The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization

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1 FERTILITY AND STERILITY Copyright 1993 The American Fertility Society Vol. 59, No. I, January 1993 Printed on acid-free paper in U.S.A. The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization Judith A. Fleetham, B.Sc. H. Anthony Pattinson, M.B., Ch.B. * David Mortimer, Ph.D.t Regional Fertility Programme, Department of Obstetrics and Gynecology, Foothills Hospital, Calgary, Alberta, Canada Objective: To determine whether the mouse embryo culture system can be sensitized to provide improved differentiation of suboptimal culture media for in vitro fertilization. Design: Mouse embryo development in media prepared from one of three water sources were compared using embryos from two mouse strains, culturing embryos from either zygote or two-cell stage, and pretreating with either zona removal and/or cryopreservation. Setting: Academic research department, tertiary care referral center. Results: Embryos from CD 1 mice were able to develop in suboptimal culture conditions, even when pretreated with zona removal or cryopreservation. Embryos from BeCBA/F IJ mice were more sensitive to suboptimal culture conditions when harvested at the zygote stage than at the two-cell stage, and this sensitivity was improved after zona removal before culture. Conclusions: The mouse embryo culture system has deficiencies as an assay of culture medium quality, but the sensitivity of the assay can be optimized by harvesting at the zygote stage from an appropriate strain and by zona pellucida removal before culture. Fertil Steril1993;59:192-6 Key Words: Mouse embryo culture, in vitro fertilization culture medium, water quality The culture of mouse two-cell embryos to the blastocyst stage has often been recommended as a standard bioassay for assessing culture medium quality (1-3). However, it has frequently been suggested that zona pellucida (ZP)-intact two-cell mouse embryos may not be sensitive to suboptimal culture conditions that may affect the outcome of human in vitro fertilization and embryo transfer (lvf-et). It has been shown that two- or four-cell mouse embryos will develop to the blastocyst stage in medium made with tap water, even in the absence of protein (4,5). Previous attempts to sensitize the assay involved culturing the embryos from the zygote Received June 15, 1992; revised and accepted September 15, * Reprint requests: H. Anthony Pattinson, F.R.C.S.(C.), Regional Fertility Programme, 162, 29 Street NW, Calgary, Alberta, Canada T2N 4L 7. t Present address: Sydney In Vitro Fertilization Pty Ltd, Sydney, New South Wales, Australia. stage (6) or removing the ZP at the two-cell stage before culture (7). Mouse IVF-ET may be a better test of the culture conditions but is not practical as a routine quality control assay. Our objectives in this study were to compare the two methods of improving the sensitivity of the assay, to introduce a possible third method, cryopreservation, and to compare the sensitivities of embryos from two strains of mice. The test media were prepared using water of three different purities; tissue culture grade, reverse osmosis (RO) purified, and municipal tap water. The aim was to determine whether a sensitized assay could detect presumed suboptimal culture conditions, i.e., the difference in quality at least between media prepared from tissue culture grade water and tap water. Water Sources MATERIALS AND METHODS Modified Whittingham's T6 culture medium (8) supplemented with 3 mgjml bovine serum albumin 192 Fleetham et al. IVF culture quality control

2 (BSA) was prepared with water from the three sources. The first culture medium was prepared using chlorinated municipal water taken fresh from the laboratory faucet. Tap water was passed through an activated carbon cylinder (P/N 36 CRF; Biolabs, Oakville, Ontario, Canada) and then an RO system (Milli-RO 2 system; Millipore, Mississauga, Ontario, Canada) comprising a carbon prefilter and a polyamide RO membrane. Fresh RO purified water was used to prepare RO medium. Milli-Q purified water (MQ) was prepared as previously reported (9) by passing RO water through the Milli-Q five-cartridge system (Millipore) and a final.22-m filter, and this was used to prepare MQ medium. Endotoxin levels were assayed using the Pyrotell Limulus Amebocyte Lysate kit (Associates of Cape Cod, Inc., Woods Hole, MA) (1). The resistivity of the MQ water was monitored using the meter on the Milli-Q system. Conductivity of MQ and RO water was measured using a Pure H 2 Test, range.1/99.9 mho, (Cole Parmer, Chicago, IL). The conductivity of tap water was measured using the DIST 3 total dissolved solids tester, range 