FERTILITY AND STERILITY
Vol. 58, No.3, September 1992
Printed on acid-free paper in U.S.A.
Copyright 1992 The American Fertility Society
Comparison of results obtained with human serum and a protein solution as a supplement for in vitro fertilization culture medium
Gerritdina J. Huisman, M.Sc.*t Nadia M. Lo-A-Njoe, M.T.A.* Albert Th. Alberda, M.D., Ph.D.*
Robert A. Leerentveld, M.D., Ph.D.* Arie Verhoeff, M.D., Ph.D.t Gerard H. Zeilmaker, Ph.D.*§
Academic Hospital Rotterdam-Dijkzigt and Erasmus University, Rotterdam, The Netherlands
The culture medium for human in vitro fertilization (IVF) and embryo transfer (ET) is usually supplemented with inactivated human serum (1). Patient serum is a safe supplement because no foreign protein will be introduced into the patient during ET. In the present study, the use of a human pasteurized plasma protein solution as a protein supplement for IVF culture medium was investigated in a prospective controlled study. MATERIALS AND METHODS
As culture medium, a (17:3) mixture of Earle's balanced salt solution and Ham's F-I0 medium (GIBCO, Paisley, UK) was used (2). Patient serum was used after heat inactivation (56°C for 30 minutes) in a concentration of 7.5% (vol/vol) during all phases of the IVF laboratory procedure. The protein solution (Central Laboratory of the Blood Transfusion Service, Amsterdam, The Netherlands) is manufactured according to international standards (3, 4) and commonly used in hospitals for medical purposes. Women with tubal infertility participated in the study and were prospectively divided into two
Received January 8, 1992; revised and accepted May 14, 1992. * Department of Obstetrics and Gynaecology. t Reprint requests: Gerritdina J. Huisman, M.D., In Vitro Fertilization Laboratory, Department of Obstetrics and Gynaecology, Academic Hospital Rotterdam Dijkzigt, Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. § Department of Endocrinology and Reproduction.
Vol. 58, No.3, September 1992
groups. If fertilization occurred, ET was performed 2 or 3 days after ovum pick-up. Depending on the age of the patient and the quality of the embryos available for transfer, up to five embryos were transplanted in the patient. One or more days after ET supernumerary embryos of good quality were cryopreserved. A clinical pregnancy was recorded with a positive test on urinary human chorionic gonadotropin (sensitivity 20 IU /L) 17 days after ovum pick-up followed by the recording of fetal heart activity seen by ultrasound (US) 7 weeks after ovum pick-up. Data were analyzed using X2 and two-way analyses. RESULTS
The protein solution supplemented medium was used for 215 treatments and patient serum supplemented medium for 151 treatments. The groups are comparable with respect to age of the patients and number of oocytes retrieved (Table 1). In the serumtreated group, 151 oocyte retrievals resulted in 136 ETs, in the protein solution-treated group, 193 transfers were done out of 215 ovum pick-ups. Total fertilization failure occurred in 10% of the treatments in both groups. The number of blastomeres on the day of the transfer was not significantly different in embryos cultured in protein solution and serum supplemented medium (Table 2). Good quality supernumerary embryos were cryopreserved 1 or more days after transfer. Embryos judged not suitable for cryopreservation sometimes developed into expanded blastocysts. Subsequently, hatching was
Huisman et aI.
Communications-in-brief
637
Table 1 Comparison of Serum and Protein Solution Addition to IVF Culture Medium: Patient Age and Results of the IVF -ET Treatment
Protein supplement
Serum
Protein solution
No. of ovum pick-ups Age (y)* Outcome of IVF treatment per patient No. of oocytes* No. of transfers Fertilization rate (%) No. of pregnancies:j: PR(%) Outcome of ET per embryo No. of embryos replaced No. of fetal hearbeats:j: Implantation rate (%)
151 33.6 ± 4.3
215 33.5 ± 3.6
9.3 ± 6.4 136 90 44 32
10.6 ± 8.0 193 89 53 28
432 63 15
576 82 14
NSt
NS NS NS
NS
* Values are means ± SD. t NS, not significant. :j: Confirmed by US, 7 weeks after ovum pick-up.
observed both in serum and in protein solution supplied culture medium. There was no difference between the use of serum and protein solution in the implantation rate of the individual embryos, as judged by US. Although the clinical pregnancy rate (PR) is somewhat lower in the protein solution-treated patients, the difference is not statistically different (Table 1).
