Evaluation of a synthetic serum substitute to replace fetal cord serum for human oocyte fertilization and embryo growth in vitro*

Transcription

1 FERTILITY AND STERILITY Copyright<> 1989 The American Fertility Society Printed on acid-free paper in U.S.A. Evaluation of a synthetic serum substitute to replace fetal cord serum for human oocyte fertilization and embryo growth in vitro* lv Psalti, M.Sc.t Ernest Loumaye, M.D. Manuel Pensis, B.Sc. Suzy Depreester, B.Sc. Karl Thomas, M.D. Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynaecology, University of Louvain, Brussels, Belgium A comparison was made between a serum substitute, UltroSer G (Gibco, Ghent, Belgium) (2%) (medium B) and 10% human fetal cord serum (medium A), as regards their ability to support 1-cell and 2-cell mice embryo development in vitro. Sixty percent and 56% ofthe 1-cell embryos reached the expanded blastocyst stage when cultured in media A and B, respectively. Eighty-four percent and 88% of 2-cell embryos reached the expanded blastocyst stage when cultured in media A and B, respectively. A prospective randomized study was then performed to evaluate this synthetic serum substitute in human in vitro fertilization. Among 141 ovum pick-up (OPU), oocytes retrieved in 74 cases were processed in medium A and oocytes retrieved in 67 others in medium B. In media A and B, the fertilization rate was 67% and 44.3% respectively, and the pregnancy rate/ OPU 23% and 9%, respectively. The pregnancy rate/transfer was 28.8% and 12.2% respectively, and the implantation rate/transferred embryo 9.5% and 4.2%. In the human sperm survival assay, the vitality and residual motility after 24 hours of incubation were significantly lower in medium B. In conclusion, UltroSer G successfully sustained the development in vitro of mouse embryos. However in human, it reduced sperm survival, oocyte fertilization, and embryo viability. Fertil Steril52:807, 1989 Culture media for in vitro fertilization (IVF) and embryo development are usually supplemented with 7.5% to 15% maternal serum or fetal cord serum (FCS). The potential advantages ofthis procedure -are the addition of undefined growth factors, the support of spermatozoa motility, the buffer capacity of serum, and the increased viscosity of medium which eases embryos handling. There are also disadvantages with the use of sera, such as the time-consuming and cumbersome collection and handling of these samples, or inconstancy in sera Received April10, 1989; revised and accepted July 13, * Presented in part at the VI World Congress, In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, Israel, April2 to 7, t Reprint requests: Mr. Iv Psalti, Physiology of Human Reproduction Unit, University of Louvain 5330, Av. E. Mounier 53, B-1200 Brussels, Belgium. quality. Some sera are even embryo toxic. 1 Furthermore, sera use exposes to putative contamination by various viral agents such as human immunodeficiency virus (HIV) and hepatitis B. Recently indeed, exposure of 177 IVF patients to hepatitis B antigen due to the use of a contaminated lot of human serum has been reported. 2 Finally, the presence of serum makes it difficult to study embryo metabolism, given its unstandardized complexity. 3 It is therefore of interest to find alternatives to the use of serum for IVF. An initial possibility is to omit serum. Human embryos have been successfully cultured in serum-free defined media. 3-5 However, other studies 6 7 have shown that the omission of serum leads to impaired development of human embryos, but so far there is no definitive agreement on this point and the majority of IVF laboratories continue to supplement their media with serum. A second alternative is the use of a standardized se- Psalti et al. Serum substitute for IVF 807

2 rum substitute. Recently, a serum substitute (UltroSer G) was shown to be able to successfully sustain the development in vitro of two cell mouse embryos.8 The aims of this study were (1) to evaluate the ability of this serum substitute to support one cell and two cell mouse embryo development in vitro. The two cell model was chosen to compare our results with previous work.8 The 1-cell model was used for its higher sensitivity to detect toxics in human IVF culture media. 