- Email: [email protected]
Pregnancy following in vitro fertilization and embryo transfer using pure human serum as culture and transfer medium
Vol. 41, No.6, June 1984 Printed in U.SA.
FERTILITY AND STERILITY Copyright © 1984 The American Fertility Society
Pregnancy following in vitro fertilization and embryo transfer using pure human serum as culture and transfer medium
Peter Kemeter, M,D.* Wilfried Feichtinger, M.D. Institute of Reproductive Endocrinology and In Vitro Fertilization, Vienna, Austria
Various culture media have been successfully used for in vitro fertilization (IVF) and embryo transfer (ET) since the first full-term pregnancies were achieved with this method. 1-3 The preparation of these clearly defined culture media is rather difficult and time-consuming or, if they are commercially available, rather expensive. All of these media are supplemented with proteins in the form of bovine or human albumin, inactivated human serum, fetal calf serum, or human cord serum. 4 The reported concentrations of these proteins in the culture medium vary between 10% and 30%, but none of these media and/or protein concentrations was superior with respect to fertilization and pregnancy rates. Menkin and Rock, 5 on the other hand, showed as early as 1948 that human oocytes could be fertilized and cultured in pure human blood serum to the 2- and 3-cell stages. Trounson6 found the same fertilization and cleavage rate of mouse 00cytes in pure serum as in conventional culture medium. We therefore found it justified to test pure human serum as a culture medium for human oocytes in our IVF-ET system. 7
transferred in pure heat-inactivated serum of the patients adjusted to 280 mOsm. Only patients with more than two oocytes were included in the study, and the oocytes for serum cultivation were randomly selected. A maximum of two oocytes per patient were cultured in serum, whereas all of the remaining oocytes were cultured in B2 medium (API-System, S.A., Montalieu-Vercieu, France). Blood for the serum preparation was drawn on the day before oocyte recovery, - 10 h,Ours after the administration of 5000 IU of human chorionic gonadotropin (hCG) intramuscularly. The steps of serum preparation are listed in Table 1.
RESULTS
The 15th patient of this study became pregnant after a transfer of two serum-cultured oocytes. In these 15 patients, 17 of 20 oocytes cultured in pure serum and 32 of 44 oocytes cultured in B2 medium were fertilized. The successful case is reported here. CASE REPORT
MATERIALS AND METHODS
In a pilot study including 15 patients, 20 of 64 oocytes were preincubated, inseminated, and Received November 2,1983; revised and accepted February 28,1984. *Reprint requests: Dr. Peter Kemeter, Institute of Reproductive Endocrinology and In Vitro Fertilization, Trauttmansdorffgasse 3A, A-H30 Vienna, Austria. 936
Kemeter and Feichtinger Communications-in-brief
In a 31-year-old woman with bicornuate uterus and primary tubal infertility, three oocytes were recovered by laparoscopy. After ovarian stimulation with clomiphene, 100 mg from days 5 to 9 of her cycle, and human menopausal gonadotropin, 225 IU from days 10 to 13, the midcycle luteinizing hormone (LH) surge started on day 14, when the diameter of the leading follicle measured by ultrasound was 23.5 mm. Follicle aspiration was Fertility and Sterility
1 Table 1. Preparation of Pure Human Serum as Culture Medium for IVF Take blood the morning after the hCG injection Allow to clot for 20 minutes; centrifuge twice (1 blood, 2 serum) Inactivate at 55° C for 30 minutes Adjust osmolarity to 280 mOsmlkg using deionized water (Analar-BDH) containing 0.2 gmJl ampicillin Sterilize using a 0.22-fL filter
performed 26 hours after the first increase of urinary LH, and three preovulatory oocytes were found. One oocyte was put into B2 medium and two into pure human heat-inactivated serum (280 mOsm). They were all incubated in 5% CO2 in air at 37 0 C. Blood for the serum preparation was taken 8 hours after the initial LH increase in urine. Insemination with the husband's sperm prepared with B2 medium was performed 32 hours after the initial LH increase. Sixteen hours after insemination the two oocytes cultured in pure serum showed two pronuclei each and were considered to be fertilized, whereas the oocyte in B2 showed no pronucleus and only one polar body and was considered to be unfertilized. The two fertilized oocytes were transferred in the same pure serum to the uterus of the patient 18% hours after insemination. At that time, the patient received another 5000 IU of hCG. On the 15th day after laparoscopy, menstruation was missed and the urinary hCG level was 40 lUll. Three days later, hCG was 2500 lUll, and two amniotic sacs were detected in the uterus by ultrasound. Urinary hCG levels showed a high increase to 54,000 IU during the eighth week of pregnancy, when one gestational sac and fetus with positive heart action was detected by ultrasound in one uterine hom. The pregnancy has now reached the 20th week and is considered absolutely normal. DISCUSSION
