spermatozoa/mL will be alerted to a potential infertility problem.
In a fourth aspect, the invention features sperm diagnostic kits or systems
3s which comprise a number of simple reagents and devices packaged in a box. In one
embodiment for use in identifying a high ple~llallcy potential sperm sample, the kit can
include a sperm isolation means and a means for identifying a high pregnancy potential
sperm sample. Optionally, the sperm isolation means is of an applol~,;ate conformation (e.g.
funnel shaped) to facilitate collection. Also optionally, a reagent for liquefying semen (e.g.
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pronase or chymotrypsin) is included in the kit for use in sperm isolation. Preferably the
means for identifying sperm with high pregnancy potential is based on lipid peroxidation and
the means for screening for pregnancy potential (e.g. infertility or effective male
contraception) is based on sperm count.
s
The instant disclosed assays, devices and kits are easy to perform and can be
completed in less than about 15 minutes' time. The assays, devices and kits provide
information which is useful, for example, in monitoring the impact of changes in such factors
as diet, sleep, exercise, exposure to smoke or other carcinogens, and intake of alcohol or
0 drugs on the fertility of sperm samples produced thereafter. Also, the devices, kits and
methods can indicate whether a particular male fertility or contraceptive treatment has been
effective or whether sperm subsequently obtained from the same male will initiate a
pregnancy upon contact with an oocyte. In addition, the assays, devices and kits are useful
for determining whether a particular sperm sample is suitable for use in an ART.
Certain of the assays, devices and kits are appro~liate for use in a reference
laboratory, while others can be used in a doctor's office and/or at home. Other features and
advantages will become readily a~palelll from the following Detailed Description and
Claims.
Brief Description of the Figures
Figure 1 is a perspective view of one embodiment of a sperm isolation device,
which includes a tube for receiving and caplu,;llg motile sperm.
2s Figure 2 is a perspective view of another embodiment of a sperm isolation
device, which includes a less dense fluid layer in contact with a more dense sperm-containing
sample.
Figure 3 is a perspective view of yet another embodiment of a sperm isolation
device, in which a porous membrane separates the sperm sample from a motile sperm
collection area.
Figure 4 is a graph plotting the motility recovery after cryopreservation for
sperm samples having a stress test score of greater than about 0.8 and samples having a stress
3s test score of less than about 0.8.
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Detailed Description
The invention pertains to easy to use devices that can rapidly recover motile
sperm from a sperm co.~ g sample (e.g. semen) and assays and kits, which employ the
s devices for use in identifying a high pregnancy potential sperm sample. The invention also
pertains to assays and kits for screening males for infertility and for confinning whether a
male contraceptive is effective or has taken effect.
Preferred sperm isolation devices are based on sperm motility and comprise a
0 housing means for holding sperm, an isolation means selectively disposable within the
housing means for isolating motile sperm and a means for testing the motile sperm to
determine pregnancy potential. One embodiment is illustrated in Figure 1. A container 10
that has side walls 12, 14, 16 and 18 and a bottom portion 20 forming a sample well 21 for
receiving a sample 30 collt~il~illg ejaculate from a male donor. Although illustrated as
1S cubical, the container 10 can have any selected shape or size suitable for the capture and
retention of a sperm COI~ i.lg sample. A retainer 24 can be positioned within the sample
well 21 of the container, and is preferably placed in fluid communication with the sample 30.
Those of ordinary skill will recognize that the retainer 24 can be mounted to the floor portion
20 of the container 10 by a number of known methods, including the use of an adhesive, or
20 can be supported within the container 10 by any suitable support means. Although illustrated
as cylindrical, the retainer 24 can have any selected shape or size suitable for the isolation and
retention of motile sperm, and can include commercially available instruments, such as tubes,
capillaries, straws, pipettes, and other suitable receptacles having an internal conduit.
Additionally, the container 10, although illustrated as having a substantially rectangular
2s shape, can be configured to have any selected shape, such as a funnel-like configuration, to
assist the male donor in collecting and ca~uling a sperm sample. The container 10 and
retainer 24 can be made from any suitable biocompatible material, such as glass or plastic.
The illustrated sample 30 can contain both motile and non-motile sperm.
30 According to a plerell~d practice, the retainer 24 is positioned within the well 21 or
constructed to allow the motile sperm to migrate into the conduit 22 of the receptacle 24. The
receptacle 24 captures the motile sperm, which are then analyzed to determine the pregnancy
potential of the sperm sample. For example, after a sufficient period of time has elapsed to
ensure that motile sperm present in the sample 30 have migrated into the retainer 24, the
3s retainer can be removed from the container 10 and placed within a second container 31. e.g.,
a test tube, cont~inin~ a means for testin~ the motile sperm to deter~nine the pregnancy
potential of the sarnple 34.
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Alternatively, a detection mech~ni~m 36 can be mounted within the conduit 22
of the retainer 24. The motile sperm that migrate into the conduit 22 preferably contact the
detection mech~ni~m 36. The detection mech~ni~m preferably includes a suitable membrane
or substrate co~ illg a means for testing motile sperm to determine the pregnancy potential
of the sperm col~ -g sample.
Motile sperm can alternatively be isolated from non-motile cells in a sperm
sample based on contact with a mechanical or fluid barrier or gradient. For example, as
shown in Figure 2, a relatively dense sperm-co~ -g sample can be contacted with a less
0 dense fluid layer, which is preferably of a similar t~ pel~Lu~e, pH and salt concentration as
the sperm-col~ g sample. In Figure 2 and in all subsequent figures, like parts are
represented throughout with the same reference numerals plus a superscript prime. The
illustrated container 10' includes a first fluid layer 40 and a second fluid layer 46. The first
fluid layer 40 includes a sperm sample from the male donor and optionally an app-opl;ate
reagent for liquefying the semen sample, e.g. pronase or chymotrypsin. The layer 40
typically includes motile and/or non-motile sperm. The second fluid layer 46 is preferably a
fluid having a density that is less than the density of the first fluid layer 40, thus creating a
density gradient along the height of the container 10'. The decreasing density between layers
40 and 46 promote the migration of motile sperm from the sample fluid layer 40 into the
collection fluid layer 46. This axial layered construction of the fluids 40 and 46 can be
accomplished by selecting a~ uliate fluids that are emissible or partially emissible relative
to one another.
According to a preferred practice, a sperm sample is placed within the
2s container or is mixed with a reagent for liquefying ejaculate prior to introduction into the
container 10'. A second fluid layer 46 having a lower density is then introduced into the
container 10' and is separated from and disposed axially above the first fluid layer 40. After a
sufficient amount of time, the motile sperm present in the sample layer 40 migrate into the
second fluid layer 46 in response to the ~liminishing density gradient along the height of the
container 10'. Thus, the motile sperm are isolated in the second fluid layer 46. After a
selected period of time (e.g., five minutes) a sample of fluid from fluid layer 46 is removed,
as by a pipette 50, and is introduced into a test tube 52 which holds a test solution 54. The
solution preferably contains a hypotonic solution having peroxidase-conjugated anti-SOD
and/or anti-GPx IgG polyclonal antibodies. A test strip col-t~il-il-g bound sheep anti-SOD
3s and/or anti-GPx IgG polyclonal antibodies is placed into the test solution, removed and then
placed in a final solution cont~illillg a peroxidase substrate (e.g. hydrogen peroxide and
DAB). In response to the presence of motile sperm, the test strip changes to a selected color.
