Uvod: V ramci jubilejniho 25. sympozia asistovane reprodukce byl pracovni oběd již posedme organizovaný formou diskuse, ktera se zabývala vybranými tematy asistovane reprodukce. U diskusnich stolů se seslo vice než 150 ucastniků sympozia, kteři diskutovali o aktualnich tematech reprodukcni mediciny. Řesena temata: Vybrana temata se týkala aktualnich otazek z oblasti gynekologie a embryologie. Během oběda byla řesena tato temata: (1) přeshranicni zdravotni pece v asistovane reprodukci, (2) indikace k PGS (preimplantacniho genetickeho screeningu) ve světle nových poznatků, (3) patři stimulace ovarii do ambulanci registrujicich gynekologů? (4) lecba klomifenem – jen pro odborniky v reprodukcni medicině? (5) jak a komu směrovat psychologickou podporu při IVF, (6) stimulace žen s nizkou ovarialni rezervou, (7) je vysetřeni spermiogramu dostacujici? (8) relevantni markery embryonalniho vývoje – time laps system, (9) jak se zorientovat v nabidce medii a spotřebniho materialu v IVF laboratoři a (10) freeze all – nový trend v kryokonzervaci vhodný pro vsechny? Zavěr: Veskere zavěry jednotlivých diskusi byly prezentovany vsem ucastnikům konference v odpolednim bloku a jsou uvedeny v teto praci. Tato cast byla zajimava pro mnoho ucastniků konference a měla hojnou ucast. Některa temata přinesla konkretni výsledek. Některa nebylo možne jednoznacně uzavřit a budou předmětem dalsich jednani v budoucnu.

Introduction: During the 25th symposium of assisted reproduction in Brno was lunch time organised as the lunch table discussion on the selected topics of assisted reproduction. More than 150 specialists reviewed themes related to gynecology and embryology.Discussed topics: Lunch table discussion covered the following topics: (1) Cross-border health care in assisted reproduction; (2) Indication for PGS (preimplantation genetic screening) in the context of actual information; (3) Does ovarian stimulation belong to the ambulance of registering gynecologists? (4) Therapy with clomifen - only for IVF specialists? (5) How and with whom should psychological support be directed during IVF? (6) Stimulation in women with low ovarian reserve; (7) Is basic semen analyses sufficient? (8) Time-lapse systems as relevant markers of embryonic development; (9) How to be oriented with choices of media and consumables in the IVF lab, and (10), Freeze All" - is this new trend in cryopreservation suitable for all?Conclusions: Panel conclusions were presented during the afternoon session, which had great attendance, featured lively commentary, and produced some definitive consensus. Certain issues remained inconclusive, and these matters will be the subject of further discussion in the future. Specific summation of all deductions is presented in this paper.

OBJECTIVE:The evaluation of the developmental abilities of human embryos according to the timing of their early mitotic cleavages.DESIGN:Retrospective study.SETTING:Prague Fertility Centre and Institute for Care of Mother and Child, CAR, Prague.METHODS:The embryos obtained in IVF program were used for further observations and subjected to automated time-lapse monitoring (PrimoVision, Cryo-Innovation, 1 picture/10 min, intermittent white-light illumination) under standard cultivation conditions (37.0 degrees C, 5% CO2 in humid air). Image sequences were digitally recorded for later use. For intravital spindle detection we used polaryzing microscopy (Oosight, Research Instruments) and Hoechst 33342 fluorescent dye for intravital chromatin visualization. A total number of 180 human embryos which gave a vital pregnancies (FHB, fetal heart beat) were analysed retrospectively for timing of early cleavages. In our study, the exact timing of the four interphases (IP) and synchrony of sister cell divisions (ID, interval division) occurring after fertilization were identified and manually recorded. Interphases: IP1 was defined as the period from fertilization till 2 cell stage. IP2 between 2 and 3 cells stages, IP3 between 3 and 5 and IP4 between 5 and 9 cells embryo. INTERVAL DIVISION: ID2 was recorded as a time interval between 3 and 4 cells, ID3 between 5 and 8 cells and ID4 between 9 and 16 cells stage embryos.RESULTS:In the embryos giving viable pregnancies, the durations of IP1 was 20-26 hrs. IP2 was 10-12 hrs, IP3 was 14-16 hrs and IP4 was 20-26 hrs. In these embryos, the sister blastomeres cleaved in a very synchronous manner. The duration of ID1 was recorded to varry from 120 to 210 min. ID2 from 20 to 60 min., ID3 from 120 to 240 min. and ID4 from 230 to 360 min.CONCLUSION:The viable embryos cleave in a very similar time pattern which can be defined and applied as referencial value. Non-invasive monitoring of the timing of early embryo cleavages can be used as an objectively measurable predictor of human embryo.