Like our preliminary results, this case shows that IVF of human oocytes can be performed successfully using heat-inactivated human serum for
Vol. 41, No.6, June 1984
the main steps, including preincubation, insemination, and transfer. It would be the least expensive, most easily available culture medium for human IVF in clinical routine. Our early results show that fertilization rates seem to be similar to, if not better than, those in chemically defined media. A controlled randomized study for validating these data, which could further simplify the IVFET technique, is under way. SUMMARY
Pure heat-inactivated serum of patients adjusted to 280 mOsm was used for preincubation, insemination, and transfer of oocytes in an IVFET program. In patients where the oocyte recovery yielded more than two oocytes, two oocytes were chosen randomly for serum cultivation. The 15th patient of that series became pregnant after a transfer of two serum-cultured embryos. The follow-up weekly clinical investigation and ultrasound revealed a normal single pregnancy, now in the 20th week. REFERENCES 1. Edwards RG, Steptoe PC, Purdy JM: Establishing full
2.
3.
4. 5. 6. 7.
term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:737, 1980 Lopata A, Johnston IWH, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117,1980 Feichtinger W, Kemeter P, Szalay S: The Vienna program of in vitro fertilization and embryo-transfer-a successful clinical treatment. Eur J Obstet Gynaecol Reprod BioI 15:63,1983 Lopata A: Success and failure in human in vitro fertilization. Nature 288:642, 1980 Menkin MF, Rock J: In vitro fertilization and cleavage of human ovarian eggs. Am J Obstet Gynecol 55:440, 1948 Trounson A: Unpublished data Feichtinger W, Kemeter P: A simplified technique for fertilization and culture of human preimplantation embryos in vitro. Acta Eur Fertil14:125, 1983
Kemeter and Feichtinger Communications-in-brief
937
FERTILITY AND STERILITY Copyright © 1984 The American Fertility Society
Pregnancy following in vitro fertilization and embryo transfer using pure human serum as culture and transfer medium
Peter Kemeter, M,D.* Wilfried Feichtinger, M.D. Institute of Reproductive Endocrinology and In Vitro Fertilization, Vienna, Austria
Various culture media have been successfully used for in vitro fertilization (IVF) and embryo transfer (ET) since the first full-term pregnancies were achieved with this method. 1-3 The preparation of these clearly defined culture media is rather difficult and time-consuming or, if they are commercially available, rather expensive. All of these media are supplemented with proteins in the form of bovine or human albumin, inactivated human serum, fetal calf serum, or human cord serum. 4 The reported concentrations of these proteins in the culture medium vary between 10% and 30%, but none of these media and/or protein concentrations was superior with respect to fertilization and pregnancy rates. Menkin and Rock, 5 on the other hand, showed as early as 1948 that human oocytes could be fertilized and cultured in pure human blood serum to the 2- and 3-cell stages. Trounson6 found the same fertilization and cleavage rate of mouse 00cytes in pure serum as in conventional culture medium. We therefore found it justified to test pure human serum as a culture medium for human oocytes in our IVF-ET system. 7
transferred in pure heat-inactivated serum of the patients adjusted to 280 mOsm. Only patients with more than two oocytes were included in the study, and the oocytes for serum cultivation were randomly selected. A maximum of two oocytes per patient were cultured in serum, whereas all of the remaining oocytes were cultured in B2 medium (API-System, S.A., Montalieu-Vercieu, France). Blood for the serum preparation was drawn on the day before oocyte recovery, - 10 h,Ours after the administration of 5000 IU of human chorionic gonadotropin (hCG) intramuscularly. The steps of serum preparation are listed in Table 1.