This color is matched to a color chart to determine the pregnancy potential of the sperm
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sample. Alternatively, the pipette co~ i"g motile sperm can be directly applied to a test
strip, which contains a sperm reagent.
According to an ~ltern~te embodiment, as illustrated in Figure 3, the second
5 collection fluid layer 46 can be replaced with a porous membrane 60. The permeable porous
membrane 60 introduces or presents a mechanical resistance to the migration of non-motile
sperm from the sample layer 40' into or through the membrane 60. However, the porous
membrane 60 facilitates the migration and hence the isolation of motile sperm in the isolation
layer 10' by allowing motile sperm to pass between sample layer 40' and the membrane 60.
0 The porous membrane 60 can be composed of any suitable biocompatible material that
facilitates the passage of motile sperm without introducing damage thereto.
In another aspect, the invention relates to pl~;relled methods for determining
the pregnancy potential of a sperm sample. A sperm sample with "high pregnancy potential"
5 as described herein has a greater than about 50% probability for initiating a pregnancy upon
contact with an oocyte. Although a sperm sample having high pregnancy potential has an
increased probability of initiating a pregnancy, contact of a high pregnancy potential sperm
sample with an oocyte does not guarantee a successful pregnancy. Other factors, such as
inability of an oocyte to decondense human sperm chromatin, defective oocyte DNA (e.g.
20 due to age), two-cell embryo block or early embryo demise, may prevent the initiation of a
pregnancy even by a high plegnallcy potential sperm sample.
A sperm sample with "low pregnancy potential", on the other hand, has very
little or no chance of initiating a pregnancy upon contact with an oocyte. Indication that a
25 sperm sample provided by a particular male has low pregnancy potential does not indicate
that subsequent sperm samples will also have low potential. Since the pregnancy potential of
the sperm samples produced by a given male can change over time and can be influenced by
such factors as diet, sleep, exercise, exposure to smoke or other carcinogens, or intake of
alcohol or drugs, the pregnancy potential of a particular sperm sample is in general only
30 accurate for a period of about 48 hours.
Preferred sperm CO~ ,g samples (e.g. semen) for use in the disclosed
assays are obtained from a human or animal (e.g. a bull, stallion, ram or other domesticated
animal or an endangered animal). To ensure accuracy, tests are preferably performed on
35 freshly collected ejaculate.
Preferred tests for identifying sperm samples with high pregnancy potential
are lipid peroxidation tests, which measure an indicator of lipid peroxidation or a change in
an indicator over a period of time under defined conditions. The value obtained is then
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g
compared with a standard value for that particul. indicator to det~rmine the pregnancy
potential of that sperm sample.
A stress test measures a change in a particular indicator of lipid peroxidation
s over a period of time in response to a stress (a stress test). A stress test score can be obtained
by dividing a measured post-stress value of an indicator of lipid peroxidation by a measured
pre-stress value. Examples of al,plopl;ate stresses or stressing agents for obtaining a test
score include radiation (such as thermal (e.g. as described in Example 2), electromagnetic),
freeze-thawing, oxidation or exposure to chemical agents (e.g. oxidizing agents such as the
o ferrous iron/ascorbate system described in Example 3).
Examples of lipid peroxidation indicators that change in response to a stress
include:
Motility: When motility is used as an indicator of lipid peroxidation
and the stress is thermal (e.g. incubation for at least about 1 hour at a
temperature in the range of about 27-45C), a test score of greater than about
0.8 indicates high pregnancy potential. A test score of less than about 0.8
indicates low potential.
Lipid peroxidation breakdown products: Samples that have a relatively
high post-stress value of lipid peroxidation breakdown products (such as lipid
hydroperoxides, malonaldehyde, pentane and ethane) relative to pre-stress
value (test score < 0.5) have a low probability of resulting in a successful
2s pregnancy, while test scores of about 0.5 or greater increase the probability of
initiating a pregnancy. The lipids can be analyzed spectrophotometrically. For
example, lipid hydroperoxides can be extracted with hexane and detected at
233nm. Malonaldehyde can be measured at 532nm following reaction with
the thiob~bilulic acid (Tapel, A.L. et al., (1959) Arch Biochem Biophys,
80:326). Pentane and ethane can be measured by head-space gas
chromatography .
Ratio of membrane phospholipids: Samples that oxidize at high rates
have phosphatidylethanolamine (PE)/ phosphatidylcholine (PC) ratios < about
0.5, while samples that oxidize at low rates (test scores > about 0.8 as
indicated by loss of motility) have PE/PC ratios of greater than about 0.7.
Therefore, sperrn ~vith high pregnancy potential have PE/PC ratios greater
than about 0.7, but less than about 1.5, sperm with some pregnancy potential
have PE/PC ratios of greater than about 0.5 and less than about 0.7, and sperm
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with low pregnancy potential have PE/PC ratios of less than about 0.5. PE
and PC can be measured, for example, by high-performance thin-layer
chromatography (Alvarez, J.G. et al., (1987) JLiquid Chromatogr 10: 3557).
Table I
P~ Potential Based on Test Score
Indicator Stress Test Score
Lipid peroxidation breakdown products oxidation,
(e.g. Iipid hydroperoxides,increased ~ .,.aLulc > 0.5 High Pregnancy Potentialmalonadehyde, pentane, ethane) <0.5 Low ~ Potential
PE/PC oxidation
>0.7 - <1.5 = High ~C~ y Potential
>0.5 - 0.7 = Some Plt:g~ y Potential
<0.5 = Low Pregnancy Potential
Motili[y increased ~ aIul~
> O 8 = High Pregnancy Potential
< 0.8 = Low Pregnancy Potential
Examples of lipid peroxidation indicators that can be directly measured as an
indication of pregnancy potential include:
Oxidation of DNA: Samples that oxidize at high rates have higher
levels of oxo-8-deoxyguanosine (oxo8dG), which can be measured, for
example, by high-~es~ule liquid chromatography (HPLC) using
electrochemical detection (Fraga, C.G. et al., (1991) Proc Natl Acad Sci
88:11003.). Sperm with high pregnancy potential have less than about 20
fmol of oxo8dG/ ~g of DNA, while sperm with low pregnancy potential have
greater than about 40 fmol of oxo8dG/ ~g of DNA.
Superoxide dismutase (SOD) activity: Solid phase-bound anti Cu/Zn-
SOD antibodies and enzyme labeled anti-Cu/Zn-SOD antibodies can be used
to detect SOD activity as described in Example 3. Sperm samples with SOD
activities greater than or equal to about 9U/108 cells have been shown to be
associated with high pregnancy rates after IVF (i.e. have high pregnancy
potential), while activities less than about 7U/108 cells are associated with low
pregnancy rates (i.e. Iow pregnancy potential).
