Patent Abstract:
An embryo from a female animal is transferred to another animal by determining presence of an embryo in the uterus of a donor animal by ultra-sonic imaging and inserting an endoscope vaginally into the uterus to a location adjacent the embryo. A tool of the endoscope projects to a position to extract the embryo washed into a container of the tool which is then closed by moving a closure part to enclose the embryo and extracting the endoscope to remove the embryo for transfer to a recipient animal. The fluid into the container can be controlled in pressure to maintain a required pressure generally matching that inside the uterus.

Full Description:
This application is a continuation in part of Application PCT/CA2012/050407 filed Jun. 19, 2012 and claims the benefit under 35 USC 119 (e) of Provisional Application 61/499,351 filed Jun. 2, 2011. 
     This invention relates to an apparatus and method for embryo transfer from one female mammal to another. The description hereinafter primarily relates to mares where the commercial operation of transfer methods is desirable, but ineffective; but can relate to any female mammal. 
    
    
     BACKGROUND OF THE INVENTION 
     Embryo transfer (ET) is the process of harvesting an embryo or embryos from a donor and transferring it to a recipient. The process can be done surgically or non-surgically with the latter being the preferred method in bovine and equine species and the former being the preferred technique in smaller species such as porcine, ovine, caprine and canine. 
     Application for ET in the mare is commercially done for three main reasons; a donor mare in competition can produce and transfer an embryo and still compete while a recipient mare carries her genetics to a term pregnancy, to produce multiple pregnancies in one year, to transfer to a recipient mare when the donor mare is considered a high risk for pregnancy complications. 
     In cattle superovulation is a successful procedure where with hormone therapy multiple (average 6 but numbers as high as 40 reported) embryos can be harvested (flushed) in any one procedure. Unfortunately mares do not respond successfully to superovulation so a single procedure yields at best only one embryo. The exception is when a mare naturally double ovulates and thus two potential embryos might be retrieved. The average success of achieving a pregnancy through embryo transfer in the mare is 25%. The average cost per attempt is from $7000 to $12000 and the industry reports embryo transfer in mares to be approximately $250 000 000 annually worldwide. This means that $187 500 000 is spent with no results. This poor success has hindered the process of embryo transfer in mares from becoming a more main stream procedure. 
     The traditional method for transferring an embryo in mares is to aseptically pass a catheter through the vulva, vagina and cervix and into the uterine body. A cuff is inflated to seal the cervical uterine junction. The uterus is flushed with approximately 4 liters of specialized solution. The solution is filtered through a 20 micron filter. The filter is emptied into petri dishes and then the dishes are searched with microscopy to find an embryo. If found the embryo is isolated and washed in another specialized solution and then loaded into a transfer pipette. The recipient mare is aseptically prepared for transfer and the transfer pipette is passed through the vulva, vagina and cervix and into the uterine body where the embryo is deposited. 
     Special concern for timing is required for a successful pregnancy from an ET procedure. The procedure is considered to start at day 0 which is when the mare is observed to have ovulated. The sperm must be present in the fallopian tubes prior to ovulation. Fertilization takes place in the fallopian tubes shortly after ovulation and the embryo remains there for 5 days after which time it moves into the uterus. Flushing or retrieving the embryo is normally done at day 7 which allows for adequate time for the embryo to reach the uterus for it cannot be retrieved from the fallopian tube. After day 8 the embryo hatches from its protective shell called the zona pellucita which then makes the embryo more fragile to handle. So 7 days post ovulation achieves the highest success rates thus far. A uterus is dynamic and changes through the female cycle. For this reason a recipient mare must be synchronized with the donor mare and her uterus must be close to 7 days post ovulation which adds another level of difficulty to the procedure. At the time a flush is performed it is unknown whether there is a viable embryo present or not. 
     Attempts have been made to transfer embryos at later stages of development such as 11 to 14 days post ovulation. At 11 days post ovulation the embryo is visible to a highly trained practitioner using ultra-sonography. This would seem to be ideal as retrieval would only be attempted if there was a pregnancy visualized. Unfortunately no success has been achieved at this stage. It is hypothesised that the embryos were too fragile and didn&#39;t survive the procedure. 
     SUMMARY OF THE INVENTION 
     It is one object of the invention to provide an improved method and/or apparatus for use in embryo transfer. 
