A prospective comparison of `in house' and commercially prepared Earle's balanced salt solution in human in-vitro fertilization

C. Karamalegos and V.N. Bolton1

Assisted Conception Unit, Department of Obstetrics and Gynaecology, King's College School of Medicine & Dentistry, Denmark Hill, London SE5 8RX, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A prospective, randomized study was undertaken to compare the use of Earle's balanced salt solution (EBSS) prepared `in house' with that produced commercially, in 448 cycles of therapeutic in-vitro fertilization. Outcome was assessed in terms of fertilization and cleavage rates, embryo morphology, and implantation rates following embryo transfer. The only differences that were found between the two media in any of the outcome parameters were in the number of cycles with failed fertilization (1/218 in `in house' medium compared with 10/230 in commercially prepared medium; P = 0.0186), and in the rate at which embryos cleaved. Thus, while the median number of blastomeres per embryo was no different in the two groups at 46–49 h post insemination (three in embryos cultured in `in-house' medium, compared with four in those cultured in commercially prepared medium; P > 0.1), the number of embryos per cycle that had cleaved to the 4-cell stage by 46–49 h post insemination was significantly greater in the Medi-Cult than in the EBSS medium (P < 0.001).

Key words: culture medium/Earle's balanced salt solution/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The culture media used for therapeutic in-vitro fertilization (IVF) may be prepared either `in house' or commercially, when they are supplied ready to use and specified for use in IVF. The disadvantages of preparing media `in house' include batch-to-batch variation, the difficulty of establishing reliable quality control, and the labour-intensive nature of its preparation. Although these disadvantages are offset by the fact that the composition of `in-house' media is defined, they have led to increasingly widespread use of commercially-prepared media. However, while the convenience of commercial preparations is undisputed, they are costly and their composition may not be clearly defined. Moreover, a direct comparison of media from each source, in terms of IVF outcome, has yielded inconclusive results (Staessen et al., 1994Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
All couples undergoing therapeutic IVF at the Assisted Conception Unit of King's College Hospital were included in the study provided the female partner was less than 38 years old, the male partner had semen that was normal according to WHO (1992) criteria (count >=20x106/ml; progressive motility >=40%; normal forms >=30%), and had achieved fertilization of at least one oocyte in any previous cycle of IVF. Pituitary suppression and ovarian stimulation, using luteinizing hormone-releasing hormone analogue, human menopausal gonadotrophin and human chorionic gonadotrophin (HCG), and oocyte retrieval were carried out as described previously (Waterstone and Parsons, 1992Go). Couples were randomized by random sampling, with replacement prior to administration of HCG, to IVF and embryo culture either in `in house' prepared EBSS, or in the commercially prepared nearest equivalent (Medi-Cult).

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., 1976Go), and insemination was performed 40 h post-HCG using 100 000–200 000 motile spermatozoa/ml. Fertilization was assessed at 18–20 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., 1989Go), and those with `poor morphology' had irregular-shaped blastomeres and/or >10% fragmentation (grades 2 and 1, Bolton et al., 1989Go).

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 {chi}2 test with Yates' correction, and the normal approximation for the two-tailed Mann–Whitney 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 448 treatment cycles was included in the study, the oocytes from 218 of which were cultured in EBSS, and from 230 of which were cultured in Medi-Cult; the characteristics of these cycles and the outcome of insemination in vitro are summarized in Table IGo. The two groups did not differ significantly either in terms of the age of the female partner, or in the number of oocytes collected per cycle.


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Table I. Characteristics of the 448 treatment cycles included in the study
 
Culture medium batches
The study took place during a period of 13 months, during which a total of 29 batches of EBSS and 19 batches of Medi-Cult were used. The mean number of treatment cycles per batch was 7.5 (range 2–14) in the EBSS group, and 12.1 (range 4–23) in the Medi-Cult group. There were no significant differences between the batches of each medium in terms of fertilization, cleavage rate, embryo morphology, implantation or pregnancy rates (data not shown).

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 IGo). 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 IIGo. 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 46–49 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 46–49 h of insemination in Medi-Cult than in EBSS (P < 0.001; Table IIGo), although the median number of blastomeres per embryo was not different.


