Search

The aim of this study was to explore the effect of three pro-nuclei (3PN) incidence on clinical outcomes in the fresh cleavage-stage embryo transfer (CSET) and blastocyst-stage embryo transfer (BSET) cycles.This retrospective cohort study included 1427 CSET cycles, 632 BSET cycles, and 313 elective single BSET cycles from January 2013 to June 2015. The patients were divided into two groups as follows: Group 1 included patients with no 3PN zygotes and Group 2 included patients with20% 3PN zygotes.We observed that the fertilization rate was significantly lower in 3PN = 0% than 3PN 20% group (p 0.05), but the day-3 grade I + II embryo and day-3 grade I + II + III embryo rates were not significantly different between 3PN = 0% and 3PN 20% group (p 0.05). Interestingly, in the CSET, the implantation (42.87% and 41.76%, p = 0.585) and clinical pregnancy (59.94% and 58.25%, p = 0.538) rates were not significantly different between two groups. In the BSET, the implantation (61.93% and 49.62%, p 0.001) and clinical pregnancy rates (69.45% and 61.02%, p = 0.043) were significantly higher in 3PN = 0% than 3PN 20% group. In the elective single BSET, the implantation (68.91% and 61.33%, p = 0.223) and clinical pregnancy rates (68.48% and 61.33%, p = 0.251) were higher in 3PN = 0% than 3PN 20% group, but there was no significant difference.We concluded that a high 3PN incidence may predict poor outcomes in BSET but not CSET cleavage-stage.

Item does not contain fulltext

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Resume Objectif Depuis fin 2010, apres expertise de la litterature faisant etat a la fois de la superiorite de cette methode en termes de survie des embryons et de son innocuite pour les enfants nes, l’Agence de Biomedecine a valide la vitrification embryonnaire comme amelioration technique de la congelation lente (CL). Afin de constituer notre experience de cette technique, nous avons collecte a partir d’une serie prospective observationnelle les issues biologique et clinique apres decongelation des embryons vitrifies comparees aux resultats d’une serie retrospective faisant etat de la decongelation d’embryons issus de CL. Nous rapportons egalement dans cette etude la premiere naissance en France apres utilisation de la technique de vitrification au stade precoce du developpement embryonnaire. Patientes et methodes Cette etude prospective a concerne les 58 premiers cycles de decongelation d’embryons vitrifies a J2/3 pour lesquels l’issue clinique etait documentee. Les donnees obtenues ont ete comparees a une serie retrospective de 189 cycles de decongelation apres CL. Seuls les rangs 1 et 2 de decongelation embryonnaire ont ete inclus. Le critere de jugement principal etait le taux de survie (TS) (% d’embryons ayant ≥ 50 % de blastomeres intacts apres decongelation), associe au taux de survie intact (TSI) (% d’embryons ayant 100 % de blastomeres intacts apres decongelation) et a l’indice de survie blastomerique (ISB) (% de blastomeres intacts par embryon ayant survecu). Le critere de jugement secondaire etait le taux de grossesse clinique (TGC), defini comme la presence d’un sac gestationnel intra-uterin avec activite cardiaque. Nous rapportons le cas de la premiere naissance francaise apres vitrification embryonnaire. Resultats Au total, 87 et 412 embryons ont ete decongeles apres vitrification et CL. Nous avons observe une difference hautement significative respectivement des TS, TSI et ISB apres decongelation des embryons vitrifies lorsque compares aux embryons decongeles issus d’une CL (98,3 ± 13,1 % vs 77,3 ± 32,0 %, p −4 ; 88,2 ± 28,3 % vs 47,7 ± 41,4 %, p −4 ; 97,7 ± 6,1 % vs 87,3 ± 14,4 %, p −4 ). Par ailleurs, le TGC par cycle de decongelation embryonnaire etait respectivement de 32,7 % (19/58) et 18,5 % (35/189) ( p = 0,03) en faveur du groupe des embryons decongeles apres vitrification. La naissance a 38 semaines d’amenorrhee de deux enfants bien portants de sexe feminin a eu lieu par cesarienne le 8 aout 2011. Discussion et conclusion Nous avons verifie dans cette serie preliminaire que les embryons vitrifies resistaient significativement mieux au processus de congelation/decongelation que ceux ayant ete congeles par CL. Par voie de consequence, ce resultat etait associe a une amelioration du TGC par decongelation.

