MALE FACTOR. Fabienne Devreker, M.D.,* Kate Hardy, Ph.D., Marc Van den Bergh, M.T., Joel Winston, Jamila Biramane, M.T., and Yvon Englert, Ph.D.

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1 FERTILITY AND STERILITY VOL. 73, NO. 5, MAY 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MALE FACTOR Noninvasive assessment of glucose and pyruvate uptake by human embryos after intracytoplasmic sperm injection and during the formation of pronuclei Fabienne Devreker, M.D.,* Kate Hardy, Ph.D., Marc Van den Bergh, M.T., Joel Winston, Jamila Biramane, M.T., and Yvon Englert, Ph.D. Faculty of Medicine, Free University of Brussels, Erasme Hospital, Brussels, Belgium; and Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom Received August 9, 1999; revised and accepted November 23, Presented at the 14th annual meeting of the European Society of Human Reproduction and Embriology (ESHRE), Göteborg, Sweden, June 21 24, Supported by the Fondation Erasme, Free University of Brussels, Belgium. Reprint requests: Fabienne Devreker, M.D., Clinic of Fertility, Department of Obstetrics and Gynaecology, Erasme Hospital, Route de Lennik 808, 1070 Brussels, Belgium (FAX: ; * Scientific collaborator to the Belgian National Funds for Scientific Research, Brussels. Laboratory for Biology and Psychology of Human Fertility, the Faculty of Medicine, Free University of Brussels. Clinic of Fertility, Department of Obstetrics and Gynaecology, Erasme Hospital. Department of Reproductive Science and Medicine, Division of Paediatrics, Obstetrics, and Gynecology, Imperial College School of Medicine /00/$20.00 PII S (00) Objective: To improve in vitro culture conditions and human embryo selection before transfer after IVF with intracytoplasmic sperm injection (ICSI). Design: A controlled, randomized, prospective study. Setting: University hospital based IVF-ET program. Patient(s): Couples undergoing ICSI. Intervention(s): Culture of human embryos in the presence of 1 mm or 5.56 mm glucose and metabolic measurements with the use of noninvasive microfluorescence assays immediately after ICSI to the time of transfer. Main Outcome Measure(s): Embryo development, implantation rate, and glucose and pyruvate uptake. Result(s): Fertilization rates, early embryo development, and implantation rates were not significantly different between 1 mm and 5.56 mm glucose. Pyruvate uptake was significantly higher during the formation of the pronuclei, at and pmol/embryo/h for fertilized and unfertilized oocytes, respectively. Pyruvate uptake did not correlate with cleavage stage or embryo morphology. However, during the second day of incubation, pyruvate uptake was significantly higher for the untransferred embryos of pregnant women compared with nonpregnant women, at and pmol/embryo/h, respectively. Conclusion(s): The increased level of pyruvate uptake during fertilization reflects the increased demand for energy necessary for the formation of the pronuclei. However, the metabolic measurements could not improve the selection of embryos with the best implantation potential. Finally, the reduction of glucose concentration in the culture medium failed to improve embryo viability. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Embryo metabolism, low glucose, pyruvate uptake, pronuclei, implantation rate Selection of human embryos for transfer to patients undergoing IVF is routinely based on embryo morphology and cleavage rate. Despite numerous attempts to develop embryo-scoring techniques that can predict embryo viability, implantation rates remain low (1). Not all embryos with good morphology will implant, and fragmented embryos can develop to the blastocyst stage in vitro (2). To achieve acceptable pregnancy rates (PRs), more than one embryo is routinely transferred, with an unavoidable risk of multiple gestations. To improve the selection of embryos with the best developmental potential, micro-methods for assessing the metabolism of embryos have been developed. It has been shown that human preimplantation embryo viability correlates with platelet-activating factor (3) or interleukin-1 production (4). However, these tests are too time-consuming for routine use in a busy clinical program. Microfluorescence assays were developed to measure the uptake or production of energy substrates (5 7). Pyruvate and glucose uptake or lactate production by human embryos has been correlated with embryo viability and the ability to develop to the blastocyst stage (6 8). Pyruvate uptake by human embryos from the one-cell stage to the 947

