 |
US
Patent & Trademark Office |
| Patent
Number |
5,976,389 |
| Title |
Method
of semen filtration |
| Inventor(s) |
Panayiotis
M. Zavos |
| Date
Granted |
November
2, 1999 |
| United States Patent |
5,976,389 |
| Zavos |
November 2, 1999 |
Method of semen filtration
Abstract
There is disclosed a method of filtering semen comprising
admixing Sephadex beads of about 40 to 120 microns in diameter
in the column of a semen filtering device having openings
at both ends with a media, permitting the beads to hydrate,
expand, and settle in the column to form a multi-layer filter
having passages of predetermined sizes therebetween permitting
passage therethrough of motile spermatozoa, adding to the
multi-layer filter a washed semen specimen suspended in a
liquid media suitable for use with semen, and recovering the
semen that pass through the filter.
| Inventors: |
Zavos; Panayiotis M.
(2413 Vince Rd., Nicholasville, KY 40356-9343) |
| Appl. No.: |
816539 |
| Filed: |
March 13, 1997 |
| Current U.S.
Class: |
210/807 |
| Intern'l Class:
|
B01D 037/00 |
| Field of Search:
|
210/266,282,807
604/349 |
References Cited [Referenced
By]
U.S. Patent Documents
| 4151254 |
Apr., 1979 |
Gimovsky |
210/282. |
| 4244694 |
Jan., 1981 |
Farina et al. |
210/198. |
| 4430229 |
Feb., 1984 |
Yamawaki et al. |
210/263. |
| 4690678 |
Sep., 1987 |
Douglas-Hamilton |
604/349. |
Primary Examiner: Cintins; Ivars
Attorney,
Agent or Firm: Sigalos; John L.
Parent Case Text
REFERENCES TO RELATED APPLICATIONS
This application is a continuation of application Ser. No. 08/096,734,
filed Jul. 23,1993, now abandoned, which is a continuation of
application Ser. No. 07/839,042, filed Feb. 18, 1992, now abandoned,
which is a continuation of Ser. No. 07/701,320, filed May 05,
1991, now abandoned, which is a continuation of Ser. No. 07/291,960,
filed Dec. 30, 1988.
Claims
What I claim is:
1. The method of filtering semen comprising admixing a dry soluble
powder composed of microscopic beads which are about 40 to 120
microns in diameter and which are synthetic, organic compounds
derived from the polysaccharide dextran with an aqueous media
in a column of a semen filtering device having openings at both
ends, permitting said dry soluble powder to hydrate, expand,
and settle in said column to form a multi-layer filter having
passages of predetermined sizes therebetween permitting passage
therethrough of motile spermatozoa, passing through said multi-layer
filter a washed semen specimen suspended in a liquid media suitable
for use with semen, and recovering the semen that pass through
the filter.
2. The method of claim 1 wherein the concentration of sperm
in the liquid media prior to filtration is about 100.times.10.sup.6
sperm/milliliter.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a particular method of processing
semen and filtering it through a filtration column for the purpose
of selectively increasing the number of motile and morphologically
normal spermatozoa in the filtrate. The column contains a filter
device comprised of a disc made of standard insulate and numerous
dehydrated Sephadex (a dry soluble powder composed of microscopic
beads which are synthetic, organic compounds derived from the
polysaccharides dextran) beads, 40 to 120 microns in diameter,
that lay on top of the insulate disc. When hydrated with standard
laboratory media, the beads expand and settle, forming a multi-layer
filter.
2. Description of the Related Art
Several methods have been proposed wherein semen can be processed
to increase the quality and quantity of the spermatozoa population
in a semen sample, thereby increasing the probability of success
when the semen sample is used for Artificial Insemination (AI),
In Vitro Fertilization (IVF), and related clinical techniques.
