Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos

The consumption of pyruvate and glucose and the production of lactate by 40 single human preimplantation embryos has been measured using a non-invasive technique. Twelve of the embryos showed abnormal fertilization. Of the 28 normally fertilized embryos, nine (32%) developed to the blastocyst stage in culture while the remainder degenerated or arrested during cleavage. In the normal embryos, pyruvate uptake exceeded that of glucose in the early developmental stages (days 2-5 post-insemination) before glucose became the predominant substrate in the blastocyst (day 6). Considerable quantities of lactate were formed throughout development, rising from a value of 43.6 pmol/embryo/h on day 2.5 to 95.4 pmol/embryo/h on day 5.5. The values of pyruvate and glucose uptake and lactate production of those embryos which arrested were below those which developed normally. On the basis that one mole of glucose can give rise to two moles of lactate, only 50% of the lactate produced could be accounted for in terms of glucose uptake from the medium. This figure rose to 90% in the blastocyst. The remaining lactate must be derived from endogenous sources, most probably glycogen. It is proposed that the high production of lactate by human preimplantation embryos in vitro is an adaptation to the conditions of culture.     

Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos

Experiments were conducted to find a suitable cryoprotectantand suitable procedure for vitrification of 8-cell mouse embryos.The method was then applied clinically to the cryopreservationof human embryos in our assisted reproduction programme. Mouseembryos were vitrified with 30 or 40% 1,2-propanediol (PROH),dimethylsulphoxide (DMSO), ethylene glycol, glycerol, or acetamide,each diluted with a solution containing 30% Ficoll plus 0.5M sucrose. Embryos were exposed to the solutions for 0.5 or2 min at 20 or 25°C, cooled in liquid nitrogen and warmedrapidly. Embryo survival was assessed by in-vitro development.In PROH-, DMSO- and acetamide-based solutions, higher survivalrates (29–82%) were obtained with less permeating conditions,suggesting that these cryoprotectants are considerably toxic.In glycerol-and ethylene glycol-based solutions, however, highersurvival rates (74 and 92% respectively) were obtained withmore permeating conditions, suggesting that these cryoprotectantsare less toxic. Human embryos on days 2–3 were vitrifiedin an ethylene glycol-based solution (EFS40). Survival, assessedby the morphology, was higher in 4-cell embryos on day 2 and8-cell embryos on day 3 than in 2–3-cell embryos on day2 or 2–7-cell embryos on day 3. From 18 transfers, oneended with the delivery of healthy twin babies.     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse

The effect on development of early mouse embryos of making ahair-line slit in the zona pellucida of approximately one-thirdits diameter was investigated. The rate of development to mid-gestationof operated zygotes and2-cell embryos transferred directly tothe oviduct was significantly lower than that of sham-operatedor unoperated controls. However, the operation had no discernibleeffect on the development of 2-cel embryos that were culturedfor 2 days prior to transfer to the uterus, or on embryos composedof 8 or more cells transferred directly to the oviduct. Zonaslit zygotes and 2-cell embryos exhibited a significantly higherrate of anomalous development to the morula or blastocyst stagethan controls following short-term transfer to the adult orimmature oviduct. Such anomalies could not be attributed todamage of the embryos by leucocytes or bacteria entering throughthe wound in the zona. Rather, the typically non-spherical shapeof slit zonae, together with the fact that some were empty onrecovery, was consistent with operated embryos having been damagedby compression during passage through the oviduct. This suggeststhat, providing it is intact, the zona pellucida protects theearly embryo from contraction of the oviductual musculaturewhich is sufficient to lyse, arrest or extrude blastomeres priorto the formation of intercellular junctions. Hence, in experimentalmanipulations entailing damage to the zonae of early embryos,there may be a case for allowing them to form morulae in vitroprior to transfer, rather than returning them directly to theoviduct     

Non-invasive measurement of glucose and pyrovate uptake by individual human oocytes and preimplantation embryos