2 four- to eight-cell stage also has been measured before transfer. However, the wide range of values observed in these two studies decreased the possibility that pyruvate uptake could serve as the sole criterion for the selection of transferred embryos (9, 10). These metabolic studies and the analysis of secretions from the female reproductive tract also demonstrated that embryo culture media supply energy substrates or other components far in excess of what the embryo appears to require. Glucose is toxic for the early preimplantation development of embryos of several species, including mouse (11), hamster (12), rat (13), and human (14). However, in human embryos, some glucose uptake is present at very low levels during the early stages of embryo development, and increases dramatically at the transition between the morula and the blastocyst stage (7, 15). Controversial results have been observed in humans with the use of culture media with or without glucose. Some investigators (16) observed higher PRs in the absence of glucose, whereas others obtained similar PRs with or without glucose. Definite conclusions are difficult to draw because the studies were not randomized (17), included a small number of patients, or compared a combination of culture media (16, 18). No prospective and randomized study has analyzed the early development of sibling embryos allocated between a medium supplemented with high or low levels of glucose during the 2 days of culture in vitro before a transfer. The present study was performed in three steps. First, a randomized comparison of early human embryo development in 5 L or30 L of culture medium was performed to assess the safety of embryo culture in a very small volume of medium. In the second part of the study, sibling embryos from patients undergoing intracytoplasmic sperm injection (ICSI) were randomly allocated to Earle s balanced salt solution (EBSS) supplemented with either 1 mm or 5.56 mm glucose to evaluate the ability of low-glucose medium to support early embryo development. In the third part, 50 patients undergoing ICSI were allocated prospectively and randomly between two groups: embryo culture in the presence of low or high levels of glucose. Pyruvate and glucose uptake by the early embryos was measured during the 2 days of culture between the injection and the transfer to examine whether there is a correlation between substrate uptake before transfer and implantation. MATERIALS AND METHODS Source of Human Embryos Patients undergoing IVF treatment with ICSI were included in the study. Patients undergoing classic IVF were excluded to avoid the presence of cumulus cells and spermatozoa for the metabolic measurements during the first day of incubation. Women were treated with hmg (Humegon; Organon, Oss, the Netherlands) in combination with LH-releasing hormone agonist (buserelin acetate; Hoechst, Hounslow, UK). Ovulation was induced by injection of 10,000 IU of hcg (Profasi; Serono, Geneva, Switzerland) (Pregnyl; Organon), and oocyte retrieval took place hours later. Preincubation and ICSI of oocytes were carried out in EBSS containing 5.56 mm glucose and supplemented with 25 mm sodium bicarbonate (Sigma, Bornem, Belgium), 0.47 mm pyruvic acid (Sigma), and 0.5% human serum albumin (Red Cross, Belgium) under paraffin oil (Sigma) (19). Normal fertilization was confirmed hours after insemination by the presence of two pronuclei (day 1). Activated oocytes had only one visible pronucleus, unfertilized oocytes had no pronuclei, and polyspermic embryos had more than two pronuclei. Oocytes at the germinal vesicle stage were excluded from the study. On the morning of ET, hours after insemination, the embryos were examined under an inverted microscope to determine the evenness and number of blastomeres and the extent of extracellular fragmentation, and a numerical score was calculated on the basis of embryo morphology and cleavage rate (1). Four points were given for an embryo with regular blastomeres and no anucleate fragments, 3 points for an embryo with uneven blastomeres and one or two anucleate fragments, and 2 or 1 points for an embryo with uneven blastomeres and anucleate fragments of 1/3 or 1/3 of the embryonic surface, respectively. Two more points were added if the embryo had reached the four-cell stage. Good embryos were those with a score of 5 or 6 points, fair embryos had a score of 3 or 4, and poor embryos had a score of 1 or 2 points. After ET, luteal support was maintained by daily