Timing of early events during in vitro embryo development has been shown to have strong correlation with embryo viability and implantation potential. Causes of infertility, treatment and in vitro culture system were shown to influence fetal outcome. The aim of this study was to examine the effect of maternal and environmental factors on the timing of morphokinetic events during in vitro embryo development. Retrospective, multicenter trial. Four clinics from 3 countries have randomly selected time-lapse recorded cycles for this pilot study. Time-lapse recordings of 95 transferred embryos were analyzed. Embryos were cultured in microwell dishes, and monitored by automated time-lapse microscopes (PrimoVision, Hungary) inside of conventional incubators. Duration between fertilization and 2cell, 3cell, and 5cell stages, and between 3 to 4cell, and 5 to 8cell stages were recorded. The effects of patients' age, infertility diagnosis and treatment protocol, method of fertilization and culture media were investigated on the timing of these events. Mixed effects model was used for statistical analysis, P<0.05 was regarded as significant. Patients' age had no effect, while the cause of infertility affected cleavage times, e.g. in male infertility patients, the 2cell and 3cell embryonic stages were reached significantly earlier (P=0.01 and P=0.04, retrospectively). Patients with agonist short treatment had a shorter 5 to 8cell stage interval compared to the ones with agonist long treatment (P=0.02). The same interval proved to be longer in case of using Sage culture media, compared to Origio and Vitrolife (P=0.02). The type of infertility, treatment and culture media affects the timing of the first cleavages during in vitro embryo culture. These preliminary results suggest that maternal and environmental factors are to be taken into consideration when evaluating time-lapse recordings. Further factors are also being considered to potentially affect the timing of cleavages.

Chronology of three consecutive mitotic events in human pre-implantation embryos was examined by time-lapse imaging. In zygotes producing well-formed and pregnancy-yielding expanded blastocysts, uniform time-patterning of cleavage clusters (c) and interphases (i) was revealed: i2=11+/-1, i3=15+/-1, i4=23+/-1 h / c2=15+/-5, c3=40+/-10, c4=55+/-15 min. Oppositely, shortened or prolonged durations of one or more cell cycles were strongly predictive of poor implantation and development. Furthermore, trichotomic mitosis was discovered in 17 % of cases - zygotes cleaved into 3 blastomeres and 2-cell embryos into 5-6 cells (instead of normal 2 and 4). During conventional clinical assessment, such embryos are indistinguishable from normal, often considered just-in-course of the next cell cycle. Only detailed time-lapse monitoring paced at 10-minute intervals had proven all these embryos to be absolutely unviable, even in rare cases when they reduced their hypercellularity to normal cell counts via cell-cell fusion. Overall, we demonstrate that time-lapse embryo cleavage rating (ECR) as a standalone diagnostic procedure allows for effective identification of viable early embryos with 90 % specificity, while elimination of good-looking but unviable embryos can be assumed with a specificity of 100 %. Thus, making this non-invasive and contactless approach worth of addition to routine embryo screening in clinical IVF programs.

