Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal ftuid*

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1 FERTILITY AND STERILITY Copyright e 1985 The American Fertility Society Vol. 44, No, 4, October 1985 Printed in U.SA. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal ftuid* Patrick Quinn, Ph.D. t John F. Kerin, M.D., F.R.A.C.O.G. Graham M. Warnes, Ph.D. Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, South Australia, Australia Significantly more mouse zygotes developed to blastocysts in culture in a medium formulated on the composition of human tubal fluid (HTF) than in modified Tyrode's medium (T6). In a randomized 2 x 2 factorial trial of human in vitro fertilization that compared the two media and culture under oil versus culture in loosely capped tubes, significantly more clinical pregnancies (30% of 60 transfers) were obtained with HTF medium than with T6 medium (11% of 53 transfers). Decreasing the K+ content of HTF medium to that present in T6 medium significantly decreased the number of mouse zygotes that developed in culture. Modifying Ca+ + levels had no effect. It is therefore likely that the higher K+ content in HTF medium is primarily responsible for the superiority of HTF medium over T6 medium, but other differences in the composition of the two media could contribute to the results observed. Fertil Steril44:493, 1985 In vitro fertilization (IVF) of human oocytes, cleavage of embryos, and pregnancy after embryo transfer (ET) with the use of a variety of culture media have been reported.1, 2 Although there appears to be no significant difference between media for fertilization or embryo cleavage rate,1 the influence of the culture medium used for human IVF-ET on pregnancy rates is unknown. Suggestions that an elevated potassium content might be beneficia1 3, 4 have not been substantiated,1 although it has been reported 5 that more rat 00- cytes are fertilized at a faster rate with the use of a medium with a higher potassium level. Another Received May 1, 1985; accepted June 18, *Supported in part by a grant from the National Health and Medical Research Council of Australia. treprint requests: Patrick Quinn, Ph.D., Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia. Vol. 44, No.4, October 1985 variable between different IVF groups is the type of culture vessel used for IVF and embryo culture. The two main techniques employed are IVF and culture in 1 ml of medium in loosely capped plastic culture tubes 6 or in 0.1- to 0.5-ml drops of medium under oil in plastic culture dishes. 7 No significant difference was found between these two culture techniques for the development of mouse zygotes to fully expanded blastocysts when the medium was adequately equilibrated with the culture atmosphere. However, the oil technique was superior when the equilibration conditions were suboptimal. 8 Approximation of the culture conditions as close as possible to those found in the natural environment of the gametes will most likely yield the best results. Therefore, based on this rationale, Tervit et al.9 formulated a culture medium similar in biochemical composition to sheep oviduct fluid. This medium supports the development of a high proportion of cattle,9 sheep, and Quinn et al. Media and IVF pregnancy rate 493

2 Table 1. Composition of HTF and T6 Media Component mm NaCI KCI MgS04 7H20 KH 2P0 4 Na2HP04 CaCI2 2H20 NaHC03 Glucose Na pyruvate Na lactate Penicillin Streptomycin S04 Phenol red HTF Vlml 50 ILg/ml % (wt/vol) T Vlml 50 ILg/ml % (wt/vol) goat lo embryos to the blastocyst stage. We have applied this concept to human gametes and formulated a culture medium based on published analyses of human fallopian tube fluid. 