i.m. injection of 100 mg of oily P or with intravaginal pessaries (three times 200 mg/d of micronized P; Laboratories Piette, Drogenbos, Belgium). Early vaginal ultrasound scans were performed approximately 28 days after oocyte retrieval to assess the number of gestational sacs. Clinical pregnancy was defined either by a gestational sac seen on the ultrasound scan or by villosity present in the miscarriage material. The ratio between the number of ongoing pregnancies and the number of transfers represents the PR. The implantation rate was defined as the total number of fetal sacs divided by the total number of transferred embryos. Embryo Culture and Metabolic Measurements After ICSI, all embryos included in the study were placed individually for sequential 24-hour incubation periods in either 30 L (control) or 5 L of culture medium until the time of transfer. Embryos were washed in three changes of the appropriate culture medium and were cultured under paraffin oil (Sigma) in a gas phase of 5% CO 2,5%O 2, and 90% N 2 (19). For metabolic measurements in the second and third parts 948 Devreker et al. Human embryo metabolism during fertilization Vol. 73, No. 5, May 2000

3 of the study, identical microdroplets of 5 L of medium alone incubated adjacent to the embryo-containing drops served as controls. Individual 2- L aliquots of the spent and control drops of medium from the recently terminated culture were diluted with 398 L of water. The concentrations of pyruvate and glucose in the control and incubation drops were determined by the method described by Devreker et al. (20) on the basis of a technique developed for the noninvasive assessment of substrate uptake by mouse and human embryos (5, 6). All of these assays are based on the fluorescence of the coenzyme NAD(P)H when oxidized. The depletion of substrates by the embryo was calculated by analyzing the difference between substrate concentrations in the control and incubation drops. Embryo morphology, cleavage rate, implantation rate, and PR were compared for each of the culture protocols. Experimental Designs Embryo Development in 5- L Drops This step was undertaken to assess the safety of culturing human embryos in a reduced volume of medium immediately after ICSI to the time of transfer. After completion of the ICSI procedure, 586 sibling zygotes, from 50 patients were randomly allocated to one of two protocols: culture in 30- L drops of medium (standard condition) or culture in 5- L drops of medium. Zygotes were cultured in EBSS supplemented with 5.56 mm glucose, and development was analyzed as described previously. On day 2, a maximum of three embryos were selected for transfer. Embryo Development in the Presence of 1 mm Glucose After the ICSI procedure, 385 sibling zygotes from 41 patients were randomly allocated to one of two protocols: culture in 30- L drops of EBSS with 5.56 mm glucose (standard condition) or culture in 5- L drops of EBSS with 1 mm glucose. Zygotes were cultured and development was analyzed as described previously. On day 2, a maximum of three embryos were selected for transfer. Metabolic Measurements From the Time of Insemination Until the Time of Transfer On the day of oocyte retrieval, 50 patients were randomly allocated to one of two protocols: standard culture conditions or embryo culture in 5 L of EBSS supplemented with 1 mm glucose. Maternal age, duration and causes of infertility, duration of stimulation, number of oocytes collected, and number of transferred embryos were not different between the groups (Table 1). On day 2, a maximum of three embryos were selected for transfer. Embryo development, implantation rates, and delivery rates were analyzed to examine whether the reduction of glucose in the culture medium has a beneficial effect on embryo developmental potential. Pyruvate and glucose uptake by early embryos included TABLE 1 Characteristics of the patients included in the two treatment groups in the third experiment. Characteristic 5.56 mm glucose in the second and the third parts of the study was measured during the 2 days of culture between the injection and the transfer to examine whether there is a correlation between substrate uptake before transfer and implantation. This work was approved by the research ethics committee of the Free University of Brussels, Erasme Hospital. Statistical Analysis Variations of pyruvate uptake between groups were compared using analysis of variance and the Wilcoxon rank-sum (Kruskall-Wallis, Mann-Whitney U) test. Chi-square