     According to one aspect of the invention there is provided a method for transfer of an embryo from a female animal comprising: 
     determining presence of an embryo in the uterus of a donor animal by ultra-sonic imaging; 
     inserting an endoscope vaginally into the uterus to a location adjacent the embryo; 
     operating a tool of the endoscope to extract the embryo into a container of the tool; 
     closing the container of the tool to enclose the embryo and extracting the endoscope to remove the embryo for transfer to a recipient animal. 
     Preferably the container includes two parts which move to a fully closed position. 
     This is preferably done by providing two hemi-spherical parts which rotate, or one of which rotates relative to the other from a first open position with one cupped inside the other to a closed spherical position sealing around the edges of the two parts. Other closure systems can be used for example a sliding sleeve arrangement around an inner tube which has a hole to collect the embryo. 
     Optionally there is provided a fluid supply duct for supplying fluid to the tool where the fluid supply duct opens into the closed container. In this case, there can be provided a pressure sensor for controlling pressure of the fluid inside the container to match that in the uterus. Pressure control may or may not be necessary within the closed scoop. 
     Preferably the container has a transverse dimension of at least 1.0 cm and preferably of the order of 1.5 cm. 
     Preferably the presence of the embryo is detected at a time period of the order of 11 days after insemination. 
     Preferably the endoscope is guided to a position within the uterus by passing through a separate guide tube inserted into the uterus through the vagina. 
     Preferably the guide tube is held against bending during operation of the tool so as to locate an end of the guide tube at a fixed position within the uterus. That is the guide tube is either rigid so that it cannot bend at all, or is semi-rigid so that it is adjustable in shape by bending at one or more points along its length but that it maintains that shape when in use, that is as it is inserted into the uterus through the vulva, vagina and cervix and during insertion and operation of the endoscope and tool. 
     It will be appreciated that the guide tube is typically inserted and guided manually by the veterinarian in many cases by feeling the position of the guide tube within the vagina of the animal by a hand inserted through the rectum. That is the end of the guide tube can be carefully guided and moved to its position within the uterus by the veterinarian feeling exactly where that end is in relation to the cervix. 
     The shape and arrangement of the guide tube is arranged so that the guide tube is held fixed relative to the uterus during operation of the tool so as to locate an end of the guide tube at a fixed position within the uterus. This provides a fixed point or basis for the functioning of the operating components of the endoscope for moving an end of the endoscope relative to an end of the guide tube with the end of the guide tube held in fixed position relative to the uterus. In this way the skilled veterinarian can operate the conventional operating components of bend, orientation and displacement of the end of the endoscope to accurately locate the end of the endoscope at a required position relative to the wall of the uterus. 
     Preferably the guide tube is held fixed relative to the uterus by locating the guide tube at the cervix. 
     Preferably the guide tube is located at the cervix by first and second inflating balloons with one inside the uterus at the cervix and the other outside the cervix in the vagina. 
     According to a second aspect of the invention there is provided a method for transfer of an embryo from a female animal comprising: 
     inserting an endoscope vaginally into the uterus to a location adjacent the embryo; 
     operating a tool of the endoscope to extract the embryo into a container of the tool; 
     guiding the endoscope to a position within the uterus by a guide tube inserted into the uterus through the vagina; 
     holding the guide tube fixed relative to the uterus during operation of the tool so as to locate an end of the guide tube at a fixed position within the uterus; 
     and operating components the endoscope for moving an end of the endoscope relative to an end of the guide tube. 
     Preferably the guide tube is held against bending during operation of the tool so as to locate an end of the guide tube at a fixed position within the uterus. 
     Preferably the guide tube is held fixed relative to the uterus by locating the guide tube at the cervix. 
     Preferably the guide tube is located at the cervix by first and second inflating balloons with one inside the uterus at the cervix and the other outside the cervix in the vagina. 
     Preferably the endoscope and the tool are inserted into the guide tube when the guide tube is in fixed position with the end of the guide tube within the uterus. 
     Based on the augmentation and refinement to the method of embryo described herein, retrieval at 9 to 13 days and more preferably 11 or 12 days can achieve success rates in excess of 80%. 