1/1,99 S. The osmolarity and ph of each test medium were measured. Media were filter-sterilized using a.22-m Millex-GV filter (Millipore) and equilibrated with 5% CO 2 before each experiment. Mouse Superovulation and Embryo Collection Embryo development in test media was compared between two strains of mice: CDl outbred (Charles River Canada Inc., Quebec, Canada) and B 6CBA/ FlJ (57BL/J female X CBA/J male; Jackson Laboratories, Bar Harbor, ME). Embryos from outbred strains are known to exhibit a two-cell block in vitro and were therefore not used in culture from the zygote stage. Five-week-old females were injected intraperitoneally with 5 IV of human menopausal gonadotropin (Gestyl; Organon, Oss, Holland) followed 48 hours later with 5 IV human chorionic gonadotropin (hcg, Sigma Chemical Co, St. Louis, MO). Zygote stage embryos within the cumulus masses were excised from the oviducts of B 6CBA/FlJ females approximately 2 hours after hcg. The cumulus was digested with.1 % hyaluronidase (Sigma Chemical Co, H-3884, 1, units/mg solid) in Hepes-buffered T 6 medium with 3 mg/ml BSA. Zygotes were pooled, rinsed twice, and divided equally between the test media in 35 X 1-mm Falcon culture dishes (Falcon Plastics, Oxnard, CA). Two-cell stage embryos were flushed from the oviducts of CDl and B 6CBA/F lj females approxi- mately 42 hours after hcg. They were collected, pooled, rinsed, and divided equally between the test media in 5-mL Falcon culture tubes (Falcon Plastics). Embryo Treatment Untreated The first group of embryos were used fresh with the ZP intact (fresh ZP+). Two-cell embryos from both strains were cultured to blastocyst stage in 1 ml of test medium in 5-mL Falcon culture tubes (15 to 2 embryos per tube) at 37 C under 5% CO 2, 5% O2,9% N2 (5/5/9). Zygote stage embryos were cultured in test media in 35 X 1-mm Falcon culture dishes until the twocell stage when the undivided embryos were discarded. The baseline number of embryos was then taken as the number of two-cell embryos that developed from zygote stage in test medium. The twocell embryos were then placed in 1 ml of the corresponding test medium in 5-mL Falcon tubes for the duration of the culture period. Zona Pellucida Removal Two-cell embryos, either harvested at two-cell or cultured in vitro from zygote stage, were placed in 1 ml of Hepes-buffered T 6 medium containing.5 % Protease P-5147 (Pronase; Sigma Chemical Co) and 3 mg/ml BSA at room temperature. This solution had been predigested for 3 minutes at room temperature, filter sterilized, and stored in aliquots at -2 C. The embryos were observed until the zonae were no longer visible. These embryos were designated fresh ZP-. The embryos were then rinsed twice in medium and cultured one per well in Lux 526, 6-well HL-A plates (Miles/Canlab, Mississauga, Canada) containing 8 ml of test medium at 37 C under 5/5/9. Parallel fresh ZP+ experiments were carried out, thereby providing controls for the fresh ZP- experiments. Fresh ZP+ embryos were not cultured in HL-A plates because they do not adhere to plastic and can be easily recovered from culture tubes, which is not possible with ZP- embryos. Also, ZP- embryos were cultured individually to prevent their adhering to each other and fusing to form giant blastocycts. Cryopreservation Embryos, both cultured from two-cell and zygote stages, were cryopreserved at the two-cell stage using the ultrarapid freezing method developed by Vol. 59, No.1, January 1993 Fleetham et al. IVF culture quality control 193

3 Trounson and colleagues (11). Embryos were placed in Dulbecco's phosphate-buffered medium (PBl) containing 3.5 M dimethyl sulfoxide and.25 M sucrose for 2 to 2.5 minutes, during which time they were loaded into.25-ml straws, up to 5 per straw. After sealing, the straws were plunged directly into liquid nitrogen and stored for 1 day to 4 weeks. Rapid thawing was performed in a 37 C water bath. The embryos were expelled into PBl containing.25 M sucrose. After 1 minutes they were rinsed twice and cultured in 1 ml of test medium in 5-mL Falcon tubes. These embryos were designated cryo ZP+. Parallel experiments with fresh ZP+ embryos from the same embryo pools were carried out in the same batches of test media. Cryopreservation and ZP Removal After thawing and equilibrating with culture medium, embryos were placed in Hepes-buffered Ts medium containing.5% Pronase until the zonae were no longer visible. Embryos were rinsed and cultured singly in Lux 6-well HL-A plates. These embryos were designated cryo ZP-. Parallel experiments were carried out with cryo ZP+ embryos. The embryo pools were previously assessed for viability by culturing fresh ZP+ embryos in the test media. Embryo Assessment In all cases, embryo