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Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the composition of embryo culture media. This work was funded by The European Society for Human Reproduction and Embryology. No competing interests to declare.

uman organs and tissues differ in regard to their clinical and research uses and the regulatory legislation controlling their use. Organs such as livers and hearts are usually taken from donors who are brain‐dead; in addition, kidneys can also come from live volunteers. Organs decay rapidly and need to be transplanted quickly. Surgeons and coordination teams in transplantation centres control the procurement, while dedicated national and international organisations facilitate their allocation. Human tissues such as bone, skin and heart valves are usually removed from cadavers in hospitals, morgues or even funeral homes and, unlike organs, can be stored—sometimes for years—in tissue banks. These tissues can be used in numerous recipients as and when they are needed. In the early days of human tissue banking, not‐for‐profit banks, mostly located in hospitals, dominated the field. The tissues they stored—heart valves and skin, for example—saved many lives.

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This study is a long-term evaluation of the total pregnancy potential of cohorts of fresh and cryopreserved sibling embryos from in-vitro fertilization (IVF) cycles stimulated with either the gonadotrophin-releasing hormone analogue buserelin (BUS) (long protocol) or clomiphene citrate (CC) both in combination with human menopausal gonadotrophin (HMG). Therefore a retrospective analysis was performed on patients who entered the IVF programme between January 1986 and July 1987 and who had triple embryo transfer in the collection cycle. Significantly more fertilized oocytes developed to good-quality embryos in the CC-HMG group (86.1%) than in the BUS-HMG group (80.8%). Transfer of the three morphologically best-looking embryos was performed in day 2 post-insemination in 106 CC-HMG and 80 BUS-HMG cycles. Supernumerary embryos were cultured for a further 24 h and multicellular embryos with up to 20% of fragments were frozen slowly with 1.5 M dimethylsulphoxide on day 3 post-insemination (162 embryos in CC-HMG cycles, 102 embryos in BUS- HMG cycles). Outcome was measured by embryo survival rate, embryo implantation rate and delivery rate in fresh and frozen embryo transfers. Delivery rates were 31.3 and 21.7% per fresh embryo transfer in BUS-HMG and CC- HMG cycles respectively. Fresh embryo implantation rates were significantly higher in collection cycles stimulated with BUS-HMG (17.9%) than in cycles stimulated with CC-HMG (11.3%). Implantation rates were significantly enhanced in embryos transferred in excess of one in cycles leading to pregnancy, perhaps indicative of higher embryo quality in BUS-HMG cycles. Almost all cryopreserved embryos have by now been thawed, so the contribution of frozen embryos to overall pregnancy rates can be evaluated. Overall morphological survival rates of frozen-thawed embryos have by now been thawed, so the contribution of frozen embryos to overall pregnancy rates can be evaluated Overall morphological survival rates of frozen-thawed embryos were similar for 140 embryos from CC-HMG cycles (50%) and 100 embryos from BUS-HMG cycles (46%). The percentage of fully intact embryos was, however, significantly lower in the BUS-HMG group (19%) than in the CC-HMG group (39.5%). Delivery rates were significantly lower following 30 transfers of frozen-thawed embryos from BUS-HMG-stimulated cycles (3.3%) than following 42 transfers of frozen-thawed embryos from CC-HMG cycles (19.1%). Embryo implantation rates were lower for frozen-thawed embryos from BUS-HMG cycles (2.3%) than from CC-HMG cycles (12.7%). Here we demonstrate that ovarian stimulation with the long protocol BUS-HMG instead of the CC-HMG protocol led to higher embryo implantation rates in collection cycles but to lower intact embryo survival rates and to lower embryo implantation rates for frozen sibling embryos. Despite the lower implantation rates with frozen embryos originating from the BUS-HMG protocol, there was no significant difference between total delivery rate per transfer from cycles stimulated with CC-HMG (30.2%) compared with BUS-HMG (33.8%).

