Role of cathepsins in blastocyst hatching in the golden hamster

The mammalian embryo is encased in a glycoproteinaceous coat, the zona pellucida (ZP) during preimplantation development. Prior to implantation, the blastocyst must undergo 'hatching' or ZP escape. In hamsters, there is a thinning of the ZP followed by a focal lysis and a complete dissolution of the ZP during blastocyst hatching. Earlier studies from our laboratory have indicated a role for cysteine proteases in the hatching phenomenon. In this study, we tested the effect of specific inhibitors of the three classes of cysteine protease on blastocyst hatching. Cystatin, an endogenous cathepsin inhibitor, blocked blastocyst hatching. Similarly, Fmoc-Tyr-Ala-diazomethane, a synthetic cathepsin inhibitor, blocked hatching. Both showed dose-dependent and temporal inhibition of hatching. However, Z-Val-Ala-Asp-fluoromethylketone, a synthetic caspase inhibitor, and calpastatin, an endogenous calpain inhibitor, had no effect on hatching. The cathepsins were localized to blastocyst cells. Exogenous addition of cathepsins L, P or B to cultured 8-cell embryos caused a complete ZP dissolution. The expression of mRNA and protein of cathepsins L and P was observed in peri-hatching blastocysts. Cathepsins L and P were detected in trophectodermal projections and in the ZP of peri-hatching blastocysts. These data provide the first evidence that blastocyst-derived cathepsins are functionally involved as zonalytic factors in the hatching of blastocysts in the golden hamster.     

Fertilization and early embryology: Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium

The formulation of chemically defined culture media that supportprimate embryo development would facilitate studies on primatepreimplantation embryogenesis. The specific aims of this studywere (I) to evaluate the development of macaque embryos in asimple, chemically defined, protein-free medium developed fora rodent embryo model, and (ii) to determine if a two-step progressiveculture system could enhance blastocyst development and zonaescape. In experiment 1, in-vitro-fertilized pronucleate stageembryos (n=81) from nine monkeys were randomly allocated toone of three treatments: (a) hamster embryo culture medium-6(HECM-6; chemically defined, protein- free medium), (b) CMRL-BCSmedium (modified CMRL 106 medium containing 20% bovine callserum; BCS), and (c) a two-step culture procedure (HECM-6 throughto the 8- to 12-cell stage, and CMRL-BCS medium beyond thatstage). Optimal development was attained equally (P0.05) withembryos cultured in CMRL-BCS medium or the two-step procedure(48 and 61% blastocysts respect ively). HECM-6 alone supporteddevelopment to the morula stage (72%) equally as well as CMRL-BCSmedium (80%) or the two-step procedure (69%), but not to theblastocyst stage (22 versus 48 and 61% respectively). Hatchingof the blastocysts was essentially limited to the serum-containingmedia (CMRL-BCS medium, 31% two-step procedure, 44%). In experiment2, in-vitro-fertilized pronucleate-stage embryos (n=87) fromnine monkeys were randomly placed in each of four two-step treatments:(a) HECM-6 through to the 8- to 12-cell stage and CMIRL-BCSmedium beyond that stage, (b) HIECM-6 through to the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HIECM 6 throughto the morula stage and CMIRL-BCS medium beyond that stage,and (d) HECM-6 through to the morula stage and HECM-6-BCS beyondthat stage. Greater (P 0.05) percentages of embryos developedinto blastocysts, expanded blastocysts and hatched blastocystswhen switched at the 8- to 12-cell versus the morula stage inthe second step medium. When transferred into BCS containingmedium at either the 8- to 12-cell or morula stage, embryosunderwent blastulation and expansion equally well in CMIRL-BCSmedium versus HECM-6-BCS. However, when embryos were switchedto the second step medium at the 8- to 12-cell stage, hatchedblastocysts 1690 were obtained more (P0.05) frequently in CMRL-BCSmedium (50.9%) than in HECM-6-BCS (37%). This work is the firstto produce in-vitro-fertilized primate blastocysts culturedfrom the pronucleate stage in chemically defined, protein-freemedium, and demonstrates that while primate embryos can formmorulae in such a medium, their requireents for blastocoel formationand zona escape appear to be more demanding, and may be acquiredas early as the 8-cell stag.     