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.

Preimplantation genetic diagnosis (PGD) is the screening of embryos at the cleavage stage in order to select and transfer only the desired embryo. The main indications of PGD are monogenic disorders and aneuploidy. This article reviews the recent advances in PGD methodology and presents their advantages, disadvantages and challenges. Ethical considerations arising with more widespread use of PGD for physical traits is also discussed. PGD for monogenic disorders remains the most successful indication, but is technically more challenging. PGS for aneuploidy has passed through a period of reconsideration and is now re-emerging with complete genome screening.

Objective To determine the optimum pH in sequential media for embryo culture around the fertilization-zygote stage and cleavage stage, with use of a mouse embryo assay. Design Experimental laboratory study. Setting University Hospital and University Research Unit. Animal(s) F1 hybrids between CD1 female and BDF male mice. Intervention(s) Fertilized, one-cell mouse embryos were cultured 5 days in test media where pH was changed at defined time intervals. Main Outcome Measure(s) Percentage of good-quality embryos, defined by strict morphology. Result(s) A significantly improved development was observed when pH was as high as 7.30 before the pronuclear stage and lowered to pH 7.15 during the cleavage period. Conclusion(s) Good embryo development is consistent with two different pH values in sequential culture media. This could have important implications for embryo culture in human IVF.

Objective Occasionally, submission of blastomere(s) or trophectoderm cells for preimplantation genetic screening (PGS) may be interpreted as inconclusive (“no results”). The main reasons for those results include failure of DNA amplification, degraded DNA, or failure to load cell(s) into the biopsy tube. In a particular case at our clinic, blastomere biopsies were submitted for PGS and all embryos were labeled as “no results”. Interestingly, the zona pellucida (ZP) of all embryos was extremely elastic and blastomere displacement was difficult. We theorize that the high elasticity and extended micromanipulation procedures may have affected the DNA in such a way as to interfere with the accurate assessment/interpretation of results. Design Case Report. Materials and Methods A total of 43 oocytes were harvested at retrieval. Oocytes were inseminated via standard IVF or ICSI. Ten cleavage stage embryos with a minimum of 6 blastomeres were selected for biopsy/PGS screening. Assisted hatching (AH) via partial zona dissection (PZD; tridimensional) was performed prior to blastomere biopsy. Embryo biopsy was performed via blastomere displacement. Results The ZP of all biopsied embryos was highly elastic. In addition, the inner layer of the ZP appeared to be detached in certain areas for some embryos. Those embryos acquired an oval-elliptical shape. The lack of rigidity prevented generation of internal pressure inside the embryo, which is necessary to displace the blastomere(s). The ZP of each embryo had to be probed at different angles in order to displace the selected blastomere(s). The time required to harvest blastomeres was extended to approximately ≥5 min per embryo. Selected blastomeres were eventually isolated without evidence of damage to the remaining blastomeres. All embryos were labeled as “no results” after assessment by the genetic laboratory. The report and additional options were discussed with the patients, and it was decided to proceed with embryo transfer (D5) based on blastocyst morphology. Two high quality blastocysts were transferred, resulting in a singleton term pregnancy. The remaining viable embryos were considered for vitrification. Conclusions Zona pellucida elasticity and extended manipulation of embryos/blastomeres may result in a form DNA degradation that interferes with the accuracy or interpretation of results during genetic assessment. Further analysis of similar cases may shed light into the relationship between difficult biopsies and inconclusive PGS results. Disclosures Nothing to disclose.

The effect of cleavage-stage group culture (CGC; embryos cultured in groups of three or more for the first 3 days and then individually to blastocyst) was compared to extended single embryo culture (ESC; embryos cultured individually to the blastocyst stage). While implantation and ongoing pregnancy rates were similar between groups, the blastocyst utilization rate (number of blastocysts suitable for freezing and thawing/total number of embryos cultured to Day 5 and 6) was significantly higher when embryos were cultured in CGC for women ≤35 yrs thereby increasing the number of embryos available for clinical use for the younger women. This strategy of group culture to Day 3 would seem an ideal protocol to capitalize on an overall embryo quality in two particular settings, namely programmes wishing to (i) undertake Day 3 transfers, and (ii) keep embryos separate from Day 3 to Day5/6 for the purposes of selection. The culture system can also be applied to the embryos of older women without adverse effect, enabling the same system to be used for all embryos.