Mouse 1-cell embryos were frozen ultrarapidly at a rate of approximately 2500 degree C/min in solutions containing 0.25 M sucrose, 0.5% (w/v) bovine serum albumin (BSA) and 3 or 4.5 M dimethyl sulphoxide (DMSO) or 3 or 4.5 M 1,2-propanediol (PROH) in HEPES-buffered modified Earle's medium. We investigated the effect of pre-freeze equilibration for 1, 3, 5 or 10 min at 22 degrees C and for 1, 3, 5, 10, 15 or 20 min at 4 degrees C. After thawing in a 22 degrees C water bath at a rate of approximately 2500 degrees C/min and dilution in 1 M sucrose in HEPES-buffered modified Earle's medium, embryos were cultured in vitro in bicarbonate-buffered modified Earle's medium with 0.5% (w/v) crystalline BSA. Embryo viability was expressed as the percentage of hatching or hatched blastocysts resulting from the initial number of frozen-thawed 1-cell embryos. To determine the toxicity of the freezing solutions, embryo viability was evaluated after equilibration without freezing. Our results demonstrated that the concentration, the equilibration temperature and time are very important factors in ultrarapid freezing of mouse 1-cell embryos. Optimal viability was found when equilibration was done in 4.5 M DMSO for 3-5 min at 22 degrees C and in 4.5 M PROH for 3-5 min at 4 degrees C. The results with regard to exposure to the freezing solutions indicated that the loss of viability beyond an optimum is not due solely to cryoprotectant toxicity, in particular not at 4 degrees C and not for DMSO.

This study was designed to assess the effect of pentoxifylline (PTX) on fertilization and early embryonic development in the mouse. Oocytes from superovulated B6CBA female mice were inseminated in vitro with spermatozoa from B6CBA males incubated with PTX according to different protocols, i.e. (i) 3.6 and 7.2 mM PTX washed out prior to insemination, (ii) 3.6 and 7.2 mM PTX diluted six times in the insemination medium and (iii) PTX present at 3.6 and 7.2 mM in the insemination medium. After insemination and washing, fertilization was assessed by the presence of 2-cell stage embryos. These were further cultured up to the blastocyst or egg-cylinder stage to assess embryonic development. Parthenogenetic activation was evaluated by exposing post-ovulatory oocytes to 3.6 and 7.2 mM PTX. If spermatozoa were washed free from PTX before insemination, no effect on either fertilization or subsequent development was found. If PTX was not washed out, fertilization was reduced significantly, yet development of fertilized oocytes was unaffected. If insemination was performed in the presence of PTX both fertilization and development were impaired. Parthenogenetic activation was not increased by PTX exposure. We conclude that if used in in-vitro fertilization, exposure of oocytes and/or zygotes to PTX has to be avoided by washing out the compound thoroughly to prevent adverse effects on early embryonic development.

In order to optimize the in-vitro fertilization (IVF) procedure and to avoid the batch-to-batch fluctuations of in-house culture medium, we investigated the use of a commercially available medium. In our human IVF programme, a controlled comparison was performed from July 1991 to February 1992 between in-house Earle's medium and commercially available Ménézo B2 medium. The major difference between the two tested media is the additional presence of amino acids and water-soluble vitamins in Ménézo B2 and the different origins of the proteins source, i.e. bovine serum albumin (BSA) in Ménézo B2 and human serum albumin (HSA) in Earle's medium. In the first, auto-controlled study, sibling oocytes cultured in Ménézo B2 showed a significantly higher fertilization rate than those cultured in Earle's medium: 58.1 +/- 37.2% and 52.1 +/- 36.9% respectively. After further culture of the fertilized oocytes, there was no difference in the morphological characteristics of the embryos cultured in the different media, although the rate of development was different. In Ménézo B2 medium, a higher proportion of the embryos had reached at least the 4-cell stage at transfer. In the second, randomized study, effects on the pregnancy and implantation rates of both media were analysed. In the group of oocytes cultured in B2, more excellent or good-quality embryos were available for transfer at a more advanced stage of development. No significant difference in pregnancy rate was observed: 39.1% per transfer in Earle's medium versus 40.5% per transfer in Ménézo B2.

Attempts to increase the probability of a successful pregnancy in in-vitro fertilization (IVF) treatment by increasing the number of embryos transferred automatically also increase the probability of multiple pregnancies and their attendant risks. Even where the number of transferred embryos is limited to a maximum of three as in this and other centres, there is a high incidence of twins and triplets. The question therefore arises whether the number of transferred embryos should be further limited to a maximum of two in cases where the prognosis is otherwise good. The only objection to this idea is a possible lowering of pregnancy rate. The present study set out to investigate this question. No significant lowering of pregnancy rate was found, so that limiting the number of transferred embryos to two where the prognosis is otherwise good has now become standard practice in our centre. A good IVF prognosis was defined by the following criteria: first attempt for IVF, less than 37 years old, and good embryo development. From 183 patients fulfilling these criteria, 80 agreed to the transfer of two embryos (group 1) and 103 opted for a triple transfer (group 2). Patient characteristics and embryology results were similar in the two groups. In group 1, 34 patients (42.5%) became pregnant and in group 2, 50 (48.5%). This difference is not significant. Similarly, twin pregnancy rates in both groups were high; eight twin pregnancies (23.5%) in group 1 and 12 (24%) in group 2.

