Serum is more effective than albumin in promoting human embryo development and implantation

Transcription

1 FERTLTY AND STERLTY Vol. 64, No.6, December 1995 Copyright 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Serum is more effective than albumin in promoting human embryo development and implantation Corinne A. Hargreaves, M.R.C.O.G.* Faiza Rahman, M.R.C.O.G.* Dickinson Cowan, F.R.C.O.G.t Meryl Santis*t Tracy Keefe, H.N.C. *t Richard J. S. Howell, F.R.C.O.G.*t Tim Chard, M.D., F.R.C.O.G.* Sheryl T. Homa, Ph.D.*t+ St. Bartholomew's Hospital, and the Portland Hospital for Women and Children, London, United Kingdom Objective: To compare the effects of serum with those of Albuminar-5 (Armour Pharmaceutical Co., Eastbourne, Sussex, United Kingdom) as medium supplements to Earle's balanced salt solution (EBSS) for VF and subsequent embryo development. Design: A retrospective study. Gametes and embryos from 318 patients were cultured in the presence of serum (group 1). Gametes and embryos from 130 patients were cultured in the presence of Albuminar-5 (group 2). Embryos obtained from VF were replaced into the uterus within 48 hours after insemination. Surplus bipronucleate embryos were cultured up to 14 days with either serum or Albuminar-5. Setting: Two tertiary referral fertility clinics; university teaching hospital. Patients: Four hundred forty-eight patients with a wide spectrum of causes of subfertility, ranging in age from 24 to 43 years. Main Outcome Measures: Fertilization rate, pregnancy rate (PR), implantation rate, and surplus embryo development in vitro. Results: The PR for group 1 patients was higher than that of group 2 (27.0% versus 15.4%, respectively). Although fertilization rates were identical in the two groups, cumulative embryo scores and implantation rates were significantly higher in group 1. There was no difference between the groups in age distribution, types of ovarian stimulation, numbers of patients with day 1 or day 2 transfers, or luteal phase support. Of 31 embryos cultured with serum, 54.8% reached the fully expanded blastocyst stage and 25.8% hatched. Of 19 embryos cultured with Albuminar-5, only 5.3% reached the fully expanded blastocyst stage and none hatched. Conclusions: The results suggest that, under certain conditions, serum supplementation yields better results than protein supplementation alone. The latter may be suitable only in conjunction with additional components. Fertil Steril 1995;64: Key Words: Serum, albumin, culture medium, VF, embryo development, implantation, pregnancy Media currently used for VF range from human tubal fluid or simple balanced salts solutions to more complex solutions supplemented with energy substrates, amino acids, and vitamins. Although early Received March 22, 1995; revised and accepted June 12, * Department of Obstetrics, Gynaecology and Reproductive Physiology, St. Bartholomew's Hospital. t The Portland Hospital Fertility Unit, BM Portland Hospital for Women and Children. t Reprint requests: Sheryl T. Homa, Ph.D., The Portland Hospital Fertility Unit, Portland Hospital for Women and Children, 209 Great Portland Street, London WN 6AH, United Kingdom (FAX: ) Hargreaves et al. Serum versus albumin in VF human embryo development can occur under certain defined conditions in the absence of protein (1), in most cases a protein supplement in the form of maternal or fetal cord serum, amniotic fluid, or serum albumin is used to support gamete culture and embryo development (2-4). However, serum that is commonly used as a protein source for VF is highly variable. Although it provides constituents that are stimulatory for embryonic growth, equally it contains factors that have been shown to have an adverse effect on embryo development at different developmental stages or in combination with other components (5-8). For these reasons, VF in the presence of a protein