RESULTS
The 15th patient of this study became pregnant after a transfer of two serum-cultured oocytes. In these 15 patients, 17 of 20 oocytes cultured in pure serum and 32 of 44 oocytes cultured in B2 medium were fertilized. The successful case is reported here. CASE REPORT
MATERIALS AND METHODS
In a pilot study including 15 patients, 20 of 64 oocytes were preincubated, inseminated, and Received November 2,1983; revised and accepted February 28,1984. *Reprint requests: Dr. Peter Kemeter, Institute of Reproductive Endocrinology and In Vitro Fertilization, Trauttmansdorffgasse 3A, A-H30 Vienna, Austria. 936
Kemeter and Feichtinger Communications-in-brief
In a 31-year-old woman with bicornuate uterus and primary tubal infertility, three oocytes were recovered by laparoscopy. After ovarian stimulation with clomiphene, 100 mg from days 5 to 9 of her cycle, and human menopausal gonadotropin, 225 IU from days 10 to 13, the midcycle luteinizing hormone (LH) surge started on day 14, when the diameter of the leading follicle measured by ultrasound was 23.5 mm. Follicle aspiration was Fertility and Sterility
1 Table 1. Preparation of Pure Human Serum as Culture Medium for IVF Take blood the morning after the hCG injection Allow to clot for 20 minutes; centrifuge twice (1 blood, 2 serum) Inactivate at 55° C for 30 minutes Adjust osmolarity to 280 mOsmlkg using deionized water (Analar-BDH) containing 0.2 gmJl ampicillin Sterilize using a 0.22-fL filter
performed 26 hours after the first increase of urinary LH, and three preovulatory oocytes were found. One oocyte was put into B2 medium and two into pure human heat-inactivated serum (280 mOsm). They were all incubated in 5% CO2 in air at 37 0 C. Blood for the serum preparation was taken 8 hours after the initial LH increase in urine. Insemination with the husband's sperm prepared with B2 medium was performed 32 hours after the initial LH increase. Sixteen hours after insemination the two oocytes cultured in pure serum showed two pronuclei each and were considered to be fertilized, whereas the oocyte in B2 showed no pronucleus and only one polar body and was considered to be unfertilized. The two fertilized oocytes were transferred in the same pure serum to the uterus of the patient 18% hours after insemination. At that time, the patient received another 5000 IU of hCG. On the 15th day after laparoscopy, menstruation was missed and the urinary hCG level was 40 lUll. Three days later, hCG was 2500 lUll, and two amniotic sacs were detected in the uterus by ultrasound. Urinary hCG levels showed a high increase to 54,000 IU during the eighth week of pregnancy, when one gestational sac and fetus with positive heart action was detected by ultrasound in one uterine hom. The pregnancy has now reached the 20th week and is considered absolutely normal. DISCUSSION
Like our preliminary results, this case shows that IVF of human oocytes can be performed successfully using heat-inactivated human serum for
Vol. 41, No.6, June 1984
the main steps, including preincubation, insemination, and transfer. It would be the least expensive, most easily available culture medium for human IVF in clinical routine. Our early results show that fertilization rates seem to be similar to, if not better than, those in chemically defined media. A controlled randomized study for validating these data, which could further simplify the IVFET technique, is under way. SUMMARY
Pure heat-inactivated serum of patients adjusted to 280 mOsm was used for preincubation, insemination, and transfer of oocytes in an IVFET program. In patients where the oocyte recovery yielded more than two oocytes, two oocytes were chosen randomly for serum cultivation. The 15th patient of that series became pregnant after a transfer of two serum-cultured embryos. The follow-up weekly clinical investigation and ultrasound revealed a normal single pregnancy, now in the 20th week. REFERENCES 1. Edwards RG, Steptoe PC, Purdy JM: Establishing full
2.
3.
4. 5. 6. 7.
term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:737, 1980 Lopata A, Johnston IWH, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117,1980 Feichtinger W, Kemeter P, Szalay S: The Vienna program of in vitro fertilization and embryo-transfer-a successful clinical treatment. Eur J Obstet Gynaecol Reprod BioI 15:63,1983 Lopata A: Success and failure in human in vitro fertilization. Nature 288:642, 1980 Menkin MF, Rock J: In vitro fertilization and cleavage of human ovarian eggs. Am J Obstet Gynecol 55:440, 1948 Trounson A: Unpublished data Feichtinger W, Kemeter P: A simplified technique for fertilization and culture of human preimplantation embryos in vitro. Acta Eur Fertil14:125, 1983
Kemeter and Feichtinger Communications-in-brief
937