Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Cleavage-stage or blastocyst transfer: what are the benefits and harms?

ET is a critical step in an assisted reproduction cycle. Over the past decade there has been an increasing trend to extending culture from cleavage-stage to blastocyst transfer. There has also been a trend to single ET and reporting the success of an assisted reproductive cycle as a cumulative live-birth rate after using both fresh and frozen embryos. There is low evidence that fresh blastocyst transfer is associated with improved live-birth rates compared with fresh cleavage-stage embryos. However, in the few studies that report cumulative pregnancy rates after fresh and frozen transfers, no significant difference was found. Cleavage-stage transfer is associated with greater numbers of embryos available for freezing, and blastocyst transfer is associated with increased number of cycles with no embryos to transfer. Further well-designed studies are warranted to evaluate the outcomes for blastocyst transfer including cumulative live-birth rate after fresh and frozen transfers, time to live birth, costs of the different transfer strategies, and perinatal mortality and severe perinatal morbidity.

A pilot study to evaluate a device for the intravaginal culture of embryos.

The aim of this comparative randomized embryology trial was to determine if an intravaginal culture device (IVC) can provide acceptable embryo development compared with conventional IVF. Ten women between the ages of 27 and 37 years with an indication for IVF treatment were included in this study. After ovarian stimulation, oocytes were randomized to fertilization in the IVC device or using conventional IVF. Fertilization rates were higher in the IVF group compared with the IVC device (68.7% ± 36 % versus 40.7% ± 27%), respectively, whereas cleavage rates were similar (93% ± 1.5% versus 97% ± 6%) for both groups. A significantly lower number of embryos of suitable quality for transfer was obtained from the IVC device compared with conventional IVF (OR, 0.47; 95% CI, 0.26 to 0.87). The clinical pregnancy rate from transfer of IVC device embryos was 30%. Satisfaction questionnaires were also completed by all participants. Most women (70%) placed high importance on having had fertilization and embryo development occur while carrying the device. Overall, the IVC device produced reasonable pregnancy rates suggesting this technology may have a place under certain circumstances. Cost-benefit analysis, psychological factors and future studies must be considered.

Value of transferring embryos that show no evidence of fertilization at the time of fertilization assessment.

OBJECTIVE: To determine the value of transferring embryos formed from nonpronuclear (0PN) zygotes. DESIGN: A case-control study. SETTING: Not applicable. PATIENT(S): The current study was a retrospective analysis of embryo transfers of just 0PN embryos using fresh cleavage-stage embryos (0PN cleavage fresh), frozen-thawed cleavage-stage 0PN embryos (0PN cleavage frozen), and frozen 0PN blastocyst-stage embryos (0PN blast frozen). INTERVENTION(S): To study the effect of 0PN transfer, comparison groups were used: fresh cycles of 2PN (2PN cleavage fresh-C) and frozen-thawed cycles cleavage-stage (2PN cleavage frozen-C) and blastocyst-stage (2PN blast frozen-C). Comparison groups were matched for cycle and patient characteristics to the 0PN group. MAIN OUTCOME MEASURE(S): Implantation rate (IR), pregnancy rate, and transferable embryo rate. RESULT(S): For fresh cycles, the IR in the 0PN cleavage fresh was lower than that in the 2PN cleavage fresh-C (8.04% vs. 19.50%, respectively). For frozen-thawed cycles, the IR in the 0PN cleavage frozen was lower than that in the 2PN cleavage frozen-C (15.38% vs. 28.24%, respectively), but the IR in 0PN blast frozen was comparable to that of 2PN blast frozen-C (39.56% vs. 48.18%, respectively). CONCLUSION(S): Transfer of 0PN embryos from fresh or frozen-thawed cycles results in pregnancies and live births. Nonpronuclear embryos have a lower IR than 2PN embryos, but if the embryos are cultured to the blastocyst stage and then are frozen, their IRs approach that of 2PN embryos in subsequent frozen-thawed cycles. The culture of 0PN embryos to the blastocyst stage may select for embryos with a near-normal IR.

