Assisted Conception Unit, Department of Obstetrics and Gynaecology, King's College School of Medicine & Dentistry, Denmark Hill, London SE5 8RX, UK
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Abstract |
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Key words: culture medium/Earle's balanced salt solution/IVF
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Introduction |
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We report the results of a prospective, randomized study comparing two culture media based on Earle's balanced salt solution (EBSS), one prepared `in house' (EBSS) and the other commercially (IVF Medium; Medi-Cult, Redhill, Surrey, UK), used for therapeutic IVF. These media differ from each other only in terms of the concentration of sodium pyruvate, the antibiotic content, and the nature of the protein supplement used. The outcomes of treatment cycles were compared in terms of fertilization and cleavage rates, embryo morphology, and implantation rates following embryo transfer.
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Materials and methods |
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Preparation of EBSS
EBSS was prepared by the dilution of concentrated stock Earle's balanced salt solution (x10; GibcoBRL, Life Technologies, Inchinnan, UK) with sterile water for injection (Fresenius, Basingstoke, UK) and the addition of 1.0% (w/v) sodium bicarbonate (AnalaR®; BDH, Poole, UK), 0.11 mg/ml sodium pyruvate (Sigma, Poole, UK), 0.02 mg/ml gentamicin (ICN Flow, High Wycombe, UK) and 0.06 mg/ml penicillin (Sigma). The osmolarity was adjusted to 284 ± 1 mOsmol, and the medium was supplemented with 10% (v/v) Albuminar (5% human albumin solution; Immuno, Sevenoaks, UK). The EBSS was filtered through a 0.01 µm filter (Acrocap®; Gelman Sciences Ltd, Northampton, UK) and stored at 4°C for up to 2 weeks.
The composition of `in-house' EBSS differed from that of Medi-Cult only in terms of the supplementary antibiotics used (0.06 mg/ml penicillin and 0.05 mg/ml streptomycin in Medi-Cult), the concentration of sodium pyruvate (0.1 mg/ml in Medi-Cult), and the protein supplement (1% human serum albumin and 0.001% synthetic serum replacement, the composition of which is not disclosed, in Medi-Cult)
Oocyte culture and insemination
Oocytes were placed individually into 1ml drops of the appropriate, pre-equilibrated culture medium overlaid with light paraffin oil (Sigma) and incubated at 37°C in an atmosphere of 5% CO2 in air. Semen samples were prepared in the same medium using the `swim-up' technique (Lopata et al., 1976), and insemination was performed 40 h post-HCG using 100 000200 000 motile spermatozoa/ml. Fertilization was assessed at 1820 h post-insemination (hpi) and oocytes showing two pronuclei (2PN) were transferred individually to fresh, pre-equilibrated 20 µl drops of the appropriate medium overlaid with light paraffin oil for further culture.
Embryo morphology
Embryos were scored between 45 and 48 hpi for cleavage stage and morphology, according to the symmetry of the blastomeres and degree of extracellular fragmentation. Embryos with `good morphology' had regular, spherical blastomeres and <10% fragmentation (grades 4 and 3, Bolton et al., 1989), and those with `poor morphology' had irregular-shaped blastomeres and/or >10% fragmentation (grades 2 and 1, Bolton et al., 1989
).
IVF outcome
Embryo transfer was performed between 46 and 49 hpi with a maximum of three embryos. In cases where three or more embryos with good morphology were available, couples either elected to have only two embryos transferred, or, as part of a separate study, were randomized to have either two or three embryos transferred. A urine pregnancy test was performed 14 days later using a commercial kit (Clearview®; Unipath, Bedford, UK), an ultrasound scan was performed 3 weeks following embryo transfer to establish the number of intrauterine gestation sacs, and clinical pregnancy was confirmed 2 weeks later by the identification of one or more fetal hearts using ultrasound.
Statistical analysis
Statistical analysis was performed using the 2 test with Yates' correction, and the normal approximation for the two-tailed MannWhitney test. Power analysis was used to ensure that a sufficient number of couples was studied in order to demonstrate a significant difference in the pregnancy rates if one existed. Significance was set at P < 0.05.
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Results |
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Fertilization
There was a significant difference between the EBSS and Medi-Cult groups in the incidence of cycles in which no oocytes were fertilized (failed fertilization; 1/218 and 10/230 respectively), although there was no difference in the overall fertilization rate per cycle (Table I). Among the cycles in which fertilization was achieved, there was no significant difference between the two groups in the number of oocytes fertilized per cycle, or in the fertilization rate per cycle. The number of oocytes per cycle that developed more than two PN, and the number of cycles in which polyploid embryos developed, did not differ between the two groups.
In the case of the single cycle which resulted in failed fertilization in EBSS, a further 13 cycles were undertaken in the same batch of medium. Of the 10 cycles that resulted in failed fertilization in Medi-Cult, four were undertaken using the same batch of medium; however, a further 12 cycles in which the same batch of medium was used resulted in fertilization. The remaining six failed fertilization cycles were distributed among five different batches of Medi-Cult.