development was evaluated after 72 hours in culture. Less than 75% development to blastocyst was considered to indicate an impairment of embryo growth. Experiments were performed in triplicate, except in the case of the CDl experiments in which blastocyst development was obviously not affected by any ofthe treatments. In all other experimental groups, a minimum of 75 embryos were assessed. RESULTS There were significant differences (P <.1) between the total dissolved solids content and endotoxin levels in tap water compared with MQ and RO (Table 1). The differences between MQ and RO were not significant. The osmolarity and ph measurements were not significantly different between the three test media. In all experiments, :2:.75% ofthe embryos cultured in MQ medium developed to blastocyst stage. There were no differences in the numbers of zygotes that developed to the two-cell stage in the three media. Table 1 Comparison of Water Quality Between RO Plus Milli-Q Purification, RO Only, and Tap Water Obtained Directly From the Municipal Supply* Osmolarity Conductivity Endotoxins ofts ph ofts,.mho/em ng/ml MQ < RO 1 to 2.16 to Tap 23 to 35 > * Values are means for the duration of the experiments. The effects of embryo treatments and culture in each test medium on the development of two-cell CDl, two-cell BsCBA/FlJ, and zygote stage BsCBA/ F lj embryos are represented graphically in Figure 1. Two-cell CDl embryos did not exhibit impairment of blastocyst development whether they were untreated, or zonae-free, cryopreserved or both, and cultured in any of the test media (Fig. la). Intact fresh or cryo two-cell BsCBA/FlJ embryos demonstrated no significant decrease in blastocyst development in tap medium compared with MQ and RO media (Fig. IB). A significant decrease in development was observed with both fresh and cryo two-cell embryos cultured in tap medium when the zonae were removed; 48% (46/95) development to blastocyst in tap medium versus 75% (71/95) and 96% (68/71) in MQ and RO, respectively, for fresh ZP- embryos (P <.1); 44% (33/75) development in tap medium versus 79% (59/75) and 78% (59/76) in MQ and RO for cryo ZP- embryos (P <.1). Untreated BsCBA/FlJ embryos harvested at the zygote stage and cultured in test medium to blastocyst stage demonstrated impaired development in tap medium (57% [82/143]) compared with MQ (84% [132/158]) and RO (87% [135/156], P = <.2, Fig. lc). When the zonae were removed at the twocell stage after culture in test media, development to blastocyst stage was further impaired in tap medium (6% [8/137]) versus 76% (18/142) in MQ and 76% (16/14) in RO (P <.1). Cryopreserved BsCBA/F lj embryos harvested at the zygote stage from which the zonae were removed showed impaired development even in MQ media. DISCUSSION The problems involved in the development of a sensitive quality control assay for the human IVF ET culture system must be overcome both for the purposes of the standardization of culture conditions and the assurance that all IVF laboratories perform 194 Fleetham et al. IVF culture quality control

4 ,, I, 1.. Q..!l " > " " 1 g-...' g- ] '3.!l " > 6 c:: A. 2-ce 11 CO, embryos - HQ RO TAP Cul ture Medium B. 2-ce11 B 6CBA/F,J embryos HQ RO TAP Culture Medium C. B 6CBA/F, J zygotes. G -G..... '\\\ -:... MQ RO TAP Culture Medium fresh. yos _zp+.. ZPcryo eoiryos -- ZP+ --ozpfresh. yos _ZP+ --"ZP-._-ozpfresh. yos _ZP+ --"ZPcryo eoiryos --OZP+ _-ozp-. P<O.2.. P<O.OOI Figure 1 Blastocyst development of (A) two-cell CDl embryos, (B) two-cell B 6CBA/FlJ embryos, and (C) B 6CBA/FlJ zygotes, cultured in MQ, RO, and tap medium; untreated two-cell embryos (fresh ZP+); after ZP removal from fresh two-cell embryos (fresh ZP-); cryopreserved at two-cell stage, thawed, zonae intact (cryo ZP+); cryopreserved at two-cell stage, zonae removal post-thaw (cryo ZP-). the procedure to the highest known standards. The standard mouse two-cell embryo development to blastocyst culture system quality control assay has some basic problems. Because it bypasses the IVF step, its use is limited to testing for the embryotoxicity of materials and reagents used in human IVF ET. Embryos exposed to toxic substances may still develop to blastocyst because toxic exposure at early stages may not be manifested until later stages of embryonic development, i.e., implantation and beyond. Absence of embryotoxicity using this assay is not particularly meaningful because of a lack of sensitivity to presumed suboptimal culture conditions: in this study, > 75% of intact two-cell mouse embryos from either outbred or F 1 hybrid strains develop to the blastocyst stage in culture medium made with tap water that contains appreciable amounts ofbacterial endotoxin. Human embryos have been