DISCUSSION
To evaluate the use of protein solution as a protein supplement in IVF culture medium, a prospective controlled study was conducted. Fertilization rates and PRs are similar with protein solution or serumsupplemented medium. Embryos cultured in Albuminar-5 (Armour Pharmaceuticals, UK) supple-
Table 2
mented medium, as described by Ashwood-Smith et al. (5), did not develop well beyond the four-cell stage. Results published by Staessen et al. (6) also showed an impaired development of supernumerary embryos in Albuminar-20 supplemented medium. The relatively poor support of embryonic development after the four-cell stage was explained by the lack of additional factors that are present in human serum but not in the commercial protein supplements. In the present study, the good embryo development can possibly be explained by the availability in the supplement of more serum proteins than albumin alone. Even excess embryos considered not to be suitable for cryopreservation sometimes developed into an expanded blastocyst. On the basis of the results obtained and the above mentioned considerations, it can be concluded that a pasteurized plasma protein solution addition to the culture medium provides safe and optimal conditions for culturing embryos.
SUMMARY
A prospective controlled study was performed to compare the PRs obtained after use of a uniform IVF culture medium containing a pasteurized serum protein solution or patient serum. The ongoing PRs per ET in the serum and the protein solution group were 32% and 28%, respectively (not significant). Culture of supernumerary embryos showed blastocyst formation and even hatching with both supplements. The PR will not drop when this protein solution is used as a protein supplement in IVF culture medium instead of patient serum. Key Words: In vitro fertilization, culture medium, serum replacement.
Cleavage Stage of Embryos at the Time of Transfer in the Two Treatment Groups No. of cells on the day of ET
Protein supplement
Culture period
2 to 4 cell
5 to 6 cell
7 to 8 cell
9 to 10 cell
d
Serum* Protein solution Serum Protein solution
2 2 3 3
143 208 11 38
* Results in the two groups are not significantly different.
638
Huisman et al.
Communications-in-brief
(71)t (73) (7) (18)
32 62 36 28
(17) (22) (21) (13)
7 (4)
13 (5) 107 (64) 125 (59)
12 (6) 2 (1)
14 (8) 21 (10)
t Values in parentheses are percents.
Fertility and Sterility
Acknowledgment. We gratefully thank the technical assistance of Ms. Jacqueline Termeulen, Ms. Els Slappendel, and Mr. Pierre Blommers (lVF Laboratory, Academical Hospital Rotterdam, Dijkzigt Rotterdam, The Netherlands).
3. 4.
REFERENCES 1. Edwards RG, Purdy JM, Steptoe PC, Walters DE. The growth of human preimplantation embryos in vitro. Am J Obstet Gynecol1981;141:408-16. 2. Zeilmaker GH. The laboratory procedure of in-vitro fertilization: some observations and conclusions. In: Nunez J, Dumont JE, King RJB, editors. Hormones and cell regula-
Vol. 58, No.3, September 1992
5.
6.
tion. London, Paris: John Libbey Eurotext Ltd., 1986;139: 43-54. Plasma protein fraction (human). In: Code of federal regulations. Washington, D.C.: Food and Drugs, 1990;21:133-5. Human plasma protein solution. In: European pharmacopoeia, 2nd ed. Maisonneuve, Sainte Ruffine, France: Vol. 14, 1990;14:287.1-287.5. Ashwood-Smith MJ, Hollands P, Edwards RG. The use of Albuminar (TM) as a medium supplement in clinical IVF. Hum Reprod 1989;4:702-5. Staessen C, van den Abbeel E, Carle M, Khan I, Devroey P, van Steirteghem AC. Comparison between human serum and Albuminar-20 (TM) supplement for in vitro fertilization. Hum Reprod 1990;5:336-41.
Huisman et al.