Surface SOD immllnofluorescence Samples that oxidize at high rates
have low surface-SOD immunofluorescence and low total SOD activity.
Surface-SOD immunofluorescence can be measured, for example, by flow
cytometry using sheep anti-Cu/Zn-SOD IgG polyclonal antibodies and FITC-
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conjugated rabbit anti-sheep secondary antibodies (Alvarez, J.G. 18th Annual
Meeting American Andrology Society, Tampa, FL., 1993, abstract 170).
Greater than about 300 FITC units is indicative of high pregnancy potential,
while less than about 300 FITC units is indicative of low pregnancy potential.
s
Ratio of unsaturated fatty acids to saturated fatty acids Unsaturated
fatty acids are prone to oxidation while saturated fatty acids are insensitive to
this process. Determination of the ratio of the unsaturated fatty acid,
docosahexaenoic acid (22:6) to the saturated fatty acid palmitic acid (16:0) or
lo of other ulls~ d~ed fatty acids to saturated fatty acids can be used to predict
pregnancy potential. Samples that oxidize at high rates will have low
docosahexaenoic acid/palmitic acid ratios. Unsa~ulaled/saturated fatty acids
can be measured by gas chromatography following ~lk~line methanolysis with
lN sodium methoxide at 40C for 1 hour (Alvarez, J.G. and J.C. Touchstone,
Practical Manual on Lipid Analysis. Series of Monographs: I- Fatty Acids.
Norell Press (New Jersey), 1991). A ratio of unsaturated to saturated fatty
acids of greater than or equal to about 1.0 indicates high pregnancy potential; a
ratio greater than or equal to about 0.5 and less than about 1.0 indicates
borderline pregnancy potential; and a ratio of less than about 0.5 indicates lowpregnancy potential.
Creatine kin~ce activity The concentration of creatine kinase in sperm
reflects the degree of cytoplasmic extrusion during the last phase of
spermatogenesis. Samples exhibiting abnormally high levels of creatine
2s kinase activity have also been found to have high rates of lipid peroxidation as
determined by the PE/PC ratios (PE/PC ratios ' 0.5 and increased
malonaldehyde production). This correlation supports the detection of
creatine kinase activity as a means for determining pregnancy potential of a
sperm sample. Creatine kinase activity in human sperm can be measured by
spectrophotometric analysis at 365 nm of the NADPH generated during
reaction of creatine kinase with ADP to produce creatine and ATP, followed
by reaction of hexokinase with ATP and glucose to produce glucose-6-
phosphate, followed by reaction of glucose-6-phosphate dehydrogenase with
glucose-6-phosphate and NADP to produce NADPH. The NADPH generated
under these conditions is proportional to the activity of creatine kinase in
human sperm (Huszar, G. (1988) Gamete Res 19:67,). Creatine kinase (CK)
exists in two isoforrns: MM and BB. Sperm with high pregnancy potential
have a CK-MM/CK-MM+CK-BB ratio of greater than or equal to about 10%.
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Table 2
Pregnancy Potential or Col-lr ~ e,l' ~e
Efficacy Values from Direct Indicators
of Lipid r~ r
Indicator High Pregnancy Potential Low Pregnancy Potential
SOD Activity > 9 U/10~ <7U/10~
oxo~dG ~20 f~nol/ ',lg DNA >40 fmol/ ~lg DNA
Surface SOD >300 FITC <300 FITC
Immunofluu. ~c~l.ce
Ullsalu,al~dlsaturated > 1.0 ~0.5
Fatty Acid Content
CK-MM/CK-MM + CK-BB > 10% ~ 10%
Lipid peroxidation indicator values or changes in value in response to a stress
can be quantitated by techniques and devices, which are well-known to one of skill in the art.
The particular lipid peroxidation indicator chosen may be dictated by the degree of accuracy
5 required and the availability of instruments to detect the results.
In a ~fcrellcd embodiment for use as a kit, which is described in detail in the
following Example 4, the indicator of sperm lipid peroxidation is based on sperm superoxide
dismutase (SOD) activity. In a particularly plcrellcd embodiment, sperm SOD activity is
0 tested using an SOD antibody. As used herein, an antibody for use in detecting a sperm lipid
peroxidation indicator can be any material that binds an antigen (e.g. polyclonal, monoclonal
or single chain antibody or antibody fragment, such as an Fab of Fab'2 fragment).
Immunodetection of sperm SOD or another antigenic indicator of lipid
15 peroxidation can be accomplished using any of a number of competitive or non-competitive
assay procedures. In general collll~e~ilive immunoassays are performed by adding SOD to a
sperm contaillillg sample, so that the sperm and the SOD compete for a limited number of
antibody binding sites resulting in the formation of sperm-antibody and labeled SOD-
antibody complexes. By m~ g the concentration of labeled SOD and SOD antibody
20 constant, the amount of labeled SOD-antibody complex formed is inversely proportional to
the amount of sperm present in the sample. A quantitive det~rmin~tion of the sperm SOD
can therefore be made based on the labeled SOD-antibody complex. Competitive assays can
be homogeneous (i.e. not requiring separation of antibody bound tracer (e.g. labeled SOD)
from free tracer, since the antigen-antibody interaction causes, directly or indirectly, a
25 measurable change in the signal obtained from the label group of the tracer). Alternatively,
competitive assays can be heterogeneous (i.e. requiring separation of bound tracer from free
tracer prior to determining the amount of ligand in the sample).
In contrast to competitive immunoassays, non-competitive assays involve
30 incubating a sperm cont~ining sample with an immobilized SOD antibody for a period of
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time sufficient to reach equilibrium with regard to the formation of antibody-sperm
conjugates. The SOD antibody can be directly or indirectly labeled. For example, indirect
labeling can be carried out after a wash step to remove unbound sperm by contacting the
immobilized antibody-sperm complexes with a second, labeled antibody that is specific for
5 the antibody-sperm complex. Following a second wash step to remove unbound second
antibody, the amount of bound second antibody can be detected and measured as anindication of bound sperm. An example of this procedure is described in the following
Example 5.
0 Exemplary competitive and non-competitive immunoassays include
fluorescence polarization immlmc)assay (FPIA), fluorescence immllnnassay (FIA), enzyme
immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked
immunosorbent assay (ELISA) and radioimmunoassay (RIA). General techniques for
performing the various immunoassays are known to one of skill in the art. Moreover, a
general description of most procedures is provided in U.S. Patent No. 5,051,361, which is
incorporated herein by reference. The antibodies can be labeled in any manner that facilitates
detection. Preferred labels include enzymes (e.g. horseradish peroxidase, alkaline
phosphatase, urease, ,B-galactosidase), enzyme co-factors, radioisotopes (e.g. 3H, 14C, 125I,
32p, 131I and 35S), fluorescent compounds (e.g. fluorescein, rhodamine, allophycocyanin,
phycoerythin, erythrosin, europian, luminol, luciferin and coumarin) and colored or
uncolored beads or particles (e.g. silica gel, controlled pore glass, magnetic,
Sephadex/Sepharose, cellulose, metal (e.g. gold) or latex). Preferred supports for
immobilizing antibodies include membranes (e.g. polyethylene, polypropylene, polyamide,
polyvinylidenedifluoride, glass fiber, paper), beads or particles and tubes, (e.g., glass, plastic
or metal capillaries, straws or pipettes).