2 source other than serum has its advantages. Furthermore, it is not always possible to use maternal serum for VF because it may contain antisperm antibodies. n such cases, human serum albumin (HSA) is used widely in place of serum. n the present study, we show that supplementation with HSA (Albuminar-5; Armour Pharmaceutical Co., Eastbourne, Sussex, United Kingdom) yields a lower pregnancy rate (PR) than does supplementation with maternal serum. This suggests that HSA is not universally suitable as an alternative supplement for VF. Patients MATERALS AND METHODS A total of 448 patients undergoing VF-ET were recruited from two fertility centers in London, United Kingdom. Three hundred thirteen patients were recruited from St. Bartholomew's Hospital and 135 patients were recruited from the Portland Hospital. All procedures were carried out separately at the two centers. Ovarian Stimulation n 399 cases, the pituitary was down-regulated with intranasal buserelin acetate (GnRH analogue; Suprefact, Hoechst UK Ltd., Hounslow, United Kingdom). Both the flare-up (87 patients) and long regime (312 patients) were used. The ovaries were stimulated by M injection ofhmg (Pergonal; Serono Laboratories Ltd., Welwyn Garden City, United Kingdom) or urofollitropin (Metrodin; Serono Laboratories Ltd.). n the remaining cases, there was no prior down-regulation; these patients received either clomiphene citrate (Marion Merrell Dow Ltd., Uxbridge, Middlesex, United Kingdom) and hmg or urofollitropin, or hmg or urofollitropin alone. Transvaginal oocyte aspiration was carried out approximately 34 hours after injection ofhcg (Profasi; Serono Laboratories Ltd.). n Vitro Fertilization Oocytes were cultured in 1.5 ml medium in the center well of organ culture dishes (Falcon 3037; Becton Dickinson, Cowley, Oxfordshire, United Kingdom) (up to 5 oocytes per dish) with approximately 100,000 partner's spermlml at 37 C under 5% CO2 in air. The medium was Earle's balanced salts solution (EBSS; Life Technologies, Ltd. Paisley, United Kingdom) containing 100 /LM sodium pyruvate (9), 25 mm NaHC0 3, penicillin, and gentamicin, supplemented with serum or HSA as described below. The difference in osmolarity of the medium in the presence of serum compared with HSA was < 1 %. Vol. 64, No.6, December 1995 Patient Study Groups n this retrospective study, allocation to treatment study groups at both clinics was carried out between November 1992 and April n group 1, gametes from 318 patients (234 from St. Bartholomew's Hospital and 84 from the Portland Hospital) were prepared and cultured in medium supplemented with 10% maternal serum, which had been previously sterilized through a 22-/Lm filter and heat inactivated at 56 C for 45 minutes. n group 2, in 130 subjects (80 from St. Bartholomew's Hospital and 50 from the Portland Hospital), either the partner's semen or maternal serum contained antisperm antibodies, or blood was unobtainable or hemolyzed. Gametes were prepared and cultured in medium supplemented with 10% Albuminar-5 (containing 5% HSA; Armour Pharmaceutical Co., Eastbourne, United Kingdom). Embryo Transfer and Luteal Support Mter 18 hours, cumulus cells were removed mechanically to determine formation of pronuclei. Up to three embryos were transferred either at the pronucleate stage or after a further 24 hours in culture. n the latter case, embryo quality was assessed as described previously (10), and the embryos with the highest scores were replaced. Luteal phase support was given in the form of P (Cyclogest; Hoechst UK Ltd.) pessaries vaginally, hcg injections M, or dydrogesterone (Duphaston; Duphar Laboratories, Ltd., Southampton, United Kingdom) tablets orally. n patients who were not down-regulated during treatment, no luteal support was given. Embryo Culture As part of a study licensed by the Human Fertilisation and Embryology Authority and approved by the local Ethical Committee, surplus bipronucleate embryos were cultured for up to 14 days. These surplus embryos were those that either were considered to be unsuitable for freezing because of fragmentation on day 2 postinsemination or those that the patient did not wish to be frozen. On day 2 after insemination, 50 surplus embryos were placed in 1 ml fresh EBSS, containing 100 /LM pyruvate, penicillin, and gentamicin supplemented with 10% maternal serum (group 1; 31 embryos) or Albuminar-5 (group 2; 19 embryos). All embryos except for four in group 1 had been cultured in medium supplemented with 10% maternal serum up until this time. The remaining four embryos were cultured initially in 10% Albuminar-5. The proportion of embryos that was fragmented was the same in both groups (21%). Embryos were cultured at 37 C under 5% CO2 in Hargreaves et al. Serum versus albumin in NF 1163