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Table II. Development of human embryos following IVF and formation of two pronuclei during culture in EBSS or Medi-Cult
 
Embryo transfers and pregnancies
A total of 419 embryo transfers was performed, 205 of which were in the EBSS group, and 214 of which were in the Medi-Cult group, the details of which are shown in Table IIIGo. Twelve cycles were abandoned before embryo transfer in the EBSS group, compared with six cycles in the Medi-Cult group, because the embryos which developed were not suitable for transfer. The reasons for this included: failure to undergo the first cleavage division (arrested at pronuclear stage), and inability to identify blastomeres because there were too many extracellular fragments, caused by disintegration. This difference was not significant. When cycles with no fertilization were combined with those in which no embryo transfer was performed despite fertilization, a total of 13/218 cycles failed to reach embryo transfer in the EBSS group, compared with 16/230 in the Medi-Cult group. This difference was not significant ({chi}2 test). There were no differences between the two groups in terms of the number of cycles with one, two or three embryos transferred, the number of embryos available per cycle with good morphology (data not shown), the number of cycles in which three or more embryos with good morphology developed, or the number of cycles in which four or more embryos with good morphology developed. There was no difference in the incidence of positive HCG (P = 0.25; {chi}2 test; power = 0.86), pregnancy confirmed by ultrasound (P = 0.14; {chi}2 test; power = 0.78), or clinical pregnancy (P = 0.15; {chi}2 test; power = 0.79) between the two groups.


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Table III. Outcome of IVF cycles following oocyte and embryo culture in EBSS or Medi-Cult
 
Implantation
The implantation rates of embryos from the two groups are shown for all patients in Table IVGo, and for patients who had a positive urinary HCG 14 days post embryo transfer in Table VGo. There were no significant differences between the EBSS and Medi-Cult groups, either among all patients or among only those with positive HCG, in terms of the number of embryos that formed a gestation sac, a fetal heart, or a live infant.


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Table IV. Implantation of embryos after culture in EBSS or Medi-Cult and embryo transfer
 

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Table V. Implantation of embryos after culture in EBSS or Medi-Cult and embryo transfer in patients who conceived
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of commercially prepared media is increasingly widespread in all aspects of assisted conception, with a number of products now available for procedures ranging from egg collection, embryo culture and semen separation, to semen and embryo cryopreservation. Although the increased use of commercial products has largely been due to practical and economic considerations, since the preparation of a number of different media `in-house' can be very labour-intensive and requires expensive equipment, there is also a powerful argument in favour of better quality control with commercial mass production than is possible in small institutions. Auto-controlled studies have been undertaken, comparing first, `in-house' and commercially prepared media (Staessen et al., 1994Go), and second, three different commercially-prepared media (Staessen et al., 1998Go). These have shown no differences between the media tested, in terms of fertilization rates and implantation rates, or in embryo morphology. However, these studies are limited by the fact that while each medium was based on Earle's balanced salt solution, they all differed considerably, yet in undefined ways, in composition, for example in terms of the presence or absence of supplementary metabolites, DNA-RNA precursors, vitamins and amino acids.

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., 1991Go), 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; {chi}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, 1994Go). 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; {chi}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; {chi}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., 1989Go; Giorgetti et al., 1995Go). 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 IVGo).

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.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bolton, V.N., Hawes, S.M., Taylor, C.T. and Parsons, J.H. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates and development to the blastocyst. J. In vitro Fert. Embryo Transf., 6, 30–35.[ISI][Medline]

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, 2427–2431.[Abstract]

Lopata, A., Patullo, M.J., Chang, A. and James, B. (1976) A method for collecting motile spermatozoa from human semen. Fertil. Steril., 27, 677–684.[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, 285–289.[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, 1915–1919.[Abstract]

Staessen, C., Janssenswillen, C., De Clerck, E. and Van Steirteghem, A. (1998) Controlled comparison of commercial media for human in-vitro fertilization: Menezo B2 medium versus Medi-Cult universal and BM1 medium. Hum. Reprod., 13, 2548–2554.[Abstract]

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. 283–326.

Waterstone, J.J. and Parsons, J.H. (1992) A prospective study to investigate the value of flushing follicles during transvaginal ultrasound-directed follicle aspiration. Fertil. Steril., 57, 221–223.[ISI][Medline]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.

Submitted on September 28, 1998; accepted on April 8, 1999.





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