Introduction: Human embryonic stem cells (hESC) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos.While the majority of hESC lines originate from good quality embryos donated after cryogenic storage, poor quality embryos (PQEs) not suitable for clinical use have also been shown to generate hESC.This provides a newfound function for embryos that would otherwise be discarded following IVF or ICSI.Due to their lack of clinical importance, however, data on the poorest embryos in a cohort go largely unreported in the literature.It is therefore of interest to better understand the availability of PQEs and their ability to develop to blastocysts with good quality ICMs for use in hESC derivation.We have previously shown that IVF based embryo grading can be predictive for hESC derivation efficiency from PQEs.In this study, we investigate the influence of patient parameters and embryo cohort on blastocyst development, ICM quality and successful hESC derivation from PQEs.Material and Methods: Donated PQEs on day 3 of development that did not meet the IVF laboratory's criteria for transfer or cryopreservation were included in this study if they had ≥ 4 blastomeres and ≤ 50% fragmentation.Embryos were cultured individually in Cook Blastocyst Media from day 3 until day 6 at 37°C, 6% CO2, and 5% O2.On day 6, blastocysts were graded for expansion status and the quality of the ICM and trophectoderm.Only blastocysts with good quality ICMs were used for hESC derivation.Embryo history, cohort parameters, and anonymous patient data were then retrospectively compiled and analyzed.Results: Overall, 3690 PQEs from 777 patient cycles did not meet clinical criteria for transfer or cryopreservation.Of these, 2785 (75.6%) met the criteria for inclusion into this study, resulting in 895 blastocysts.385 blastocysts originating from 250 cycles contained good quality ICMs on day 6 of development.The cycles that resulted in blastocysts with good quality ICMs had significantly more oocytes retrieved, mature oocytes, fertilized oocytes and embryos cryopreserved than those that did not develop to blastocysts or had only poor quality ICMs (all P < 0.005).Blastocyst development in this group was also significantly higher than the overall mean (49.5% vs. 32.1% P < 0.001).Blastocysts with good quality ICMs came from significantly younger patients than those without (32.6 ± 4.3 vs. 33.6 ± 4.8y P < 0.005).132 blastocysts with good quality ICMs were successfully plated for hESC derivation purposes with 16 normal lines derived.There were no differences observed in their cycle parameters for pregnancy rate, gonadotropin dose, number of previous cycles, or fertility diagnosis for those that did or did not generate hESC.There was, however, a further influence of age, with hESCproducing-ICMs originating from significantly younger patients (30.7 ± 3.9y vs. 33.3± 4.7y; P < 0.05).There were no differences in the mean number of embryos donated per patient between these groups but the PQEs in the hESCmaking group had significantly higher blastocyst development (61.6% vs. 44.6%;P < 0.05) and the percentage of these blastoscyts having good quality ICMs was also higher (75.6% vs 57.4%; P < 0.05). Conclusion:PQEs are an abundant source of embryos capable of developing to blastocysts with good quality ICMs and generating hESC.We have shown that IVF cycle characteristics known to correlate with IVF success can also help predict which PQEs have the best hESC developmental potential.In particular, PQEs arising from younger patients have a better chance of making good quality ICMs and these ICMs have more potential for hESC generation.In addition, the development of embryos within a cohort is correlative of PQE hESC potential.Experiments designed to compare HESC derivation techniques or efficiency using PQEs should consider clinical IVF parameters to establish groups with equal developmental competence.

10.1093/humrep/26.s1.93

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Proportional immunofluorescent analysis of phospholipase C zeta (PLC ?) is a promising indicator of oocyte activation capability in human sperm

Human Reproduction (2012)

B. Heindryckx Junaid Kashir Céline Jones Ginny Mounce Walaa M. Ramadan

Oocyte activation

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Embryology: experimental

Human Reproduction (2009)

Marko Došen Veljko Vlaisavljević Borut Kovačič M Ci ek-Sajko Dos ˇen

Introduction: Spontaneous miscarriage (SM) is possible adverse outcome in pregnancy achieved with assisted reproduction technology (ART).SM is a very common event in the population of healthy cycling women younger than 35 years.It was showed that the incidence of SM was slightly increased in the ART pregnancies after adjusting for maternal age and previous SM.Other variables were marked as potentially linked to the risk for SM.The objective of this study was to examine the possible influence of quality of embryos on SM in ART pregnancies.Materials and Methods: The total of 1433 hormonally stimulated IVF and ICSI cycles achieved in our center in the period from 2001 to 2002 after single or double embryo transfer (SET and DET, respectively) were retrospectively analyzed.Of these, in the final analysis were included only those cases with SET or DET on day 5 for which complete data on spontaneous miscarriages (ongoing pregnancies) existed -418 cycles in total.The level of statistical difference between groups of patients with specific combination of observed variables was determined by x 2 test, Student's t-test for independent samples, Kruskal-Wallis test and ANOVA.Results: The mean age of patients was 32,2+4,5 years (range from 22 to 43 years).On Day 5, in 247 cases only one blastocyst was transferred and in 634 cases two blastocysts were transferred.After SET, SM rate was 11,6% (8/69) and after DET 12,0% (42/349).Live birth rate per per embryo transfer after SET was 23,5% (58/247) and after DET was 46,2% (293/634).Investigating effects of embryo quality on SM after SET, we didn't find significant statistical difference between groups SM vs. pregnancy without SM (subgroups optimal vs. suboptimal quality of transferred blastocyst( (p.0,05), but the observed groups were very small (two of four groups with frequencies !5).After DET, statistically significant difference was shown between groups SM vs. pregnancy without SM (subgroups: both blastocysts of optimal quality (SM in 8,5%) vs. one blastocyst of optimal and the other of suboptimal quality (SM in 10,1%) vs. both blastocyst of suboptimal quality (SM in 25,4%)) (p , 0,05).Observing possible effects of technique used in ART on SM, we didn't find any statistically significant difference between groups SM vs. pregnancy without SM (subgroups IVF vs. ICSI) (p.0,05).Also, investigating possible effects of the number of transferred blastocysts on SM, in our data set we didn't find statistically significant difference between groups SM vs. pregnancy without SM (subgroups SET vs. DET) (p.0,05).The age of the patients was the single most important factor connected to SM (,35 yo vs. !35 yo) (p , 0,0001).Conclusions: The overall rate of SM in pregnancies achieved by IVF or ICSI after SET or DET on Day 5 is smaller than in general population (app.12%), which is probably the consequence of embryo selection before ET.Quality of transferred embryos after DET represents a variable which influence SM rate.We couldn't show this effect after SET most probably because of small sample size.Maternal age strongly influences the outcome of pregnancy after ART.The choice between IVF or ICSI or the number of transferred embryos didn't show the influence on SM rate in our data set.

Embryology

10.1093/humrep/dep791

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PLC zeta is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence

Genetics Research (2018)

A Hachem Jonathan Godwin Margarida Ruas H C Lee Minerva Ferrer Buitrago

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In-vitro matured oocytes for human cloning

Reproductive BioMedicine Online (2006)