Morphological assessment of embryo viability.

Morphological assessment is discussed in the context of significant literature at all stages of in vitro development, beginning with the oocyte and culminating at the blastocyst stage. Current evidence is used to debate the inclusion of commonly observed morphological features in grading schemes. The biological rationale behind observed phenomena such as multinucleation and fragmentation are also explored. Current limitations as well as technological advancements that increase our ability to assess viability are highlighted. Particular attention is paid to the relationship between developmental timing and assessment schemes. Failure to standardize assessment timing and inclusion criteria is glaring weaknesses of the literature that currently make consensus unattainable. Mounting evidence suggests that the future of static assessment is very likely to be influenced by information gathered from preimplantation genetic screening and other invasive techniques as well as from continuous monitoring tools such as time lapse.

Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles.

OBJECTIVE: To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome. DESIGN: Observational study. SETTING: Private university-affiliated IVF center. PATIENT(S): Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons. INTERVENTION(S): Vitrification by the Cryotop open device. MAIN OUTCOME MEASURE(S): Delivery rate (DR) per warming cycle. RESULT(S): Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%-95.3%) for day 2 and 95% (95% CI 94.3%-95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%-34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%-96.8%) for day 2, 94.2% (95% CI 93.4%-94.9%) for day 3, 95.7% (95% CI 94.5%-96.9%) for day 5, and 97.6% (95% CI 96.9%-98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%-38.5%), 41.7% (95% CI 39.9%-43.4%), 32.6% (95% CI 31.0%-34.2%), and 38.1% (95% CI 36.4%-39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912-1.277) showed no impact on the DR. CONCLUSION(S): Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect on the survival and delivery rates.

Implementation of an inexpensive method of vitrification and warming of human cleavage-stage embryos using cut standard straws.

The aim of this report is to describe our experience and results with implementation of a cut standard straw technique for vitrification and warming of day 3 cleavage-stage human embryos. Detailed description of the method and results of 63 frozen embryo transfers performed with this technology are discussed, and it is concluded that this method provides a reliable, inexpensive, and effective option of embryo vitrification at a cleaved stage.

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

This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Following background presentations about current practice, the expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. 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 dataset required for the accurate description of embryo development. This paper reports the proceedings and outcomes of an international consensus meeting on human oocyte and embryo morphology assessment. An expert panel developed a series of consensus points to define the minimum criteria for such assessments. The definition of common terminology, and standardization of laboratory practices related to these morphological assessments, will permit more effective comparisons of treatment outcomes around the world. This report is intended to be referenced as a global consensus to allow standardized reporting of the minimum descriptive criteria required for routine clinical evaluations of human embryo development in vitro.

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.

Morphological embryo assessment: reevaluation.

OBJECTIVE: To assess whether the pronuclear score and embryo nuclear scoring have an additive value to day 2 morphology criteria in the prediction of the implantation rate (IR). DESIGN: Retrospective clinical study. SETTING: IVF Unit, Soroka University Medical Center. PATIENT(S): Analysis of all homogenous IVF fresh embryo transfers from 2008-2010. INTERVENTION(S): Morphological evaluation of pronuclear scoring, embryo nuclear scoring, and day 2 embryo morphology scoring on embryos obtained from IVF or intracytoplasmic sperm injection (ICSI) cycles. MAIN OUTCOME MEASURE(S): Implantation rate of homogeneous transfers. RESULT(S): No statistical association was found between the pronuclear scoring system (Z) and IR. The pronuclear score Z3 had a significantly better performance than Z1, Z2, and Z4 (IR = 25.2%, 22%, 21.4%, and 11.8%, respectively). Assessment of the second day embryo morphology scoring (index [IDX]) was found to associate with IR. IDX1, IDX2, IDX3, IDX4, and IDX5 had an IR of 36.1%, 28%, 21.9%, 14.2%, and 0, respectively. The nuclear scoring including: 1 nucleus in all blastomeres, 1 nucleus in part of the blastomeres, no visualization, and multinucleation showed high association with IR = 32.7%, 22.9%, 14.8%, and 9.1%, respectively. The effect of the nuclear scoring on IR was lost in a multivariable analysis that included day 2 embryo morphology scoring. CONCLUSION(S): Our results suggest that there is no additive value in grading the zygote on day 1 or embryo nuclear scoring on day 2 to day 2 embryo morphology for the prediction of IR.