Among these methods are washing and storing spermatozoa in media,
and the "rise" or "swim up" procedure, a technique that takes
advantage of the swimming abilities of a small percentage of
spermatozoa within a population. See e.g. Russell and Rogers,
J. Androl., 8:25-33, 1987.
None of these methods are simple and short and all of them are
susceptible to technician error and accident. However, these
disadvantages are overcome by the present invention, which provides
for a simple apparatus and procedure that enhances normal morphologic
spermatozoa forms and increases the number of motile spermatozoa.
Semen that is processed and filtered in accordance with this
invention demonstrates qualitative and quantitative spermatozoa
characteristics that are as good or better than any other semen
processing method. Data from tests made on the procedure and
apparatus claimed in this invention are set forth in Table 1,
which is attached hereto.
SUMMARY OF THE INVENTION AND OBJECTS
The present invention is a sterile, non-hydrated, disposable
non-spermicidal filtration column to be manufactured in accordance
with strict quality controls, packaged in sterile wrapping,
and made available for wide distribution to clinical locations.
It consists of a disposable column, a top stopper, a removable,
nonporous upper disc situated horizontally across the column
approximately two-thirds from the top, a bottom non-spermicidal
filter disc made of standard insulate material that allows for
the free flow of elements less than 50 to 100 microns in size,
numerous dehydrated non-spermicidal beads, 40 to 120 microns
in diameter, that lay on top of the insulate disc, and a reuseable
bottom closure cap. The device is used to filter spermatozoa
after it has been prepared for filtration in accordance with
a semen processing method that is part of the invention.
It is the primary object of the present invention to provide
a method and apparatus by which to process semen and filter
spermatozoa for the purpose of selectively increasing the normal
morphology, motility, sperm concentration, and fertilization
potential of semen samples for use in AI and related clinical
techniques.
A second object of the present invention is to provide a simple
method and apparatus by which to filtrate spermatozoa that is
easily and quickly accomplished with good repeatability and
with little likelihood of technician error or accident.
A third object is to provide a sterile, non-hydrated, disposable
filtration column that can be manufactured and distributed widely
to clinical locations, where it can be hydrated and used with
relative ease.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a lateral perspective of the present invention.
DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENT
FIG. 1 depicts generally the present invention, which includes
a top stopper (1), a nonporous upper disc (2) located horizontally
across the column approximately two-thirds of the column height
from the top, a lower disc (3) made of standard insulate material
that allows for the free flow of elements less than 50 to 100
microns in size, numerous dehydrated Sephadex beads (4), 40
to 120 microns in diameter, that lie on top of the insulate
disc, and a reuseable bottom closure cap (5).
Use of the present invention involves two major steps:
(A) Mixing and washing the semen in order to prepare the spermatozoa
for filtration; and
(B) Filtering the spermatozoa through the Semen Filter Column.
The spermatozoa are prepared for filtration by obtaining a freshly
ejaculated and liquified semen specimen and evaluating it for
volume, sperm count per milliliter, motility and presence of
debris. These findings should be noted for later reference.
Next, the semen specimen is diluted in a media. Any standard
laboratory media accepted in the literature for use with human
semen is acceptable. The liquid should be mixed gently and centrifuged
at 400 g for 6 minutes. The supernatant is then removed and
discarded. The resulting sperm pellet is resuspended in media
to a final concentration of 100.times.10.sup.6 sperm/milliliter.
With the mixing and washing of the semen complete, the spermatozoa
is now ready for filtration.
Prior to or at the beginning of semen preparation, a Semen Filter
Column (SFC) should be removed from its sterile package. One
or more SFCs may be needed per ejaculate to be processed. At
this same time, a 15 ml. test tube should be placed upright
in a 37 degree centigrade water bath.
Holding the SFC upright and with the bottom closure cap in place,
the top stopper should be removed and 3.0 ml. of media placed
into the SFC. The media should be at room temperature. The media
will flow onto the nonporous upper disc, which should then be
gently removed by nudging it with a pipette so as to push the
disc against the walls of the SFC, and pulling it upward. The
media can now drip down the column and mix with the dehydrated
Sephadex beads on the lower insulate disc. A Pasteur pipette
should be used to mix the beads with the media and remove any
air bubbles that may exist or have formed in the bottom of the
SFC. Caution should be taken not to disturb the bottom disc.