3 air in a humid atmosphere and assessed daily for blastocyst formation. Statistics The results are described as medians together with the range or interquartile range. The significance of the difference between medians was assessed by the Wilcoxon-Mann-Whitney U-test. The significance of the differences between proportions was assessed by the X 2 test. RESULTS The total PR was almost double in group 1 patients (medium supplemented with maternal serum) than in group 2 patients (medium supplemented with Albuminar-5) (Table 1). The proportionate difference in PR between the two groups was reflected in both fertility centers (Table 1). The PR still was decreased significantly in group 2 when only pregnancies past 24 weeks were included (Table 1) or when the comparison was limited to patients who had three embryos transferred (30.7% [80/261] group 1 versus 18.6% [19/102] group 2; P < 0.02). The distribution of diagnoses of the causes of subfertility within the two study groups is shown in Table 2. n both groups, the most common cause was tubal defects. Although there was a higher proportion of these patients in group 1 than in group 2 (P < 0.001), the PRs within this subgroup showed a similar doubling trend for group 1 compared with group 2 (22.6% [36/159] and 10.2% [5/49], respectively). There was a higher proportion of male and female patients with antisperm antibodies in group Table 2 Relative Distribution of the Different Causes of Subfertility Within Study Groups* Sperm defects Male antis perm antibodies Female antis perm antibodies Tubal disease Endometriosis Anovulatory Polycystic ovary syndrome diopathic Cervical mucus hostility Group 1 Group 2 Combined 15.0 (59) 2.6 (10) 0.3 (1) 49.0 (192) 8.2 (32) 4.8 (19) 3.8 (15) 14.0 (55) 2.3 (9) 14.7 (25) 7.6 (13) 5.9 (10) 37.6 (64) 8.2 (14) 4.7 (8) 2.4 (4) 15.9 (27) 2.9 (5) 14.9 (84) 4.1 (23) 2.0 (11) 45.6 (256) 8.2 (46) 4.8 (27) 3.4 (19) 14.6 (82) 2.5 (14) * Values are expressed as a percentage of the total number of causes with number of patients in each category in parentheses. n many cases, there was more than one cause for subfertility. n such instances, each cause was counted separately. 2 (P < 0.01) as a result of selection, however, they constituted only 10% (13/130 subjects) of the total patient population within this group. nterestingly, the PRs for individuals in whom male or female antibodies were the sole known cause for subfertility were lower in group 1 (12.5% [118]) than in group 2 (38.5% [5113]), although this was not significant. There was no difference in the range or means of female age between groups, in the distribution of the types of ovarian stimulation protocol, or whether luteal phase support was used or whether the embryos were transferred on day 1 or day 2 after insemination (Table 3). Fertilization rates did not vary with type of supplement (66.7% and 66.6% in groups 1 and 2, respectively). The overall cumulative embryo score of the replaced embryos, which characterizes morphological Table 1 Pregnancy Outcomes Within Study Groups Group 1 Group 2 P value Table 3 Distribution of Treatment Variables Within Study Groups Group 1 Group 2 Total PR* Portland Hospital St. Bartholomew's Hospital Combined Centers Pregnancy outcomes for combined centers*:j: (30/84) 23.9 (56/234) 27.0 (86/318) 0.6 (2) 1.9 (6) 3.5 (11) 0.6 (2) 20.4 (6) 17.6 (9/51) 13.9 (11/79) 15.4 (20/130) o 1.5 (2) 0.8 (1) o 13.1 (17) <0.05 t <0.01 <0.05 * Values are expressed as a percentage of total patients who reached an ET in each study group with number of patients in each group in parentheses. t, not significant. :j: 1, ectopic pregnancy; 2, biochemical or anembryonic pregnancy; 3, sac with fetal pole with or without heart beat and lost before 13 weeks; 4, fetal heartbeat but lost by 24 weeks; 5, past 24 weeks or delivered Hargreaves et al. Serum versus albumin in VF No. of patients Mean (range) female age (y) 34 (24 to 42) 33 (25 to 43) Stimulation regimen* Day 21 buserelin acetate + hmg or urofollitropin 71.1 (226) 66.2 (86) Day 1 buserelin acetate + hmg or urofollitropin 19.5 (62) 19.2 (25) Clomid + hmg or urofollitropin 6.3 (20) 10.8 (14) Other 3.1 (10) 3.8 (5) Luteal support* None 9.0 (29) 11.5 (15) RCG 20.8 (66) 18.5 (24) Progesterone 66.7 (212) 66.2 (86) Dydrogesterone 3.5 (11) 3.8 (5) No. of embryos transferred* (19) 10.8 (14) (38) 10.8 (14) (261) 78.4 (102) Day ofet* (27) 10.8 (14) (291) 89.2 (116) * Values are expressed as a percentage oftotal patients in each category with numbers of patients in each category in parentheses.