Based on the above-described procedures for identifying sperm samples with
high pregnancy potential, the invention also features processes for increasing the success rate
for initiating a preganancy using an assisted reproductive technology (ART) (i.e. a procedure
for contacting a sperm with an ovum to initiate a pregnancy). Examples of ARTs include in
vitro fertilization (IVF), gamete intrafallopian transfer (GIFT) intrauterine insemination (IUI)
and intracytoplasmic sperm injection (ICSI). Procedures for performing ARTs are well-
known to practitioners. It is expected that additional ARTs will be developed over time.
A preferred process of the invention involves obtaining multiple sperm
samples from a donor (e.g. a male partner from a couple undergoing an ART) over time. For
example, a donor can provide a new sperm sample once every other day. An aliquot (e.g. lx
1 o6 cells) of each sample can then be obtained for testing, while the remainder can be banked
(e.g. cryopreserved) for potential future use in an ART. Preferably the process includes a
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means for correlating a particular aliquot with the banked sample from which it was obtained.
For example, an aliquot taken from a sample, as well as the sample itself obtained on day I
can be labelled #1. A subsequently obtained sample from the same donor and an aliquot
taken from that sample can be labelled #2, etc..
All aliquots provided by a particular donor can then be tested to identify spermsamples having "high pregnancy potential" (e.g. sperm having a greater than 50% probability
of initiating a pregnancy upon contacting an oocyte). The sperm sample indicated as having
the higher pregnancy potential can then be used in an ART and the rem~ining, lower
0 pregnancy potential samples can be discarded.
The following Example 2 provides the results of a blind prospective cohort
study of 33 couples undergoing in vitro fertilization (IVF) and 11 couples undergoing gamete
intrafallopian transfer (GIFT), no successful pregnancies resulted from sperm sarnples which
5 exhibited a stress test score of less than about 0.8. In contrast, when sperm samples having a
stress test score of about 0.8 or greater were used in an ART, a pregnancy resulted 55% of the
time. Therefore, where the indicator of lipid peroxidation is loss of motility, a stress test
score of less than about 0.8 indicates low pregnancy potential, while a stress test score of
about 0.8 or above indicates high pregnancy potential and is suitable for use in an ART.
Table 3 shows how the stress test score and therefore the pregnancy potential
of sperm obtained from the same male can vary with time.
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Table 3
ST scores for IVF male partners
Patient Date of IVF ST score
1. 11/05/93 0.35
04/20/94 0.88
2. 11/15/93 0.88
09l27l94 0.88
3. 11/17/93 0.88
06l28l94 0.88
4. 01/24/94 1.00
02/16/94 0.50
5. 01/05/94 0.20
06/20/94 0.92
6. 05/03/94 1.00
06/29l94 0.40
7. 05/05/94 1.00
06l27l94 0.50
8. 02/22/94 0.80
10/05/94 1.00
9. 06l28l94 1.00
10/18/94 1.00
In addition, Figure 4 shows that sperm samples with stress test scores greater
than about 0.8 have a high motility recovery, even after cryopreservation. Therefore it
appears that a further benefit of using sperm samples indicated as having high pregnancy
potential in an ART results from the fact that such samples are not damaged by
o cryopreservation to the same degree as sperrn with low pregnancy potential.
In contrast to high pregnancy potential sperm samples, identification of sperm
samples with low pregnancy potential can also be useful, for example, as a screen for
detecting potentially infertile males. In addition, since even fertile males can occassionally
15 generate ejaculates with low pregnancy potential, by identifying a sperm sample as having
low pregnancy potential, use of the sample in an ART can be avoided. Further, byidentifying sperm sarnples with low pregnancy potential, the user can determine whether a
particular male contraceptive is effective or has taken effect. For example, a vasectomy
typically requires a phase in period in which to take effect. The use of assays to identify
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sperm samples with low pregnancy potential, as disclosed herein, is therefore useful for
confirming the efficacy of a male contraceptive.
Low pregnancy potential sperm samples can be identified by lipid
s peroxidation tests as described above. In addition, low pregnancy potential sperm samples
can be identified based on quantification of spermatozoa (non-motile and motile) in a sperm
cont~ining sample (e.g. ejaculate). If the number obtained is less than about 20 million
spermatozoa/mL, the sperm sample is considered to have low pregnancy potential.
0 Examples of sperm targets or sperm components that can be quantitated as an
indication of the number of sperm present in a sperm containing sample, include a sperm
protein (e.g. a sperm flagella protein, a glycolytic enzyme, an antioxidant enzyme (e.g.
glutathione peroxidase or superoxide dismutase), a nuclear protein, an acrosomal protein, a-
tubulin, lactate dehydrogenase (LDH-X), protamine, acrosin or a mitochondrial protein.); or a
sperm lipid (e.g. cholesterol, a phospholipid, a glycolipid, a triglyceride, a fatty acid,
phosphatidylglycerol, seminolipid, and a docosahexaenoic acid). Preferred targets for sperm
quantitation are selective for and abundant on sperm cells.
Preferably a sperm reagent (e.g. sperm antibody, ligand, lectin or substrate) isused to quantitate sperm. Preferred sperrn reagents include labeled (e.g. enzyme, tracer (e.g.
radioactive), dye or color particle labeled) or unlabeled anti-sperm antibodies (e.g. anti-
hurnan sperm polyclonal antibody; Arnel Products Co., Inc, Cherokee Station, New York,
N.Y.; Chemicon International Inc., Temecula, California) or labeled or unlabeled antibodies
against a sperm component (e.g. a sperm protein or sperm lipid). Further preferred sperm
reagents include labeled (e.g. dye or tracer labeled) or unlabeled reagents that interact with a
sperm protein (e.g. Protein Reagent (0.3% tetrabromophenol, Miles Scientific, Connecticut)
or rhodamine 123, which accumulates within sperrn mitochondria. Alternatively, the labeled
reagent (e.g. rhodamine) can interact with a sperm lipid.
Preferred methods for counting sperm employ detectably labeled antibodies
that specifically bind a sperm antigen (e.g. anti-human sperm polyclonal antibodies and
antibodies specific to an epitope of the sperm flagellum, nuclear proteins, glycolytic
enzymes, acrosome etc.). One method for quallLil~lillg sperm involves incubating a sperrn
sample with colored particles cont~ining anti-sperm antibodies for an ~plopliate period of
time to allow the sperm antigens to react with the antibody bound colored particles; ii)
filtering the sample of step i), so that spermtcolored particletantibody complex is retained on
the filter and unbound, colored particle/antibody and seminal plasma protein passes through
the filter; and iii) vis~ ing the color intensity.