Once the media has begun mixing with the dry beads, five minutes
or more should be allowed for the beads to expand and settle
thereby forming a multi-layer filter permeable to liquids at
atmospheric pressure on the insulate disc, the filter having
passages of predetermined sizes between the beads permitting
passage therethrough of motile spermatozoa. Next, remove the
bottom cap from the SFC in order to allow two to three drops
of media to drip through the bottom opening. The opening is
quickly closed with the bottom closure cap and the SFC is placed
into the 37 degree centigrade water bath until the semen is
ready for filtration.
When ready to start the actual filtration, remove the SFC from
the water bath. If the initial evaluation of the semen showed
little or no debris and motility greater than 50%, place 0.75
ml. of the well mixed-washed semen preparation into the top
of the SFC. If, however, debris is excessive and motility is
less than 50%, only 0.5 ml. of the semen preparation should
be placed into the SFC.
Once the semen preparation is in the filtration column, the
bottom closure cap should be removed and the SFC placed into
the 15 ml. test tube so that as the filtrate flows through the
SFC it can be collected in the test tube. The semen preparation
should be filtered for 15 minutes, with more media periodically
being added to the SFC in order to maintain the media at its
original 3.0 ml. level. Occasionally during filtration, it may
be necessary to aspirate media from the SFC using a pipette
and then expirating it directly onto the top of the filter beads
so as to prevent the build up of a film of dead sperm and debris
which could block or interfere with proper flow of filtrate.
When filtration is complete, remove the SFC from the test tube
and replace the bottom closure cap. Pull the filtrate from the
test tube, centrifuge it, and reconstitute the spermatozoa at
the desired level of dilution in a buffer of choice. The level
of sperm dilution, or the sperm concentration in the resuspended
preparation, shall be determined by the clinical purpose that
the spermatozoa will be used for. For example, the filtered
spermatozoa may be used for In Vitro Fertilization (IVF), Artificial
Insemination (AI), Intrauterine Insemination (AIH-IU), Gamete
Intrafollopian Transfer (GIFT) and other clinical techniques.
Prior to actual use of the filtered-resuspended semen specimen,
it should be assessed for sperm count per milliliter, motility,
the presence of debris and other parameters routinely used in
semenology. The filtered spermatozoa are now ready for use.
TABLE 1
__________________________________________________________________________
Qualitative and quantitative characteristics of spermatozoa before and
after filtration
recovered through the Sephadex filtration process, as well as, recovered
via the swim-up
procedure using human spermatozoa
Qualitative - Quantitative Sperm Characteristics (N = 12)
Motility
Grade
Morphology
Acrosomes
Debri Presence.sup.2
Sperm Recovered
(%) (0-4) (% Normal) (% Normal) (Subjective) (.times.
10.sup.6 /ml)
__________________________________________________________________________
Before filtration
57.6 .+-. 5.1
3.1 .+-. 0.6
61.3 .+-. 7.0
57.4 .+-. 7.1
3.0 .+-. 0.9
78.3 .+-. 9.1
After filtration 84.7 .+-. 3.7 .+-. 0.4 87.4 .+-. 4.1 88.3 .+-.
5.1 0.8 .+-. 0.1 31.2
.+-. 5.3
Swim-up.sup.3 86.1 .+-. 3.7 .+-. 0.5 82.4 .+-. 5.2
81.0 .+-. 6.2 0.7 .+-. 0.1
17.1 .+-. 4.4
__________________________________________________________________________
.sup.1 According to Zavos and Cohen, Fertil. Steril., 1980.
.sup.2 Subjective evaluation; 0 = comp1ete absence; 4 = severe. (Cohen et
al., 1985).
.sup.3 According to Cohen et al., Fertil. Steril., 1985.
* * * * *
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