     In the equine uterus a cascade of events begins in which, if no embryo is present, the uterus undergoes changes and the reproductive system starts the process toward ovulation. If a viable embryo is present in the uterus it blocks the hormone and chemical pathways that initiate the cascade to ovulation thus pregnancy is maintained. At the time of transfer on day 11 or 12 the recipient&#39;s reproductive track is already undergoing changes that may make it unable to maintain a pregnancy. 
     In the present method, the recipient and the donor both are bred at the same time and an embryo is removed from the recipient mare and exchanged for an embryo from the donor mare. The delicate nature of the embryo is a very important consideration for success of this procedure so highly specialized equipment has been designed to overcome this. 
     Using this modified embryo transfer technique the procedure is cost effective and is a more attractive method of breeding in the equine industry. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       One embodiment of the invention will now be described in conjunction with the accompanying drawings in which: 
         FIG. 1  is a vertical cross sectional view through a tool for use in an endoscope for extraction of an embryo, showing the tool in an initial open position. 
         FIG. 2  is a cross-sectional view along the lines  2 - 2  of  FIG. 1 . 
         FIG. 2A  is a cross-sectional view along the lines  2 - 2  of  FIG. 1  showing the tool in the closed position after collection of an embryo. 
         FIGS. 3 and 4  show the operation of the tool in a method of extraction. 
         FIG. 5  is a vertical cross sectional view through a second embodiment of tool for use in an endoscope for extraction of an embryo, showing the tool in an initial open position for insertion through the tube of the endoscope. 
         FIG. 6  is a top plan view through of the tool of  FIG. 5  in the closed position after insertion through the tube of the endoscope and collection of the embryo. 
         FIG. 7  is a vertical cross sectional view through a tube for locating within the uterus of the animal the endoscope and tool of  FIG. 1  for extraction of an embryo. 
         FIG. 8  is a vertical cross sectional view on an enlarged scale through the remote end of the tube of  FIG. 1  showing the insertion and operation of the endoscope. 
         FIG. 9  is a vertical cross sectional view on an enlarged scale through the proximal end of the tube of  FIG. 1  showing the control system of the endoscope. 
         FIG. 10  is a vertical cross sectional view on a reduced scale the tube of  FIG. 1  together with the endoscope and the tool of  FIG. 1  showing the components in position inserted through the vulva, vagina and cervix of the animal into the uterus. 
     
    
    
     In the drawings like characters of reference indicate corresponding parts in the different figures. 
     DETAILED DESCRIPTION 
     Endoscopes are a well known device widely used in surgery and other procedures and comprise a tube with a camera and illumination which can be passed through an opening into the interior of the body and which can be manipulated to different positions. A central bore allows a tool to be passed through the tube for acting on the interior, many different tools are available. Typically a collar is provided which can be inflated to locate the tube at a required portion and to seal the opening relative to the tube. Arrangements of this type are widely used and well known to persons skilled in the art so that further details are not required. 
       FIG. 1  shows a recovery tool for use as part of a modified endoscope  40  including a tube  10  with a camera lens  11  and an illumination source  12  carried on the tube and including fiber optic communication from a distal end  10 A of the tube  10  to control systems of the endoscope and the near end for operation by the user. The tube  10  is arranged so that it can be passed through an opening, in this case the vagina into the interior of the body. The tube includes components (not shown) which allow the end  10 A to be manipulated to different positions. One or more central bores  13  or ports allow a tool  14  to be passed through the tube. Typically a collar  15  is provided which can be inflated to locate the tube at a required portion and to seal the opening, in this case the uterus, relative to the tube. 
     Through one of the working ports  13  of the endoscope is inserted a grasping tool  30 . The grasping tool is small globe  20 , approximately 1.5 cm in diameter. The globe has two hemispherical halves  21  and  22  one of which rotates by sliding slides inside the other. The components are made out of surgical grade stainless steel. 
     When closed as shown in  FIG. 2  the two parts  21  and  22  form a sealed unit or globe  20  with a sealing edge  23 . The inner part  22  is rotated around the axis of the sphere by an operating element  24  at the end of the tube  10  which is operated by a control at the near end of the tube from the open position where the inner part is wholly within the outer part to form a hemi-spherical scoop to a closed position in which the globe is closed and sealed. This globe also has a fluid port  25  within it so fluid can be added or withdrawn from the globe  20 . The fluid supply system  26 A of a control unit  26  for supply to the port  25  passes through the tube  14  and includes an inline pressure sensor  27  sensitive to the internal pressure in the line and therefore within the closed globe  20 . The supply  26 A of the control unit  26  can be operated so that holding fluid can be supplied or removed to adjust the internal pressure in the globe  20  to match the same pressure as that of a normal uterine environment for that stage of embryo. The pressure within the uterus can be measured in situ or can be predetermined from historical measurements. 