11, 12 The pregnancy rate obtained in our human IVF-ET program has been analyzed in a randomized 2 x 2 factorial trial, which compared this medium and modified Tyrode's T6 medium and culture in tubes, contrasted with culture under oil. MEDIA MATERIALS AND METHODS The formulation of the medium based on the composition of human tubal fluid (HTF medium) is given in Table 1. Fresh medium was prepared each week from stock solutions of the various components, as previously described,s with the use of water that had been processed through a Millipore reverse osmosis unit and Milliq treatment system (Millipore Corporation, Sydney, New South Wales, Australia). Modified Tyrode's T6 mediums was prepared with the use of the same procedures. IN VITRO FERTILIZATION AND EMBRYO CULTURE Mouse zygotes from (C57BLl6 x CBA) Fl mice were collected and cultured in groups of up to 20 in approximately 30-I.Ll drops of medium containing 5 mg/ml bovine serum albumin (fraction V, Sigma Chemical Company, St. Louis, MO) under oil in plastic culture dishes.s The effect of modifying the ionic composition of the medium on the development of mouse zygotes was tested by varying N a + /K + or Ca + + /Mg + + levels. Five media were used in each experiment. 494 Quinn et al. Media and IVF pregnancy rate Initially, two media containing bovine serum albumin were prepared. One was HTF medium and the other was similar in all respects except that its N a + /K + or Ca + + IMg + + content was like that of T6 medium. The osmolarity of this second medium was adjusted to that of the HTF medium by modifying the NaCl content. Three other media with intermediate levels of N a + /K + or Ca + + / Mg+ + (25%, 50%, or 75% of the levels present in HTF medium) were then prepared by mixing appropriate aliquots of the two media. Approximately 30-f,Ll drops of the five different media were then placed in plastic culture dishes (#301 V, Sterilin, Teddington, Middlesex, UK) under oil and equilibrated in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 37 C for 18 hours before the addition of fertilized I-cell mouse zygotes. The protein supplement in the media and culture techniques for human IVF-ET have been described previously.s Each batch of medium used for the culture of human gametes was tested for its ability to support the development of fertilized mouse zygotes to fully expanded blastocysts over 5 days of culture with the use of 40 to 60 zygotes on each occasion. From September 1983 to April 1984 all patients who entered our IVF program were used in a randomized 2 x 2 factorial trial of media and culture conditions in a cross-over design. For any 4-week period, the gametes of all patients were cultured in HTF medium for the first 2 weeks; then T6 medium was used in the second 2 weeks. In the first and third weeks, culture was carried out under oil; in weeks 2 and 4, loosely capped 5-ml plastic culture tubes (#2058, Falcon Plastics, Oxnard, CA) containing 1 ml of medium were used. STATISTICAL ANALYSES Differences between treatments for the proportion of human embryos fertilized and cleaved or mouse embryos developing were compared with the use of Student's t-test or chi-square analysis, Table 2. Development of I Cell Mouse Zygotes in T6 and HTF Media a Medium T6 HTF No. of zygotes No. of blastocysts % Range apooled results from three replicate experiments. X2 = 8.28, P < Fertility and Sterility

3 Table 3. Human IVF Data from a Randomized 2 x 2 Factorial Trial of Media and Culture Techniques Medium Culture Mouse zygote No. of pregnancies/ technique growth Collected Fertilized Cleaved no. of transfers (o/c) HTF Oil (3.62) HTF Tube (3.61) T6 Oil (3.76) T6 Tube (3.78) Total (3.69) 'If 117 (3.16; 87) 94 (2.54; 80) 11/37 (30) 72 (3.13; 87) 64 (2.78; 89) 7/23 (30) 53 (3.12; 83) 44 (2.59; 83) 2/17 (11) 118 (3.28; 87) 104 (2.89; 88) 4/36 (11) 360 (3.19; 86) 306 (2.71; 85) 24/113 (21) as appropriate. The effect of different ratios of Na + IK + or Ca + + IMg+ + on the development of mouse zygotes was assessed by analysis of variance after arc sine transformation of the proportion of embryos developing. RESULTS The number of mouse zygotes developing to blastocysts when cultured in RTF or T6 medium under oil is given in Table 2. Significantly more embryos (X2 = 8.28, P < 0.005) developed to fully expanded blastocysts over 5 days of culture in RTF medium than in T6 medium. The numbers of human oocytes collected, fertilized, and cleaved and the subsequent