Zusammenfassung 2zellige Kaninchenembryonen, die 26 Stunden nach der Paarung gewonnen wurden, konnten sidi nach 4–6stundiger Cytodialasin-B-Behandlung (10 μg/ml) und anschliesender 8stundiger Kultivierung im Kontrollmedium zu 87,5% zu mutmaslich tetraploiden 4-Zellern entwickeln. Wahrend der sich anschliesenden bis zu 48stundigen Kultivierungsperiode konnten sich 74% weiterentwickeln. 29,7% der Embryonen waren tetraploid, 18,9% wiesen sowohl tetraploide als auch diploide Metaphasen auf. Bei 16,2 % waren die Blastomeren wahrend der Furchungen nach der CB-Behandlung zweikernig geblieben, und die Kerne befanden sich in einem synchronen Stadium. Die restlichen Embryonen waren nicht analysierbar. Summary In vitro development of cytochalasin — B — induced tetraploid and diploid/tetraploid rabbit embryos before implantation Two-cell rabbit embryos, obtained 26 hr after coitus were found, after 4–6 hr treatment with cytochalasin-B (10 μg/ml) and then 8 hr cultivation in control medium, to have developed in the case of 87.5% of them, into probable tetraploid 4-cell embryos. During subsequent culture up to 48 hr some 74% developed further. 29.7% of the embryos were tetraploid; 18.9% had both tetraploid and also diploid metaphases. In 16.2% the blastomeres remained binucleate during CB treatment and the nuclei were in a synchronous state. The remaining embryos could not be analysed. Resume Developpement in vitro avant l'implantation d'embryons tetraploides et diploides/tetraploides de lapins induits par la cytochalasine B Des embryons a deux cellules de lapins obtenus 26 heures apres la fecondation se sont developpes apres un traitement de 4–6 heures a la cytochalasine B (10 μg/ml) et apres 8 heures en milieu de controle en 4 cellules probablement tetraploides dans une proportion de 87%. Le 74% s'est ensuite developpe jusqu'a 48 heures durant la periode de culture. Le 29,7% des embryons fuent tetraploides et le 18,9% presenterent une metaphase aussi bien tetraploide que diploide. Le 16,2% monta des blastomeres restees a deux noyaux durant la segmentation apres le traitement a la CB et les noyaux etaient a un stade synchrone. Le reste des embryons n'a pas pu etre analyse. Resumen Desarrollo in vitro de embriones tetraploides y diploides/tetraploides de coneja, inducidos por citochalasina B (CB), antes de la implantacion Se pudieron desarrollar embriones bicelulares de conejas, los cuales se habian logrado recoger 26 horas despues del apareamiento, tras tratamiento durante 4–6 horas con citochalasina B (10 μg./ml.) y cultivo ulterior durante 8 horas en medio de control hasta dar un 87,5% de tetracelulares tetraploides presuntos. Durante el periodo de cultivo que seguia y que duraba hasta 48 horas se pudo desarrollar el 74%. 29,7% de los embriones eran tetraploides, 18,9% presentaban tanto metafases tetraploides como diploides. En el 16,2% se habian quedado los blastomeros binucleares durante las segmentaciones tras el tratamiento con CB, y los nucleos se hallaban en un estadio sincronico. Los embriones restantes no pudieron ser analizados.

Aim: Using oocyte donation cycles as an ideal model, we sought to compare pregnancy and implantation rates in cleavage stage (day 3) versus blastocyst stage (day 6) embryo transfers (ET); assess the predictive value of blastocyst formation rates based on cleavage cell stage and morphology grade; and evaluate the ability to predict formation of high quality (HQ) blastocysts. Methods: Ninety three consecutive oocyte donation cycles from July 2003 to August 2005 were retrospectively evaluated and analyzed to determine if either resulted in a cleavage stage (n = 30) or blastocyst (n = 45) ET. The primary outcomes measured pregnancy rates, the percent development of HQ blastocysts based on day 3 embryo status, and the ability to select day 3 embryos suitable for transfer among four blinded evaluators by assessing their day 6 embryo outcome. Results: Cleavage stage ET resulted in significantly lower pregnancy rates, clinical pregnancy rates, and implantation rates (47% [n = 14/30]; 40% [n = 12/30] and 27 ± 7%) compared to blastocyst stage (82% [n = 37/45]; 73% [n = 33/45] and 64 ± 6% [±SE], P