test or Fisher s exact probability test was used to look for a possible relation between qualitative variables with the use of Yates correction. Analysis was completed using SPSS 7.0 for Windows 95 (Microsoft Inc., Redmond, WA). RESULTS 1mM glucose n Maternal age (y) Infertility duration (y) No. of attempts Duration of stimulation No. of ampules hmg E 2 max 2, , No. of oocytes No. in metaphase II Fertilization rate (%) Embryo score No. of transferred embryos Score of transferred embryos Note: Values are means SE, n, or %. Differences are not statistically significant. Embryo Development in 5- L Drops For the 50 patients included in this part of the study, the delivery rate was 28%. One hundred thirty-six embryos were transferred and 26 gave rise to a gestational sac, giving an implantation rate of 19.1%. The fertilization rates were similar between the groups: 72% (196/272) with 5 L and 75% (228/304) with 30 L. A similar proportion of embryos reached at least the four-cell stage 42 hours after insemination in both protocols: 52.7% for 5 L and 63.7% for 30 L (P.534). The distribution of embryo quality according to the cleavage stage (i.e., 2 3-cell, 4-cell, or 4-cell) was not significantly different between culture with 5 Lor30 Lof medium (Fig. 1). The proportions of transferred embryos FERTILITY & STERILITY 949

4 FIGURE 1 Distribution of embryo quality according to the stage reached by the embryos on day 2 and the amount of culture medium. Values are not significantly different. Poor embryos ( ), fair embryos ( ), good embryos (^) FIGURE 2 Distribution of embryo quality according to the stage reached by the embryos on day 2 and the amount of glucose in the medium. Values are not significantly different. Poor embryos ( ), fair embryos ( ), good embryos (^). were not significantly different between 5 Lor30 L, at 55 (33%) and 81 (39%), respectively (P.227). Eight transfers that gave rise to a pregnancy (two miscarriages and six deliveries) combined embryos cultured in 5 L or30 L. Thus, for 9 embryos in 5 L and 15 embryos in 30 L, it is not possible to unequivocally relate culture conditions with implantation. Among the remaining 42 transfers, 9 gave rise to a pregnancy, including one miscarriage and eight deliveries. The implantation rate was 17.4% and 26% for embryos cultured in 5 L or30 L, respectively (P.414). Embryo Development in the Presence of 1 mm Glucose In this part of the study, 41 couples were included, and the delivery and implantation rates were 36.6% and 24%, respectively. After ICSI, 385 sibling oocytes were randomly distributed between media containing 1 mm or 5.56 mm glucose. Fertilization rates were not significantly different between the groups: 74% and 75% for embryos cultured with 1 mm or 5.56 mm glucose. The proportions of activated and unfertilized oocytes were not significantly different between the groups. Sixteen percent and 20% of the oocytes were unfertilized, and 7.6% and 3.3% were activated oocytes in the presence of 1 mm or 5.56 mm glucose, respectively. The distributions of cleavage stages and embryo scores on day 2 after fertilization were not significantly different between the media (Fig. 2). The proportions of transferred embryos also were similar between the groups. Thirteen of 20 pregnancies (including miscarriage) were observed after the transfer of a combination of embryos cultured in both media (36 embryos), and it was not possible to determine which embryo implanted. The proportions of embryos that failed to implant were not significantly different between the groups: 62% with 5 L and 44% with 30 L (P.091). Metabolic Measurements From the Time of Insemination Until the Time of Transfer In this prospective and randomized comparison, 499 oocytes were collected from 50 patients. After ICSI, 248 oocytes from 25 patients were cultured in the presence of 1 mm glucose in 5- L drops, and 251 oocytes from 25 patients were cultured in the presence of 5.56 mm glucose in 30- L drops. The proportions of normally fertilized oocytes were not significantly different between the protocols: 67.7% and 70.5% with 1 mm and 5.56 mm glucose, respectively. However, significantly more unfertilized oocytes were observed in the presence of 1 mm compared with 5.56 mm glucose (27.8% vs. 18.7%, respectively; P.021), and significantly fewer activated embryos were observed in presence of 1 mm glucose compared with 5.56 mm glucose (3.2% vs. 8.4%, respectively; P.023). The proportions of polyspermic embryos were not significantly different between the protocols: 1.2% and 2.4% with 1 mm and 5.56 mm glucose, respectively. The proportions of embryos reaching at least the four-cell stage and the distribution of embryo scores were not significantly different between the groups. The mean scores of the transferred embryos were similar in the presence of 1 mm or 5.56 mm glucose ( Devreker et al. Human embryo metabolism during fertilization Vol. 73, No. 5, May 2000

