Claims:We claim:
1. A sperm washing method, the method comprising:
layering a first density medium, a second density medium, and semen in a main tube; and
forming a sperm pellet of washed sperm by centrifuging the contents of the main tube.
2. The method as claimed in claim 1, wherein density of the first density medium is higher than density of the second density medium.

3. The method as claimed in claim 2, wherein the layering comprises:
forming a first layer of the first density medium at the bottom of the main tube;
forming a second layer of the second density medium under the first layer; and
forming a third layer of semen over the first layer.
4. The method as claimed in claim 3, wherein forming the second layer comprises releasing of the second density medium at the bottom of the first layer, whereby the first density medium is replaced and pushed upwards by the second density medium.

5. The method as claimed in claim 4, wherein the second density medium is released in a laminar flow manner with respect to the first density medium.

6. The method as claimed in claim 1, further comprising forming a final inseminate volume of washed sperm by adding fresh culture medium to the sperm pellet.

7. The method as claimed in claim 6, further comprising carrying out artificial insemination using the final inseminate volume.
, Description:FIELD OF THE INVENTION
The present invention relates to a method of sperm washing and apparatus for sperm washing.
BACKGROUND
State of the art includes many centrifugation methods and separation of various solutions into its constituent parts. In particular, separation of seminal fluid into concentrated semen and serum is still a very precise method conducted in laboratory conditions. However, sperm count enhancement in semen of human male is a concern particularly in the field of artificial insemination in humans.
Sperm washing is used to separate individual sperms from semen. Washed sperm may be used in artificial insemination. Artificial insemination may be carried out using the intrauterine insemination (IUI) technique or in-vitro fertilization (IVF). Sperm washing may be used to decrease risk of HIV transmission by an HIV-positive male, in which case the washed sperm is injected into a female using an artificial insemination technique. Sperm washing involves removing any mucus and non-motile sperm in the semen to improve the chances of fertilization and to extract certain disease-carrying material in the semen.
The present invention provides an easy to use apparatus and method for sperm washing.