shown to be sensitive to as little as 1 ngjml endotoxin in the culture system (12). The present study again demonstrates the unsuitability of CDl embryos for assessing the quality of IVF culture media. This study has shown that the sensitivity of the mouse embryo quality control assay can be increased by using embryos from BsCBAjF1J hybrid mice instead of outbred strains and by culturing those embryos from the zygote stage in test medium. Removal of the ZP from either embryos harvested at the twocell stage or those developed from zygote and continued in culture to blastocyst in test medium also resulted in improved sensitivity, as did cryopreservation and zonae removal from BsCBA/FlJ embryos obtained at the two-cell stage. The combination of cryopreservation and zona removal from BsCBAj F lj zygotes rendered them unable to develop even in MQ media, and this combination would therefore not be suitable as a quality control. The best discrimination of suboptimal culture conditions in this study was obtained using BsCBAj F lj zygotes whose zonae had been removed after development to the two-cell stage in test medium. If a mouse embryo culture system is to be used for IVF quality control, this would seem to be the most reliable method. However, even this sensitized system was unable to identify any difference between media made with MQ or RO water. A hamster sperm motility bioassay has been used as an alternative to the mouse embryo development assay as a quality control for IVF media, and it has been suggested (13) that the sensitivity of this assay can be increased by excluding protein from the culture medium. Eliminating protein from the mouse embryo culture system may have some value in increasing the sensitivity of the assay, but this was not attempted in this study because the embryos become very difficult to handle in the absence of protein. The ultimate validation of any system to assess IVF and embryo culture conditions requires that the test be used routinely so that the sensitivity and specificity can be determined on the basis of IVF success or failure and the occurrence of pregnancies. However, a direct chemical validation of the method by the addition of endotoxin to the culture media could be considered before the clinical use of this method. The modified mouse embryo culture system Vol. 59, No.1, January 1993 Fleetham et al. IVF culture quality control 195

5 as described would seem to address some, if not all, ofthe requirements for a quality control assessment but represents a significant improvement over the traditional mouse embryo system. REFERENCES 1. Ackerman SB, Stokes GL, Swanson RJ, Taylor SP, Fenwick L. Toxicity testing for human in vitro fertilization programs. J In Vitro Fert Embryo Transf 1985;2: Parinaud J, Reme J, Monrozies X, Farrin S, Sarramon M, Pontonnier G. Mouse system quality control is necessary before the use of new material for in vitro fertilization and embryo transfer. J In Vitro Fert Embryo Transf 1987;4: Ackerman SB, Swanson RJ, Stokes GK, Veeck LL. Culture of mouse embryo preimplantation embryos as a quality control assay for human in vitro fertilization. Gamete Res 1984;9: Silverman IH, Cook CL, Sanfilippo JS, Yussman MA, Schultz GS, Hilton FH. Ham's F-1 constituted with tap water supports mouse conceptus development in vitro. J In Vitro Fert Embryo Transf 1987;4: George MA, Braude PR, Johnson MH, Sweetnam DG. Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse. embryos is unaffected by the quality of water used in culture media. Hum Reprod 1989;4: Davidson A, Vermesh M, Lobo RA, Paulson RJ. Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model. Fertil Steril 1988;49: Fleming TP, Pratt HPM, Braude PRo The use of mouse preimplantation embryos for quality control of culture reagents in human in vitro fertilization programs: a cautionary note. Fertil Steril1987;47: Mahadevan MM, Fleetham J, Church RB, Taylor PJ. Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, HEPES or phosphate. J In Vitro Fert Embryo Transf 1986;3: Fleetham J, Mahadevan MM. Purification of water for in vitro fertilization and embryo transfer. J In Vitro Fert Embryo Transf 1988;5: Harada T, Morita T, Iwanaga S. A new assay method for bacterial endotoxins using horseshoe crab hemocyte lysate. J Med Enz 1978;3: Trounson AO, Peura A, Kirby C. Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation. Fertil Steril 1987;48: Fishel S, Jackson P, Webster J, Faratian B. Endotoxins in culture medium for human in vitro fertilization. Fertil Steril 1988;49: Bavister BD, Andrews JC. A rapid sperm motility assay procedure for quality control testing of water and culture media. J In Vitro Fert Embryo Transf 1988;5: Fleetham et al. IVF culture quality control