A mature animal body contains a variety of different cell types, and these cells are distributed in a well-organized fashion along the body axes. One of the major questions in developmental biology is how cells acquire different characteristics. In addition, it is important to understand how the embryo forms the body axes and how cells are allocated along these axes during development. Among mammalian species, the molecular mechanisms that regulate embryonic development have been well analyzed and characterized in mice. Here, mouse preimplantation embryonic development is briefly summarized and our current understanding of this complex process based on recent observations is reviewed.

To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator.We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators.No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%).These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

To analyze using hypergeometric probability statistics the impact of performing preimplantation genetic screening (PGS) on a cohort of day 3 cleavage stage embryos.Statistical mathematical modeling.We find the benefit of performing PGS is highly dependent on the number of day 3 embryos available for biopsy. Additional hidden variables that determine the outcome of PGS are the rates of aneuploidy and mosaicism, and the probability of a chromosomally mosaic embryo to test "normal". If PGS is performed, our analysis shows that many combinations of the number of biopsiable embryos, and the rates of aneuploidy and mosaicism results in a marginal benefit from the intervention. Other combinations are detrimental if PGS is actually undertaken. Finally, increases in PGS error rates lead to a rapid loss in the ability of PGS to provide useful discriminatory information.We set out the statistical framework to determine the limits of PGS when a specific number of day 3 preimplantation embryos are available for biopsy. In general, PGS cannot be recommended a priori for a specific clinical situation due to the statistical uncertainties associated with the different hidden variable quantitative parameters considered important to the clinical outcome.

Objective To compare and evaluate the neonatal outcome in infants born after vitrified day 3 embryo transfers with that of fresh day 3 embryo transfers. Design Retrospective analysis. Setting Private assisted reproductive center. Patient(s) Sixty-nine women, who delivered 89 babies. Intervention(s) Surplus day 3 cleavage stage embryos were vitrified using ethylene glycol-based solution by cryoloop carrier system and transferred after warming. Main Outcome Measure(s) Post-thaw survival of embryos, implantation and pregnancy rates (PR), neonatal outcome, and congenital birth defects. Result(s) A total of 907 vitrified day 3 embryos from 285 cycles were warmed and 817 survived (90.37%), which were used for embryo transfer. The pregnancy, implantation, miscarriage, and live birth rates achieved were 36.84%, 18.11%, 7.71%, and 24.21%, respectively. Of the 69 deliveries, singleton, twins, and triplets comprised of 72.46%, 26.08%, 1.44%, respectively, and 8 (11.59%) were preterm deliveries. The mean Apgar scores were 8.3, 9.2, 9.4 at 1, 5, and 10 minutes. The congenital birth defect rate was 1.18%. These results were comparable with pregnancies using fresh embryo transfers. Conclusion(s) This preliminary study shows that vitrification is an efficient method of cryopreservation of human day 3 embryos with neonatal outcome comparable to fresh embryo transfers.

Chromosome instability is a hallmark of tumorigenesis. This study establishes that chromosome instability is also common during early human embryogenesis. A new array-based method allowed screening of genome-wide copy number and loss of heterozygosity in single cells. This revealed not only mosaicism for whole-chromosome aneuploidies and uniparental disomies in most cleavage-stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders.

The investigation into lineage allocation and early asymmetries in the pre-and peri-implantation mouse embryo is gaining momentum. As we review here, new insights have been gained into the cellular and molecular events that lead to the establishment of the three lineages of the blastocyst, to the determination of the origin and the fates of the visceral endoderm in the peri-implantation mouse embryo, and to the generation of cellular and molecular activities that accompany the emergence of asymmetries in the pre-gastrulation embryo. We also discuss the continuing debate that surrounds the relative impacts of early lineage bias versus the stochastic allocation of cells with respect to the events that pattern the blastocyst and initiate its later asymmetries.

