Abstract:
A multi-chamber module ( 10 ) for preparing sperm for assisted reproductive techniques comprises a first chamber ( 40 ) for holding a fluid medium, a second chamber ( 52 ) for receiving semen, and a third, harvesting chamber ( 50 ) into which the fluid medium flows from the first chamber ( 40 ) and from which that fluid medium passes into the second chamber ( 52 ), thereby to permit sperm to move against the fluid flow and pass from the second chamber ( 52 ) into the third chamber ( 50 ) from which they are harvested. A flow-restricting barrier composed of a stack of ribbed plates ( 44 ) defining radial passages therebetween is positioned between the second chamber ( 52 ) and the third chamber ( 50 ).

Description:
[0001]    The present invention relates generally to infertility, and more particularly to apparatus for and methods of preparing sperm for assisted reproductive technology, including IUI (intra-uterine insemination), IVF (in vitro fertilization), GIFT (gamete intra-fallopian transfer) and ICSI (intra-cytoplasmic sperm insertion).  
         BACKGROUND OF THE INVENTION  
         [0002]    To increase the chances of fertilization, several kinds of treatment including IUI, IVF, GIFT and ICSI are performed with prepared sperm composed of viable, motile sperm, free of seminal plasma and debris. A variety of methods have been developed to separate motile sperm from semen. The most commonly used sperm isolation techniques, including “Swim up” and “Percol”, that involve washing and centrifugation, may result in some damage to the sperm (J. Kerlin and W. Byrd “Human Reprod.” 1991; 6: pages 1241-1246; R. J. Aitken and J. S. Clarkson: “J. Reprod. Fertil.” 1987; 81: pages 459-469.  
           [0003]    More traditional methods for the preparation of sperm may lead to iatrogenic damage to sperm (D. Mortimer “Human Reprod.” 1991; 6: pages 173-176).  
           [0004]    Aitken and Clarkson have shown that centrifugal force generates the production of reactive oxygen species that may damage sperm and impair their fertility potential. “Percol” has been used largely in the setting of laboratory research and its clinical use is associated with certain disadvantages. Some batches have been found to contain high levels of endotoxin, making them unsuitable for clinical use (C. Y. Andersen, and J. Grinsted: “J. Assisted Reprod. Gent.” 1997; 14: pages 624-628). In late 1996, “Percol” was withdrawn from clinical use as a sperm separation medium (Guneet Makkar et al. “Fertil. Steril.” 1999; 72: pages 796-802).  
           [0005]    An ideal isolation technique would be rapid, inexpensive and isolate all motile sperm without damaging them.  
           [0006]    It has been reported that when sperm are put into a fluid flow, the motile sperm rapidly align themselves and swim against the flow (F. Abed. “ The new finding of a phenomenon in sperm motility: the spermatozoa swims against flow  ” —from “ In vitro fertilization and assisted reproduction ”, edited by V. Gomel and P. C. K. Leung, Monduzzi Editore, 1997: pages 13-15). Non-motile and sluggish sperm, along with other cellular components, are washed downstream away from the motile sperm. Cilia have been shown to be present in endometrial cells of many mammals. Ciliary currents in both the fallopian tubes and the uterus move in the same direction and extend towards the external os. One may expect that this flow performs two functions. Firstly, this flow acts as a guide for sperm, leading sperm with the correct motility parameters towards the site of fertilization at the ampoule of the fallopian tubes. Secondly, this flow acts as a natural selection mechanism to optimize the quality of sperm able to reach the fertilization site.  
         SUMMARY OF THE INVENTION  
         [0007]    It is an object of the present invention to utilise the known phenomenon of sperm alignment against flow in order to be able to prepare sperm for assisted reproductive techniques and procedures.  
           [0008]    In accordance with the present invention there is provided a multi-chamber module for preparing sperm for assisted reproductive techniques, comprising a first chamber for holding a fluid medium, a second chamber for receiving semen, and a third, harvesting chamber into which the fluid medium is arranged to flow from the first chamber and from which that fluid medium is arranged to pass into the second chamber, thereby to permit sperm to move against the fluid flow and pass from the second chamber into the third chamber from which they are harvested as sperm.  
           [0009]    In a preferred embodiment, the second and third chambers are separated by a mechanical flow-restricting barrier through which fluid is arranged to flow from the third chamber to the second chamber and through which sperm are arranged to pass from the second chamber to the third chamber.  
