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On the transfer of conceptuses from oocytes fertilized in vitro
FERTILITY AND STERILITY Copyright c 1983 The American Fertility Society
Vol. 39, No.2, February 1983 Printed in U.SA.
On the transfer of conceptuses from oocytes fertilized in vitro Howard W. Jones, Jr., M.D. * Anibal A. Acosta, M.D. Jairo E. Garcia, M.D. Bruce A. Sandow, Ph.D. Lucinda Veeck, M.L.T. Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
In response to many requests for the details of our method of transfer of conceptuses into the uterus after fertilization of oocytes in vitro, the following details are supplied. There is no claim that this is a superior method. However, the instrumentation and technique have not varied from January 1, 1981, through June 30, 1982, and the pregnancy rate after transfer is comparable to the rate of other programs. Generally speaking, transfer has been carried out at the 3- to 6-cell stage at about 44 hours after insemination. In the event the transfer is of more than one conceptus, the second or third conceptus could be in a much earlier stage of development due to a longer period of incubation of an immature oocyte prior to fertilization. The transfer catheter is made from American Wire Gauge #20 Teflon tubing ofthin wall thickness. The internal diameter is 0.034 inches (0.8636 mm), and the outside diameter is 0.058 inches (1.4732 mm). The tip is closed with a bullet-shaped plug of nylon without cement (Fig. 1). A side notch is cut in the tubing in such a way that a stream offluid forced from the catheter will eject at an angle of 45 degrees to the axis of the catheter. The side opening is at least equal in diameter to the internal diameter of the tubing. Received August 9, 1982; revised and accepted October 7, 1982. *Reprint requests: Howard W. Jones, Jr., M.D., Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 603 Medical Tower, Norfolk, Virginia 23507. Vol. 39, No.2, February 1983
Each catheter measures about 40 cm in length, but there is nothing critical about the length. Culture medium and conceptus are drawn into the catheter by a 1-ml tuberculin syringe with a #18 needle that fits snugly into the lumen of the catheter. The catheter is inserted through a carrying tube with a slightly curved tip so that the catheter can be ejected into the axis of the endocervical canal (Fig. 2). The carrying tube is made from #12 steel needle tubing with an internal diameter of 0.085 inches (2.157 mm). Prior to loading the conceptus, the catheter is flushed one or two times with medium from the moat of the 60-mm organ culture dish in which the conceptus has developed. The flushing extends for a length of about 25 cm of the catheter and has as its purpose the wetting of the inside of the catheter and the testing of the integrity of the aspiration system. For convenience in loading and to have a flat surface to reduce aberration for photography, we transfer the conceptus from the well of the organ culture dish to a 60-mm tissue culture dish in a small drop of medium. After a photograph is taken, the remainder of the medium from the well of the organ culture dish is now added to the tissue culture dish. Under the microscope, the conceptus is drawn into the catheter with 30 I-LI of culture medium in the following sequence: 30 I-LI of medium without the oocyte is first drawn into the catheter. This is followed by 20 I-LI of air. The next 30 I-LI of meJones et al. Communications-in-brief
241
Figure 1 Photograph of the tip of the transfer catheter, showing a bullet-shaped plug of nylon and a side notch.
Figure 2 Photograph of the nylon transfer catheter and the I-ml syringe attached to the distal end, together with the carrying tube, the tip of which is placed just within the endocervical canal for the transfer.
dium contains the conceptus. This is followed by 20 j.LI of air. This is followed by a final 30 j.LI of medium. Thus, the conceptus will lie about 11 cm from the side opening near the tip ofthe catheter, and the entire column of medium with the two air gaps will occupy 22 cm of the catheter length. In the event that more than one conceptus is transferred, they will be treated as a clutch and all transferred together in the middle 30 j.LI of medium. Transfer has been carried out with patients in the knee-to-chest position when their uteri were in the normal anterior position, in the operating room adjacent to the laboratory. Because diminution of infection by the use of antibiotics has seemed to improve the "take" rate in transcervical embryo transfer in cattle, patients have received 250 mg tetracycline four times daily during the day prior to and during the day of the transfer. No vaginal cleaning is used. Excess mucus and debris may be removed from the cervix by a cotton-tipped applicator. A single-bladed vaginal retractor is preferred, and there is no hesitation in using a single-tooth tenaculum on the anterior lip of the cervix to draw the cervix down into the vagina if this should prove necessary. The tip of the metal catheter is inserted barely into the mucus at the external os, and the Teflon catheter is then passed through the cervix for a predetermined distance, usually about 6 cm, marked with a piece oftape on the Teflon tubing 6 cm distal to the distal end of the metal carrier with the anterior tip of the Teflon catheter flush with the anterior tip of the metal catheter.