Udgivelsesdato: null-null PURPOSE: The aim was to elucidate if the nuclear size and number are indicative of aberrant chromosome content in human blastomeres and embryos. METHODS: The number of nuclei and the nucleus and blastomere size were measured by a computer controlled system for multilevel analysis. Then the nuclei were enumerated for 13 chromosomes by a combination of PNA and DNA probes. RESULTS: In the mononucleated embryos there was no difference in the mean size of chromosomally normal and abnormal nuclei but a significant difference in the mean nuclei size of nuclei that had gained chromosomes compared to nuclei that had lost chromosomes. The nuclei from multinucleated blastomeres had a significant smaller mean size and the frequency of chromosomally aberrant blastomeres was significantly higher. CONCLUSION: The mean nuclear size is not a marker for the chromosome content in mononucleated embryos. However, it seems that the nuclei size can be related to multinucleation and maybe to the chromosome content.

Excess embryos obtained from intracytoplasmic sperm injection cycles may be cultured and observed until day 5 if the couple receiving treatment do not want them to be cryopreserved. In order to investigate the correlation between blastocyst formation in extended culture and pregnancy outcome, 194 patients treated in two separate IVF units were examined retrospectively. The patients were separated into two groups: group 1 with at least one blastocyst formed in culture, and group 2 with no blastocyst formation. The pregnancy rates were 60.0% and 41.7% for groups 1 and 2, respectively. The pregnancy rate in group 1 was statistically significantly higher than in group 2 (P = 0.01). The results suggest that the developmental potential of embryos obtained from a single assisted reproduction treatment cycle may be similar and that blastocyst formation in vitro may help to predict the pregnancy outcome of that cycle.

Background Both cleavage-stage and blastocyst-stage embryo transfer policies have advantages and drawbacks. The number of embryos transferred, however, is a crucial parameter that needs to be considered before attempting any comparison. Methods An extensive literature search yielded initially 282 studies from which 8 randomized controlled trials met the inclusion criteria: (i) truly randomized design (ii) policy to transfer equal number of embryos in both the cleavage-stage and the blastocyst-stage groups and (iii) published as full text in a peer-review journal. Primary outcome was the live birth rate and secondary outcomes were clinical pregnancy rate, multiple pregnancy rate, cancellation rate and cryopreservation rate. Results A total of 1654 patients were reviewed. Live birth rate per randomized patient was significantly higher (n = 6 studies) in patients who had a blastocyst-stage transfer as compared to patients with cleavage-stage embryo transfer [odds ratio (OR): 1.39, 95% confidence interval (CI): 1.10-1.76; P = 0.005]. Clinical pregnancy rate (OR: 1.27, 95% CI: 1.03-1.55; P = 0.02) and cancellation rate per patient randomized (OR: 2.21, 95% CI: 1.47-3.32; P = 0.0001) were significantly higher in patients with a blastocyst-stage embryo transfer as compared to patients in whom a cleavage-stage embryo transfer was performed. The cryopreservation rate was significantly higher in the cleavage-stage group (OR: 0.28, 95% CI: 0.14-0.55; P = 0.0002). Conclusions The best available evidence suggests that the probability of live birth after fresh IVF is significantly higher after blastocyst-stage embryo transfer as compared to cleavage-stage embryo transfer when equal number of embryos are transferred in the two groups compared.

In fresh IVF cycles, embryos reaching the eight-cell stage on day 3 of development are thought to have a higher chance of implantation than those reaching this stage on day 4. To determine whether this difference persists after cryopreservation, we compared pregnancy and implantation rates between frozen embryo transfer (FET) cycles using delayed cleavage-stage embryos (cryopreserved day 4) and normal cleavage-stage embryos (cryopreserved day 3).Participants underwent FET between 2008 and 2012 using embryos cryopreserved on either day 3 (n = 76) or day 4 (n = 48), depending on the length of time needed to achieve the eight-cell stage. All embryos, regardless of day of cryopreservation, were thawed and transferred on the 4th day of vaginal progesterone following endometrial preparation with oral estradiol. Chi-square and Mann-Whitney U tests were used to compare patient demographics and cycle outcomes.More women in the day 4 group had diminished ovarian reserve (44 vs 16 %, p = 0.003). Pregnancy outcomes in preceding fresh cycles were not different between the two groups. Pregnancy, implantation, and live birth rates following FET did not differ between the day 3 and day 4 groups.This is the first study to address outcomes using day 3 versus day 4 cryopreserved embryos. Despite a higher prevalence of diminished ovarian reserve (DOR) in the day 4 group, delayed cleavage-stage embryos utilized in FET cycles performed as well as embryos growing at the normal rate, suggesting delayed embryo development does not affect embryo implantation as long as endometrial synchrony is maintained.