     The special tool described above can in some embodiments be used with a stock endoscope. The tool can alternatively be a permanent “biopsy tool” which is manufactured by assembly into place in an endoscope from a typical supplier, but where the tool is not be able to be removed after it is manufactured into the scope. This is due to the fact that the typical globe is too large for the portal through the tube of a typical endoscope. 
     The tool operates in a similar manner to an ice cream scoop. In the open position one half of the globe  20  is rotated inside the other half as shown in  FIG. 2A . The neck  29  of the tool that passes through the endoscope portal  13  is formed from two flexible tubes  25  and  24  one inside the other. The outer tube  25  is fixed to the bottom half  21  of the globe and the inner tube  24  is fixed to the top half of the globe. At the operator end the user operates the device  26  by activating a turning movement to the inner tube  24  so that it rotates the top half of the globe to close it. The inner tube  24  also provides the fluid port which is optional. 
     When the tool is first inserted as shown in  FIG. 4  in the open position and the collar  15  inflated to hold the tool in place, once the tool is passed into visual proximity of the embryo it can be used to pick up the embryo. 
     Endoscopes have the ability to pass fluid through the port  13  or through a separate special port (not shown) to dilate the inside of the tube  14  or open the lumen of the uterus. 
     Thus most endoscopes have a small port adjacent to the lens. This port typically has a very small metal deflector that directs water across the lens to clean it should it become obscured with mucus or other debris. The air required in the present method can also be passed through this port. There is a pump on the power unit that works the scope. At the operator end there is a two stage valve that is normally worked by the index finger. With light depression air is pumped through the port adjacent to the lens that is normally used for insufflation to allow for dilation which enhances passage of an endoscope. If the valve is fully depressed fluid is pumped through to clean the lens. 
     This fluid supply through the endoscope is used to open the uterus and to infuse a small amount of fluid into the uterus to float the embryo away from the tissue of the uterus wall so that it can be simply picked up with the scoop. Air or air and fluid may be used to insufflate the uterus to allow for better visualization and pull the majority of the endometrium away from the embryo. Fluid may then be used to completely free the embryo or the tool can be used to pick up the embryo at that point, if its positioning is good and endometrial contact is minimal. 
     Once the embryo is in view the cuff  15  will be inflated so that if further fluid is infused the embryo will not float away. When insufflation is normally done there is constant loss of air along the outside of the scope but once the embryo is in view, the cuff  15  is inflated so constant insufflation is no longer needed and dilation of the uterus can be static. 
     It is necessary to control the supply and volume of fluid to prevent the embryo from floating too far away. In normal instances, because the pressure in the inflation collar  15  is kept low, the natural closure/collapse of the tissue of the uterus around the collar and the tool keeps a partial seal around the instrument and provides a slope running away from the collar  15  to prevent the embryo from falling into the area of the collar  15  where it become impossible to retrieve. The injection of fluid through the endoscope typically is required because of the fragile and movable nature of the embryo. In  FIG. 4 , the inflation collar  15  is close to the end of the endoscope at the location of the tool since this better locates the tube  14  and allows better control over movement of the tool. The third fluid supply tube  25  is optional but when provided acts to bathe the embryo. 
     When the embryo has been picked up, the tool is retracted from the donor animal and moved to the recipient. Once the embryo is placed in the recipient and the globe re-opened to release the embryo, fluid can be infused into the bottom of the globe and the embryo floated out. 
     The complete procedure is as follows: 
     1. The donor mare is synchronized in her estrous cycle with recipient mare sufficiently that they are in synchronism; or the recipient can be as much as 24 hours ahead or 72 hours but preferably not more than 48 hours behind the donor mare in her ovulation. 