4 60 -f! >- u 30 &:: Q) :::l 20 :::T Q) u Stage of development Figure 1 Embryo development in culture. The results are expressed as a percentage of the total number of embryos cultured in each study group. 1, Embryos that underwent several cleavage divisions but failed to cavitate; 2, embryos that began to form a blastocoele cavity and then arrested; 3, embryos that became fully expanded blastocysts but failed to hatch; 4, partially hatched embryos; 5, fully hatched embryos. 0, group 1;., group 2. quality (10), was significantly higher for embryos cultured in serum compared with those cultured in Albuminar-5 (34.8 ± 0.8 versus 27.8 ± 1.3, respectively; P < ). The implantation rate, defined as the percentage fetal sacs of the total number of embryos transferred, also was increased significantly for group 1 compared with group 2 patients (13.8% [119/865] versus 7.8% [27/346], respectively; P < 0.01). n the in vitro study of embryo development, 31 embryos were cultured in 10% serum, and 17 (54.8%) reached the fully expanded blastocyst stage by day 6 (Fig. 1). Of these blastocysts, eight (25.8% of all embryos) successfully hatched in culture by day 7. Of 19 embryos cultured with Albuminar-5, only 1 (5.3%) reached the fully expanded blastocyst stage by day 6 of culture and none hatched. However, seven embryos did begin to cavitate on days 5 to 6 but did not progress to fully expanded blastocysts. DSCUSSON The results clearly show that, under the conditions used in this study, serum is superior to Albuminar-5 as a supplement to EBSS medium for VF. This is in contrast to two previous studies that have compared serum and Albuminar-5 (11) or Albuminar-20 (12) as supplement to EBSS medium. These studies showed either no statistical difference in implantation or pregnancy rates (11) or a highly significant increase in implantation and pregnancy rate in the presence of Albuminar (12). Both Ashwood-Smith et al. (11) and Staessen et al. (12) used pyruvate concentrations (0.3 and 0.33 mm, respectively) higher than that used in this study (0.1 mm) and lactate was included in the study Vol. 64, No.6, December 1995 by Staessen et al (12). Pyruvate is the primary substrate for embryo metabolism during the initial cleavage stages of mouse and human embryos (13, 14). t is possible that the culture medium used in the present study may have contained insufficient pyruvate to sustain embryo growth in the absence of serum, which itself contains approximately 0.08 mm pyruvate (15). When embryos were cultured in a combination of EBSS and the complex medium Ham's F-10, supplemented with either serum or a plasma protein solution (16), there was no difference in implantation rate. Although the PR per ET was 5% less in the plasma protein group, this difference was not significant (16). Another study compared Plasmanate (a plasma protein fraction containing primarily human albumin and globulins) and serum as a supplement to human tubal fluid for VF (17). mplantation and pregnancy rates were higher in Plasmanate, but the distribution of diagnoses in each group was biased considerably. However, when patients were matched in a subsequent prospective study, they found the PRs were reversed (17), consistent with the results presented in the present retrospective study. Embryo development is compromised in the absence of serum, because there is a significant decrease in overall embryo quality of the embryos cultured in Albuminar, which may have contributed to the decreased implantation rate observed in group 2 patients in this study. n addition, the ability of embryos to develop to the