Various time-lapse studies have postulated embryo selection criteria based on early morphokinetic markers. However, late paternal effects are mostly not visible before embryonic genome activation. The primary objective of this retrospective study was to investigate whether those early morphokinetic algorithms investigated by time-lapse imaging are reliable enough to allow for the accurate selection of those embryos that develop into blastocysts, while of course taking into account the correlation with the type of injected spermatozoa. During a period of 18 months, a total of 461 MII oocytes from 43 couples with severe male factor infertility and previous “external” IVF failures after cleavage-stage embryo transfer (ET) were fertilized by intracytoplasmic morphologically selected sperm injection (IMSI). Thereof, 373 embryos were monitored in a time-lapse incubator until ET on day 5. Blastocyst outcome in combination with three previously postulated MKc (cc2: t3–t2, 5–12 h; t3, 35–40 h; t5, 48–56 h) and the morphology of the selected sperm were analyzed. A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

Early mammalian development is regulative - it is flexible and responsive to experimental intervention. This flexibility could be explained if embryogenesis were originally completely unbiased and disordered; order and determination of cells only arising later. Alternatively, regulative behaviour could be consistent with the embryo having some order or bias from the very beginning, with inflexibility and cell determination increasing steadily over time. Recent evidence supports the second view and indicates that the sequence and the orientations of cell divisions help to build the first asymmetries.

The paper provides a review of the current state of knowledge on apoptosis during normal preimplantation development based on the literature and on the authors' own findings. Information is focused on the occurrence and the characteristics of spontaneous apoptotic processes. Reports concerning the chronology and the incidence of programmed cell death in mouse, cow, pig and human embryos in early preimplantation stages up to the blastocyst stage are summarized. In addition, specific attributes of the apoptotic process in mammalian preimplantation development are provided, including the description of both morphological and biochemical features of cell death.

Phosphatized fossils from the Neoproterozoic Doushantuo Formation have provided valuable insight into the early evolution of metazoans, but the preservation of these spectacular fossils is not yet fully understood. This research begins to address this issue by performing a detailed specimen-based taphonomic analysis of the Doushantuo Formation phosphatized metazoan embryos. A total of 206 embryos in 65 thin sections from the Weng'an Phosphorite Member of the Doushantuo Formation were examined and their levels of pre-phosphatization decay estimated. The data produced from this examination reveal a strong taphonomic bias toward earlier (2-cell and 4-cell) cleavage stages, which tend to be well-preserved, and away from later (8-cell and 16-cell) cleavage stages, which tend to exhibit evidence for slight to intense levels of organic decay. In addition, the natural abundances of these embryos tend to decrease with advancement in cleavage stage, and no evidence of more advanced (beyond 16-cell) cleavage stages or eventual adult forms were found in this study. One possible explanation for this taphonomic bias toward early cleavage stages is that later cleavage stages and adult forms were more physically delicate, allowing them to be more easily damaged during burial and reworking, allowing for more rapid decay. The spectacular preservation of these embryos was probably aided by their likely internal enrichment in phosphate-rich yolk, which would have caused their internal dissolved phosphate levels to reach critical levels with only miniscule organic decay, thereby hastening phosphatization. If internal sources of phosphate did indeed play a role in the phosphatization of these embryos, it may explain their prolific abundance in these rocks compared to other phosphatized fossils as well as indicating that metazoans lacking such internal phosphate sources were likely much more difficult to preserve. The phosphatic fossils of the Doushantuo Formation, therefore, provide an indispensable, yet restricted, window into Neoproterozoic life and metazoan origins.