     2. Both mares are bred on their synchronized ovulation as per normal breeding methods. 
     3. At earliest possible time post ovulation an embryo is searched for via ultrasonography in both the donor and recipient mares. Currently this is carried out at day 11 post ovulation when the embryo is sufficiently large to be determined by this method. 
     4. Once pregnancy is confirmed by the ultra-sound image in both the donor and recipient the embryo transfer and exchanged is commenced. 
     5. The donor and recipient mares are prepared pre-embryo recovery for a normal aseptic embryo recovery technique. Ideally the recipient mare is pregnant but that is not absolutely necessary. This transfer can still be attempted if the recipient is not pregnant but still in synchrony with the donor. 
     6. The procedure starts with the recipient where the recipient is sedated for ease of recovery and transfer. 
     7. In the recipient, a first technician operates the ultrasound imaging system to locate and document where the embryo is residing. 
     8. A second technician passes the recovery scope vaginally using normal aseptic palmed delivery to the cervix and the cervix is digitally enlarged and the scope is then advanced through the cervix and the operators hand is removed. The scope is then advanced until it appears on the ultrasound adjacent to the embryo. The ultrasonographer may or may not stop at this time. One the embryo is found via ultrasound the ultrasound is removed and the perineum washed thoroughly and the scope is passed into position. 
     9. Once the scope is in view with the embryo, the uterine horn is insufflated by air or air and fluid supply enough to free the majority of endometrial contact with the embryo. The uterus is infused with the fluid through the supply tube  25  with a fluid, such as a commercially available embryo recovery medium, to float the embryo. The inflation cuff  15  on the end of the recovery scope is arranged to prevent washing the embryo away. The embryo is captured with the grasping tool  20  on the recovery scope. The grasping tool  20  on the working end  10 A of the recovery scope  10  is now a closed and is infused with the commercially available embryo holding fluid. The recovery scope is withdrawn from the uterus. From the recipient animal, the embryo is discarded or kept for other purposes. 
     10. The step  9  is repeated with the donor mare. 
     11. The recovery scope is washed with warmed alcohol and then 1 liter of warmed saline 
     12. The recipient mare is sedated again if necessary and her perineum washed again. 
     13. The recovery scope, now containing the donor&#39;s embryo is passed using normal aseptic palmed delivery to the cervix and the cervix is digitally enlarged 
     14. The recovery scope is then advanced to the location from where recipients own embryo was removed. The embryo is deposited in the uterus at the bifurcation of the uterine horns. The grasping tool  20  is opened and the embryo is either dumped by turning the whole tool by the base tube  29  to invert the cup or expelled with fluid. The recovery scope is withdrawn and the procedure is complete. 
     The mare is checked via ultra sound immediately after the procedure for embryo placement. The mare is checked by ultrasound imaging at 6 and 24 and 48 hours post-transplant for embryo viability and procedure success. 
     Turning now to  FIGS. 5 and 6 , a second embodiment of the tool  50  is shown which is formed of an outer sleeve  51  slideable on the outer surface of a tube  52  inside the sleeve  51 . The tube  52  has an end portion  52 A projecting beyond an end of the sleeve  51  which connects to a cylindrical stainless steel tip member  54 . The tip member is typically 2 to 3 cms long and has an elongate slot  53  in one side leading to a hollow interior  53 A. The tip member includes a portion  53 B which is necked down to a reduced diameter onto which the end portion  52 A of the tube  52  is engaged. The hollow interior  53 A of the tip member  54  communicates with the interior of the tube  52  allowing access to the interior of the tube  52 . The tube  52  and the sleeve  51  are both formed of a medical grade plastics material allowing some flexibility. The stainless steel tip member  54  has an end closure portion  54 A which closes the front end of the tip member. The metal tip member thus has a hole in the hollow interior that communicates with the slot  53  so that fluid can be passed from the inner tube  52  into the slot  53 . The outer face  54 B of the closure portion  54 A is domed or hemi-spherical to provide a smooth rounded surface of a transverse diameter or the order of 1.0 cms. The diameter of the tip member is lightly larger than that of the outer surface of the cylindrical portion of the tip member  54  to provide a shoulder  55  surrounding the end of the tube to provide an abutment for the end of the sleeve. In operation the endoscope is maneuvered to the required position and the tool  50  inserted through the bore  13  to the location of the embryo. As described previously, fluid can be supplied through the tube  52  through the opening  53  to the uterus to wash out the embryo from its position. When exposed, the embryo is scooped by rotating the tool so that the opening  53  is moved to the embryo to allow it to enter into the interior of the metal tip within the slot and not enter the tube. When the embryo is captured, the sleeve is moved longitudinally to close the hole by covering the tip portion  52 A up to the tip member  54  allowing the tool and captured embryo to be extracted. The fluid control systems previously described are used to ensure the protection of the captured embryo. 