number of patients clinically pregnant after ET together with the quality control assay of mouse zygote development for each batch of medium used are given in Table 3. The data are presented separately for the different culture techniques and media used in Tables 4 and 5, respectively. There was no difference in the numbers of oocytes collected, fertilized, and subsequently cleaved per patient between any of the treatments. The culture technique did not affect the subsequent pregnancy rate or mouse embryo quality control assay (Table 4). There was, however, a significant difference between the two media in the number of mouse embryos developing (t Ul = 4.14, P < 0.001) and also the number of pregnancies initiated after transfer (X 2 = 5.87, P < 0.025). Almost three times as many pregnancies occurred when fertilization and culture were carried out with RTF medium (18/60, 30%), compared with T6 medium (6/53, 11%). The data for numbers of oocytes collected, fertilized, cleaved, and transferred for pregnant and nonpregnant patients are given in Table 6. The pregnant patients had significantly more oocytes collected at laparoscopy than the nonpregnant patients and maintained this advantage until transfer of the resulting embryos. There was no difference between the pregnant and nonpregnant patients in the number of mouse embryos developing in the media used for fertilization and embryo culture. In an endeavor to discover which components of the two media might be responsible for the observed differences in mouse embryo development in vitro and initiation of human pregnancies, the composition of the two media was inspected (Table 1). The greatest differences in composition of the two media are their N a + IK + and Ca Mg+ + ratios. When media were tested with Ca + + IMg+ + levels varying from 1.78/0.71 mm to 2.04/0.20 mm in five equally spaced increments, there was no significant difference in the number of mouse zygotes developing to blastocysts in any of the media. An average of96% (257 Table 4. Effect of Culture Technique on the Results of Human IVF-ET Culture Mouse zygote No. of pregnancies/ technique growth Collected Fertilized Cleaved no. of transfers (%) 'If Oil (3.67) 170 (3.15; 86) 138 (2.56; 81) 13/54 (24) Tube (3.71) 190 (3.22; 87) 168 (2.85; 88) (19) Total (3.69) 360 (3.19; 86) 306 (2.71; 85) 24/113 (21) t-test till X 2 = 0.50 P > 0.2 > 0.5 > 0.5 > 0.2 > 0.5 Vol. 44, No.4, October 1985 Quinn et al. Media and IVF pregnancy rate 495

4 Table 5. Effect of Culture Medium on the Results of Human IVF-ET Medium Mouse zygote growth No. of pregnancies/ Collected Fertilized Cleaved no. of transfers (%) 'k HTF (3.62) T (3.77) Total (3.70) t-test hll P < > (3.15; 87) 158 (2.63; 84) 18/60 (30) 171 (3.23; 86) 148 (2.79; 87) 6/53 (11) 360 (3.19; 86) 306 (2.71; 85) 24/113 (21) X 2 = 5.87 > 0.5 > 0.5 < of 267; range, 92% to 100%) of the zygotes developed to blastocysts in four replicated experiments testing the five different media. The results from a similar experiment, which involved variations in the Na+/K+ ratio from 150.5/1.42 mm to 148.3/5.06 mm, are shown in Figure 1. Analysis of variance showed that there were significant linear and quadratic responses in the number of embryos developing to expanded blastocysts with increasing levels of K +. In medium containing the K+ levels oft6 medium (1.4 mm), 75% of the zygotes developed, which was significantly fewer (F1 = 38.0, P < 0.01) than the 95% to 100% of embryos developing when the K + level was 2.3 to 5.1 mm. DISCUSSION The greater number of mouse zygotes that developed to expanded blastocysts when cultured in HTF medium, compared with T6 medium, was paralleled by the threefold increase in the number of pregnancies initiated in those patients whose gametes had been fertilized and cultured in HTF medium, rather than T6 medium. The absence of any difference in mouse embryo development or human pregnancy rates when culture was performed under oil or in tubes is in agreement with our previous findings that there is no difference between the two culture techniques for the development