Undisturbed culture: a clinical examination of this culture strategy on embryo in vitro development and clinical outcomes.

OBJECTIVE: To compare the effect of a fully undisturbed culture strategy over a sequential one on embryo in vitro development and clinical outcomes in intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Retrospective cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): This study included 4,564 ICSI cycles performed over 5 years, including autologous and oocyte donation treatments with extended embryo culture until blastocyst in one of the two defined culture strategies. INTERVENTION(S): Embryo cohorts were cultured in one of two culture systems: a fully undisturbed culture, including an incubator with integrated time-lapse technology, a one-step culture medium and embryo selection assisted by semi-automatic tools on the basis of embryo morphokinetics, or a sequential culture, using a conventional benchtop incubator, sequential media and traditional morphological evaluation under optical microscope. The effect of the culture strategies on embryo development and clinical outcomes was quantified by generalized estimated equations, controlling for possible confounders through the inverse probability of the treatment weighting method. MAIN OUTCOME MEASURE(S): Weighted odds ratios (ORs) and 95% confidence intervals (CIs) for live birth rate after fresh single embryo transfer and the cumulative live birth rate. In addition, blastocyst development and morphology and other intermediate outcomes were also assessed. RESULT(S): A significant positive association was found between the employment of undisturbed embryo culture and higher live birth rate in the first embryo transfer in both autologous (OR, 1.617; 95% CI, 1.074-2.435) and oocyte donation cycles (OR, 1.316; 95% CI, 1.036-1.672). Cumulative live birth rate after 1-year follow-up was also positively associated with the undisturbed culture strategy in oocyte donation cycles (OR, 1.5; 95% CI, 1.179-1.909), but not in autologous cycles (OR, 1.051; 95% CI, 0.777-1.423). Similarly, blastocyst rate, good morphology blastocyst rate, and utilization rate were positively associated with the employment of undisturbed culture in oocyte donation cycles, but not in autologous cycles. CONCLUSION(S): These findings imply that a culture system combining integrated time-lapse incubators with a one-step culture medium may enhance the success rates of patients undergoing ICSI treatment by increasing the production of higher quality blastocysts and improving embryo selection while streamlining laboratory procedures and workflow.

Elevated serum progesterone does not impact euploidy rates in PGT-A patients.

PURPOSE: Some women undergoing stimulated cycles have elevated serum progesterone (P) on the day of ovulation trigger, but its effect on embryo quality is unclear. We analyze embryo quality among patients with high and low serum P undergoing preimplantation genetic testing for aneuploidy (PGT-A). METHODS: This retrospective study included 1597 patients divided into two groups by serum P values: < 1.5 ng/mL or ≥ 1.5 ng/mL. A gonadotrophin-releasing hormone (GnRH) antagonist protocol was established for each patient. Serum P levels were measured on the day of triggering. Propensity score matching and Poisson regression were done. Age, body mass index, and ovarian sensitivity index were also compared. RESULTS: Elevated serum P was not significantly associated with euploid embryo rate or other embryo-quality variables evaluated in our study. Age was the only variable associated with euploidy rate (per MII oocyte, P < 0.001; per biopsied embryo, P = 0.008), embryo biopsy rate (P < 0.001), absolute number of euploid embryos (P = 0.008), and top-quality embryo rate (P = 0.008). Categorical variables decreased in value for every year of increased age in patients with high serum P. CONCLUSIONS: Elevated serum P did not affect the number of euploid and good-quality embryos for transfer in GnRH antagonist intracytoplasmic sperm injection (ICSI) cycles. Contrary to the clear influence of premature P elevation on endometrial receptivity based on literature, our results may help to tip the balance towards the absence of a negative effect of P elevation on embryo competence. More studies are needed to fully understand the effect of P elevation on reproductive outcomes.

The influence of oxygen concentration during embryo culture on obstetric and neonatal outcomes: a secondary analysis of a randomized controlled trial.