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     

Fertilization and early embryology: Timing of development is a critical parameter for predicting successful embryogenesis

Development of embryos from the 1-cell stage into blastocystsin vitro is generally slower than the time-course for developmentin vivo. It was the objective of this work to determine whetherembryos that reach the 8-cell stage within a normal time-framehave a developmental advantage (both in vitro and post-embryotransfer) over slower embryos. Hamster 1-cell embryos were collected10 h post-egg activation (PEA) and cultured for 48 h (58 h PEA= t⁵⁰ for 8-cell embryo development in vivo) in hamster embryoculture medium-6. Embryos were sorted according to stage reached,culture was continued in fresh medium and stage of developmentwas observed at 78, 82 and 86 h PEA. At 58 h PEA, embryos were<4-cell (4%), 4-cell (19%), 5- to 7-cell (16%) or 8-cell(61%). The 58 h 8-cell embryos had a significantly greater abilityto develop to the blastocyst stage than 58 h 4-cell embryosat 78, 82 and 86 h PEA (74 versus 13%, 69 versus 25% and 65versus 37% respectively). The percentages of 14-day-old fetusescollected after embryo transfer indicated that morulae and blastocystsderived from 58 h 4-cell embryos were, on average, less viable(26% fetuses) than morulae and blastocysts from 58 h 8-cellembryos (51% fetuses). Thus morulae and blastocysts developingin vitro from faster or slower cleaving embryos can be qualitativelyas well as quantitatively different. These data indicate thatthe timing of development in vitro, specifically the timingof completion of the third cell cycle, is a critically importantparameter for predicting successful embryogenesis in the hamster.     

Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage

Energy substrate requirements (pyruvate, lactate and amino acids) were determined for in-vitro development of hamster 1- and 2-cell embryos to blastocysts, using a chemically defined, protein free medium (hamster embryo culture medium, HECM). One-cell embryos were very sensitive to energy substrate type and concentration. Pyruvate alone could not support development of 1-cell embryos to greater than 4-cells, whereas lactate as sole energy substrate supported 14% development into morulae/blastocysts. Pyruvate, with lactate and 20 amino acids, inhibited 1-cell embryo development into blastocysts relative to lactate and 20 amino acids. The highest development of 1-cell embryos to blastocysts (up to 27%) occurred with reduced lactate concentration (less than 10 mM) and either 20 amino acids or 0.2 mM glutamine. Hamster 2-cell embryos were much less sensitive to energy substrates, requiring only lactate for development to blastocysts (53%). Lactate with 20 amino acids supported 70-75% of 2-cell embryos to blastocysts. Glutamine as sole energy and nitrogen source supported development to morulae and blastocysts of some 2-cell, but not 1-cell, embryos. Pyruvate did not enhance development of 2-cell embryos. We conclude that (i) altering the types and concentrations of available energy substrates drastically changes the developmental responses of 1-cell hamster embryos in vitro and (ii) energy substrate requirements for hamster embryo development in vitro are markedly different from those of mouse embryos, the standard model for studies on preimplantation development. This is the first report of successful in-vitro culture of hamster 1-cell embryos to the blastocyst stage.     

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha- chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.      

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.     

Implantation: Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts

The behaviour of bovine, equine and human blastocysts was studiedin vitro by time-lapse videomicrography and computer imaging.This study revealed that cytoplasmic extensions of the trophectoderm[‘trophectoderm projections’ (TEP)] were expressedby embryos of all three species, prior to or during zona escape.Bovine and human blastocysts escaped their zonae with a combinationof blastocoele expansion, collapse and re-expansion coupledwith the penetration of the zona pellucida by TEP. In equineembryos, after several cycles of blastocoele expansion and collapse,trophectoderm ruptured the zona with the concomitant appearanceof TEP. This study provides documentation that TEP are expressedby a diverse range of mammalian species, bringing the totalnumber of species in which this phenomenon is found to six,since TEP are also known to be expressed by guinea-pig, hamsterand rhesus monkey blastocysts, representing rodents, ungulatesand primates. In all species studied, the dynamic nature (extension,retraction, and angular movement) of the TEP was similar, movingin an undulating manner with rapid cycles of extension and retraction.Because TEP appear to be a genera] feature of mammalian blastocysts,they are implicated in one or more key events in early development,namely zona escape, attachment and/or implantation.     

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel and embryoblast. GLUT3 was exclusively localised in the apical membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.     

Production of normal mice from oocytes fertilized and developed without zonae pellucidae.

Mouse oocytes, freed from zonae pellucidae, were inseminated and cultured in vitro. When inseminated with a modest concentration of spermatozoa (approximately 5/microliter), 79% of 151 oocytes were fertilized normally, of which the majority developed into blastocysts. When 177 blastocysts were transferred to 12 pseudopregnant foster mothers, 10 (83%) of the mothers became pregnant and 37 live pups were obtained. Although the overall rate of development of zona-free embryos was slightly inferior to that of zona-intact (control) embryos, these results nevertheless indicate that the oocyte and embryo are potentially capable of normal fertilization and development in the total absence of the native zona pellucida. Since the acrosome reaction is essential for successful sperm-oocyte fusion, zona-free oocytes must have been fertilized by spermatozoa which had undergone the reaction either in the medium or on the surface of the oocyte plasma membrane. Such acrosome reactions, which are not mediated by the native zona pellucida, can be called 'unphysiological' or 'spurious' but the spermatozoa with such reactions were certainly functional, as they produced healthy offspring.     

Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development.

Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [(3)H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2-cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 +/- 75, 43.0 +/- 28.3 and 22.0 +/- 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cell embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.     

Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development

The developmental potential in vitro and in vivo of preimplantation mouse embryos biopsied at the 4-cell, 8-cell and morula stages were investigated. Biopsy had the least impact when performed at the 8-cell stage. There was no effect of biopsy on the development of 8-cells of blastocysts in vitro (95% compared with 99% of controls) or the implantation rate after transfers (82 versus 87%, P greater than 0.05); however, fewer embryos (52 versus 71%, P less than 0.05) resulted in viable fetuses. There was no effect of biopsy at the 8-cell stage on fetal weight on day 17. Blastocyst formation in vitro was significantly less for 4-cell biopsies compared with their controls (76 versus 90%, P less than 0.001) and biopsy also affected the implantation rate (44 versus 59%, P less than 0.01). Biopsy was most detrimental when performed on morulae, reducing the implantation rate from 65% for controls to 21% for biopsies (P less than 0.001). Fetal viability was also markedly affected with a reduction on day 17 from 42 to 26% accompanied by a significant reduction (24%, P = 0.02) of the mean fetal weight. Handling of embryos for biopsy at the morula stage, which involved removal of the zona pellucida, was a significant but not complete cause of the reduced implantation potential observed (sham-controls and intact-controls: 34 and 65%, P less than 0.001), while puncture of the zona during the biopsy of 4-cell and 8-cell embryos had no effect. Therefore, the 8-cell mouse embryo is the most suitable state for embryo biopsy.     

Stereological study of mouse uterine and in vitro grown blastocysts: Cell numbers and volumes

Mouse blastocysts were studied to determine if there were differences in cell number and volumes between those that were (1) derived from the uterus prior to implantation on the afternoon of day 4 of pregnancy and (2) those that were cultured for 72 hr from two-cell-stage embryos. Blastocysts were fixed, embedded in resin, and serially sectioned at 1.5 or 2 μm. Photographic prints of alternate sections were used to count the numbers of inner cell mass (ICM) and trophectoderm cells. Cavalieri's direct estimator was applied to the same prints to estimate the volume of the whole blastocyst. Point counting was used to determine the volumes of the ICM, trophectoderm, and zona pellucida. The number of cells and size of the ICM were similar between the two groups of blastocysts, although it was found that the ICM of uterine embryos that did not have a zona pellucida were smaller than the ICM of those that did. There were twice as many trophectoderm cells in the blastocysts that were cultured from two-cell embryos, and these cells were also found to be larger. Furthermore, the volume of the zona pellucida was less in the uterine blastocysts. This study indicates that, while trophectoderm proliferation is enhanced in vitro, the ICM is more constant and thus may be self-regulating and independent of the growth conditions of the blastocyst as a whole. This study also suggests partial zona lysis occurs in utero and occurs either at a reduced rate or not at all in vitro. © 1993 Wiley-Liss, Inc.     

Preimplantation sexing and diagnosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay

Hypoxanthine phosphoribosyl transferase (HPRT)-deficient male embryos derived from heterozygous (carrier) female mice were diagnosed by biochemical microassay of X-chromosome-coded HPRT activity in a single cell taken from the 8-cell embryo or in 5-10 cells sampled from the blastocyst. In the latter procedure, carrier female blastocysts could also be distinguished from affected males, and normal males and females, as having intermediate HPRT activity in the sampled trophectoderm cells. During the assay procedures, the operated preimplantation embryos were cultured. They were then transferred, in batches as diagnosed, to recipient females. The resulting fetuses were sexed by gonad morphology and assayed for HPRT activity. All those identified as HPRT-negative embryos by biopsy at the 8-cell or blastocyst stages were indeed HPRT-negative males. The heterozygous females were also correctly identified by the trophectoderm biopsy procedure. The sex of an embryo can also be diagnosed by HPRT activity dosage in a single blastomere taken from 8-cell embryos from a normal mating and cultured for 12 hours before assay. Both X chromosomes are active in female morulae and the blastomeres sampled from female preimplantation embryos have twice the X-coded HPRT activity compared to those from the male embryos. The accuracy of this procedure for sexing was again verified by transfer of the putative male and putative female embryos into recipient females.     