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For example, if colored particles are used, sperm/colored particle/antibody complexes
can be 4uanLilaled by coml,a~ g the color of the filter to a color chart, which depicts various
color possibilities for various quantities of sperm. Less than about 20 million
spermatozoa/mL indicates low pregnancy potential. Alternatively, the filter membrane can
5 be configured to indicate a "+" (i.e. fertility), if a sufficient amount of sperm/detectable
particle/antibody complex is retained on the filter membrane to indicate that the sample is
suitable for initiating a pregnancy (greater than about 20 million spermatozoa/mL) and a "-"
(i.e. infertility) if an insufficient amount of sperm/detectable particle/antibody complex is
retained on the filter membrane (less than about 20 million spermatozoalmL).
In order to use antibodies or reagents that may also react with seminal plasma
components present in a sperm COI~t~ sample (e.g. ejaculate), sperm can first be isolated.
Seminal plasma-free sperm can then be contacted with anti-sperm antibody coated colored
particles. After a sufficient period of time to allow antibodies and antigens to react, unbound
5 antibody coated colored particles can be removed from the mixture and sperm/colored
particle/antibody complex detected and quantitated. For example, the quantity of sperm in
the sample can be 4ual~ d using a color chart or a "+" or "-" filter as described above.
Alternatively, sperm can be 4ualllil~led by detecting the appearance of agglutination in a drop
of sample following addition of anti-sperm antibodies with bound latex particles.
Alternatively, low pregnancy potential can be determined by quanlil~ling only
motile sperm in a sperm sample. Motile sperm can be isolated from a sperm Cont~ining
sample, for example, using the devices and procedures described above. Preferred methods
for quanlilalillg sperm employ detectably labeled antibodies that specifically bind a sperm
25 antigen. Motile sperm can be quantitated using detectably labeled, sperm specific antibodies,
such as anti-glutathione peroxidase (GPx) antibody. As with sperm SOD, immunodetection
of sperm GPx or other sperm antigen can be accomplished using any of a number ofcompetitive or noncompetitive assay procedures. In addition, sperm specific antibodies can
be labeled (e.g. enzymatically) and if necessary immobilized by procedures that are well
30 known in the art or as described above for SOD antibodies.
In a further aspect, the invention features sperm diagnostic kits or systems
comprising a number of simple reagents and devices packaged in a box. The kit components
can include one or more of the devices or components illustrated in Figures 1-3, and reagents
35 for detennining the pregnancy potential of sperm samples. Preferable kits include reagents
for liquefying semen (e.g. chymotrypsin or pronase). For identifying sperrn samples with
high pregnancy potential (i.e. Male Fertility kit), preferable kits are based on measuring an
indicator of lipid peroxidation or a change in an indicator over a period of time under defined
conditions. Alternatively, as further described in the following Examples, a kit can be based
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on det~.,ni.~ g the number of spermatozoa present in a sperm sample, wherein greater than
about 40 million motile spermatozoa/mL indicates high pregnancy potential; less than about
20 million motile spermatozoa/mL indicates low pregnancy potential; and greater than about
20 million, but less than about 40 million indicates borderline pregnancy potential.
s
Preferable kits for identifying a sperm sample with low pregnancy potential,
(e.g. to confirm the effectiveness of male contraception) are based on d~.. inil~g the number
of spermatozoa/mL present in the sample, wherein less than about 50,000 spermatozoa/mL
indicates low pregnancy potential or successful contraception. Reagents and devices for use
0 in clçtennining sperm pregnancy potential can include dispensing devices (e.g. capillaries or
pipettes) for delivering a defined volume of isolated sperm from a collection container into a
container which includes a means for deterrnining the pregnancy potential of the sample, for
example by quanlil~lillg the extent of sperm lipid peroxidation or change in lipid peroxidation
resulting from a stress; and/ or sperm count.
In a ~refell~d embodiment, the means for detecting sperm lipid peroxidation is
based on sperm superoxide dismutase (SOD) activity and/or the means for detecting sperm
count is based on sperm protein concentration. In a particularly preferred embodiment, the
means for detecting sperm SOD activity employs a colorimetrically labeled SOD antibody
20 and the means for detecting sperm protein employs gold particles. The kit also preferably
includes a color chart against which concentrations of SOD and/or protein can be compared
against known values. The kit can be optimized to develop color based on the protein levels
colle~uollding to sperm concentrations greater than or equal to about 50,000
spermatozoalmL .
2s
A plefellcd kit for home deterrnination of the pregnancy potential of a sperrn
sample can include: i) a collection container (optionally conformed to capture ejaculate,
and/or co~ ining a solution (e.g. enzymatic) that facilitates semen liquefaction); ii) a second
container in which is contained anti-sperm antibodies covalently bound to colored particles in
30 a hypotonic solution; iii) a membrane or filter for capturing sperm/colored particle or
sperm/colored particle/antibody complexes and allowing passage of unbound, colored
particle or colored particle/antibody; and iv) a chart for interpreting the results obtained based
on the color of the filter. Indication of less than about 20 million spermatozoalmL indicates
low pregnancy potential when using the Male Fertility approach; or less than about 50,000
35 spermatozoalmL when using the Male Contraceptive approach.
The present invention is further illustrated by the following Examples which
are intçndçd merely to further illustrate and should not be construed as limiting. The entire
contents of all of the references (including literature references, issued patents, published
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patent applications, and co-pending patent applications) cited throughout this application are
hereby expressly incorporated by reference.
Example 1: Kit for Isolating Motile Sperm From a Semen Sample
s
Kit Component~:
1) (1) container (tube #1) for collecting and liquefying ejaculate cont~ining 30mg/mL of
dextran and Smg/mL of chymotrypsin in an isotonic solution;
2) (1) see-through container (tube #2), which indicates three volumes: lower (1 mL), middle
o (1.5 mL) and upper (2 mL). The three volumes can be indicated by black and red marks (the
top and the bottom mark being of the same color)7
3) 0.5 mL container of isotonic solution (tube #3)
3) (1) 0.5 mL container of bovine serum albumin (BSA) at 0.2 mg/mL in an isotonic solution
(tube #4);
15 4) (1) empty container;
5) (at least 3) disposable sterile pipettes or capillaries;
6) (1 ) capillary pipette to remove and transfer motile sperm
7) (at least 1) test strip co..~ i.,g a means for qual~ lhlg sperm (e.g. protein reagent);
8) instructions for use
20 9) (1) heating block at 37C (optional)
Procedure:
The ejaculate was collected in the collection tube (tube #1) C~ g
25 Smg/mL of chymotrypsin and 30mg/mL of dextran in an isotonic solution. The mixture was
allowed to stand at room temperature for about 5 minutes.
Using a dropper, 1 mL aliquots of the semen/dextran/chymotrypsin mixture
were added to the calibrated see-through container (tube #2), so that the mixture reached the
30 first marked level. The pipette was disposed of.