of mouse zygotes as long as the culture medium is adequately equilibrated with the culture atmosphere. These observations confirm and extend our previous conclusions, 13 that the mouse embryo development test can be used to detect differences in culture techniques that have a significant influence on the rate of pregnancy initiation in human IVF-ET. However, the mouse embryo test can be used only for detection of major suboptimal conditions in the culture procedures and cannot predict the likelihood of individual pregnancies because there is no difference in the rates of mouse embryo development in media used for the gametes of patients who did or did not become pregnant (Table 6). Our data are in agreement with those of others l4, 15 and indicate that pregnancy is more likely if more oocytes are collected, fertilized, and transferred. The fertilization and cleavage rate of human embryos up to the 2- to 4-cell stage was not affected by the culture conditions, and similarly the rate of development of mouse embryos for the first 2 days of culture to the 4- to 8-cell stage was similar under the various culture regimens.16 It is between the 4- and 8-cell stage in mouse embryos that major changes occur in embryonic genome expression,17 metabolism, IS totipotency of blastomeres,19 and growth rates in vitro, com- Table 6. Embryo Development in Pregnant and Nonpregnant Patients Patients Mouse zygote No. of growth patients Collected Fertilized Cleaved Transferred Not pregnant Pregnant 'he 302 (3.39) 115 (4.79) 266 (2.99; 88) 94 (3.92; 82) 220 (2.47; 83) 86 (3.58; 91) 204 (2.29; 93) 76 (3.17; 88) Total t-test tlll 0.57 P > (3.69) 3.52 < (3.19; 86) 2.61 < (2.71; 85) 3.55 < (2.48; 92) 4.01 < Quinn et at. Media and IVF pregnancy rate Fertility and Sterility

5 go 0. o 90 ~ ~80, ; ; ~ 60 '~'4----~2'3-----'~'----~'-' ----" K+(mEq/l) Figure 1 Effect of K + concentration on the development of mouse zygotes in vitro. Mean and range from three replicates with 20 embryos in each replicate. pared with in vivo, development.2o It would be reasonable to assume that similarly in human embryos, little difference in growth rates would be apparent before the 8-cell stage. However, by then ET has occurred, and differences in viability entrained by suboptimal culture conditions for the first 2 days of culture after fertilization can only be detected by differences in the rates of pregnancy initiation. The results obtained with mouse zygotes (Fig. 1) indicate that differences in the Na + IK + levels between HTF and T6 media are most likely responsible for the observed increase in pregnancy rates in patients whose gametes had been cultured in HTF, rather than T6, medium. Wales21 was the first to show that mouse embryos required a critical level of potassium ions for maximal rates of development, but the level of 0.8 mm he observed as a minimum for optimal development is below the level of 1.42 mm K + present in T6 medium. These experiments however were conducted with 2-cell mouse embryos, which are less sensitive to suboptimal culture conditions than I-cell embryos.22 Lopata 3 obtained more human blastocysts when the level of potassium in culture medium was increased from 5 mm to 10 mm. The Na + IK + ratio of HTF medium is nearly fourfold lower than that of T6 medium (Table 7) and is closer to the values reported for oviduct fluid and blood serum of a variety of mammals, including humans. 4, 11,25 The Na + IK + ratio for three other culture media commonly used for human IVF-ET is also in the range of 20 to 35 (Table 7), and it is therefore likely that these media would be just as suitable for human IVF ET as HTF medium. In conclusion, our data support the proposal that the ratio of N a + to K + ions in the culture medium used for human IVF-ET is important for the determination of the quality of the embryos produced by in vitro procedures and therefore the subsequent clinical pregnancy rate. This study also demonstrates the potential for obtaining meaningful data from carefully controlled trials in an IVF program with a moderate number of patients. Table 7. Composition of Media Used for Human IVF-ET WM, (Hopge Modified Earles' Component and Pitts ) (Edwards') mm NaCI KCI MgS04.7H 2O KH 2P NaH 2P04 2H2O 1.01 Na2HP04 CaCI2.2H20 (or Ca lactate) NaHC Na lactate 21.6 Na pyruvate Glucose Penicillin 100 Vlml 100 Vlml Streptomycin S04 50 IJ.g/ml 50 IJ.g/ml Phenol red 0.001% % Na+/K Ca+ +IMg Ham's F-I0" (Edwards; Lopata et al. 24) T6 HTF Vlml 100 Vlml 100 Vlml 50 IJ.g/ml 50 IJ.g/ml 50 IJ.g/ml % 0.001% 0.001% aalso contains CUS04, FeS04, ZnS04, amino acids, and vitamins. Vol. 44, No.4, October 1985 Quinn et ai. Media and IVF pregnancy rate 497