Pyruvate and glucose uptake by 73 individual human oocytes andpreimplantation embryos was measured non-invasively, using anultramicrofluorescence assay to analyse changes in substratelevels in mkrodroplets of culture medium. The uptake of bothsubstrates was measured over successive daily incubations betweendays 1 (unfertilized oocytes) or 2 (‘spare’ embryoswhich were not transferred) and day 6 (day 0 = day of insemination).Under these conditions, 58% (25/43) of fertilized embryos withtwo pronuclei on day 1 developed to the blastocyst stage byday 6. The pyruvate uptake of these embryos increased from –28to a maximum of 40 pmol/ embryo/h between days 2.5 and 4.5.Similarly, glucose uptake increased from 8 to 14 pmol/embryo/hbetween days 2.5 and 4.5, but then increased further to 24 pmol/embryo/hon day 5 at the blastocyst stage. The pyruvate uptake of fertilizedembryos which arrested at cleavage stages was significantlylower than for those which developed to the blastocyst stage.Polyspermk and parthenogenetic embryos, and unfertilized oocytesalso had lower pyruvate uptakes at later stages. The glucoseuptake of unfertilized oocytes and abnormal embryos never reachedthe level of fertilized embryos at the blastocyst stage on day5.5. Non-invasive measurement of pyruvate uptake before embryotransfer may provide a valuable functional criterion for theselection of viable embryos capable of developing to the blastocyststage.     

An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion

Mammalian preimplantation embryos are conventionally grown in small, static droplets of medium. By contrast, embryos within the oviduct are subject to mixing forces arising from the action of cilia and the contractions of the myosalpinx. Such forces will minimize the buildup of unstirred layers around the embryos and facilitate the exchange of gases and metabolites. We have devised a mathematical model to investigate whether preimplantation mouse and human embryos grown in static culture can satisfy their requirement for oxygen solely by diffusion i.e. in the absence of stirring. The model incorporates the diffusion coefficients for oxygen in the medium outside and within the embryo, the oxygen tension of the culture medium, the size of the embryo and its oxygen uptake. Solutions of the model are provided for mouse oocytes and 2-cell embryos, mouse blastocysts and human morulae. In each case, the model is solved for two different values of the diffusion coefficient of oxygen within the cell and of the oxygen content of the medium (5 and 20%). The conclusion is that mouse embryos in static culture are likely to be able to satisfy their demands for oxygen by diffusion alone, but that human embryos may become marginally hypoxic, especially at lower oxygen levels.     

The chromosome constitution of human preimplantation embryos fertilized in vitro

The chromosome constitution of five haploid, 178 diploid and11 triploid embryos fertilized in vitro was determined afterfixation on day 2 or day 3 of development. Karyotype analysisof 178 diploid embryos revealed abnormalities in 40 (22.5%)cases: 34 (19.1%) aneuploids, four (2.2%) mosaic embryos andtwo (1.1%) structural anomalies were identified. The majorityof aneuploid karyotypes (28/34) involved a single chromosomebut six embryos had aneuploidy of two or three chromosomes.The E group was most frequently involved in aneuploid karyotypes(10/23 hyperdiploid embryos) and trisomy 16, the most commonsingle anomaly in diploid embryos, was detected in 2.2% (4/178)of cases. Only one case of sex chromosome monosomy was identified.An excess of female karyotypes was detected in abnormal cases(sex ratio 0.48); this ratio was significantly (p< 0.05)different from that observed in normal cases (74: 64, XY: XX).The incidence of aneuploidy increased with maternal age butthis did not reach statistical significance. Embryo morphologyand growth rate, assessed by embryo development rating (EDR),did not distinguish between normal (mean score 7.9; mean EDR96.1) and aneuploid (mean score 8.1; mean EDR, 92.1) embryos.Numbers of hyperploid (n = 17) and hypoploid (n= 11) embryos(non-mosaic cases involving single chromosomes) were not statisticallydifferent. The relative proportions of chromosomes involvedin trisomic karyotypes showed a remarkable similarity to thepattern in spontaneous abortions. Pronuclear status was an unreliablepredictor of ploidy. Small numbers of karyotyped triploid embryosrevealed equal proportions of XXX, XXY and XYY embryos     

New advances in human embryology: implications of the preimplantation diagnosis of genetic disease