Using a second droppel7 an applopl;ate volume of the isotonic solution
(container #3) was used to overlay the semen/dextran/chymotrypsin ~ lule to the upper
mark of each see-through container, resulting in the formation of a two layer density gradient:
3s the lower layer consisting of the semen/dextran/chymotrypsin mixture and the upper layer
consisting of the isotonic solution. The dropper was disposed of.
The mixture was allowed to stand for at least S minutes at room temperature or
optionally in a 37C heating block.
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A sterile capillary pipette was inserted to reach the middle mark of the
container and applied to a test strip which contained a means for 4~ Ling sperm, e.g.,
protein reagent, lipid reagent.
Example 2: Temperature Stress for Predicting Pregnancy Potential of a Sperm Sample
Human sperm (HS) samples were obtained from 44 male partners of couples
undergoing in vitro fertilization (IVF) (n=33) or gamete intrafallopian transfer (GIFT)
lo (n=11). All male partners had normal semen by conventional analysis (By convention, a
normal semen sample is defined as having greater than 20 million cells/mL, greater than 60%
motility, a rate of progression greater than 2 (on a scale of 1 to 4), greater than 60% normal
forms, 1.5 to 5.0 mL of ejaculate, no significant sperm agglutination, no significant number
of leucocytes, and no hyperviscosity). Sperm samples were washed using the standard
Percoll procedure and resuspended in HTF medium cont~ining 10% pl~m~n~te. An aliquot
of the sperm suspension was utilized for artificial reproductive technologies (ART, e.g. IV~
and GIFT) and a 1 OO~L aliquot of the same sperm suspension was incubated at 40C for 4h
(stress test). Stress test scores were expressed as the ratio of final to initial motility. Analysis
was performed by stepwise multiple regression. The independent variables were: women's
age; HS motility before and after the stress test; stress test score; and the number of embryos
transferred per IVF cycle. The dependent variables were: pregnancy and fertilization
outcome.
In a blind cohort study, of the 44 ART cycles, l I (25%) resulted in pregnancy.
Of the 44 sperm samples used in the study, 24 (55%) had stress test scores < 0.8 and 20
(45%) > 0.8. Controlling for all variables, the stress test score was significantly correlated
with pregnancy outcome (P = 0.0001). In sharp contrast, the motility before the stress test
was not correlated with pregnancy outcome. All pregnancies occurred in sperm samples with
stress test scores > 0.8. Therefore, a cut-off value > 0.8 was selected to predict pregnancy
outcome. The sensitivity of the stress test was 100% and the specificity was 73%. The
negative predictive value of the test, defined as the absence of pregnancy with stress test
scores < 0.8, was 100% and the positive predictive value, defined as the occurrence of
pregnancy with stress test scores > 0.8, was 55%. Although the stress test score is highly
predictive of pregnancy outcome, the fertilization rate was better predicted by the motility
after the stress test (P = 0.007).
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Example 3: Oxidative Stress for Pre~lictin~ Pregnancy Potential of a Sperm Sample
A similar test as described in Example 4 was carried out, except that the stresswas provided by adding 10',1L of 0.125mM ferrous iron in PBS (phosphate based saline) and
0.6mM ascorbate also in PBS to 3011L sperm suspensions for a total volume of 50 ~L and
incubating the llliXLUlC for 0.5h at 37C with gentle .~h~king. Results indicate that the values
obtained correlate with the stress test score at 40C for 4h.
10 F.Y~rle 4: Superoxide Dismutase (SOD) Activity in Sperm Cor,lat~s with
Pregnancy Rate
Human spermatozoa release oxygen radicals and undergo spontaneous lipid
peroxidation resulting in extensive damage to the sperm plasma and acrosomal membranes
s and loss of their pregnancy potential. Cu/Zn-superoxide dismutase (SOD) protects sperm
against oxygen radical-mediated toxicity and endogenous lipid peroxidation. Surface SOD
immunoreactivity is t;xplcssed over the mid piece and post equatorial regions of human
sperm (Alvarez and Storey, (1992) J. Andrology 13(3):232-241, the contents of which are
incorporated herein by reference).
Whether SOD activity correlates with sperm pregnancy potential and
pregnancy rate was studied using sperm samples obtained from 27 male partners from
couples undergoing in vitro fertilization (IVF). The age of the female partner ranged from 24
to 49 years. The number of oocytes inseminated ranged from 3 to 20 and the number of
embryos transferred from 1 to 7. Sperm samples were washed using the standard mini
Percoll procedure, and resuspended in HTF medium co~ g 10% pl~cm~n~te. An aliquot
of the sperm suspension was utilized for IVF and another aliquot was used to measure the
SOD activity of that particular sample.
SOD activity in the sperm samples examined ranged from 1.5 to 12 U/1 o8
cells (P < 0.001). All six sperm samples with SOD activities less than or equal to 3.3U/108
cells resulted in failed fertilization (P < 0.001). Between 4.7 and 7.1U/108 cells fifteen out
of sixteen oocytes were fertilized but no ongoing pregnancies resulted.
These results show that sperm samples with high SOD activity (i.e. greater
than about 9 units/ 108 cells) have a high pregnancy potential, while samples with low SOD
activity (i.e. less than about 7 units/ 108 cells) have a low pregnancy potential. Samples
having an SOD activity in the range of greater than or equal to about 7 and less than about 9
units/108 cells have borderline pregnancy potential.
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Example 5: Assay for Identifying a High Pregnancy Potential Sperm Sample
The ejaculate was collected in the collection tube (tube #1) cont~ining
5 5mg/mL of chymotrypsin. After about three to five minl-tes (at which time the semen should
be liquefied), an aliquot (about 5011L) of the liquefied semen was added to another test tube
(tube #2), which contained horseradish peroxidase-conjugated sheep anti-human superoxide
dismutase (SOD) IgG polyclonal antibodies (The Binding Site, Inc., 5889 Oberlin Drive, San
Diego, CA 92121) and horseradish peroxidase-conjugated sheep anti-human glutathione
10 peroxidase (GPx) IgG polyclonal antibodies (The Binding Site, Inc., 5889 Oberlin Drive, San
Diego, CA 92121) dissolved in a hypotonic solution. After five minutes, a dip test strip with
bound sheep anti-human SOD and anti-human GPx Ig G polyclonal antibodies (The Binding
Site, Inc., 5889 Oberlin Drive, San Diego, CA 92121) was inserted into test tube#2. After
five minutes, the test strip was removed from the tube and rinsed with tap water.
The test strip was then dipped into another test tube (tube #3), which
contained the peroxidase substrate, hydrogen peroxide (H2O2) and diaminobenzidine (DAB);
(Sigma Chemical Co., St. Louis, MO 63178). The color ratio of SOD/GPx provides an
indication of pregnancy potential by comparison with a chart provided with the kit. Color in
20 the GPx pad indicates equal or greater than about 20 million spermatozoa/mL. If the color in
the SOD pad is higher than the color in the GPx pad, a high pregnancy potential of the sperrn
is indicated. If the color in the SOD pad is equal to or lower than the color in the GPx pad, a
low pregnancy potential is indicated.