9 (2) To test, in a randomized prospective study, the ability of this serum substitute to replace FCS for human oocyte fertilization and embryo growth in vitro. Medium and Sera MATERIALS AND METHODS The culture medium was modified Earle's medium (Gibco, Ghent, Belgium). One single batch of medium was used throughout the study. The medium was supplemented either with 10% heat inactivated human FCS (Medium A) 10 or with 2% VltroSer G (Medium B). FCS were tested for HIV and hepatitis B before use. Medium supplemented with 10% heat inactivated maternal serum (Medium C)11 was used for sperm preparation. Embryo transfers (ET) were performed in medium supplemented with 50% of maternal serum. UltroSer G is a serum substitute which contains growth and adhesion factors, mineral trace elements, steroids, binding proteins, vitamins, and aprotinin, a trypsin-inhibiting factor. UltroSer G is supplied as a sterile lyophilized powder controlled for bacteria, fungi, mycoplasma, and viruses by manufacturer. Mice Superovulation and Embryo Development F1 hybrid (C57 Black X CBa Brown) female mice were superovulated with 10 IU pregnant mare serum gonadotropin (PMSG) (Folligon, Intervet, Brussels, Belgium) and 10 IU human chorionic gonadotropin (hcg) (Preg~yl, Organon, Oss, The Netherlands) 48 hours later. They were then mated with F1 male. Oviduct dissection was carried out 16 hours or 40 hours after hcg injection to collect 1-cell or 2-cell mice embryos, respectively. These embryos were cultured in conditions similar to human embryos (37oC, 5% C0 2). Seventy-two hours after the two cell stage, the proportion of embryos having reached the stage of expanded blastocyst was recorded. 9 ' 12 ' 13 Human in Vitro Fertilization All the patients (n = 141) who underwent an OPU for IVF from October 1988 to December 1988 were included in this study. Patient randomization between FCS and UltroSer G was performed on a weekly basis. Controlled ovarian hyperstimulation was performed with daily nasal administration of 3 X 300 JLg of gonadotropin releasing-hormone agonist (GnRH-a) (Buserelin, Suprefact nasal spray, Hoechst, Frankfurt, West Germany) started on day 21 of the cycle preceding the treatment. Daily injection of 225 IU of human menopausal gonadotropin (hmg) (Humegon, Organon, Oss, The Netherlands) was started on the 3rd day of menstruation. Ovulation was induced with 5000 IU hcg. The OPU, IVF, ET, and luteal phase support were performed as previously described. 14 ' 15 Fertilization was assessed by visualization of 2 or more pronuclei 18 to 20 hours after insemination. An ET was performed when at least one normally fertilized oocyte reached the two to eight cell stages, 50 hours after insemination. Human Sperm Survival Assay A swim-up technique was used for sperm preparation.11 The effect of UltroSer G on sperm survival was studied by washing one divided semen sample simultaneously with media B and C. Nine samples of human semen were selected for a normal count (99.6 ± 69.2 M/mL, range: 39 to 243 M/mL) (mean ± Standard Deviation [SD]) and normal progressive motility (41.7 ± 5.6%, range: 35% to 50%). The parameters used to define normal semen was World Health Organization's values (sperm concentration: ~20 X 10 6 spermatozoa/ml, motility: ~50% with forward progression, and morphology: ~50% normal morphology). 16 Quantitative progressive motility was determined by counting motile spermatozoa in a Biirker haemocytometer (Assistent, Sondheim Rohn, West Germany). After washing, the spermatozoa were cultured at 37oC in a 5% C0 2 atmosphere. Twenty-four hours later, the spermatozoa residual motility as well as their viability(% spermatozoa excluding eosin Y) were measured. Statistical Analysis Statistical analysis was carried out using the x 2 test for mouse embryo and human IVF results, and 808 Psalti et al. Serum substitute for IVF Fertility and Sterility

3 Table 1 Comparison of IVF of Human Oocytes and Embryo Development in Media A and B Medium A Medium B x 2 No.ofOPU No.ofintactoocytes Fertilization rate (%) P<0.001 ET/OPU (%) NSa Pregnancy rate/opu (%) 23 9 P<0.05 Pregnancy rate/et (%) P<0.05 Implantation rate/ transferred embryo(%) P= 0.05 a NS; not significant. Fisher's F-test for human sperm analysis. Results were expressed as mean± standard deviation (SD). RESULTS Mouse Embryo Development in Vitro Two hundred thirty-one one-cell mice embryos were cultured in medium A and 128 in medium B. Sixty percent and 56% of these embryos respectively, reached the stage of expanded blastocyst not significant (NS). One hundred eighty-one two cell mice embryos were cultured in medium A and 107 in medium B. Eighty-four and 88% of them respectively, reached the stage of expanded blastocyst (NS). Human IVF and ET Five hundred and one oocytes retrieved in 7 4 of the 141 ovum pick-up (OPU) were processed in medium A, whereas 506 oocytes retrieved in the other 67 were processed in medium B (Table 1). The fertilization rate was 67% for the oocytes inseminated in medium A and only 44.3% for the oocytes insemin~ted in medium B (P < 0.001). The number of embryos per patient available for transfer were similar with medium A (3.97 ± 2.44) and with medium B (3.65 ± 2.25) (F-test: NS). An ET was possible in 59/74 cycles for which oocytes and embryos were cultured in medium A, and in 49/67 cycles for which oocytes and embryos were cultured in medium B. The overall pregnancy rate per OPU was 23% with medium A and 9% with medium B (P < 0.05). The pregnancy rate per transfer was 28.8% and 12.2% for media A and B respectively, (P < 0.05). The implantation rate per transferred embryo was 9.5% and 4.2% respectively, for media A and B (P = 0.05). In addition, to avoid any bias due to a putative Table 2 Comparison of IVF of Human Oocytes and Embryo Development in Media A and B When OPU Performed for Male Indication Were Excluded for Data Analysis Medium A Medium B x 2 No.ofOPU No. of intact oocytes Fertilization rate (%) P<0.001 ET/OPU (%) NSa Pregnancy rate/opu (%) P<0.05 Pregnancy rate/et (%) P<0.02 a NS, not significant. difference in the male factor between the 2 groups of OPU, the results were also analysed after exclusion of cycles performed for male indication (Table 2). In these conditions 48 attempts (leading to 41 ET) were performed with medium A and 44 attempts (leading to 40 ET) with medium B. The fertilization rate was lower in medium B than in medium A (61.2% versus 74.1%) (P < 0.001). The pregnancy rate per OPU was 27.1% with medium A and 9.1% with medium B (P < 0.05). The pregnancy rate per transfer was 31.7% and 10% for media A and B respectively, (P < 0.02). Human Sperm Survival Assay The recovery of progressive motile spermatozoa by sperm washing was 5.21 ± 3.07 M/mL in medium B and 4.48 ± 2.39 M/mL in medium C (NS). Residual motility after an incubation period of 24 hours was higher in medium C than in medium B (85.3 ± 15.7% versus 42.7 ± 27.4%) (P < 0.001). Vitality was also higher in medium C than in medium B (79.9 ± 7% versus 56.6 ± 23.9%) (P < 0.05) (Table 3). DISCUSSION It is possible to grow human embryos in vitro in various culture media which may or may not be Table 3 Comparison of Sperm Washing Procedure in Media C and B a MediumC MediumB F Motile spermatozoa recovered (M/mL) 4.48 ± ± 3.07 NSb Residual motility after 24 hours (%) 85.3 ± ±27.4 P<0.001 Vitality after 24 hours(%) 79.9 ± ± 23.9 P < 0.05 a The washing procedure was performed simultaneously on 9 normal ejaculates. b NS, not significant. Psalti et al. Serum substitute for IVF 809

4 supplemented with serump However, the addition of serum to culture media seems to accelerate the cleavage rate of human embryos 7 as it does for mouse embryos, 6 when compared with medium without protein supplementation. Moreover, Saito et al. 6 have reported that serum supplementation decreases the sister chromatid exchange in embryos, which is considered to be a sensitive measure of deoxyribonucleic acid damage due to culture conditions. 6 This study also suggests that serum is a safe condition for embryo growth and FCS provides better conditions for culturing embryos than various serum fractions and than 0.5% human serum albumin. The large-molecular-weight fraction of this serum seems to contain the beneficial components. 6 The fertilization rate in media supplemented with maternal serum or FCS is no different FCS does however, have some advantages when patients present circulating antisperm antibodies.19 Moreover, many maternal sera were found to inhibit the development of mouse embryos in vitro.1 The substitution of serum by bovine serum albumin or human serum albumin, as is the case in B3 medium, seems to be appropriate for human oocyte fertilization and embryo development in vitro. 3 4 Additional extensive confirmatory study will be welcome, and it should be noted that the cost of this medium is still relatively high. The promising results obtained by Pope et al.8 with UltroSer G supplementation in mouse embryo culture encouraged us to study this synthetic medium in our IVF program. The manufacturer recommends a final concentration of 2%. This corresponds to the data of Pope et al.8 in which optimal concentration for mouse embryo development in vitro was indeed 2%. We did not test higher concentrations of UltroSer G given that this did not improve human spermatozoa survival rate in vitro and reduced the proportion of two cell mouse embcyos reaching the blastocyst stage. 