The diagnosis of genetic disease in preimplantation embryosis discussed. The typing of spermatozoa may be feasible forfactors such as the presence of an X and Y chromosome. Embryosmight be typed by non-invasive methods, by assessing their uptakeof metabolites although the widest opportunities may arise bythe use of invasive methods which involve the removal of oneor a small number of cells. The methods of diagnosis are discussed,including enzyme assays and the use of DNA probes, preliminaryresults with human embryos are presented and the difficultiesrelated to these techniques are debated. The low rate of implantationof replaced embryos will mean that many embryos will have tobe diagnosed, and certain embryological factors such as thehigh incidence of chromosomal imbalance and the problems of‘imprinting’ might obscure certain diagnoses. Theadvantages and disadvantages of the method are discussed.     

Variation in the dry mass of mouse embryos throughout the preimplantation period.

Information on the net balance of embryonic metabolism during the preimplantation period is provided by measurements of embryo dry mass. The dry mass of mouse embryos at 10 defined stages of preimplantation development was measured using the Vickers M86 scanning microinterferometer. The results demonstrate that there is an overall loss of dry mass from the unfertilized ovum (39.10 +/- 0.6 ng) to the late blastocyst stage (32.80 +/- 0.53 ng) with these differences being highly significant. However, there is a significant increase in embryonic dry mass from the 1-cell (36.88 +/- 0.34 ng) to 2-cell stage (40.48 +/- 0.40 ng). These results suggest that protein synthesis exceeds degradation during the 2-cell stage but that from the 4-cell to morula stage the reverse is true. In addition, the variation in dry mass between embryos at the same developmental stage is extremely small, suggesting that this may be a useful indicator of embryonic viability.     

Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos

BACKGROUND: Cleavage stage embryos as well as postimplantation embryos have been studied extensively over the years. However, our knowledge with respect to the chromosomal constitution of human embryos at the blastocyst stage is still rudimentary. METHODS: In the present paper, a large series of human blastocysts was examined by means of fluorescent in situ hybridization (FISH). RESULTS: It was found that only one in four blastocysts (25%) displayed a normal chromosomal pattern. We defined a group of blastocysts (26%) displaying a simple mosaic chromosome pattern (different cell lines resulting from one chromosomal error), an about equally large group of blastocysts (31%) displaying a complex mosaic chromosome pattern, and a smaller group of blastocysts (11%) showing a chaotic chromosome distribution pattern. Six per cent of all blastocysts analysed could not be assigned one of the previously mentioned chromosomal patterns. CONCLUSION: Anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage.     

Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial

BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and < 0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology.     

Paternal effects acting during the first cell cycle of human preimplantation development after ICSI.

BACKGROUND: The ability of human embryos to undergo normal development has been shown previously to be subject to strong paternal (sperm-derived) effects. This study was undertaken to determine whether paternal influences on human embryo quality are detectable as early as the first cell cycle after fertilization. METHODS: The quality of zygotes and cleaving embryos resulting from sibling donor oocytes fertilized by sperm from different patients were compared in a donor oocyte-sharing programme. RESULTS: Fertilizations with sperm from certain individuals repeatedly resulted in the formation of high proportions of zygotes with abnormal pronuclear morphology that subsequently tended to cleave slowly and to show extensive fragmentation and blastomere irregularities. This phenomenon was observed with oocytes from two different donors for each of these individuals and contrasted with normal developmental performance of embryos resulting from sibling oocytes fertilized by sperm from other men with similar basic sperm characteristics. Fertilization rates were not related to these differences. CONCLUSIONS: These data point to a very early onset of paternal effects that condition human embryo development. These effects may be both of genetic (related to the minor gene activity of the male pronucleus) or epigenetic (related to the sperm-derived oocyte-activating factor or sperm centrosome) origin.     