2s Example 6: Assay for Monitoring the Efficacy of Male Contraception (Color Chart
Approach)
The ejaculate from a male who had taken an a~lopliate dose of a male
contraceptive that suppresses spermatogenesis (e.g. a GnRH antagonist) or who had had a
30 vasectomy, was collected in the collection tube (tube #1) co~ it~ g 5mg/mL ofchymotrypsin in an isotonic solution. After three to five minutes (at which time the semen
should be liquefied) about 500 IlL of the liquefied semen was added to another test tube (tube
#2) cont~ining anti-human sperm polyclonal antibodies (Arnel Products Co., Inc., Cherokee
Station, New York, N.Y.; Chemicon International Inc., Temecular, CA) covalently bound to
35 gold particles (having an intrinsic pink color) or to colored latex particles (having a blue or
yellow color) in a hypotonic or isotonic solution. The mixture was incubated for up to about
10 minutes to allow the sperm antigens exposed after the hypotonic treatment to react with
the antibodies. Following incubation, the contents of test tube #2 was transferred to a filter
membrane, so that sperm/colored particle/antibody complex was retained and unbound,
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colored particle/antibody passed through the filter into a reservoir. The color of the filter can
then compared to various color possibilities depic,ted on a chart that is included with the kit to
determine the number of sperm present in the sample and thereby determine the efficacy of
the male contraceptive treatment. This approach allows one to detect in semen a
s concentration of at least SO,OOO spermatozoa/mL. This sensitivity is comparable to that
obtained with the regular light microscope commonly used in andrology laboratories.
Example 7: Assay for Monitoring the Efficacy of Male Contraception (Plus (+) or
Minus (-) Approach)
The ejaculate was collected in the collection tube (tube #1) Co~ lg
Smg/mL of chymotrypsin in an isotonic solution. After about 3 to S minutes (at which time
the semen should be liquefied) a drop (SO~l) (Male Fertility Kit) or 0.5 mL (Male
Contraceptive Kit) of the liquefied semen was added to another test tube (tube #2) cont~ining
5 a hypotonic solution. After a period of incubation of S minutes, a drop (about SO~lL) (Male
Fertility Kit) or the total contents (Male Contraceptive Kit) was transferred to a membrane
provided with two windows: window #l was used to apply the sample where after the sample
was applied, the soluble sperm antigens reacted with anti-human sperm antibodies conjugated
to gold or colored latex particles included in a reservoir in the membrane; window #2 had a
horizontal colored line. As the sperm antigen/antibody/particle complex migrated through the
membrane, a second anti-human sperm antibody bound in the vertical position to the
membrane underlying window #2 (capture antibody) reacted with the sperm
antigen/antibody/particle complex producing a colored vertical line when the sperm
concentration in semen was above SO,OOO spermatozoa/mL in which case a plus (+) sign
2s appeared. If the concentration of sperm in semen was below 50,000 spermatozoa/mL, a
minus (-) sign appeared.
Example 8: Male Fertility & Contraceptive Screening Kits (Filter Approach)
30 Kit Components
1 ) ( 1 ) calibrated semen collection tube;
2) (1) squeeze bottle cont~ining the chymotrypsin solution;
3) (1) test tube cont~ining the isotonic or hypotonic solution;
35 4) (2) droppers;
S) (1) filter mounted on a liquid reservoir;
6) (1) squeeze bottle cont~ining anti-sperm antibody-coated colored latex or gold particle
solution;
7) instructions for use.
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Procedure
Using the squeeze bottle, chymotrypsin solution (dextran-free) was added to
5 the semen collection tube in a volume equal to the semen volume. The llliX~ ; was allowed
to stand at room telll~ e for about S minutes in order for the semen to liquefy.
Using a dlu~l~el, one drop (Male Fertility Kit) or 0.5 mL (Male Contraceptive
Kit) of the semen/chymotrypsin mixture in the semen collectiûn tube was added to another
0 test tube (test tube #2) c~ ing an isotonic solution (e.g. phosphate buffered saline or
Earle's Medium) or a hypotonic solution (e.g. distilled water) .
One drop (5011L) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) ûf
test tube #2 was added to a filter (e.g. blocked nitrocellulose, 5~1m pore size or microfiber
5 glass filter, 2.7 ,um pore size) mounted on a liquid reservoir. Seminal plasma proteins, which
were about 10-20 nm in size passed through the filter, while sperm cells which were about
1000 times bigger (50~1m in size) were retained.
One drop of anti-sperm antibody-coated colored particles (e.g. gold particles
20 that have an intrinsic pink color, or a colored latex particle) was added onto the filter and the
appearance of color on the filter indicated a potentially fertile sperm sample. By using this
filter procedure, the antibody used to coat the latex particle did not have to be highly specific
for the sperm cell (i.e. it could exhibit some cross-reactivity with seminal plasma proteins).
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Example 9: Male Fertility & Cohtr~ e Screening Kits (Filter Approach with
Preincubation of the Antibody and the Sperm Antigens)
s Kit Components
1) (1) calibrated semen collection tube;
2) (1) squeeze bottle cot~ the chymotrypsin solution;
3) (1) test tube co.l~ ;.lg the isotonic or hypotonic solution;
0 4) (2) dlo~
5) (1) filter mounted on a liquid reservoir;
6) (1) squeeze bottle co~ lg anti-sperm antibody-coated colored latex or gold particle
solution;
7) instructions for use.
Procedure
Using the squeeze bottle, chymotrypsin solution (dextran-free) was added to
the semen collection tube in a volume equal to the semen volume. The mixture was allowed
to stand at room ~elllpel~ e for about 5 minutes in order for the semen to liquefy.
Using a dl~p~er, one drop (about 50,~L) (Male Fertility Kit) or 0.5 mL (Male
Contraceptive Kit) of the semen/chymotrypsin mixture in the semen collection tube was
added to another test tube (test tube #2) cont~ining anti-sperm antibody-coated colored
particles (e.g. gold particles that have an intrinsic pink color; or a colored latex particle) in an
isotonic solution (e.g. phosphate buffered saline or Earle's Medium) or hypotonic solution
(e.g. distilled water) .
One drop (50~1L) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of
30 the contents of test tube #2 was added to a filter (e.g. nitrocellulose, 511m pore size or
microfiber glass filter, 2.7 ~m pore size) mounted on a liquid reservoir. Seminal plasma
proteins/color particle antibody/complex, which were about 10-20 nm in size passed through
the filter, while sperm cells/colored particle antibody/complex which were about 1000 times
bigger (50~m in size) were retained. By using this filter procedure, the antibody used did not
35 have to be highly specific for the sperm cell (i.e. it may show some cross-reactivity with
seminal plasma proteins).