     Turning now to  FIGS. 7 to 10 , there is shown the tool  14  inserted through the endoscope  40  which is guided to a position within the uterus  62  by a guide tube  50  inserted into the uterus  62  through the vulva  60 , the vagina  61  and the cervix  63 . 
     The guide tube is rigid during insertion and during operation of the endoscope  40  and tool  14  so as to locate an end  51  of the guide tube  50  at a fixed position within the uterus  62 . Guiding to the required position is controlled by the veterinarian by holding the proximal end  52  and by feeling the location of the distal end  51  relative to the cervix through the bowel wall. 
     After insertion, the guide tube  50  is held fixed relative to the uterus during operation of the tool so as to locate an end of the guide tube at a fixed position within the uterus by locating the guide tube at the cervix. The guide tube is located at the cervix by first and second inflating balloons  53 ,  54  on an exterior surface of the tube  50  with one  54  inside the uterus at the cervix and the other  53  outside the cervix in the vagina. Thus the cervix is located between the two balloons to prevent longitudinal movement of the tube when the balloons are inflated. Also the cervix is relatively stiff and positioned at a fixed location relative to the interior wall of the uterus so that the tube is held at a fixed location to allow the veterinarian to operate the endoscope to locate its end at a required position adjacent the wall of the uterus. 
     The endoscope  40  includes operating components for moving an end of the endoscope relative to an end of the guide tube with the end of the guide tube held in fixed position relative to the uterus. 
     The balloons  53 ,  54  are defined on an outside of the body of the tube  50  by a layer of a resilient material  55  covering the body of the tube  50  which is cast in place or applied to define a passage  56  from the end  52  to the first of the balloons which then communicates with a passage  56 A to the end balloon. The balloons are formed by thinner annular sections of the covering  55  so that the annular sections inflate preferentially relative to the remainder of the covering to form annular balloons surrounding the tube body and extending over a limited extent longitudinally of the tube. The spacing between the balloons is designed to match approximately the thickness of the cervix to hold the cervix between them. In this way the balloons are inflated by an inflation pump  57  when the veterinarian has determined that the tube is at the required location to hold the tube at fixed position longitudinally and radially. 
     The balloons can be inflated independently by separate passages if required which can be used to locate the tube more effectively by locating it from one side of the cervix before the second balloon is inflated. 
     The endoscope is then inserted into the guide tube when the guide tube is in fixed position with the end of the guide tube within the uterus. 
     The endoscope  40  includes a conventional control system  45  operable by the veterinarian including operating components of the endoscope. These include a fluid supply  41 A for supply of a fluid to the end of the endoscope at a nozzle  41 ; a gas supply  42 A for supply of a gas to the end of the endoscope at a nozzle  42 ; a camera control  11 A for operating the camera  11  and a light control  10 A for operating eh illumination  10 . The endoscope also includes a manually operable control  43 A for operating bending elements (not shown) for bending the end of the endoscope to sides of an axis of the tube  50 . Typically this is effected by a wire pulling system which pulls on the end differentially to effect bending to one side. In addition the control system can be manually moved longitudinally as indicated at  47 A to push the end in and out of the tube  50  longitudinally as indicated at  47 . Also the control system can be manually rotated angularly around the axis of the tube  50  as indicated at  48 A to rotate the end as indicated at  48 . These controls thus allow movement of the end of the endoscope to required positions within the uterus relative to the fixed or stable end of the guide tube which is held in fixed position relative to the uterus. The tool  14  can be inserted after the required adjustment movement or can be in place while that movement is being effected. 
     The ultrasound system for guiding the extraction of the embryo by the veterinarian is schematically illustrated in  FIG. 10  at  80  and includes a probe  81  and a display  82 . This enables the veterinarian to view the position of the embryo and to use the tool to extract the embryo as previously described.

Technology Classification (CPC): 0