SUMMARY OF THE INVENTION
The following presents a simplified summary of the subject matter in order to provide a basic understanding of some aspects of subject matter embodiments. This summary is not an extensive overview of the subject matter. It is not intended to identify key/critical elements of the embodiments or to delineate the scope of the subject matter. Its sole purpose is to present some concepts of the subject matter in a simplified form as a prelude to the more detailed description that is presented later.
An improved method of sperm washing is disclosed in the present disclosure. Specifically adapted apparatus for sperm washing is also disclosed. The apparatus includes a main tube, and three functionally distinct caps for the main tube. The three caps are used at different stages of the method to achieve accuracy and efficiency in sperm washing.
BRIEF DESCRIPTION OF THE DRAWINGS
For a better understanding of the embodiments of the apparatus and method described herein, and to show more clearly how they may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, wherein like reference numerals represent like elements/components throughout and wherein:
FIGs. 1A-1C illustrate a main tube according to an embodiment of the present invention;
FIGs. 2A-2C illustrate a layering cap according to an embodiment of the present invention;
FIGs. 3A-3C illustrate a centrifuge cap according to an embodiment of the present invention;
FIGs. 4A-4C illustrate a segregation cap according to an embodiment of the present invention;
FIGs. 5A-5F illustrate a layering process using the main tube and the layering cap, according to an embodiment of the present invention; and
FIGs. 6A-6G illustrate a segregation process using the main tube, centrifuge cap, and the segregation cap, according to an embodiment of the present invention.
DETAILED DESCRIPTION
Exemplary embodiments now will be described with reference to the accompanying drawings. The disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art. The terminology used in the detailed description of the particular exemplary embodiments illustrated in the accompanying drawings is not intended to be limiting. In the drawings, like numbers refer to like elements.
The specification may refer to “an”, “one” or “some” embodiment(s) in several locations. This does not necessarily imply that each such reference is to the same embodiment(s), or that the feature only applies to a single embodiment. Single features of different embodiments may also be combined to provide other embodiments.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and/or “comprising” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It will be understood that when an element is referred to as being “connected” or “coupled” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. Furthermore, “connected” or “coupled” as used herein may include operatively connected or coupled. As used herein, the term “and/or” includes any and all combinations and arrangements of one or more of the associated listed items.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The present invention discloses an apparatus and method for sperm washing. The apparatus includes a main tube, and three caps each of which can be removably coupled to an open end of the main tube. A layering cap is coupled to the main tube to carry out process of layer formation of different density media in the main tube. A centrifuge cap is coupled to the main tube to carry out centrifugation of the layered media of the main tube using a centrifuge. Further, a segregation cap is coupled to the main tube to carry out separation of a supernatant and sperm pellet. Fresh culture medium is added to the sperm pellet to get final inseminate volume that may be used for artificial insemination.
FIG. 1A illustrates a main tube 102 in accordance with one embodiment of the present invention. The main tube has a body 110 having an open end 104 and a closed end 106. The exterior surface of the body 110 is smooth and has a circular cross section. At the open end 104, external threads 108 are provided on the exterior surface of the body 110. FIG. 1B illustrates a cross section of the main tube 102 and an internal surface 112 thereof. FIG. 1C illustrates a top view of the main tube 102. The main tube 102 may be made of medical grade plastic. In one embodiment, the main tube 102 may be formed of glass.
FIG. 2A illustrates a layering cap 200. The layering cap 202 has a cap portion 202 and a tube portion 204. The cap portion 202 is circular in shape and has a flat top surface (see FIG. 2C). The cap portion 202 has internal threads thereon for securing the layering cap 200 onto the main tube 102. The tube portion 204 is a longitudinal hollow and narrow channel having its first end as the hole in the cap portion 202 and second closed end opposite to the first end. In proximity to, the second closed end of the tube portion 202, there are one or more holes 206-208 in the body of the tube portion 202.
FIG. 2B illustrates a cross section view of the layering cap 200 and FIG. 2C illustrates a top view of the layering cap 200. As can be seen, in the top view, the cap portion 202 has top surface 210 having a central hole 212 therein. When the layering cap 200 is coupled to the main tube 102, the central hole 212, the tube portion 204, and the one or more holes 206-208 form a continuous channel for fluid flow. A fluid may come out of the one or more holes 206-208 in a direction perpendicular to the axial direction of the tube portion 204.
FIG. 3A illustrates a centrifuge cap 302. The centrifuge cap has a top flat surface 304. The surface 304 is uniform and continuous without any holes or protrusions. The cap 302 may have internal threads on the inside walls thereof. The internal threads secures the cap 302 on to the main tube 102 by engaging with the external threads 108 of the main tube 102. FIG. 3B illustrates a cross section view of the cap 302.
