G. Calderon

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

BACKGROUND Many variations in oocyte and embryo grading make inter-laboratory comparisons extremely difficult. This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. METHODS Background presentations about current practice were given. RESULTS The expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. CONCLUSIONS It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.

Impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes

A prospective study was performed to assess the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. The study population included 178 couples (62 cycles of IVF, 116 of intracytoplasmic sperm injection (ICSI)) with own (n=77) and donor (n=101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Correlation between DNA damage to oocyte fertilization, embryo quality and clinical pregnancy, implantation and miscarriage rates was evaluated. DNA fragmentation was not related to fertilization rates in either IVF (r=0.08) or ICSI (r=-0.04) cycles. DNA fragmentation was similar in patients with <50% embryo utilization rate compared with ≥50%, in cancelled and in embryo transfer cycles and in miscarriages and in successful deliveries. Moreover, DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the multivariable analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation threshold, results did not vary. It is concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes.

Evaluation of cytogenetic analysis for clinical preimplantation diagnosis

OBJECTIVE To evaluate the feasibility of using cytogenetic analysis in preimplantation diagnosis. DESIGN Two different biopsy protocols (chemical drilling and zona cutting) and two fixation methods were tested in a mouse model. Afterwards, the efficiency of obtaining chromosome preparations from untransferable human embryos depending on the method used to obtain the blastomeres (embryos biopsy or removal of the zona pellucida and blastomere disaggregation) was determined. The chances of obtaining chromosome preparations depending on the type of embryo (haploid, diploid, triploid, and apparently unfertilized) were also evaluated. RESULTS Results from the mouse model showed that chemical drilling yields better results than cutting in terms of metaphases per biopsied embryo and surviving rate after biopsy. In human embryos, biopsy of diploid embryos produced 46.6% chromosome preparations, while 29% were obtained after blastomere disaggregation and 20.4% when biopsying triploid embryos. CONCLUSIONS These results suggest that the disaggregating procedure and triploid embryos cannot be considered as good models to assess the feasibility of cytogenetic analysis in preimplantation diagnosis. Poor chromosome quality and loss during fixation are the main problems to use cytogenetics in preimplantation diagnosis; a combination of cytogenetics and other techniques is suggested in cases of balanced translocations.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator

OBJECTIVE To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). DESIGN Prospective cohort study on sibling donor oocytes. SETTING University-affiliated in vitro fertilization (IVF) center. PATIENT(S) Embryos from 59 patients. INTERVENTION(S) Culture in a TLI in a single medium with or without renewal of the medium on day-3. MAIN OUTCOME MEASURE(S) Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. RESULT(S) The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. CONCLUSION(S) Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory.

Antimitotic Treatments for Chemically Assisted Oocyte Enucleation in Nuclear Transfer Procedures

Chemically assisted enucleation has been successfully applied to porcine and bovine oocytes to prepare recipient cytoplasts for nuclear transfer procedures. In this study, the antimitotic drugs demecolcine, nocodazole, and vinblastine were first assessed for their ability to induce the formation of cortical membrane protrusions in mouse, goat, and human oocytes. While only 2% of the treated human oocytes were able to form a protrusion, high rates of protrusion formation were obtained both in mouse (84%) and goat oocytes (92%), once the treatment was optimized for each species. None of the antimitotics applied was superior to the others in terms of protrusion formation, but mouse oocytes treated with vinblastine were unable to restore normal spindle morphology after drug removal and their in vitro development after parthenogenetic activation was severely compromised, rendering this antimitotic useless for chemically assisted enucleation approaches. Aspiration of the protrusions in mouse oocytes treated with demecolcine or nocodazole yielded 90% of successfully enucleated oocytes and allowed the extraction of a smaller amount of cytoplasm than with mechanical enucleation, but both enucleation methods resulted in the depletion of spindle-associated gamma-tubulin from the prepared cytoplasts. Treatment of mouse oocytes with demecolcine or nocodazole had no effect on their in vitro development after parthenogenetic activation, or on their ability to repolymerize a new spindle after the removal of the drug or the reconstruction of the treated cytoplasts with a somatic nucleus. Therefore, demecolcine- and nocodazole-assisted enucleation appears as an efficient alternative to mechanical enucleation, which can simplify nuclear transfer procedures.

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate

OBJECTIVE To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. DESIGN Prospective cross-sectional study. SETTING Multicenter study. PATIENT(S) Seven hundred embryo transfers and 1,028 early-stage human embryos. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Implantation according to the presence of EC and embryo quality. RESULT(S) The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. CONCLUSION(S) At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation.

Two years of assisted fertilization by partial zona dissection in male factor infertility patients

OBJECTIVE To assess partial zona dissection in our routine IVF-ET program over a 2-year period. DESIGN Partial zona dissection before insemination on the day of oocyte collection or 24 hours after unsuccessful conventional IVF. In a subgroup of patients, oocytes were randomized to either partial zona dissection before insemination or IVF. SETTING University infertility clinic. PATIENTS Couples who suffered principally from male factor infertility or who had failed fertilization previously. INTERVENTIONS Micromanipulation of oocytes with partial zona dissection. MAIN OUTCOME MEASURES Comparison of fertilization rate, embryo morphology, and implantation rate between partial zona dissection inseminated oocytes and conventional IVF inseminated oocytes (controls). RESULTS Five pregnancies were established in 199 patients. The incidence of polyspermy was significantly higher in the partial zona dissection group than in conventional IVF (4.8% versus 1.3%). There were no significant differences in the remaining parameters. The fertilization rate of partial zona dissection and reinsemination was significantly higher than conventional IVF insemination (13.6% versus 4.5%) but similar to the rate obtained when partial zona dissection was applied before insemination (13.6% versus 15.7%). CONCLUSIONS Oocytes treated by partial zona dissection did not exhibit a greater fertilization rate than conventional IVF inseminated oocytes. Partial zona dissection may not be a useful technique for treating severe male factor infertility.

Could the addition of hp-hMG and GnRH antagonists modulate the response in IVF-ICSI cycles?

Objective. To assess if the luteinizing hormone / human chorionic gonadotropin present in some gonadotropin formulations may be of benefit in protocols with GnRH antagonists. Methods. Open, quasi-experimental, multicenter, prospective, parallel-controlled study compared 136 women undergoing in vitro fertilization – intracytoplasmic sperm injection after stimulation with highly purified human menopausal gonadotropin (hp-hMG) (n = 44), recombinant-follicle stimulating hormone (r-FSH) (n = 46), or a combination of both (r FSH + hp-hMG) (n = 46) following an antagonist protocol. Blood determinations were made on day 6 of stimulation and on the day of ovulation induction, with centralized analysis. Results. No differences were found in the ongoing pregnancy rates between groups [37.0% versus 29.5% (hp-hMG) and 23.9% (r-FSH); p = 0.688]. However, the ratio top-quality embryos / retrieved oocytes (TQE/RO) was higher in the combined therapy group (19.6%) – reaching significance versus the r-FSH group (6.5%) (p = 0.008), but not versus hp-hMG (12.3%) (p = 0.137). Conclusions. An improved TQE/RO ratio was obtained together with a greater percentage of frozen embryos in the patients that incorporated hp-hMG to their stimulation protocol. Despite good results of adding hp-hMG, non statistical differences were found in terms of ongoing pregnancy rate.

Pre-clinical validation of a closed surface system (Cryotop SC) for the vitrification of oocytes and embryos in the mouse model

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females.