Fertilization and early embryolgoy: A comparison of the effects of different biopsy strategies on the post-thaw survival of 8-cell-stage mouse embryos: implications for preimplantation diagnosis

The aim of this study was to investigate the effects of twodifferent biopsy strategies, zona slitting and zona piercing,on the post-thaw survival and subsequent in-vitro developmentof 8-cell mouse embryos. From control experiments it was determinedthat neither biopsy by zona slitting nor zona piercing adverselyaffected embryo development in vitro, as similar rates of blastocystformation and hatching were found between biopsied and zona-intactembryos; there was, however, a trend towards a lower rate ofblastocyst hatching in embryos biopsied by zona piercing (78.3%compared with 91.9% of zona-intact embryos). When biopsy wasfollowed by cryopreservation a different picture was seen: similarrates of freeze—thaw survival were found for zona-slit,zona-pierced and zona-intact embryos (84.2, 88.5 and 87.2% respectively),but this was superseded by a significant (P < 0.05) reductionin the blastocyst formation rates (61.4% zona-slit and 63.9%zona-pierced versus 78.7% zona-intact) and hatching rates (51%zona-slit and 52.5% zona-pierced versus 72.3% zona-intact) ofthe biopsied embryos. When the effects of zona slitting andpiercing were considered in isolation, i.e. without performingbiopsy, it was found that the larger holes produced by zonaslitting rendered embryos more susceptible to freeze-thaw damage.Significant (P < 0.05) reductions occurred in the blastocystformation rates (63.7% zona-slit versus 78.7% zona-intact embryos)and hatching rates (52.3% zona-slit versus 72.3% zona-intactembryos) of the zona-slit embryos.     

Caspase activity in preimplantation human embryos is not associated with apoptosis

BACKGROUND: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking. METHODS: A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos. RESULTS: Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase-positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture. CONCLUSIONS: These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.     

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.     

Requirement of preimplantation human embryos for extracellular calmodulin for development

The objective of this study was to investigate the requirementfor calmodulin in the cell division of the early human preimplantationembryo. Experiments using three agents capable of inhibitingcalmodulin activity, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide(W7), W7 linked to agarose beads (W7-agarose) and calmodulinantibody, showed that W7 and calmodulin antibody arrested divisionof embryos in a dose-dependent manner. As W7 is able to penetratethe zona pellucida and enter the cells, calmodulin antibodycan traverse the zona but not enter the cells and W7-agarosecannot traverse the zona, we have deduced that the calmodulinwhich appears relevant to embryo division may be both intracellularand intrazonal but not extrazonal. We conclude that calmodulinis specifically required for cell division in the early humanpreimplantation embryo and that the concentration of calmodulinsurrounding the embryo within the zona is particularly importantto embryo development.     

Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF

BACKGROUND: There is concern that IVF and/or ICSI might have an adverse effect on embryonic development via epigenetic alterations. Such alterations might also be involved in the sex-related growth differences in preimplantation embryos found in some animal species. In the present study we analysed cell numbers of human male and female surplus embryos that developed to the blastocyst stage after either IVF or ICSI in order to investigate possible sex-dependent differential growth rates. METHODS: Blastocysts resulting from surplus embryos obtained after either IVF or ICSI during a 5 year study period were analysed using fluorescence in situ hybridization (FISH). RESULTS: The number of cells and sex could be determined in 330 blastocysts collected from 92 IVF cycles and in 322 blastocysts collected from 121 ICSI cycles. Whereas female and male embryos originating from IVF showed comparable mean log cell numbers per embryo +/- SEM (3.76+/-0.05 in 147 female and 3.72+/-0.04 in 183 male embryos), significant differences were observed in embryos originating from ICSI (3.57+/-0.05 in 162 female and 3.90+/-0.03 in 160 male embryos). The sex-related growth difference was significantly greater in ICSI than in IVF embryos. In a subset of 84 embryos, inner cell mass (ICM) and trophectoderm (TE) were analysed separately. A significantly higher mean log cell number of TE cells in ICSI male embryos was found as compared to their female counterparts (3.44+/-0.12 in 16 female and 3.90+/-0.11 in 29 male embryos), whereas this difference was not found in IVF embryos. CONCLUSION: A clear sex-related growth difference was found in human blastocysts originating from ICSI, but not in blastocysts originating from IVF. It is as yet unknown which mechanism is responsible for our findings. We hypothesize that the ICSI procedure might interfere with the process of imprinted X-inactivation.     

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro

The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.