Communications-in-brief
639
Vol. 58, No.3, September 1992
Printed on acid-free paper in U.S.A.
Copyright 1992 The American Fertility Society
Comparison of results obtained with human serum and a protein solution as a supplement for in vitro fertilization culture medium
Gerritdina J. Huisman, M.Sc.*t Nadia M. Lo-A-Njoe, M.T.A.* Albert Th. Alberda, M.D., Ph.D.*
Robert A. Leerentveld, M.D., Ph.D.* Arie Verhoeff, M.D., Ph.D.t Gerard H. Zeilmaker, Ph.D.*§
Academic Hospital Rotterdam-Dijkzigt and Erasmus University, Rotterdam, The Netherlands
The culture medium for human in vitro fertilization (IVF) and embryo transfer (ET) is usually supplemented with inactivated human serum (1). Patient serum is a safe supplement because no foreign protein will be introduced into the patient during ET. In the present study, the use of a human pasteurized plasma protein solution as a protein supplement for IVF culture medium was investigated in a prospective controlled study. MATERIALS AND METHODS
As culture medium, a (17:3) mixture of Earle's balanced salt solution and Ham's F-I0 medium (GIBCO, Paisley, UK) was used (2). Patient serum was used after heat inactivation (56°C for 30 minutes) in a concentration of 7.5% (vol/vol) during all phases of the IVF laboratory procedure. The protein solution (Central Laboratory of the Blood Transfusion Service, Amsterdam, The Netherlands) is manufactured according to international standards (3, 4) and commonly used in hospitals for medical purposes. Women with tubal infertility participated in the study and were prospectively divided into two
Received January 8, 1992; revised and accepted May 14, 1992. * Department of Obstetrics and Gynaecology. t Reprint requests: Gerritdina J. Huisman, M.D., In Vitro Fertilization Laboratory, Department of Obstetrics and Gynaecology, Academic Hospital Rotterdam Dijkzigt, Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. § Department of Endocrinology and Reproduction.
Vol. 58, No.3, September 1992
groups. If fertilization occurred, ET was performed 2 or 3 days after ovum pick-up. Depending on the age of the patient and the quality of the embryos available for transfer, up to five embryos were transplanted in the patient. One or more days after ET supernumerary embryos of good quality were cryopreserved. A clinical pregnancy was recorded with a positive test on urinary human chorionic gonadotropin (sensitivity 20 IU /L) 17 days after ovum pick-up followed by the recording of fetal heart activity seen by ultrasound (US) 7 weeks after ovum pick-up. Data were analyzed using X2 and two-way analyses. RESULTS
The protein solution supplemented medium was used for 215 treatments and patient serum supplemented medium for 151 treatments. The groups are comparable with respect to age of the patients and number of oocytes retrieved (Table 1). In the serumtreated group, 151 oocyte retrievals resulted in 136 ETs, in the protein solution-treated group, 193 transfers were done out of 215 ovum pick-ups. Total fertilization failure occurred in 10% of the treatments in both groups. The number of blastomeres on the day of the transfer was not significantly different in embryos cultured in protein solution and serum supplemented medium (Table 2). Good quality supernumerary embryos were cryopreserved 1 or more days after transfer. Embryos judged not suitable for cryopreservation sometimes developed into expanded blastocysts. Subsequently, hatching was
Huisman et aI.
Communications-in-brief
637
Table 1 Comparison of Serum and Protein Solution Addition to IVF Culture Medium: Patient Age and Results of the IVF -ET Treatment
Protein supplement
Serum
Protein solution
No. of ovum pick-ups Age (y)* Outcome of IVF treatment per patient No. of oocytes* No. of transfers Fertilization rate (%) No. of pregnancies:j: PR(%) Outcome of ET per embryo No. of embryos replaced No. of fetal hearbeats:j: Implantation rate (%)
151 33.6 ± 4.3
215 33.5 ± 3.6
9.3 ± 6.4 136 90 44 32
10.6 ± 8.0 193 89 53 28
432 63 15
576 82 14
NSt
NS NS NS
NS
* Values are means ± SD. t NS, not significant. :j: Confirmed by US, 7 weeks after ovum pick-up.
observed both in serum and in protein solution supplied culture medium. There was no difference between the use of serum and protein solution in the implantation rate of the individual embryos, as judged by US. Although the clinical pregnancy rate (PR) is somewhat lower in the protein solution-treated patients, the difference is not statistically different (Table 1).