           [0010]    This barrier can comprise a stack of spaced plates or discs defining passageways therebetween.  
           [0011]    The module also preferably includes control means for controlling the velocity of flow of the fluid from the first chamber to the third chamber.  
           [0012]    In a preferred embodiment, the second and third chambers are located within a cup-shaped container with the chambers separated by an annular barrier through which the fluid is arranged to flow.  
           [0013]    Preferably, the module includes a waste chamber into which waste material can pass from the second chamber, for example via filter means.  
           [0014]    Also in accordance with the present invention there is provided a method of preparing sperm for assisted reproductive techniques, comprising establishing within a multi-chamber module a flow of fluid medium from a first chamber to a second chamber via a third chamber, adding fluid medium to the first chamber, adding semen to the second chamber, and harvesting from the third chamber sperm which pass, against the fluid flow, from the second chamber to the third chamber.  
           [0015]    Preferably, the flow of fluid medium from the first chamber to the second chamber is controlled so that a steady-state flow of fluid medium is arranged to pass from the third chamber to the second chamber.  
           [0016]    Also in accordance with the present invention there is provided a method of sperm preparation which comprises establishing within a processing module a fluid flow against which sperm are able to travel to a harvesting zone within the module.  
           [0017]    The apparatus and methods of the present invention have a number of advantages over conventional methods of preparing sperm. The present invention does not induce any damage to the sperm, because the procedure does not require any use of chemicals or centrifuges. The preparation process is rapid and simple. The process of sperm separation is under direct observation and can easily be controlled. The module can be used by physicians without the need for laboratory equipment. Also, the use of the module in accordance with the invention not only serves to separate sperm, but also washes the sperm, thus eliminating the need for any centrifuge process. The present invention can be used not only for the separation of motile from non-motile sperm but also can be used with motile, morphologically normal sperm, to provide sperm suitable for ICSI procedures. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWING  
       [0018]    A more detailed description of the present invention will now be given, with reference to the accompanying drawing, wherein like reference characters refer to like parts throughout the several views, and in which:  
         [0019]    [0019]FIG. 1 is a partially cut-away sectional view of a preferred embodiment of harvesting module in accordance with the invention;  
         [0020]    [0020]FIG. 2 is a view, on an enlarged scale, of part of one of the guide plates of the module of FIG. 1; and  
         [0021]    [0021]FIG. 3 is a view, again on an enlarged scale, of the two bottom guide plates of the stack shown in FIG. 1. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0022]    Referring to the drawing, the multi-chamber module of the present invention is indicated generally at  10 . The module is substantially cylindrical in shape, with an outer circumferential wall  12 , a base  14  and a cap  16 . It is preferably made of plastics material. Extending radially inwardly from the outer circumferential wall  12  is a horizontal dividing wall  18  which continues at its radially inner face as an upwardly extending vertical wall  20  which meets the underside of the cap  16 . Vertically below the wall  20  is an outer circumferential wall  22  of a cup-shaped container indicated generally at  24 . This container  24  has a base  26 . The upper edge of the outer wall  22  of the container  24  is spaced from the bottom of the vertical wall  20  to define a circumferential slot therebetween. This slot is plugged by a membrane filter  28 . The filter is preferably such as to permit the passage only of material below a figure within the range of 1.3 to 3.5 microns, preferably below 3 microns. The cup-shaped container  24  is supported within the module, for example on a support member  30  which is set on the base  14  of the module. The interior of the support member  30  connects with a first port  32  and a second port  34  in the exterior wall  12  of the module.  
         [0023]    Two holes  36  and  38  are formed through the base  26  of the container  24 , adjacent to its centre. Hole  36  communicates with outlet port  34  via a valve (not shown). Hole  38  communicates with outlet port  32  via another valve (not shown). The valve associated with outlet port  32  is also connected to the chamber  40  which is defined by the walls  12 ,  18  and  20  and by the cap  16 . This chamber  40  is hereinafter referred to as the medium chamber, i.e. a chamber which is arranged to hold a fluid medium.  
         [0024]    The chamber  42  which is located below the medium chamber  40  and which extends below the base of the container  24  is hereinafter referred to as the waste chamber. A vent hole (not shown) is provided through the upper part of the outer wall  12  of the waste chamber.  