EGCG对昆明小鼠早胚体外发育的影响

目的观察表没食子儿茶素没食子酸酯(-)(EGCG)对昆明(kunming,KM)小鼠早胚体外发育的影响,为改善小鼠早胚体外培养体系奠定实验基础。方法以空白M16培养液为对照组,M16中添加浓度为0.1μg/mL、1μg/mL、10μg/mL、20μg/mL EGCG为实验组,收集KM小鼠1-细胞胚进行体外连续培养,计数各组发育至2-、4-细胞胚、桑椹胚和囊胚等各个阶段的数目,并以2-细胞胚为基数,计算各组至不同阶段的发育率。结果添加EGCG实验组发育到桑椹胚和囊胚的比率明显高于对照组,其中以添加浓度为10μg/mL和20μg/mL的EGCG实验组的效果最显著,其桑椹胚发育率分别为77.3%和78.7%,而对照组只有43.2%(P0.01)。10μg/mL和20μg/mL的EGCG实验组的囊胚发育率分别为53.0%和47.3%,也明显高于对照组(19.2%,P0.01)。结论EGCG可以显著提高KM小鼠1-细胞胚体外发育到桑椹胚及囊胚的比率,以10μg/mL和20μg/mL浓度的EGCG效果最显著。     

In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

A prospective randomized trial of blastocyst culture and transfer in in- vitro fertilization

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co- culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.      

In-vitro development of refrozen mouse embryos

To evaluate the effects of sequential, repetitive freezing on their in- vitro development, mouse embryos at the eight- to 16-cell stage were subjected to one of five treatments. They were (i) cultured as unfrozen controls, (ii) frozen once and cultured, (iii) subjected to two consecutive freeze-thaw cycles, (iv) frozen and thawed, and then cultured for 18-30 h before being frozen a second time, and (v) frozen three times in succession without being cultured. To assess their functional survival after freezing and thawing, all embryos were cultured in vitro to the hatched blastocyst stage in Whitten's medium. In one experiment, hatched embryos that developed after one, two or three cycles of freezing and thawing were stained with Hoechst 33342 to determine their mean cell number. More embryos of the culture control group and the once-frozen group developed into hatching blastocysts than those of the refrozen groups. There was no difference in the second post-thaw rate of in-vitro development for embryos refrozen with the culture-refreeze or direct-refreeze procedure. Furthermore, there was no difference among in-vitro development rates for embryos frozen two or three times. However, among those embryos subjected to repeated cycles of freezing and thawing that did not survive, there was a considerable amount of damage to their zonae pellucidae. Furthermore, frozen mouse embryos had fewer cells per embryo at the time of hatching than the unfrozen embryos. Nevertheless, these results demonstrate that mouse embryos can survive even three successive freeze-thaw cycles yet still be capable of in-vitro development.      

Laser zona pellucida thinning--an alternative approach to assisted hatching

BACKGROUND: The purpose of this study was to assess the efficacy and hatching characteristics of in-vitro cultured human embryos subjected to laser zona pellucida thinning. METHOD: Zona thinning was performed on 110 embryos using a non-contact 1.48 microm diode laser and the hatch rate in vitro was compared with 42 control embryos. Variation of zona thickness and degree of zona expansion was assessed. Scanning electron microscopy was performed on embryos entrapped during hatching to identify the site of hatching. RESULTS: The rate of hatching was significantly higher in laser thinned blastocysts compared with control embryos (68 versus 33% respectively, P < 0.01). Laser thinning increased the variation of zona thickness in embryos from 11.6-27.3%. Natural zona thinning occurred in 92% of laser thinned hatching blastocysts and 100% of control embryos. CONCLUSION: These results suggest that laser zona thinning is effective and may provide significant advantages over conventional assisted hatching techniques, which create holes.     

Evolution of a culture protocol for successful blastocyst development and pregnancy

A cell-free culture system was designed for human embryo development to the blastocyst stage by testing a range of culture conditions in a series of protocols. The culture system that was evolved has a brief 1 h exposure to spermatozoa and then culture of the pronucleate zygote for 2 days in IVF-50 medium. Two or three embryos were cultured together in 20 microl microdrops of medium under oil. Embryos were then regrouped and two or three at a similar stage were cultured together in 50 microl microdrops of Gardner's G2 medium under oil from days 3 to 5. Embryos were transferred to fresh G2 medium on day 5 and cultured for a further 1 or 2 days (day 6 or 7). No serum was used in any of the cultures. The embryo transfer medium and G2 medium were supplemented with human serum albumin. The zonae of all blastocysts to be transferred to patients were completely removed enzymatically. Using this protocol, 52% of zygotes developed to blastocysts and 34 out of 35 patients treated received 82 blastocysts and 11 morulae on day 5 or 6. Twenty-one fetal sacs with positive heartbeats (23% implantation rate) were detected in 13 ongoing pregnancies (38% pregnancy rate/transfer or 37%/patient treated). We anticipate that further improvements in embryo development and the selection of viable embryos can be achieved using this simple and effective culture system.