DISCUSSION
To evaluate the use of protein solution as a protein supplement in IVF culture medium, a prospective controlled study was conducted. Fertilization rates and PRs are similar with protein solution or serumsupplemented medium. Embryos cultured in Albuminar-5 (Armour Pharmaceuticals, UK) supple-
Table 2
mented medium, as described by Ashwood-Smith et al. (5), did not develop well beyond the four-cell stage. Results published by Staessen et al. (6) also showed an impaired development of supernumerary embryos in Albuminar-20 supplemented medium. The relatively poor support of embryonic development after the four-cell stage was explained by the lack of additional factors that are present in human serum but not in the commercial protein supplements. In the present study, the good embryo development can possibly be explained by the availability in the supplement of more serum proteins than albumin alone. Even excess embryos considered not to be suitable for cryopreservation sometimes developed into an expanded blastocyst. On the basis of the results obtained and the above mentioned considerations, it can be concluded that a pasteurized plasma protein solution addition to the culture medium provides safe and optimal conditions for culturing embryos.
SUMMARY
A prospective controlled study was performed to compare the PRs obtained after use of a uniform IVF culture medium containing a pasteurized serum protein solution or patient serum. The ongoing PRs per ET in the serum and the protein solution group were 32% and 28%, respectively (not significant). Culture of supernumerary embryos showed blastocyst formation and even hatching with both supplements. The PR will not drop when this protein solution is used as a protein supplement in IVF culture medium instead of patient serum. Key Words: In vitro fertilization, culture medium, serum replacement.
Cleavage Stage of Embryos at the Time of Transfer in the Two Treatment Groups No. of cells on the day of ET
Protein supplement
Culture period
2 to 4 cell
5 to 6 cell
7 to 8 cell
9 to 10 cell
d
Serum* Protein solution Serum Protein solution
2 2 3 3
143 208 11 38
* Results in the two groups are not significantly different.
638
Huisman et al.
Communications-in-brief
(71)t (73) (7) (18)
32 62 36 28
(17) (22) (21) (13)
7 (4)
13 (5) 107 (64) 125 (59)
12 (6) 2 (1)
14 (8) 21 (10)
t Values in parentheses are percents.
Fertility and Sterility
Acknowledgment. We gratefully thank the technical assistance of Ms. Jacqueline Termeulen, Ms. Els Slappendel, and Mr. Pierre Blommers (lVF Laboratory, Academical Hospital Rotterdam, Dijkzigt Rotterdam, The Netherlands).
3. 4.
REFERENCES 1. Edwards RG, Purdy JM, Steptoe PC, Walters DE. The growth of human preimplantation embryos in vitro. Am J Obstet Gynecol1981;141:408-16. 2. Zeilmaker GH. The laboratory procedure of in-vitro fertilization: some observations and conclusions. In: Nunez J, Dumont JE, King RJB, editors. Hormones and cell regula-
Vol. 58, No.3, September 1992
5.
6.
tion. London, Paris: John Libbey Eurotext Ltd., 1986;139: 43-54. Plasma protein fraction (human). In: Code of federal regulations. Washington, D.C.: Food and Drugs, 1990;21:133-5. Human plasma protein solution. In: European pharmacopoeia, 2nd ed. Maisonneuve, Sainte Ruffine, France: Vol. 14, 1990;14:287.1-287.5. Ashwood-Smith MJ, Hollands P, Edwards RG. The use of Albuminar (TM) as a medium supplement in clinical IVF. Hum Reprod 1989;4:702-5. Staessen C, van den Abbeel E, Carle M, Khan I, Devroey P, van Steirteghem AC. Comparison between human serum and Albuminar-20 (TM) supplement for in vitro fertilization. Hum Reprod 1990;5:336-41.
Huisman et al.
Communications-in-brief
639