Toxic trace metals and embryo quality indicators during in vitro fertilization (IVF).

Trace exposures to the toxic metals mercury (Hg), cadmium (Cd) and lead (Pb) may interfere with in vitro fertilization (IVF). The aim of this study is to explore biologically plausible hypotheses concerning associations between metals and embryo quality indicators during IVF. For 24 female patients, a multivariable ordinal logistic regression model suggests a 75% reduction in the odds for higher embryo cell cleavage per µg/dL increase in blood Pb (adjusted odds ratio (aOR) 0.25, 95% confidence interval (CI) 0.07-0.86). For 15 male partners, each µg/L increase in blood Hg (aOR 0.60, 95% CI 0.45-0.79) and µg/dL increase in blood Pb (aOR 0.58, 95% CI 0.37-0.91) is associated with a decrease in the analogous odds. Embryo fragmentation is reduced by higher blood Hg (aOR 0.85, 95% CI 0.72-1.00), but increased by higher blood Pb (aOR 1.47, 95% CI 1.11-1.94) in men. The magnitude of these suggested effects warrants confirmation in a larger study.

Human embryo culture and assessment for the derivation of embryonic stem cells (ESC).

The culture and critical assessment of early human embryos during the first week of human development are reviewed for the derivation of ESC. Both normal and abnormal features are assessed by phase contrast microscopy of whole embryos and in serial sections of fixed material by light and electron microscopy (TEM). Normal embryos follow a time table of development and have equal blastomeres with minimal fragmentation and nuclear defects. Abnormal embryos show more fragmentation and nuclear aberrations such as micronucleation and multinucleation, reflected by aneuploidy, polyploidy, and mosaicism. The selection of normal embryos and the hardiest of embryos that survive to blastocysts is recommended for the derivation and culture of ESC.

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos.

BACKGROUND: The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters. METHODS: Multilevel images of embryos on day 1, day 2 and day 3, were analysed using a standard scoring system. The kappa coefficient was calculated to measure intra- and inter-observer variability before and after training sessions. RESULTS: Good to excellent intra-observer agreement was present for most parameters exceptions being scoring the position of pronuclei and the presence of a cytoplasmic halo on day 1, multinucleation on day 2 and the size of fragments on day 3. Inter-observer agreement was only good to excellent for the number of blastomeres on day 2 and day 3 and the orientation of the cleavage axes on day 2. Training sessions had a positive impact on inter-observer agreement. CONCLUSION: In conclusion, assessment of morphological characteristics of early stage embryos using multilevel images was marked by a high intra-observer and a moderate inter-observer agreement. Training sessions were useful to increase inter-observer agreement.

Spindle position assessment prior to ICSI does not benefit fertilization or early embryo quality.

Intracytoplasmic sperm injection (ICSI) is traditionally performed with the first polar body at 6 or 12 o'clock, and the injection pipette inserted at 3 or 9 o'clock. This positioning aims to direct the path of the injection pipette at a distance from the presumed metaphase II spindle position. Since spindles can now be imaged directly in living oocytes using computer-assisted polarized light microscopy, the effectiveness of this positioning precaution was studied. Patients undergoing oocyte collection and ICSI had their oocytes non-invasively imaged for spindles prior to ICSI. The spindle position relative to the first polar body at 6 o'clock was assessed using an analogue clock face as an approximation. Fertilization and embryo quality were recorded blind to spindle position. Polar body displacement and spindle position at ICSI did not significantly affect fertilization or embryonic quality. The highest frequency of normally fertilized oocytes and good quality embryos developed from oocytes with spindles located in or near the plane of injection at ICSI (the 3, 4, 8 and 9 o'clock positions). This study questions the usefulness of spindle imaging and the relevance of positioning the first polar body at 6 o'clock during ICSI.

Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis.

OBJECTIVE: To examine the literature systematically in order to identify prospective comparative trials answering the following question: Is vitrification of human embryos associated with a higher postthawing survival rate as compared with slow freezing? DESIGN: Systematic review and meta-analysis. SETTING: University-based hospital. PATIENT(S): Not applicable. INTERVENTION(S): Vitrification versus slow freezing for cryopreservation of human embryos. MAIN OUTCOME MEASURE(S): Postthawing survival rate. RESULT(S): Four eligible studies were identified, three of which were randomized controlled trials. Overall, the current review summarizes information from 8,824 cryopreserved human cleavage stage embryos/blastocysts (vitrification: n = 7,482; slow freezing: n = 1,342). Survival rate of cleavage stage embryos was significantly higher after vitrification as compared with slow freezing (odds ratio 15.57, 95% confidence interval 3.68-65.82; random effects model). Postthawing survival rate of vitrified blastocysts was significantly higher compared with that observed with slow freezing (odds ratio 2.20, 95% confidence interval 1.53-3.16; fixed effects model). CONCLUSION(S): Vitrification appears to be associated with a significantly higher postthawing survival rate than slow freezing. Further prospective trials are necessary to confirm the above results and, in addition, allow the evaluation of the two cryopreservation methods in terms of pregnancy achievement.

Impact of the assessment of early cleavage in a single embryo transfer policy.

The policy of single embryo transfer (SET) adopted for women <36 years old since 1 July 2003, strongly calls for improvement of embryo selection. A total of 196 cycles in which SET was performed were randomly allocated to two groups. In the first group, early cleavage was assessed (ECA) 25 h after insemination. The embryo with the best score that cleaved early, if present, was selected for transfer. In the second group, early cleavage was not assessed (ECNA) and embryo selection was based solely on the embryo score. Ninety-eight cycles were allocated in the ECA and ECNA group respectively. Early cleavage occurred in 64% of cycles and 32.2% of embryos. Patient population and embryo morphology were similar between the two groups, and similar delivery rates were observed (27.6 versus 24.5% respectively in the ECA and ECNA groups). The assessment of early cleavage as additional parameter did not improve the delivery rate in the single embryo transfer policy.

Assessment of sperm quality parameters of six bulls showing different abilities to promote embryo development in vitro.

Experiments were conducted to investigate the possible origins of variation between six bulls showing various blastocyst rates after in vitro fertilisation. No significant difference was observed for the rates of cleavage and 5-8 cell stages, whereas blastocyst yields at Day 6, 7 and 8 post insemination were significantly different between bulls (P < 0.05). Fertilisation rates ranged from 59.5 to 79.3% (P < 0.05), with no difference in the incidence of polyspermy. The proportions of motile and progressive spermatozoa before and after Percoll separation were analysed. A positive effect of Percoll was noted on both parameters (P < 0.05), leading to the absence of difference between bulls after the separation process. Sperm viability and spontaneous acrosome reaction were assessed during 18 h incubation in fertilisation medium. A sharp decrease in sperm viability was observed for all bulls after 2 h incubation, with only 12.6-21.7% of spermatozoa still viable at 18 h. In contrast, the proportion of reacted acrosomes was low in five out of six bulls (<15% at 18 h). In conclusion, the fertilisation rate was the only parameter to show some correlation with blastocyst rate for all bulls.

Sequential embryo assessment outperforms investigator-driven morphological assessment at selecting a good quality blastocyst.

Two selection methods (morphology-only and a sequential embryo assessment algorithm) were compared within the same IVF clinic to determine which method best identifies the embryos on day 3 that will develop into the highest quality on day 5. The sequential embryo assessment algorithm was significantly better at selecting the best embryo and selecting a blastocyst compared with the morphology-only method.

Morphological assessment of preimplantation embryo quality in cattle.

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.