After the catheter has been passed through the cervix to the desired position, the entire column of culture medium containing the conceptus, or conceptuses, is quickly ejected. This is followed by an additional 50 j.LI of air to clear the catheter and to break any remaining surface tension that might make the conceptus cling to the opening of the catheter. After a moment or two, the catheter is withdrawn as it is rotated. The catheter is now returned to the laboratory, where it is examined to be sure the conceptus has been delivered. We do this examination by drawing 1 ml of medium from the moat of the organ culture dish in which the conceptus grew into the 1-ml syringe and washing the medium through the catheter from the distal end into a tissue culture dish. We then forcefully inject 1 ml of air through the catheter. This usually results in the ejection of an additional drop or two of medium into another tissue culture dish. Each of these washings is examined under the microscope, as is the tip of the catheter. If the examination fails to disclose a conceptus, it is assumed that the conceptus has been delivered into the uterus.
242
Jones et al.
Communications-in-brief
Table.l. 94 Transfers January 1, 1981, through June 30, 1982 Transfer
No.
Pregnancies
Single Double Triple Quadruple
42 39 12 1
4 (10%) 11 (28%) 6 (50%) 0(0%)
Total
94
21 (22%)
Fertility and Sterility
The patient remains prone for a minimum of 4 hours in the recovery room, after which she returns to her home or hotel. This procedure was used 94 times during the interval January 1, 1981, to June 30, 1982. The catheter was passed into the uterine cavity with relative ease in all instances except one, when it buckled in the endocervical canal. This resulted from the exertion of excessive force. If the catheter does not pass easily, it can be withdrawn a short distance and the angle of the carrying tube altered, several times if necessary, until the tip of the catheter finds its way easily through the canal. On occasion, there may be minor bleeding from the endocervical canal. This does not exclude a pregnancy.
Vol. 39, No.2, February 1983
In only one instance has a conceptus been found remaining in the catheter on examination after a transfer. In this instance, the residual conceptus was one of three and, because of damage, was not transferred. The patient became pregnant and has delivered. With the use of this method from January 1, 1981, to June 30, 1982, in 94 cycles, there were 21 pregnancies (Table 1). SUMMARY
The Norfolkian method of transfer of a human conceptus after oocyte fertilization in vitro is described. Twenty-one pregnancies resulted from the use of this method in 94 transfers.
Jones et aI. Communications-in-brief
.
243
Vol. 39, No.2, February 1983 Printed in U.SA.
On the transfer of conceptuses from oocytes fertilized in vitro Howard W. Jones, Jr., M.D. * Anibal A. Acosta, M.D. Jairo E. Garcia, M.D. Bruce A. Sandow, Ph.D. Lucinda Veeck, M.L.T. Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
In response to many requests for the details of our method of transfer of conceptuses into the uterus after fertilization of oocytes in vitro, the following details are supplied. There is no claim that this is a superior method. However, the instrumentation and technique have not varied from January 1, 1981, through June 30, 1982, and the pregnancy rate after transfer is comparable to the rate of other programs. Generally speaking, transfer has been carried out at the 3- to 6-cell stage at about 44 hours after insemination. In the event the transfer is of more than one conceptus, the second or third conceptus could be in a much earlier stage of development due to a longer period of incubation of an immature oocyte prior to fertilization. The transfer catheter is made from American Wire Gauge #20 Teflon tubing ofthin wall thickness. The internal diameter is 0.034 inches (0.8636 mm), and the outside diameter is 0.058 inches (1.4732 mm). The tip is closed with a bullet-shaped plug of nylon without cement (Fig. 1). A side notch is cut in the tubing in such a way that a stream offluid forced from the catheter will eject at an angle of 45 degrees to the axis of the catheter. The side opening is at least equal in diameter to the internal diameter of the tubing. Received August 9, 1982; revised and accepted October 7, 1982. *Reprint requests: Howard W. Jones, Jr., M.D., Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 603 Medical Tower, Norfolk, Virginia 23507. Vol. 39, No.2, February 1983
Each catheter measures about 40 cm in length, but there is nothing critical about the length. Culture medium and conceptus are drawn into the catheter by a 1-ml tuberculin syringe with a #18 needle that fits snugly into the lumen of the catheter. The catheter is inserted through a carrying tube with a slightly curved tip so that the catheter can be ejected into the axis of the endocervical canal (Fig. 2). The carrying tube is made from #12 steel needle tubing with an internal diameter of 0.085 inches (2.157 mm). Prior to loading the conceptus, the catheter is flushed one or two times with medium from the moat of the 60-mm organ culture dish in which the conceptus has developed. The flushing extends for a length of about 25 cm of the catheter and has as its purpose the wetting of the inside of the catheter and the testing of the integrity of the aspiration system. For convenience in loading and to have a flat surface to reduce aberration for photography, we transfer the conceptus from the well of the organ culture dish to a 60-mm tissue culture dish in a small drop of medium. After a photograph is taken, the remainder of the medium from the well of the organ culture dish is now added to the tissue culture dish. Under the microscope, the conceptus is drawn into the catheter with 30 I-LI of culture medium in the following sequence: 30 I-LI of medium without the oocyte is first drawn into the catheter. This is followed by 20 I-LI of air. The next 30 I-LI of meJones et al. Communications-in-brief
241
Figure 1 Photograph of the tip of the transfer catheter, showing a bullet-shaped plug of nylon and a side notch.