In 1530 consecutive patients undergoing ultrasound-guided embryotransfer (ET) the influence of patient's age, transferring person, fresh or frozen-thawed embryos, quality of transfer, quality of catheter visualisation and influence of ,,jet" phenomenon on ET results was evaluated. Embryotransfers were performed randomly by six different gynacologists using transabdominal ultrasound probe and COOK Soft EchoTip catheter. The probability of successful ET (clinical pregnancy) is dependent on age (OR 2,59, 95% CI 1,38-4,86), transfer of fresh vers. frozen-thawed embryo (OR 1,37, 95% CI 1,08-1,73), on observation of ,,jet phenomenon" (OR 1,58, 95% CI 1,07-1,73) and also on transferring person. Surprisingly the quality of ET procedure and catheter tip visualisation did not significantly influenced the achieved results.

There is a growing body of evidence to suggest that the standard technique for embryo transfer (ET) is inadequate. The aim of this prospective, descriptive, noncomparative study was to investigate if there are factors detectable during transabdominal ultrasound-guided ET influencing the results of in vitro fertilization (IVF) and ICSI. For statistic evaluation χ2 test was used. Methods: In 678 consecutive patients who were undergoing ET the influence of patient's age, transferring person, fresh or frozen–thawed embryos, quality of transfer, quality of catheter visualisation and influence of “jet” phenomenon was evaluated. Results: The use of ultrasound monitoring (using retrospective data) resulted immediately in lower variation in pregnancy rates both in fresh and frozen ET cycles compared to the old “clinical touch” technique (29.3±2 vs. 24.0±3.78). In our prospective study age is a factor significantly (p<0.001) influencing the embryo transfer results (PR/ET 36.7% in age group <30, PR/ET 16.8% in age group >38) while using the standardized technique of ultrasound guided ET the influence of transferring person can be eliminated. There was no significant difference in tranfers of fresh (PR/ET 32.7%) or frozen–thawed (PR/ET 26,9%) embryos. Surprisingly, there was also no difference in quality of the transfer procedure and in the quality of echodense catheter tip visualisation. On the other hand if “jet phenomenon” could be observed significantly better results (p=0.033) were obtained (31.3% vs. 20.2%). Conclusions: The use of ultrasound-guided COOK Soft EchoTip catheter may provide less traumatic and more precise embryo placement. Its use contributed to less variations in pregnancy rates both in fresh and frozen–thawed embryo transfers and the influence of transferring person could be eliminated. Factors influencing the results are patient's age and the observation of “jet” phenomenon. Using this technique we were not able to confirm the influence of quality of the transfer procedure and the quality of echodense catheter tip visualisation on the achieved results.

The drug-induced chromosome condensation using okadaic acid, a potent protein phosphatase inhibitor, was studied in day 1 to day 4 (D1-D4) spare human preimplantation embryos. In order to obtain cells for genetic tests, we developed a modified displacement blastomere biopsy method. During the okadaic acid treatment, approximately 40% of biopsied cells were lost due to heavy changes of the plasma membrane; this detrimental effect of okadaic acid differed markedly with the respect to the age of embryos. In comparison with the natural embryonic mitotic index, day 1 and day 2 embryonic cells gave increased yields of chromosome spreads (up to 51% of the initial D1-D2 cell number); on days 3 and 4 we were not able to obtain from surviving cells more than 31% blastomeres with condensed chromosomes (9% of total D3-D4 cell number). All chromosome spreads were successfully used for recycling in G-banding and subsequent FISH analysis.