The embryonic and larval development of Thai pangas was investigated during peak (May-July 1995) and late spawning (August-October 1995) periods. The fertilized eggs are adhesive and spherical with a yellowish or greenish-brown egg capsule. The yolk sac is yellowish-brown in color and 1.20-1.80 mm in diameter. Nine hours post-fertilization, the first cleavage stage, embryonic shield, head, tail region, neural grooves and somites were evident. The incubation period ranges from 24-36 h at a temperature of 20-30 degrees C. The newly hatched larvae are quite transparent and light yellowish in color with a body length of 2.98-3.10 mm. Eye pigments appear and the heart starts to work within 12-14 h of hatching. In 1-day-old pro-larvae, the mouth becomes well developed; barbules are elongated, prominent and look like tiny threads. The yolk sac is fairly well absorbed and the palatine teeth are fully developed during the 3 day pro-larval stage. At the end of 12 days of larval development, the stomach becomes functional and aerial respiration starts. After 2 weeks, the young fry is well-developed, and is of an adult appearance, that is, measuring up to 13.56 mm in length.

The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.

There are many morphological transformations during development of human embryos that mainly involve phenomena that can be easily assessed in living embryos by simple non-invasive microscopical observation. A clear correlation between pronuclear morphology and the ability of the resulting embryo to continue developing and to implant has been described. There is also general agreement that a positive relationship exists between early embryo morphology and implantation rate. The parameters classically involved in embryo evaluation are: cleavage rate, blastomere symmetry, cytoplasmic appearance, extent of fragmentation and blastomere nuclear status. In this paper, morphological features that have been related to embryo developmental potential are described. Furthermore, the ability of a cumulative classification scheme developed in the laboratory to predict blastocyst formation and implantation is analysed.

Geophilomorph centipedes exhibit a number of unique characteristics that make them of particular developmental and evolutionary interest. Segment numbers in geophilomorphs are higher than in any other centipedes, ranging from 27 to 191. They may be constant within a species, presenting in extreme form the "counting" problem in development, or they may vary--a situation that provides us with the opportunity to study naturally occurring variation in segment numbers. All their segments are generated during embryogenesis, a situation unlike that in the more basal centipede orders, which generate only a fraction of their 15 trunk segments in the embryo and develop the rest postembryonically. Here we provide a foundation for further developmental studies of the Geophilomorpha, building on the one study that has been conducted to date, on the coastal species Strigamia maritima. Development begins with the migration of nuclei to the surface of the egg, which then condense to form an embryonic rudiment of more than 20,000 cells, covering an entire hemisphere. During early development, the embryo can be divided into two distinct areas: a large terminal disc of apparently undifferentiated tissue and the germ-band, which has a clear anteroposterior axis and differentiated segments. The germ-band forms from the anterior of the terminal disc and extends anteriorly as the disc contracts. New segments are formed at the posterior margin of the germ-band. Once the process of segmentation ends, the germ-band folds and sinks into the yolk. We note that the classic description of centipede development, by Heymons more than a century ago, contains a fundamental error in the identification of the axes and hence in the interpretation of early segmentation.

Embryonic development of common dentex (Dentex dentex) was investigated at nine different constant water temperatures (8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C and 24°C). The observed effects were compared using of regression analysis. Constant water temperatures between 12°C and 18°C were found to support successful embryonic development. A negative relationship between the rate of embryonic development and incubation temperature was observed. While embryonic development was completed within this range (12°C–18°C), there was no cell division at water temperatures of 8°C and 24°C. Total mortalities were observed at the 128 cleavage stage at a temperature of 10°C, and after the beginning of gastrulation at 20°C and 22°C.

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.

Segmentation is a fundamental process in vertebrate embryogenesis, and one of the earliest manifestations of segmental patterning is the generation of transient, serially repeated blocks of mesodermal cells known as somites. Disruption of the normal segmentation process in humans leads to vertebral abnormalities such as spondylocostal dysostosis. In this minireview, we discuss recent advances in the dynamic molecular and cellular mechanisms governing segmentation.

Purpose: To evaluate the sex ratio in births conceived with blastocyst transfer compared to day 3-ET.

Purpose: To elucidate the appropriateness of current indications for assisted hatching (AH) in cleavage stage human embryos and to confirm our preliminary findings that only young patients (about 67%) benefit from AH.