FIG. 4A illustrates schematically a segregating cap 402. The cap 402 has a cap portion 406, a linear portion 408, and a blocking portion 410, where the linear portion is solid longitudinal part that couples the cap portion 406 with the blocking portion 410. The cap portion 406 has a circular cross section and has internal threads to couple it with the main tube 102. The linear portion 408 may be made of circular cross section having diameter less than the internal diameter of the main tube 102. The blocking portion 410 has varying diameter from its top end to its bottom end so as to fit tight just above the bottom portion 106 of the main tube 102. Once the cap 402 is secured on to the main tube 103, the blocking portion 410 divides the volume of the main tube 102 into two compartments, an upper compartment above the blocking portion 410 and a lower compartment below the blocking portion 410. FIG. 4B schematically illustrates a cross section view of the cap 402. FIG. 4C schematically illustrates a top view 414 of the cap 402. The cap portion 406 has a top surface 416 that is substantially flat. The surface 416 has two holes therein. A first hole 418 is used to drain out any fluid present in the upper compartment of the main tube 102. The second hole 420 is an air hole. The first hole 418 has a diameter larger than the second hole 420. The first hole 418 and the second hole 420 may be located on either side of the centre of the surface 416.
FIGs. 5A-5F schematically illustrates steps of a layering process 500 in accordance with one embodiment of the present invention. FIG. 5A illustrates the main tube 102 that is empty. FIG. 5B shows the layering cap 200 coupled to the main tube 102. As can be seen from the figures, when the cap 200 is secured onto the main tube 102 at its open end, the one or more holes 206-208 in the tube portion 204 are placed in proximity to the bottom end of the main tube 102. FIG. 5C illustrates addition of a low density medium 504 through the layering cap 200 using a syringe 502 couples to the hole 212 of the layering cap 200. As a plunger of the syringe 502 is pressed, the medium 504 flows from the syringe 502 to the bottom of the main tube 102, via the hole 212, the tube portion 204, the one or more holes 206-208. Thus, addition of the medium 504 is completed. As shown in FIG. 5D, a high density medium 506 is added to the main tube 102 in a similar fashion. The medium 506 is released in a laminar flow at the bottom of the main tube 102. The medium 506 replaces the medium 504 in a bottom up manner so that in a final position, the medium 504 forms a uniform layer over the layer of medium 506 which is at the bottom of the main tube 102. Layering of medias 504 and 506 in the above manner ensures that there is no intermixing of the medias with each other and uniform and stable layers are formed.
The layering cap 200 may be removed from the main tube 102 after formation of layers of media 504 and 506. As shown in FIG. 5E, semen 508 is added directly with a pipette 510 to the top of low density medium 504. The main tube 102 may be kept in an inclined position while adding the semen 508 so that semen 508 forms a layer at the top of low density medium 504 without mixing therewith. As shown, in FIG. 5F, centrifuge cap 302 is coupled to the main tube 102. At this stage, the main tube 102 has three layers of fluids, semen 508 at the top, low density medium 504 in middle, and high density medium at the bottom. The above completes the layering process. After securing the open end of the main tube 102 with the centrifuge cap 302, the centrifugation is carried out of the fluids for a predetermined period of time. In one embodiment, the predetermined period of time is 15 minutes. In another embodiment, the predetermined period of time is at least 10 minutes.
As shown in FIG. 6A, the centrifugation results in formation of sperm pellet 604 at the bottom of the main tube 102 and a supernatant 602 above the pellet 604. The supernatant 602 is needed to be discarded without mixing it up with the pellet 604 or discarding the pellet 604.
FIG. 6B, illustrates discarding of the supernatant 602 using the segregating cap 402. Once the cap 402 is secured on to the main tube 102, the blocking portion 410 of the cap 402 segregates the supernatant 602 and the pellet 604 from each other. The pellet 604 is secured in a closed compartment formed by the blocking portion 410 at the bottom of the main tube 102. When the cap 402 is secured on the main tube 102, the supernatant 602 can be drained out of the main tube 102 through the first hole 418.
FIG. 6C shows pellet 604 at the bottom of the main tube 102 after the cap 402 has been removed. There might remain some live sperm sticking on inner surface of the main tube 102. As shown in FIG. 6D, a predetermined amount of fresh culture medium 610 may be added to the pellet 604 to suspend it therein and also wash down any useful semen portion sticking to the walls of the main tube 102. After suspension of the pellet 604 in the medium 610, the centrifuge cap 302 is coupled to the main tube 102 for carrying out centrifugation. Next, centrifugation is carried out resulting in formation of pellet 612 and supernatant 614 (see FIG. 6E). Step of draining out supernatant 614 is carried out in a manner similar to that of FIG. 6B, using again the segregating cap 402.
As shown in FIG. 6F, draining out of supernatant 614, and removing the cap 402 leave behind sperm pellet 612. Fresh culture medium may be added to the sperm pellet 612 to make final inseminate volume. The final inseminate volume of washed sperm may be used in artificial insemination.
It will be apparent to those having ordinary skill in this art that various modifications and variations may be made to the embodiments disclosed herein, consistent with the present disclosure, without departing from the spirit and scope of the present disclosure. Other embodiments consistent with the present disclosure will become apparent from consideration of the specification and the practice of the description disclosed herein.