Figure 2 Photograph of the nylon transfer catheter and the I-ml syringe attached to the distal end, together with the carrying tube, the tip of which is placed just within the endocervical canal for the transfer.
dium contains the conceptus. This is followed by 20 j.LI of air. This is followed by a final 30 j.LI of medium. Thus, the conceptus will lie about 11 cm from the side opening near the tip ofthe catheter, and the entire column of medium with the two air gaps will occupy 22 cm of the catheter length. In the event that more than one conceptus is transferred, they will be treated as a clutch and all transferred together in the middle 30 j.LI of medium. Transfer has been carried out with patients in the knee-to-chest position when their uteri were in the normal anterior position, in the operating room adjacent to the laboratory. Because diminution of infection by the use of antibiotics has seemed to improve the "take" rate in transcervical embryo transfer in cattle, patients have received 250 mg tetracycline four times daily during the day prior to and during the day of the transfer. No vaginal cleaning is used. Excess mucus and debris may be removed from the cervix by a cotton-tipped applicator. A single-bladed vaginal retractor is preferred, and there is no hesitation in using a single-tooth tenaculum on the anterior lip of the cervix to draw the cervix down into the vagina if this should prove necessary. The tip of the metal catheter is inserted barely into the mucus at the external os, and the Teflon catheter is then passed through the cervix for a predetermined distance, usually about 6 cm, marked with a piece oftape on the Teflon tubing 6 cm distal to the distal end of the metal carrier with the anterior tip of the Teflon catheter flush with the anterior tip of the metal catheter.
After the catheter has been passed through the cervix to the desired position, the entire column of culture medium containing the conceptus, or conceptuses, is quickly ejected. This is followed by an additional 50 j.LI of air to clear the catheter and to break any remaining surface tension that might make the conceptus cling to the opening of the catheter. After a moment or two, the catheter is withdrawn as it is rotated. The catheter is now returned to the laboratory, where it is examined to be sure the conceptus has been delivered. We do this examination by drawing 1 ml of medium from the moat of the organ culture dish in which the conceptus grew into the 1-ml syringe and washing the medium through the catheter from the distal end into a tissue culture dish. We then forcefully inject 1 ml of air through the catheter. This usually results in the ejection of an additional drop or two of medium into another tissue culture dish. Each of these washings is examined under the microscope, as is the tip of the catheter. If the examination fails to disclose a conceptus, it is assumed that the conceptus has been delivered into the uterus.
242
Jones et al.
Communications-in-brief
Table.l. 94 Transfers January 1, 1981, through June 30, 1982 Transfer
No.
Pregnancies
Single Double Triple Quadruple
42 39 12 1
4 (10%) 11 (28%) 6 (50%) 0(0%)
Total
94
21 (22%)
Fertility and Sterility
The patient remains prone for a minimum of 4 hours in the recovery room, after which she returns to her home or hotel. This procedure was used 94 times during the interval January 1, 1981, to June 30, 1982. The catheter was passed into the uterine cavity with relative ease in all instances except one, when it buckled in the endocervical canal. This resulted from the exertion of excessive force. If the catheter does not pass easily, it can be withdrawn a short distance and the angle of the carrying tube altered, several times if necessary, until the tip of the catheter finds its way easily through the canal. On occasion, there may be minor bleeding from the endocervical canal. This does not exclude a pregnancy.
Vol. 39, No.2, February 1983
In only one instance has a conceptus been found remaining in the catheter on examination after a transfer. In this instance, the residual conceptus was one of three and, because of damage, was not transferred. The patient became pregnant and has delivered. With the use of this method from January 1, 1981, to June 30, 1982, in 94 cycles, there were 21 pregnancies (Table 1). SUMMARY
The Norfolkian method of transfer of a human conceptus after oocyte fertilization in vitro is described. Twenty-one pregnancies resulted from the use of this method in 94 transfers.
Jones et aI. Communications-in-brief
.
243