STUDY QUESTION: Does oxygen concentration during 3-day embryo culture affect obstetric and neonatal outcomes? SUMMARY ANSWER: Oxygen concentration during 3-day embryo culture does not seem to affect the obstetric and neonatal outcomes measured. WHAT IS KNOWN ALREADY: Atmospheric oxygen appears to be harmful during extended embryo culture. Embryo culture conditions might therefore be a potential risk factor for subsequent fetal development and the health of future children. No data are available concerning the obstetrics and neonatal outcomes after Day 3 transfer of embryos cultured under reduced and atmospheric oxygen tensions. STUDY DESIGN, SIZE, DURATION: A secondary analysis of a previous randomized controlled trial assessing clinical pregnancy outcomes was carried out. This analysis included 1125 consecutive oocyte donation cycles utilizing ICSI or IVF and Day 3 embryo transfers between November 2009 and April 2012. The whole cohort of donated oocytes from patients who agreed to participate in the study were randomly allocated (1:1 ratio) to a reduced O2 tension group (6% O2) or an air-exposed group (20% O2) based on a computer-generated randomization list. Fresh and vitrified oocytes were used for oocyte donation. Only those pregnancies with a live birth at or beyond 24 weeks of gestation were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Day 3 embryos were cultured in an atmosphere of 5.5% CO2, 6% O2, 88.5% N2 versus a dual gas system in air. MAIN RESULTS AND THE ROLE OF CHANCE: From the eligible 1125 cycles, 564 were allocated to the 6% O2 group and 561 cycles to the 20% O2 group. However, 50 and 62 cycles did not reach embryo transfer in the 6% and 20% O2 groups, respectively. No differences were found between 6% O2 and atmospheric O2 tension in the number of livebirths per embryo transfer (mean ± SD, 0.5 ± 0.7 versus 0.5 ± 0.7), pregnancy complications or neonatal outcomes. Both groups (6% and atmospheric O2) had similar single and twin delivery rates (40.8% versus 38.1% and 10.7% versus 12.3%, respectively). Preterm delivery rates and very preterm delivery rates (10.80% versus 13.24% and 1.25% versus 2.94%, respectively), birthweight (3229 ± 561 g versus 3154 ± 731 g), low birthweight (2.92% versus 2.45%), birth height (50.18 ± 2.41 cm versus 49.7 ± 3.59 cm), head circumference (34.16 ± 1.87 cm versus 33.09 ± 1.85 cm) and 1 min Apgar scores (8.96 ± 0.87 versus 8.89 ± 0.96) were also similar between 6% and atmospheric O2 groups, respectively. LIMITATIONS, REASONS FOR CAUTION: The number of liveborns finally analyzed is still small and not all obstetric and neonatal variables could be evaluated. Furthermore, a small proportion of the obstetric and neonatal data was obtained through a questionnaire filled out by the patients themselves. One reason for the lack of effect of oxygen concentration on pregnancy outcome could be the absence of trophectoderm cells at cleavage stage, which may make Day 3 embryos less susceptible to hypoxic conditions. WIDER IMPLICATIONS OF THE FINDINGS: Nowadays many IVF laboratories use a more physiological oxygen concentration for embryo culture. However, the benefits of using low oxygen concentration on both laboratory and clinical outcomes during embryo culture are still under debate. Furthermore, long-term studies investigating the effect of using atmospheric O2 are also needed. Gathering these type of clinical data is indeed, quite relevant from the safety perspective. The present data show that, at least in egg donation cycles undergoing Day 3 embryo transfers, culturing embryos under atmospheric oxygen concentration seems not to affect perinatal outcomes. STUDY FUNDING/COMPETING INTEREST(S): The present project was supported by the R + D program of the Regional Valencian Government, Spain (IMPIVA IMDTF/2011/214). The authors declare that they have no conflict of interest with respect to the content of this manuscript. TRIAL REGISTRATION NUMBER: NCT01532193.

Protocolo sedoanalgésico para la infiltración de toxina botulínica A en parálisis cerebral / Sedation-analgesia protocol for the injection of botulinum toxin A in cerebral palsy