Cleavage and morphology
The cleavage characteristics of the oocytes that developed 2PN are shown in Table II. There were no differences between the two groups in terms of the number of embryos that failed to cleave, the number of cycles in which at least one embryo arrested at the pronucleate stage, the number of embryos with good morphology that developed per cycle, the number of cycles in which at least one embryo developed with good morphology, or the number of embryos with good morphology that had cleaved to the 4-cell stage by 4649 h post insemination. However, there was a significant difference between the two groups in terms of the rate at which the embryos underwent cleavage, with significantly more embryos per cycle cleaving to the 4-cell stage within 4649 h of insemination in Medi-Cult than in EBSS (P < 0.001; Table II
), although the median number of blastomeres per embryo was not different.
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Discussion |
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The present study compares fertilization and cleavage rates, embryo morphology and implantation rates in comparable cycles of therapeutic IVF undertaken using either `in-house' prepared EBSS or the nearest commercial equivalent, Medi-Cult. The two media differ in composition only in the supplementary antibiotics, sodium pyruvate concentration and protein used.
The only statistically significant differences identified between the two groups were in the number of cycles with failed fertilization, and in the cleavage rate of embryos that developed 2PN. Thus, more cycles resulted in failed fertilization when oocytes were inseminated in Medi-Cult than in EBSS, despite insemination with apparently normal spermatozoa, and pronucleate stage embryos underwent the first two cleavage divisions more rapidly in Medi-Cult than in EBSS.
Regarding the former, total failure of fertilization in either culture medium is difficult to explain. Although idiopathic failed fertilization in IVF has been well documented, and in the majority of cases fertilization is achieved in a subsequent cycle (Molloy et al., 1991), the significant difference in the incidence of this phenomenon between the two culture media used in the present study must be evaluated. Of the 10 couples whose cycles resulted in failed fertilization in Medi-Cult, six achieved fertilization and embryo transfer in their subsequent treatment cycle; a further three cycles were abandoned because of poor ovarian response, while the final couple did not return for further treatment. When the three couples with failed fertilization and subsequent poor ovarian response are excluded from the analysis, on the assumption that the oocytes produced in the study cycles were of poor quality, the difference between the incidence of failed fertilization in the two culture media becomes insignificant (P = 0.14;
2 test).
However, this interpretation of the findings is only valid if the Medi-Cult group included more cycles than the EBSS group with low numbers of oocytes, that were presumably `poor quality', and had a reduced potential for fertilization, even following insemination with normal spermatozoa (Van Blerkom, 1994). Analysis of the two groups does not support this premise; both groups had 34 cycles with four or fewer oocytes, and this is not significant (P = 0.91;
2 test). Similarly, the number of cycles with only one or two oocytes was 11 and nine in EBSS and Medi-Cult respectively, and again this difference is not significant (P = 0.83;
2 test).
Regarding cleavage rate, it has been suggested that embryos that cleave to the 4-cell stage by 48 hpi have a higher implantation potential than embryos that cleave more slowly (Bolton et al., 1989; Giorgetti et al., 1995
). Thus, in view of the results reported above, it might be expected that the implantation rate following embryo transfer in the Medi-Cult group would be higher than that in the EBSS group. However, this study showed no significant difference between the implantation rate of embryos cultured in Medi-Cult compared with those cultured in EBSS (14.5 and 11.1% respectively; Table IV
).
In conclusion, it would seem that the use of the commercially available preparation of EBSS (Medi-Cult IVF Medium) as an alternative to `in house' prepared EBSS does not result in an alteration in the rate of successful outcome of IVF, as measured in terms of clinical pregnancy rate per cycle. The practical benefits of using an `off the shelf' product almost certainly outweigh any disadvantages that may arise through a possible, but very slight increase in the rate of idiopathic failed fertilization.
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Notes |
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References |
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Giorgetti, C., Terriou, P., Auquier, P. et al. (1995) Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum. Reprod., 10, 24272431.[Abstract]
Lopata, A., Patullo, M.J., Chang, A. and James, B. (1976) A method for collecting motile spermatozoa from human semen. Fertil. Steril., 27, 677684.[ISI][Medline]
Molloy, D., Harrison, K., Breen, T. and Hennessey, J. (1991) The predictive value of idiopathic failure to fertilize on the first in vitro fertilization attempt. Fertil. Steril., 56, 285289.[ISI][Medline]
Staessen, C., Van Den Abbel, E., Janssenswillen, C. et al. (1994) Controlled comparison of Earle's balanced salt solution with Menezo B2 medium for human in-vitro fertilization performance. Hum. Reprod., 9, 19151919.[Abstract]
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Van Blerkom, J. (1994) Developmental failure in human reproduction associated with chromosomal abnormalities and cytoplasmic pathologies in meiotically mature oocytes. In Van Blerkom, J. (ed.), Biological Basis of Early Human Reproductive Failure: Applications to Medically-Assisted Conception and the Treatment of Infertility. Oxford University Press, New York, pp. 283326.
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World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Spermcervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.
Submitted on September 28, 1998; accepted on April 8, 1999.