6 Acknowledgments. We wish to thank Ms. Regan Jeffrey, Ms. Christine Robertson, and Ms. Dianna Payne for skilled technical assistance and Terence J. Broom, F.R.A.C.O.G., Michael McEvoy, F.R.A.C.O.G., Colin D. Matthews, F.R.C.O.G., F.R.A.C.O.G., Robert F. Seamark, Ph.D., and Prof. Lloyd W. Cox, F.R.C.O.G., F.R.A.C.O.G. for cooperation and encouragement. REFERENCES 1. Trounson A: In vitro fertilization. Curr Top Exp Endocrinol 5:43, Jones GS: Update on in vitro fertilization. Endocr Rev 5:62, Lopata A: Factors influencing the growth of human preimplantation embryos in vitro. In Human Conception In Vitro, Edited by RG Edwards, JM Purdy. London, Academic Press, 1982, p Borland RM, Biggers JD, Lechene CP, Taymor ML: Elemental composition of fluid in the human fallopian tube. J Reprod Fertil 58:479, Toyoda Y, Chang MC: Capacitation of epididymal spermatozoa in a medium with high KINa ratio and cyclic AMP for the fertilization of rat eggs in vitro. J Reprod Fertil 36:125, Trounson AO, Leeton JF, Wood C, Webb J, Kovacs G: The investigation of idiopathic infertility by in vitro fertilization. Fertil Steril 34:431, Edwards RG: Test-tube babies, Nature 293:253, Quinn P, Warnes GM, Kerin JF, Kirby C: Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fertil Steril 41:202, Tervit HR, Whittingham DG, Rowson LEA: Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 30:493, Quinn P, Warnes GM, Walker SK, Seamark RF: Culture of preimplantation sheep and goat embryos. In Reproduction in Sheep, Edited by DR Lindsay, DT Pearce. Canberra, Australian Academy of Science and the Australian Wool Corporation, 1984, p Lippes J, Enders RG, Pragay DA, Bartholomew WR: The collection and analysis of human fallopian tube fluid. Contraception 5:85, Lopata A, Patullo MJ, Chang A, James B: A method for collecting motile spermatozoa from human semen. Fertil Steril 27:677, Quinn P, Warnes GM, Kerin JF, Kirby C: Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci 442:195, Trounson AO, Conti A: Research in human in-vitro fertilization and embryo transfer. Br Med J 285:244, Edwards RG, Steptoe PC: Current status of in-vitro fertilisation and implantation of human ej)lbryos. Lancet 2:1265, Quinn P: Unpublished data 17. Braude PR, Johnson MH, Bolton VN, Pratt HPM: Cleavage and differentiation of the early embryo. In In Vitro Fertilization and Embryo Transfer, Proceedings of the Serono Clinical Colloquia on Reproduction, No.4, Edited by PG Crosignani, BL Rubin. London, Academic Press, 1983, p Wales RG: Maturation of the mammalian embryo: biochemical aspects. BioI Reprod 12:66, Kelly SJ: Studies of the developmental potential of 4- and 8-cell stage mouse blastomeres. J Exp Zool 200:365, Harlow GM, Quinn P: Development of preimplantation mouse embryos in vivo and in vitro. Aust J BioI Sci 35:187, Wales RG: Effects of ions on the development of the preimplantation mouse embryo in vitro. Aust J BioI Sci 23:421, Quinn P, Whittingham DG: Effect of fatty acids on fertilization and development of mouse embryos in vitro. J Androl 3:440, Hoppe PC, Pitts S: Fertilization in vitro and development of mouse ova. BioI Reprod 8:420, Lopata A, Johnston IWH, Hoult IJ, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117, Bavister BD: Analysis of culture media for in vitro fertilization and criteria for success. In Fertilization and Embryonic Development In Vitro, Edited by L Mastroianni, JD Biggers. New York, Plenum Press, 1981, p Quinn et ai. Media and IVF pregnancy rate Fertility and Sterility