Objetivo: Evaluar el impacto de la técnica sedoanalgésica en el dolor experimentado por el paciente. Métodos: Estudio transversal realizado en pacientes consecutivos con parálisis cerebral (PC) que se infiltraron con toxina botulínica A (OnabotA). Los pacientes fueron divididos en 4 grupos según la estrategia analgésica asignada: Grupo I, sin sedación o crema anestésica tópica; Grupo II, inhalación de óxido nitroso; Grupo III, sedación intravenosa profunda y Grupo IV, sedación ligera con benzodiacepinas. El dolor se evaluó con diferentes escalas según la edad del paciente. Los padres clasificaron su satisfacción con la comodidad de su hijo mediante una escala tipo Likert de 5 puntos. La variable primaria de eficacia fue la proporción de pacientes que experimentaron un nivel de dolor ≤ 2, según las escalas de dolor, en los diferentes grupos de estudio. Resultados: De los 124 pacientes incluidos en el estudio, 56 (45,2%) experimentaron un nivel de dolor ≤ 2. En el Grupo III, una proporción significativamente mayor de pacientes presentó un nivel de dolor ≤ 2, p < 0,001, en comparación con todos los grupos de estudio, respectivamente. La inyección de OnabotA fue guiada por ultrasonografía en 109 (87,9%) pacientes y por referencia anatómica en 15 (12,1%). Conclusión: En los pacientes con PC tratados con infiltraciones de OnabotA, la estrategia sedoanalgésica tuvo un impacto significativo en el dolor experimentado por el sujeto. Seleccionar una estrategia analgésica apropiada es crucial para reducir el estrés asociado con la administración de inyecciones de OnabotA en niños con PC

Objective: To evaluate the impact of the sedation-analgesia technique on the pain experienced by the patient. Methods: This cross-sectional study was conducted on consecutive patients with cerebral palsy (CP) who underwent infiltration with botulinum toxin A (BoTNA). The patients were divided into 4 different groups according to the analgesic strategy assigned: Group I, without sedation or topical anaesthetic cream; Group II, inhalation of nitrous oxide; Group III, deep intravenous sedation; and Group IV, light sedation with benzodiazepines. Pain was assessed with different scales depending on patient age. Parents were asked to rate their satisfaction with their child's comfort by using a 5-point Likert-type scale. The primary end-point was the proportion of patients that experienced a pain level equal or lower than 2, according to pain scales, in the different study groups. Results: Of the 124 patients included in the study, 56 (45.2%) experienced a pain level ≤ 2. In the Group III a significantly greater proportion of patients were classified with a pain level score ≤ 2, P < .001, as compared with all the study groups, respectively. The BoTNA injection was guided by ultrasonography in 109 (87.9%) patients, and by palpation in 15 (12.1%). Conclusion: The results of this study suggested that, in patients with CP treated with BoTNA injections, the sedation-analgesic strategy had a significant impact on the pain experienced by the subject. Selecting an appropriate analgesic strategy is crucial for reducing the stress associated with the administration of BoTNA injections in children with CP

[Sedation-analgesia protocol for the injection of botulinum toxin A in cerebral palsy]. / Protocolo sedoanalgésico para la infiltración de toxina botulínica A en parálisis cerebral.

OBJECTIVE: To evaluate the impact of the sedation-analgesia technique on the pain experienced by the patient. METHODS: This cross-sectional study was conducted on consecutive patients with cerebral palsy (CP) who underwent infiltration with botulinum toxin A (BoTNA). The patients were divided into 4 different groups according to the analgesic strategy assigned: Group I, without sedation or topical anaesthetic cream; Group II, inhalation of nitrous oxide; Group III, deep intravenous sedation; and Group IV, light sedation with benzodiazepines. Pain was assessed with different scales depending on patient age. Parents were asked to rate their satisfaction with their child's comfort by using a 5-point Likert-type scale. The primary end-point was the proportion of patients that experienced a pain level equal or lower than 2, according to pain scales, in the different study groups. RESULTS: Of the 124 patients included in the study, 56 (45.2%) experienced a pain level ≤2. In the Group III a significantly greater proportion of patients were classified with a pain level score ≤2, P<.001, as compared with all the study groups, respectively. The BoTNA injection was guided by ultrasonography in 109 (87.9%) patients, and by palpation in 15 (12.1%). CONCLUSION: The results of this study suggested that, in patients with CP treated with BoTNA injections, the sedation-analgesic strategy had a significant impact on the pain experienced by the subject. Selecting an appropriate analgesic strategy is crucial for reducing the stress associated with the administration of BoTNA injections in children with CP.

Effect of oocyte vitrification on embryo quality: time-lapse analysis and morphokinetic evaluation.