         [0025]    Within the cup-shaped container  24  is positioned a stack of annular plates or discs  44 , a portion of one of which is shown in more detail in FIG. 2. These plates  44  are each annular in shape and are spaced from one another by the provision of a plurality of upstanding ribs  46  on the upper surface of each plate. As shown in FIG. 2, these ribs  46  are of constant thickness and are sector-shaped, extending from the inner radial wall to the outer radial wall of each plate  44 . In a preferred embodiment, twenty of these sector-shaped ribs  46  are provided, equispaced around each plate. When the plates  44  are stacked one upon another, this leaves radial passages through the plates, between the ribs, with each passage having a depth of, for example, 30 to 50 microns. In a preferred embodiment, twenty plates  44  are stacked together. Above the stacked plates  44  is provided a cup-shaped receptacle  48 . The plates  44  rest on the base  26  of the container  24  which, as can be seen from FIG. 1, is stepped around the perimeter. The location of the plates in this way leaves a central cylindrical chamber  50  above the holes  36  and  38 , hereinafter referred to as the harvesting chamber, and an outer annular chamber  52 , outwardly of the plates  44 , hereinafter referred to the seminal chamber.  
         [0026]    As mentioned above, the medium chamber  40  is in communication with the harvesting chamber  50  via the hole  38 . The passageway between the medium chamber  40  and the harvesting chamber  50  is substantially L-shaped, with the valve associated with outlet port  32  being located approximately at the right-angle in the passageway. In the horizontal portion of the passageway there is located a plastics material rod (not shown) which has a longitudinally extending groove in its peripheral surface, along which the fluid medium from the chamber  40  can pass to the hole  38  and thus into the harvesting chamber  50 . The groove in the rod serves to control the velocity of the fluid flow from chamber  40  to chamber  50 .  
         [0027]    The method of using the module of the present invention will now be described. A 5 ml syringe is first connected to the valve associated with port  32 . The syringe is filled with a fluid medium. The valve associated with port  34  is opened and the module is inverted. Then, approximately 1.5 ml of the medium is injected into the harvesting chamber  50  via hole  38 . This continues to ensure that there is no air remaining in the harvesting chamber. When the harvesting chamber has been filled with the fluid medium, the valve associated with port  34  is closed and the module is inverted again so that it is then as shown in the drawing. The syringe is then filled with air and is connected to the valve associated with port  32 . Next, the cap  16  of the module is opened and the medium chamber  40  is filled with fluid medium. As a result of the gravitational forces, the medium in this chamber  40  flows through the passageway which links that chamber with the harvesting chamber  50 , the fluid being regulated by the grooved rod housed within the support member  30 .  
         [0028]    Then, using a pipette, a small amount of semen is spread around the seminal chamber  52 , i.e. outside the stack of plates  44 . Then, the whole module is placed within a CO 2  incubator. During the incubation period, motile sperm move against the flow of fluid, as shown most clearly in FIG. 3, by capillary flow from the seminal chamber  52  to the harvesting chamber  50 . Because the medium chamber  40  is filled with the fluid medium before the sperm is added to the seminal chamber  52 , there is already a flow of fluid between and through the plates  44  from the harvesting chamber  50  towards the seminal chamber  52 . Because of the nature of the motile sperm, they are able to pass from the seminal chamber into the harvesting chamber, whereas sluggish and non-motile sperm cannot tolerate the flow of fluid and do not reach the harvesting chamber.  
         [0029]    The seminal chamber  52  will gradually fill with fluid. Because of the presence of the membrane filter  28 , only seminal plasma and other debris can pass through the filter from the seminal chamber  52  into the waste chamber  42 .  
         [0030]    Following the termination of the incubation period, which normally takes about  30  minutes, the cap  16  of the module is closed completely, the valve associated with port  34  is opened, and air is injected by syringe through port  32  and the associated valve and thus into the harvesting chamber  50 . The pressure of this air causes the fluid medium within the harvesting chamber, and the motile sperm within it, to pass out through port  34  and into a sterile tube.  
         [0031]    In the use of this module, normal sperm will be able to move against the fluid flow and pass the barrier which is provided by the ribbed plates. This mechanism serves to select the most qualified sperm and only permits the sperm that are capable of moving faster than the flow of fluid to reach the harvesting chamber. In this way, the motile sperm are separated from the seminal plasma, non-motile and sluggish sperm, other cellular components and bacteria.  
         [0032]    It should be noted that the module of the present invention -can also be used for the separation from semen of motile, morphologically normal sperm, as is required for ICSI procedures.