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Example 10: Male Fertility & Co~lr..ce~Jli. e Screening Kits (Agglutination Approach)
Kit Component.~
s
1) (1) calibrated semen collection tube;
2) (1) squeeze bottle cont~ining the chymotrypsin solution;
3) (1) test tube co.~ -g an isotonic or hypotonic solution and sperm-specific antibody-
coated particles (uncolored or colored);
10 4) (2) dropl~e~;
5) (1) slide having a color, which contrasts with the anti-sperm antibody-coated particles.
6) instructions for use.
Procedure
Using the squeeze bottle, chymotrypsin solution (dextran-free) was added to
the semen collection tube (tube # I ) in a volume equal to the semen volume. The mixture was
allowed to stand at room temperature for about 5 minl~te~ in order for the semen to liquefy.
Using a dlopp~l, one drop (50~1L) (Male Fertility Kit) or 0.5 mL (Male
Contraceptive Kit) of the semen/chymotrypsin mixture was added to another test tube (test
tube #2) co~ ;.lg an isotonic solution (e.g. phosphate buffered saline or Earle's Medium) or
hypotonic solution (e.g. distilled water) and sperm-specific antibody-coated particles
(uncolored or colored).
One drop of test tube #2 was deposited on a slide, which was of a color that
contrasted with the sperm specific antibody coated latex particles, so that agglutination,
indicating the potential presence of fertile sperrn could be discerned on the slide. The
absence of agglutination when using the Male Fertility approach, was indicative of less than
about 20 million spermatozoa/mL; borderline agglutination was indicative of greater than
about 20 million spermatozoa/mL and less than about 40 million spermatozoa/mL; and the
presence of high agglutination was indicative of greater than about 40 million
spermatozoa/mL. The absence of agglutination when using the Male Contraceptive approach
was indicative of less than about 50,000 spermatozoa/mL.
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Example 11: Male Fertility & Contraceptive Screening Kits (Non
Immunoreactive/Filter Approach)
5 Kit Component~
1) (1) calibrated semen collection tube;
2) (1) squeeze bottle co"l~ g the chymotrypsin solution;
3) (1) test tube co"~in;llg the isotonic or hypotonic solution;
0 4) (2) dro~pel~;
5) (1) filter mounted on a liquid reservoir;
6) (1) squeeze bottle colll~il-il-g colored reagents that react non-specifically with sperm cell
components, e.g., pink-colored gold particles (reacts with proteins) or red-colored rhodamine
(reacts with lipids).
15 7) instructions for use.
Procedure
Using the squeeze bottle, chymo~ hl solution (dextran-free) was added to
20 the semen collection tube (tube #1) in a volume equal to the semen volume. The mixture was
allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
Using a dl~ppel, one drop (5011L) (Male Fertility Kit) or 0.5 mL (Male
Contraceptive Kit) of the liquefied semen was added to another test tube (tube #2) coll~ g
2s gold particles (that have an intrinsic pink color) in a hypotonic or isotonic solution. One drop
(Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of the contents of tube #2 was added
onto the filter (nitrocellulose with 5~m pore size or microfiber glass filter with a pore size of
2.7~1m). Gold particles have an intrinsic pink color and high affinity for biomolecules (e.g.
proteins or lipids) and therefore can bind to both spermatozoa and seminal plasma proteins.
30 By using this filter procedure, the unbound gold particles and the gold particles bound to
seminal plasma proteins went through the filter into the reservoir while sperm cells with
bound gold particles were retained on the filter indicating a pink color. The presence of a
pink color when using the Male Fertility approach, was indicative of the presence of greater
than about 20 million spermatozoa/mL; and greater than about 50,000 spermatozoa/mL when
3s the Male Contraceptive approach was used.
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Example 12: Assay for Identifying a High Pregnancy Potential Sperm Sample
The ejaculate was collected in the collection tube (tube #1) cont~ining
5 Smg/mL of chymotrypsin in an isotonic solution. After about 3 to 5 minlltes (at which time
the semen should have been liquefied) a drop (about 50~L) of the liquefied semen was added
to a test tube (tube #2) cont~ining anti-glutathione peroxidase (GPx) antibodies bound to
yellow colored particles and anti-superoxide dismutase (SOD) antibodies bound to blue
colored particles in a hypotonic or isotonic solution. The lllixlule was incubated for up to
o about 10 minutes to allow the sperm antigens exposed after the hypotonic treatment to react
with the antibodies. Following incubation, the contents of test tube #2 was transferred to a
filter membrane, so that sperm/colored particle/antibody complex was retained and unbound,
colored particle/antibody passed through the filter into a reservoir. If the filter appeared
green, the SOD/GPx ratio was about 1:1 and the sample had borderline pregnancy potential;
if the filter appeared bluish, high SOD content was indicated and the sample had high
pregnancy potential; and if the filter appeared yellowish, low SOD content was indicated and
the sample had low pregnancy potential.
Example 13: Male Fertility & Contraceptive Screening Kit
Kit Con~onent~
1) (1) calibrated semen collection tube;
2) ( 1 ) squeeze bottle containing the liquefying enzyme (e.g. chymotrypsin or pronase at 5
25 mg/ml.) (squeeze bottle #1);
3) (1 ) squeeze bottle (with a removable top) cont~ining 1 50!1L of an isotonic or hypotonic
solution (squeeze bottle #2);
4) (1) squeeze bottle containing a 0.5 mg/mL solution of Rhodamine-123 (Molecular Probes?
Inc. Eugene, OR) in distilled water (squeeze bottle #3);
30 S) (1) d~op~e~,
6) ( 1 ) porous filter mounted on a liquid reservoir;
7) instructions for use.
Procedure
Using the squeeze bottle provided (squeeze bottle # 1 ) ? two drops (per
milliliter of semen) of a solution of pronase solution (Smg/mL) (Sigma Chemical Corp., St.
Louis, MO) in Earle's medium was added to the ejaculate in the collection tube. The mixture
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was allowed to stand at room te~ cldlule for about S minutes in order for the semen to
liquefy.
Using a dropper, one drop (Male Fertility Kit) or four drops (Male
5 Contraceptive Kit) of the liquefied semen was added to another squeeze bottle (squeeze bottle
#2) which had the top removed and contained 1 50~LL (Male Fertility Kit) or 250~1L (Male
Contraceptive Kit) of a hypotonic soLution. Then, one drop (Male Fertility Kit) or 4 drops
(Male Contraceptive Kit) of the Rhodamine- 123 (Molecular Probes, Inc. Eugene, OR)
solution in squeeze bottle #3 was added to squeeze bottle #2 and the top placed back on. The
0 contents were then mixed by tapping with a finger.
Two drops (Male Fertility Kit) or the total content of squeeze bottle #2 was
added to a porous filter (about 2.7 ~lm pore size) mounted on a liquid reservoir and the color
visll~li7~1 The absence of color was indicative of a concentration of less than about 20
5 million spermatozoa/mL; faint color was indicative of greater than about 20 million
spermatozoa/mL and less than about 40 million spermatozoa/mL; and the presence of color
was indicative of greater than about 40 million sperm~to7o~/mL.
Those skilled in the art will recognize, or be able to ascertain using no more
20 than routine experiment~tion, many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed by the following claims.