OBJECTIVE: To analyze whether oocyte vitrification may affect subsequent embryo development from a morphokinetic standpoint by means of time-lapse imaging. DESIGN: Observational cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): Ovum donation cycles conducted with the use of vitrified (n = 631 cycles; n = 3,794 embryos) or fresh oocytes (n = 1,359 cycles; n = 9,935 embryos) over 2 years. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Embryo development was analyzed in a time-lapse imaging incubator. The studied variables included time to 2 cells (t2), 3 cells (t3), 4 cells (t4), 5 cells (t5), morula (tM), and cavitated, early, and hatching blastocyst (tB, tEB, tHB) as well as 2nd cell cycle duration (cc2 = t3 - t2). All of the embryos were classified according to the hierarchic tree model currently used for embryo selection. The analyzed variables were compared with the use of analysis of variance or chi-square and included 95% confidence intervals (CIs). RESULT(S): The embryos that originated from vitrified oocytes showed a delay of ∼1 hour from the first division to 2 cells (t2) to the time of blastulation (tB). The embryos that originated from vitrified oocytes showed a delay of ∼1 hour from the 1st division to 2 cells (t2) to the time of blastulation (tB) (P<.05). The proportions of embryos allocated to categories A-E in the hierarchical tree were similar between groups. No differences in implantation rates between the fresh (51.3% [95% CI 47.1%-55.7%]) and vitrified (46.4% [95% CI 38.4%-54.4%]) groups were found. CONCLUSION(S): The embryo quality of vitrified oocytes was not impaired: cc2, quality according to our hierarchic morphokinetic model, and implantation rates were similar between fresh and vitrified oocytes. However, morphokinetic differences were observed from t2 to tB. Our main study limitation was the retrospective nature of the analysis, although a large database was studied.

Combination of metabolism measurement and a time-lapse system provides an embryo selection method based on oxygen uptake and chronology of cytokinesis timing.

OBJECTIVE: To evaluate correlations between oxygen consumption (OC) measurements before and after embryo cytokinesis, observing OC during embryo cleavages and combining that information with morphokinetics to relate to implantation potential. DESIGN: Prospective cohort study. SETTING: University-affiliated private IVF unit. PATIENT(S): A total of 1,150 injected oocytes in 86 first oocyte donation cycles with embryo transfer on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): We analyzed the embryo OC and combined this data with the cytokinesis event, exact timing (in hours) of blastomeric cleavages, with the use of an incubator equipped with time-lapse videography, gathering a total of 7,630 measurements during the cytokinesis (active phase) and consecutive measurements after this division (passive phase), correlating this data with embryo outcome. RESULT(S): OC was found to increase during embryo cleavage, showing high levels during first division with a strong correlation with implantation success. Moreover, those embryos with slow or fast development gave rise to lower OC levels, whereas higher levels were associated with optimal embryo division ranges linked to higher implantation potential. CONCLUSION(S): A detailed analysis of OC by time-lapse observations enhances the value that these measurements represented as markers of embryo quality, especially during the cytokinesis events produced during preimplantation development.

The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension.

Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O2). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos.

Outcome of cryotransfer of embryos developed from vitrified oocytes: double vitrification has no impact on delivery rates.

OBJECTIVE: Evaluate the outcome of cryotransfer of embryos developed from vitrified oocytes. DESIGN: Retrospective cohort study. SETTING: Private university-affiliated IVF center. PATIENT(S): Women undergoing warming cycles in which vitrified embryos were developed from vitrified or fresh oocytes. INTERVENTION(S): Vitrification by the Cryotop open device. MAIN OUTCOME MEASURE(S): Delivery rate (DR) per warming cycle. RESULT(S): A total of 471 warming cycles of 796 vitrified embryos developed from vitrified oocytes (group 1) and 2,629 warming cycles of 4,394 vitrified embryos derived from fresh oocytes (group 2) were evaluated. Overall survival rates were 97.2% [95% confidence interval [CI] 95.9%-98.6%] vs. 95.7% [95% CI 94.9-96.4], respectively. DRs per warming cycle were 33.8% (group 1) and 30.9% (group 2). Double vitrification had no effect on DR (odds ratio [OR] 0.877, 95% CI 0.712-1.080). Confounding factors (ovum donation or autologous cycles; day-3 or blastocyst embryo transfer [ET]; natural or hormonal replacement therapy for ET; single or double ET; previous cycles, number of oocytes, doses of gonadotropins and E2 levels on the day of hCG) did not modify the effect of double vitrification on DR (OR 0.872, 95% CI 0.702-1.084). CONCLUSION(S): Vitrification at early cleavage or blastocyst stage of embryos obtained from previously vitrified oocytes has no effect on DR/warming cycle.