8 Our study confirms that UltroSer G is able to successfully sustain the in vitro development of two cell mouse embryos.8 In addition, in the one cell mouse embryo model, which is more sensitive to suboptimal culture conditions,9 embryo development in vitro was also entirely satisfactory. By contrast, the IVF of human oocytes appeared to be significantly lower when this protein preparation was used, compared with FCS. This is probably related to the reduced ability of UltroSer G to sustain survival and motility of human spermatozoa in vitro. In addition to this reduced fertilization rate, the embryo viability was also lower compared with that ob- tained with FCS. Together this led to a significantly lower pregnancy rate after IVF of human oocytes and culture in media supplemented with UltroSer G than when the culture medium was supplemented with FCS. Our study provides also a cautionary lesson that the conditions required to allow mouse zygotes and embryos to develop to blastocyst in vitro are not necessarily the same as those required for successful fertilization of human oocytes in vitro. Finally, it suggests also that human sperm survival assay should be used as quality test in any medium before introducing it into a routine human IVF system. In conclusion, this study confirms the ability of UltroSer G (2%) to successfully sustain the development in vitro of mouse embryos. However, this serum substitute significantly reduces human sperm survival and human oocyte fertilization. Human embryo viability also appeared lower. UltroSer G is thus not a suitable serum replacement in culture media for human IVF and embryo development. Acknowledgments. We thank Serge de Cooman, M.D. and Wally Bianchini, B.Sc. for sperm assays. REFERENCES 1. Shirley B, Wortham JWE, Peoples D, WhiteS, Condon Mahony M: Inhibition of embryo development by some rnaternal sera. J In Vitro Fert Embryo Transfer 4:93, Alberda TH: Transmission of hepatitis B and other sexually transmitted diseases by in vitro fertilization. Abstract presented at the XII World Congress of Gynecology and Obstetrics, Rio de Janeiro, Brasil, March 13 to 19, Congress Program Book, 1988, p Menezo Y, Testart J, Perrone D: Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer. Fertil Steril42:750, Feichtinger W, Kemeter P, Menezo Y: The use of synthetic culture medium and patient serum for human in vitro fertilization and embryo replacement. J In Vitro Fert Embryo Transfer 3:87, Caro CM, Trounson A: Successfull fertilization, embryo development, and pregnancy in human in vitro fertilization (IVF) using a chemically defined culture medium containing no protein. J In Vitro Fert Embryo Transfer 3:215, Saito H, Berger T, Mishell DR Jr, Marrs RP: The effect of serum fractions on embryo growth. Fertil Steril 41:761, Kruger TF, Stander FSH, Smith K, Vander Merwe JP, Lombard CJ: The effect of serum supplementation on the cleavage of human embryos. J In Vitro Fert Embryo Transfer 4:10, Pope AK, Harrison KL, Wilson LM, Breen TM, Cummins JM: UltroSer G as a serum substitute in embryo culture medium. J In Vitro Fert Embryo Transfer 4:286, Psalti et al. Serum substitute for IVF Fertility and Sterility

5 9. Davidson A, Vermesh M, Lobo RA, Paulson RJ: Mouse embryo culture as a quality control for human in vitro fertilization: the one-cell versus two-cell model. Fertil Steril49:516, Psalti I, Loumaye E, de Cooman S, Thomas K: Les controles de qualite dans la procedure de fecondation extracorporelle d'ovocytes humains et de transferts d'embryon. Louvain Med 107:399, de Cooman S, Psalti I, Sirjacobs Y, Thomas K, Loumaye E: Interets de la fecondation in vitro dans le traitement de la sterilite masculine. Louvain Med 107:407, Ackerman SB, Swanson RJ, Stokes GK, Veeck LL: Culture of mouse preimplantation embryos as a quality control assay for human in vitro fertilization. Gamete Res 9:145, Condon-Mahony M, Wortham JWE Jr, Bundren JC, Witmyer J, Shirley B: Evaluation of human fetal cord sera, Ham's F-10 medium, and in vitro culture materials with a mouse in vitro fertilization system. Fertil Steril 44:521, Loumaye E, de Cooman S, Anoma M, Psalti I, Depreester S, Schmit M, Thomas K: Short-term utilization of a gonad- otropin-releasing hormone agonist (Buserelin) for induction of ovulation in an in vitro fertilization program. Ann NY Acad Sci 541:96, Loumaye E, Vankrieken L, Depreester S, Psalti I, de Cooman S, Thomas K: Hormonal changes induced by shortterm administration of a gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development. Fertil Steril51:105, World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p Fishel SB, Surani MAH: Changes in responsiveness of preimplantation mouse embryos to serum. J Embryol Exp Morphol45:295, Leung PCS, Gronow MJ, Kellow GN, Lopata A, Speirs AL, McBain JC, duplessis Y, Johnston I: Serum supplementation in human in vitro fertilization and embryo development. Fertil Steril41:36, Ball GD, Coulam CB, Field CS, Harms RW, Thie JT, Byers AP: Effect of serum source on human fertilization and embryonic growth parameters in vitro. Fertil Steril44:75, 1985 Psalti et al. Serum substitute for IVF 811