The incidence of monozygotic twinning appears to be increasing within the field of assisted human reproduction. Many theories have been put forward as to how and when this occurs. Whatever the cause, the normal events of embryo development, which necessarily involve axis formation, patterning and polarization, need to be adhered to in order to obtain a viable offspring. This paper describes the course of development in terms of axis formation and polarity and offers suggestions as to how either a disruption of this or duplication events in the course of the formation of these parameters could prevent or contribute to a twinning event. The likelihood of twinning occurring at any point is discussed in terms of the establishment of polarity and axes.

▪ Abstract Over the past dozen years, studies comparing the expression of orthologues of the Drosophila segmentation genes among various insects have served to broaden our view of the ways in which insects make segments. The molecular data suggest that, although the overall genetic mechanisms of segmentation during embryogenesis have been conserved, the details of this process vary both within and between various insect orders. Here we summarize comparative gene expression data relevant to segmentation with an emphasis on understanding the extent of molecular patterning prior to gastrulation. These results are discussed in embryological context with an eye toward understanding the evolution of segmentation within insects.

To analyze differences in morphokinetic parameters of chromosomally normal and aneuploid embryos utilizing time-lapse imaging and CGH microarray analysis.This retrospective cohort study included patients undergoing IVF treatment and preimplantation genetic diagnosis for sex selection. A total of 460 embryos cultured in incubators with time-lapse imaging system (EmbryoScope) were selected for biopsy on day 3 of development. Subsequently, CGH microarray analysis was performed for aneuploidy screening of 24 chromosomes. Kinetic parameters including time for appearance of second polar body (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPBf), time to division to 2(t2), 3(t3), 4(t4), 5(t5) cells, length of second and third cell cycle (CC2= t3 t2, CC3=t5-t3), synchrony of cell division from 2 to 4 cells (S2=t4-t3) and interval t5-t2 were analyzed to compare chromosomally normal and abnormal embryos.The mean time durations for tPNf, t2, t5, CC2, CC3, t5-t2 differed significantly between normal and abnormal embryos.Time-lapse imaging morphokinetics may play a role in early prediction of aneuploid embryos due to differences in kinetic behavior that may aid in improving clinical outcome.

Study question Can the equilibration steps prior to embryo vitrification be automated? Summary answer We have developed the 'Gavi' system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives in vitro outcomes equivalent to the manual Cryotop method. What is known already Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries. Study design, size, duration The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls. Participants/materials, setting, methods The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and in vitro embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe. Main results and the role of chance Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent in vitro outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (n = 176) and manual Cryotop method (n = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (n = 23) were also equivalent to Cryotop controls (n = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136°C/min and 11 239°C/min, respectively. Limitations, reasons for caution Testing of the Gavi system described here was limited to in vitro development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos. Wider implications of the findings The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic. Study funding/competing interests This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.

Purpose of review The purpose of the review is to summarize recent developments in time-lapse technologies and early embryo morphokinetics and to discuss their impact on current clinical outcomes. Recent findings Contemporary embryo culture and selection methodologies that are based on classical morphology are clearly limited in providing the most suitable embryo for a successful pregnancy. Noninvasive observation of embryo development by capturing the images with a time-lapse device has recently been proposed to be a better method of embryo viability assessment. Such methodologies have been shown to increase the quality and the quantity of information on the viability without disturbing the culture conditions. Summary Commercial availability of different time-lapse devices for human embryos facilitated the use of morphokinetics as an additional tool in human embryo selection. The application of such technologies has already shown positive results on clinical outcome by increasing our scope of traditional embryo selection, leading to higher implantation and clinical pregnancy rates. Additional benefit can come from the less-disturbed incubation environment that is created by all-in-one incubators. Such devices can also be very important research tools in order to observe and analyze the effect of different patient-specific or clinical conditions on embryo development parameters that are not available through classical embryo scoring.

Globular fossils from Doushantuo phosphorites at the Weng’an area are for the first time identified as a gastrulation stage of phosphatized embryos. They are got from the fossiliferous remains after acetic acid maceration. The fossils are found together with formerly reported animal resting eggs and embryos of the earlier cleavage stage. The oblate-shaped fossils with the same size as those reported embryos and invaginate at the middle part into the embryos, show the characteristics of the late blastula to the early gastrula stage of the embryo development. This discovery convinces the existence of animal embryos at Doushantuo age and offers new facts for the studying of the affinity of related fossils, which are still controversial at present.