hatched blastocyst stage is decreased in the absence of serum and is consistent with observations of Ashwood-Smith et al. (11) and Staessen et al. (12) using similar culture conditions. This may be attributed to the absence of vitamins and amino acids in Albuminar which are present in serum and which are known to promote blastocyst formation and hatching (18, 19). Overall, the results from this study suggest that, although HSA is used routinely and very effectively as a substitute for serum to supplement VF culture medium, under some conditions it may be inadequate as an alternative supplement. REFERENCES 1. Caro CM, Trounson A. Successful fertilization, embryo development and pregnancy in human in vitro fertilization (VF) using a chemically defined culture medium containing no protein. J n Vitro Fert Embryo Transf 1986;3: Gianaroli L, Seracchioli R, Ferraretti AP, Trounson A, Flamigni C, Bovicelli L. The successful use of human amniotic fluid for embryo culture and human in vitro fertilisation, embryo culture and transfer. Fertil Steril1986;46: Kruger TF, Stander FSH, Smith K, Van der Merwe JP, Lombard CJ. The effect of serum supplementation on the cleavage of human embryos. J n Vitro Fert Embryo Transf 1987;4: Hargreaves et al. Serum versus albumin in NF 1165

5 1 4. Ogawa T, Marrs RP. The effect of protein supplementation on single-cell mouse embryos in vitro. Fertil Steril 1987; 47: Seshagiri PB, Bavister BD. Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro. Bio Reprod 1989;40: Petters RM, Johnson BH, Reed ML, Archibong AE. Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro. J Reprod Ferti1990;89: Lawitts JA, Biggers JD. Joint effects of sodium chloride, glutamine and glucose in mouse preimplantation embryo culture media. Mol Reprod Dev 1992;31: Conaghan J, Handyside AH, Winston RML, Leese HJ. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Ferti1993;99: Trounson A. Fertilization and embryo culture. n: Wood C, Trounson A, editors. Clinical in vitro fertilization. London: Springer-Verlag, 1989: Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum Reprod 1992; 7: Ashwood-Smith MJ, Hollands P, Edwards RG. The use of Albuminar-5 (TM) as a medium supplement in clinical VF. Hum Reprod 1989;4: Staessen C, Van den Abbeel E, Carle M, Khan, Devroey P, Van SteirteghemAC. Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization. Hum Reprod 1990;5: Hardy K, Hooper MAK, Handyside AH, Rutherford AJ, Winston RML, Leese HJ. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 1989;4: Gott AL, Hardy K, Winston RML, Leese HJ. Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human pre-implantation embryos. Hum Reprod 1990;5: Leese HJ, Lenton EA. Glucose and lactate in human follicular fluid: concentrations and interrelationships. Hum Reprod 1990;5: Huisman GJ, Lo-A-Njoe NM, Alberda AT, Leerentveld RA, Verhoeff A, Zeilmaker GH. Comparison of results obtained with human serum and a protein solution as a supplement for in vitro fertilization culture medium. Fertil Steril 1992; 58: Adler A, McVicker Reing A, Bedford JM, Alikani M, Cohen J. Plasmanate as a medium supplement for in vitro fertilization. J Assist Reprod Genet 1993; 10: Kane MT, Bavister BD. Protein-free culture medium containing Polyvinylalcohol, vitamins and amino acids supports development of eight-cell hamster embryos to hatching blastocysts. J Exp Zool 1988;247: Lopata A, Hay DL. The surplus human embryo: its potential for growth, blastulation, hatching and human chorionic gonadotropin production in culture. Fertil Steril 1989;51: Hargreaves et al. Serum versus albumin in VF i ;