5 TABLE 2 Mean pyruvate uptake by oocytes from ICSI until the time of transfer. Type of oocyte n Day 0 1 Day 1 2 Unfertilized * Activated Normally fertilized Polyspermic Note: Values are means SE in pmol/embryo/h. * Significantly lower compared with activated embryos, P.010 (Mann- Whitney U test). Significantly lower compared with normally fertilized embryos, P.017 (Mann-Whitney U test). TABLE 3 Mean pyruvate uptake by normally fertilized embryos according to the stage reached at the time of transfer. Stage reached by day 2 n Day 0 1 Day cell stage to 3-cell stage cell stage cell stage * Highly fragmented embryos Note: Values are means SE in pmol/embryo/h. * Significantly higher compared with the 1-cell stage, P.010 (Mann- Whitney U test). Significantly higher compared with the 4-cell stage, P.015 (Mann- Whitney U test). 1.0 and , respectively). Finally, the proportions of transferred embryos were not significantly different: 37.5% and 33.9% of the normally fertilized oocytes were transferred with 1 mm and 5.56 mm glucose, respectively. A total of 21 pregnancies occurred, including 18 deliveries and 3 miscarriages, with the 50 transfers. In the presence of 1 mm or 5.56 mm glucose, respectively, no statistically significant differences were observed for the PRs (32% [n 8] and 40% [n 10]) or the implantation rates (16% and 25%). Similar results were observed for the pyruvate uptake by the embryos included in the second and third parts of the study, and the data were pooled for the analysis. Overall, the mean pyruvate uptake by the 340 zygotes was pmol/embryo/h during the first day after insemination and pmol/embryo/h during the second day after insemination. Normally fertilized and activated oocytes took up significantly more pyruvate than unfertilized oocytes (P.017; Table 2). The mean pyruvate uptake by the normally fertilized oocytes during the first and second days after insemination did not differ with cell stage at the time of transfer. Eight-cell embryos had a higher pyruvate uptake than one-cell or four-cell embryos (P.010 and P.015, respectively; Table 3). The differences observed in embryo morphology were not reflected by statistically significant differences in the mean pyruvate uptake. Values for mean pyruvate uptake were , , and pmol/embryo/h for poor, fair, and good embryos during the first day of incubation, respectively. During the second day of incubation, values were , , and pmol/ embryo/h for poor, fair, and good embryos, respectively. Pyruvate uptake was also compared with pregnancy outcome. The mean pyruvate uptake for the normally fertilized embryo cohort was higher for pregnant women than for nonpregnant women: compared with pmol/embryo/h during the first period and compared with pmol/embryo/h during the second day after insemination; P.004. Untransferred embryos from pregnant women took up more pyruvate than untransferred embryos from nonpregnant women: and pmol/embryo/h, respectively, during the first day (P.090), and and pmol/embryo/h, respectively, during the second day (P.010) (Fig. 3). No statistically significant difference was observed for the pyruvate uptake by transferred embryos from pregnant and nonpregnant women (Fig. 3). The levels of pyruvate uptake by the 13 embryos that implanted were and pmol/embryo/h during the first and second days of incubation, respectively. For the embryos that failed to implant, values were and pmol/embryo/h during the first and second days, respectively. Glucose uptake could not be detected during these first 2 days of culture. DISCUSSION This study reports for the first time the noninvasive measurement of pyruvate and glucose uptake during the time of pronucleus formation and early cleavage after ICSI. Glucose uptake was undetectable during the first 48 hours. Pyruvate uptake by fertilized or activated oocytes was significantly higher than that by unfertilized oocytes. However, it was not related to subsequent morphology, developmental stage at the time of transfer, or implantation. Thus, although pyruvate uptake was measured for 48 hours before transfer, it was not useful for embryo selection. Furthermore, reducing the incubation volume or the concentration of glucose during early cleavage divisions did not improve embryo viability. Pyruvate has been shown to be essential for the early preimplantation development of mouse (21) and human embryos (14). Furthermore, pyruvate uptake by human embryos FERTILITY & STERILITY 951