Molecular Reproduction and DevelopmentVolume 53, Issue 3 p. 306-317 Embryo Development Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development J. Koppel, Corresponding Author J. Koppel koppel@saske.sk Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaInstitute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.Search for more papers by this authorP. Rehák, P. Rehák Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorV. Baran, V. Baran Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorJ. Veselá, J. Veselá Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorD. Hlinka, D. Hlinka Second Clinic of Gynaecology and Obstetrics, University Hospital of L. Pasteur, Košice, SlovakiaSearch for more papers by this authorV. Manceau, V. Manceau INSERM U 440, IFM, Paris, FranceSearch for more papers by this authorA. Sobel, A. Sobel INSERM U 440, IFM, Paris, FranceSearch for more papers by this author J. Koppel, Corresponding Author J. Koppel koppel@saske.sk Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaInstitute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.Search for more papers by this authorP. Rehák, P. Rehák Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorV. Baran, V. Baran Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorJ. Veselá, J. Veselá Institute of Animal Physiology, Slovak Academy of Sciences, Košice, SlovakiaSearch for more papers by this authorD. Hlinka, D. Hlinka Second Clinic of Gynaecology and Obstetrics, University Hospital of L. Pasteur, Košice, SlovakiaSearch for more papers by this authorV. Manceau, V. Manceau INSERM U 440, IFM, Paris, FranceSearch for more papers by this authorA. Sobel, A. Sobel INSERM U 440, IFM, Paris, FranceSearch for more papers by this author First published: 21 May 1999 https://doi.org/10.1002/(SICI)1098-2795(199907)53:3<306::AID-MRD6>3.0.CO;2-YCitations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation. Mol. Reprod. Dev. 53:306–317, 1999. © 1999 Wiley-Liss, Inc. Citing Literature Volume53, Issue3July 1999Pages 306-317 RelatedInformation

In a controlled study we compared the outcome of intracytoplasmic sperm injection (ICSI) performed by two different methods. The oocytes from 20 patients were equally divided into two groups and injected either by conventional ICSI using polyvinylpyrrolidone (PVP) or by a modified PVP-free ICSI procedure. While in the conventional ICSI method the spermatozoon is aspirated into the injection pipette, in the modified ICSI procedure the spermatozoon is attached to the end of the narrow micropipette by aspirating its tail. The sperm head is never drawn into the pipette. Accordingly, even a fast-moving spermatozoon can be 'caught' easily. As a result of such an aspiration the spermatozoon loses its motility. Therefore, PVP is required neither to slow down the movement of the spermatozoon nor to facilitate the movement of the solution in the injection pipette. A total of 230 mature oocytes were injected by both methods and the results were analysed. No differences were observed in survival rate between the two ICSI procedures (89% and 91%, respectively). However, the proportion of normally fertilized oocytes was significantly higher after microfertilization by modified ICSI (74%) when compared with the outcome of the conventional ICSI method (62%). The frequency of abnormal fertilization was not influenced by the method of ICSI used. The cleavage rate and quality of resulting embryos were also comparable. In conclusion, we have demonstrated a modified ICSI method which does not require the use of PVP. When compared with the conventional ICSI procedure, even better fertilization rates can be achieved. The proposed ICSI modification may provide an alternative procedure for elimination of the potentially harmful effects which may be associated with conventional ICSI.

We have studied the effect of moderately impaired maternal insulin secretion on oocyte chromosomal constitution, fertilization and zygote DNA synthesis. Female mice were injected with a single dose of streptozotocin (65 mg/kg) 14 days before fertilization/ovulation. Zygotes/oocytes were recovered from control and subdiabetic mice on day 1 of pregnancy. Compared with control animals, subdiabetic females showed a significant difference in the proportion of zygotes/oocytes. The subdiabetic mothers had a lower percentage of zygotes and a higher percentage of unfertilized and degenerated oocytes in comparison with control animals. The investigation of [3H]thymidine incorporation did not show any influence of the maternal subdiabetes on the initial zygote DNA synthesis. An analysis of the ovulated oocytes at the metaphase II stage isolated from subdiabetic mice did not reveal increased chromosomal anomalies in comparison with the controls. Control and subdiabetic mothers had a similar percentage of oocytes with a normal haploid set of chromosomes, and the incidence of aneuploidy/diploidy did not differ significantly. These observations suggest that insulin changes in subdiabetic mothers had a deleterious influence on oocyte fertilization in mice, but apparently they did not have any effect on the nuclear events.