Human embryonic development after blastomere removal: a time-lapse analysis.

BACKGROUND: Blastomere biopsy of human embryos is performed for preimplantation genetic diagnosis (PGD). The impact on further development is largely unexplored, though studies on mice suggest an influence on the hatching process. The objective of this study was to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. METHODS: Embryos from couples undergoing PGD treatment or IVF/ICSI were included. In the PGD group, 56 human embryos had one blastomere biopsied. As controls, 53 non-biopsied IVF/ICSI embryos were selected. All embryos were cultured until 5 days after fertilization in a time-lapse incubator (EmbryoScope™). Images of embryos were acquired every 20 min. Time-points of key embryonic events were registered, and development in the two groups was compared. RESULTS: Duration of the biopsied cell-stage in the PGD group was longer than in the control group (P < 0.001), causing biopsied embryos to reach subsequent embryonic stages until hatching at significantly later time-points (P(compaction) < 0.001; P(morula) < 0.001; P(earlyblast) < 0.001; P(fullblast) = 0.01), but with unchanged intervals. Embryos in the PGD group started hatching at the same time-point as the control group, but had a smaller diameter (P < 0.001), and a thicker zona pellucida (P < 0.001) when hatching. Time-lapse videos revealed that in the control group, expansion of the blastocyst caused continuous thinning of zona pellucida until the blastocyst hatched, whereas in the PGD group the blastocyst hatched through the opening in zona pellucida artificially introduced prior to the biopsy. CONCLUSIONS: We find that blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.

A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator.

PURPOSE: Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study was to evaluate the safety of a commercially available time-lapse incubator. METHODS: In a two center, randomized, controlled, clinical trial 676 oocytes from 59 patients in their 2nd or third treatment cycle, age <38 years and ≥ 8 oocytes retrieved were cultured in the time-lapse incubator or in a conventional incubator. The primary outcome was proportion of 4-cell embryos on day 2. Secondary outcomes were proportion of 7-8 cell embryos on day 3 and proportion of blastocysts on day 5. Implantation pregnancy rates were registered based on presence of fetal heart activity visualized by ultrasound 8 weeks after embryo transfer. RESULTS: No significant difference was found between the time-lapse incubator (TLI) and conventional incubator (COI) in proportion of 4-cell embryos on day 2 irrespective of whether data was analyzed according to ITT (RR(TLI/COI): 0.81 (0.65; 1.02)) or PP (RR(TLI/COI): 0.80 (0.63; 1.01)). Nor were any significant differences detected in the secondary endpoints; i.e. proportion of 7-8-cell embryos on day three ITT (RR(TLI/COI): 0.96 (0.73; 1.26)); PP (RR(TLI/COI): 0.95 (0.72; 1.26)) and proportion of blastocysts on day five ITT (RR(TLI/COI): 1.09 (0.84; 1.41)); PP (RR(TLI/COI): 1.09 (0.83: 1.41)). We found no differences in clinical pregnancy rate or implantation rate. CONCLUSION: Culture in the time-lapse incubator supports embryonic development equally to a conventional incubator.

Distinct differences in global gene expression profiles in non-implanted blastocysts and blastocysts resulting in live birth.

Results from animal models points towards the existence of a gene expression profile that is distinguishably different in viable embryos compared with non-viable embryos. Knowledge of human embryo transcripts is however limited, in particular with regard to how gene expression is related to clinical outcome. The purpose of the present study was therefore to determine the global gene expression profiles of human blastocysts. Next Generation Sequencing was used to identify genes that were differentially expressed in non-implanted embryos and embryos resulting in live birth. Three trophectoderm biopsies were obtained from morphologically high quality blastocysts resulting in live birth and three biopsies were obtained from non-implanting blastocysts of a comparable morphology. Total RNA was extracted from all samples followed by complete transcriptome sequencing. Using a set of filtering criteria, we obtained a list of 181 genes that were differentially expressed between trophectoderm biopsies from embryos resulting in either live birth or no implantation (negative hCG), respectively. We found that 37 of the 181 genes displayed significantly differential expression (p<0.05), e.g. EFNB1, CYTL1 and TEX26 and TESK1, MSL1 and EVI5 in trophectoderm biopsies associated with live birth and non-implanting, respectively. Out of the 181 genes, almost 80% (145 genes) were up-regulated in biopsies from un-implanted embryos, whereas only 20% (36 genes) showed an up-regulation in the samples from embryos resulting in live birth. Our findings suggest the presence of molecular differences visually undetectable between implanted and non-implanted embryos, and represent a proof of principle study.

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring.

OBJECTIVE: To evaluate, using time-lapse monitoring, the temporal influence of culture in 5% O2 or 20% O2 on human embryonic development. DESIGN: Retrospective cohort study. SETTING: University-based fertility clinic. PATIENT(S): In vitro fertilized embryos from women aged <38 years with no endometriosis and ≥8 oocytes retrieved. INTERVENTION(S): Culture in 20% O2 exclusively (group 1), 20% and 5% O2 combined (group 2), or 5% O2 exclusively (group 3). MAIN OUTCOME MEASURE(S): Developmental rates and timing of developmental stages. RESULT(S): The timing of the third cleavage cycle was delayed for embryos cultured in 20% O2 (group 1) compared with embryos cultured in 5% O2 (groups 2 and 3). No difference was observed in timing of the early and full blastocyst stages. More embryos in groups 2 and 3 reached the 8-cell, early blastocyst, and full blastocyst stages than in group 1. We found that embryos in group 3 (5% O2) reached the 8-cell stage faster than embryos in group 2 (5% + 20% O2), but none of the other parameters (i.e., other time points, cumulative development, and embryo score) differed between the two groups. CONCLUSION(S): Culture in 20% O2 reduces developmental rates and delays completion of the third cell cycle. The delayed development after culture in atmospheric oxygen was seen in the precompaction embryo only and therefore appears to be stage specific. CLINICAL TRIAL REGISTRATION NUMBER: NCT01139268.

Hatching of in vitro fertilized human embryos is influenced by fertilization method.

OBJECTIVE: To describe hatching of human embryos and investigate differences in hatching between IVF and intracytoplasmic sperm injection (ICSI)-fertilized embryos with the use of time-lapse monitoring. DESIGN: Clinical observational study. SETTING: University-based fertility clinic. PATIENT(S): From February 2011 to July 2012, 161 women consented to embryo culture in a time-lapse incubator until day 6 after oocyte retrieval. The mechanism of hatching was recorded and related to method of fertilization (ICSI or IVF) and clinical pregnancy outcome. INTERVENTION(S): IVF or ICSI. MAIN OUTCOME MEASURE(S): Hatching pattern. RESULT(S): A total of 430 IVF fertilized embryos from 62 patients and 594 ICSI-fertilized embryos from 99 patients were included. We observed spontanous hatching in 165 IVF embryos and 215 ICSI embryos. Two distinct mechanisms of hatching were observed. Type 1 was characterized by penetration of the zona pellucida (ZP) by small trophectoderm projections, whereas type 2 was preceded by a regular rupture of the ZP followed by extrusion of the blastocyst. Type of hatching was significantly different between IVF and ICSI embryos, with type 2 observed more often in IVF embryos than in ICSI embryos. Furthermore, IVF embryos escaped the ZP more readily than ICSI embryos. Regardless of the type of hatching, implantation rates were similar. CONCLUSION(S): We describe two distinct mechanisms of in vitro hatching related to fertilization method and suggest that hatching pattern is associated with fertilization method. The hatching pattern has, however, no influence on future implantation. CLINICAL TRIAL REGISTRATION NUMBER: NCT01139268.