6 FIGURE 3 Pyruvate uptake by transferred ( ), frozen ( ), or untransferred (^) embryos during the first (top) and second period (bottom) of incubation. P.01. has been shown to increase between the two- and four-cell stage until the blastocyst stage and to be related to embryo viability in culture in vitro (7). Fertilization and activation increased pyruvate uptake by the human embryo by approximately 30%. This has been shown previously in mouse embryos (5, 22). Culture of isolated human blastomeres in vitro showed that those that underwent cell division had a significantly higher pyruvate uptake than those that did not cleave (Hardy, unpublished data). Thus, pyruvate uptake appears to be related to cell division, and in the zygote, it is possible that the cytoskeletal changes associated with expulsion of the second polar body and movement of pronuclei (23) require energy, which is provided by pyruvate. Oocytes with reduced levels of adenosine triphosphate (ATP) were shown to have a reduced potential to develop further (24). Pyruvate is directly metabolized to produce ATP and therefore can also reflect oocyte viability. However, the low level of pyruvate uptake and the large range of values observed made it impossible to identify viable oocytes or embryos. Although untransferred, surplus embryos from patients who became pregnant had a higher pyruvate uptake than those from patients who did not become pregnant, we could not correlate pyruvate uptake with the implantation potential of transferred embryos because pyruvate uptake was measured for 231 untransferred embryos but only for 93 transferred embryos. This confirms previous studies relating developmental potential with metabolism (7), but also confirms that this noninvasive approach of measuring embryo metabolism is not discriminatory for small numbers of embryos before transfer (9, 10) because of the high variability of pyruvate uptake. Previous studies in the human have shown that glucose uptake (7) and metabolism (15) by embryos between the two- to four-cell stage and the eight-cell stage are low. However, glucose has been shown to inhibit preimplantation development in a number of species, including mouse (11), hamster (12), rat (13), and human embryos (14). This inhibitory effect is dependent on stage, species, and medium composition (see review by Bavister [25]). Progressively reducing the glucose concentration from 1 mm to 0 mm increases total cell number in day-6 human blastocysts (14). Omitting glucose during the transition between the four- and eight-cell stages increased human preimplantation embryo development. Total cell number was significantly higher in blastocysts cultured in the absence of glucose. This positive effect was already appearing when glucose was reduced from 1 mm to 0.5 mm (14). In the light of these results, we compared early embryo development in the presence of 1 mm and 5.56 mm glucose. The concentration of 1 mm was chosen to mimic the concentration found in female genital tract fluids (26). However, both in the randomized comparison of sibling embryos and in the randomized comparison of patients, we were unable to demonstrate an improvement in embryo viability in the presence of low glucose. The high proportion of activated oocytes in the third part of the study in the presence of 5.56 mm glucose was not confirmed by the second part of the study, which randomized the oocytes between treatment groups. When pooling the data, we found that the proportion of activated oocytes was similar between 1 mm and 5.56 mm glucose. This suggests that the significant difference observed when the patients were randomized between the groups was probably oocyte dependent rather than an effect of the culture media. Furthermore, we were able to show for the first time that glucose uptake is undetectable during the first and second cleavage divisions in the human. Various possibilities could explain the absence of a ben- 952 Devreker et al. Human embryo metabolism during fertilization Vol. 73, No. 5, May 2000