Previous work from our laboratory has revealed that brief exposure of mouse zygotes to 3.6 and 7.2 mM pentoxifylline reduced cell numbers in mouse embryos reaching the blastocyst stage in vitro. Since a decrease in cell numbers may impair further development, the present study aimed to assess the implantation in vitro and the development beyond implantation of these embryos. Blastocysts cultured from the one-cell stage in the presence of 5, 10 and 50 microM pentoxifylline, i.e. concentrations expected in the secretions of the genital tract after oral administration, were shown to develop normally to the egg cylinder stage in vitro. Blastocysts, obtained after culture in vitro of zygotes exposed approximately 29 h after human chorionic gonadotrophin (HCG) for 30 min to 3.6 and 7.2 mM pentoxifylline, i.e. the concentrations used for enhancing sperm function in vitro, could implant but their development to the egg cylinder stage was found to be impaired. The same effect was noticed if blastocysts, grown in vitrofrom the one-cell stage, were exposed to 3.6 and 7.2 mM pentoxifylline for 30 min in vitro approximately 120 h after HCG. These findings imply that when pentoxifylline is used to enhance sperm function in vitro, it should be washed out of the preparation used for insemination.

We investigated the in-vitro and in-vivo development of cryopreserved and non-cryopreserved biopsied 8-cell mouse embryos, from which one to seven blastomeres were removed by micromanipulation. The results clearly indicate that the in-vitro and in-vivo development of biopsied 8-cell mouse embryos depended on the number of blastomeres removed: the more blastomeres removed, the greater the effect. When five, six, or seven blastomeres were removed, fewer blastocysts were formed. Furthermore, when these blastocysts were transferred to pseudopregnant females, no living young were formed, indicating the abnormality of these blastocysts. When up to three blastomeres were removed, there was no significant effect on the rate of in-vitro blastocyst formation. Living young were found even after the biopsy of four blastomeres, and after biopsy of only one or two blastomeres, the same percentage of living young was obtained as in the non-biopsied control embryos. Biopsied 8-cell mouse embryos were frozen and thawed in straws with 1,2-propanediol (1.5 M) and sucrose (0.1 M) with slow-freezing and rapid-thawing protocols. The survival after cryopreservation, defined as the percentage of embryos with the same number of blastomeres intact as the number of blastomeres before freezing, was excellent and no different from non-biopsied embryos, independent of the number of blastomeres biopsied. Furthermore, cryopreservation had no further impact on the in-vitro and in-vivo development of the biopsied embryos.

Previous work from our laboratory has revealed that brief exposure of mouse zygotes to 3.6 and 7.2 mM pentoxifylline reduced cell numbers in mouse embryos reaching the blastocyst stage in vitro. Since a decrease in cell numbers may impair further development, the present study aimed to assess the implantation in vitro and the development beyond implantation of these embryos. Blastocysts cultured from the one-cell stage in the presence of 5, 10 and 50 microM pentoxifylline, i.e. concentrations expected in the secretions of the genital tract after oral administration, were shown to develop normally to the egg cylinder stage in vitro. Blastocysts, obtained after culture in vitro of zygotes exposed approximately 29 h after human chorionic gonadotrophin (HCG) for 30 min to 3.6 and 7.2 mM pentoxifylline, i.e. the concentrations used for enhancing sperm function in vitro, could implant but their development to the egg cylinder stage was found to be impaired. The same effect was noticed if blastocysts, grown in vitrofrom the one-cell stage, were exposed to 3.6 and 7.2 mM pentoxifylline for 30 min in vitro approximately 120 h after HCG. These findings imply that when pentoxifylline is used to enhance sperm function in vitro, it should be washed out of the preparation used for insemination.

The effects of cryopreservation with 1,2-propanediol on two groups of 1-cell mouse embryos were studied in terms of survival after thawing, growth in vitro until the blastocyst stage and development in vivo assessed by the number of implantations and living fetuses. The two groups were divided according to different stages in the cell cycle: cells in (i) interphase with two distinct pronuclei or (ii) mitosis just prior to the first cleavage division. Zygotes in the interphase stage proved to be more resistant to freezing and thawing procedures, showing a significantly higher survival rate after thawing than zygotes in mitosis (78.5 versus 61.3%, P

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.

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