7 eficial effect on embryo viability observed in this study: [1] The glucose concentration should have been reduced further; [2] the inhibition in development could occur when glucose is present around the time of embryonic genome activation; [3] because early-stage embryos took up very small amounts of glucose, they would not be affected by high levels in the culture medium; or [4] embryos were cultured in vitro for too short a period to see any effect on development. In this comparison of 884 embryos cultured with 1 mm or 5.56 mm glucose, early embryo development did not seem to be affected by the reduction in glucose concentration. However, the resulting low number of transferred embryos could explain the absence of a statistically significant difference in the implantation rates or PRs between low or high levels of glucose. Several studies have shown that culturing mouse embryos in groups or in small volumes of medium increases blastocyst formation and cell numbers (27, 28) and reduces cell death (3, 29). This is thought to be due to the production of beneficial autocrine growth factors by the embryo, which are diluted to ineffective levels in larger volumes of medium. Human embryos have been cultured up to the blastocyst stage in volumes as small as 5 L, although under these conditions, the medium has to be changed daily to prevent nutrient depletion (7, 20). Furthermore, embryos have been cultured in 5- L drops before transfer and have given rise to successful pregnancies (9, 10). Although culturing embryos in 5- L compared with 30- L drops was not detrimental to embryo development, morphology, or implantation, it did not further improve development. Previously, Spyropoulou et al. (30) reported no beneficial effects of culturing human embryos in groups in 20 L of medium. Autocrine factors are probably already concentrated enough in drops of 30 L, explaining why no further improvement in embryo development was observed when embryos were cultured in 5 L. In conclusion, we have shown that glucose uptake during the first 48 hours is negligible and that pyruvate uptake is higher after fertilization or activation. However, analysis of oocyte and early embryo pyruvate and glucose uptake for the 48 hours before transfer did not improve the selection of viable embryos. Analysis of nutrient uptake during a longer period, for example up to the blastocyst stage, which is associated with an increase in metabolic activity, may prove to be a more successful approach. Acknowledgments: The authors thank Dr. Anne Delbaere for her help in revising the manuscript. The metabolic measurement would not have been possible without the permission of Jean-Marie Nicolas (UCB Inc., Brussels, Belgium) that allowed access to their Cobas Fara autoanalyzer (Roche, Belgium) and the help of Françoise Meriaux and Françoise Dewalcq. References 1. Devreker F, Emiliani S, Revelard P, Van den Bergh M, Govaerts I, Englert Y. Comparison of two elective transfer policies of two embryos to reduce multiple pregnancies without impairing pregnancy rates. Hum Reprod 1999;14: Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989;107: O Neill C. Role of autocrine mediators in the regulation of embryo viability: lessons from animal models. J Assist Reprod Genet 1998;15: Sheth KV, Roca GL, Al-Sedairy ST, Parhar RS, Hamilton CJCM, Jabbar FA. 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Fertil Steril 1998;69: Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci USA 1967;58: Hardy K, Handyside AH. Metabolism and cell allocation during parthenogenetic preimplantation mouse development. Mol Reprod Dev 1996;43: Payne D, Flaherty SP, Barry MF, Matthew CD. Preliminary observations on polar body extrusion and pronuclear formation of human oocytes using time-lapse video cinematography. Hum Reprod 1997;12: Van Blerkom J, Davis PW, Lee J. ATP content of human oocytes and developmental potential outcome after in vitro fertilization and embryo transfer. Hum Reprod 1995;10: Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1; FERTILITY & STERILITY 953

8 26. Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil 1990;88: Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative interactions among embryo and role of growth factors. Proc Natl Acad Sci USA 1990;87: Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod 1992;7: Brison DR, Schultz RM. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol Reprod 1997;56: Spyropoulou I, Karamelegos C, Bolton VN. A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before transfer on day 2. Hum Reprod 1999;14: Devreker et al. Human embryo metabolism during fertilization Vol. 73, No. 5, May 2000