Atmospheric oxygen (∼20%) in culture significantly impairs preimplantation embryo development. However, it is not known whether all stages of preimplantation embryo development are susceptible to oxygen toxicity. This study investigated the temporal responses of preimplantation embryos to oxygen conditions in vitro. Mouse embryos were cultured in atmospheric (∼20%) or lower (5%) oxygen concentrations for the first 48 h, followed by culture in the same or reciprocal oxygen concentrations for another 48 h: group 1 (control, 5 and 5%); group 2 (5 and 20%); group 3 (20 and 5%); and group 4 (20 and 20%). Time-lapse microscopy was performed with imaging of individual embryos at 15-min intervals. Compared with embryos cultured in 5% oxygen, embryos cultured in 20% oxygen were delayed at the 1st cleavage by 0.45 h (P < 0.05), at the 2nd cleavage by 0.84 h (P < 0.01) and at the 3rd cleavage by 3.19 h (P < 0.001). Switching from 20% to 5% oxygen after 48 h did not completely alleviate earlier induced perturbations. Partial or complete culture in atmospheric oxygen resulted in significantly fewer blastocyst cell numbers compared with control (P < 0.05). Oxygen can influence mouse embryo development at both the cleavage and post-compaction stages.
Introduction
Historically, preimplantation embryos of the human and other eutherian mammals have been cultured under atmospheric oxygen (∼20%) (Edwards et al., 1981), a protocol derived from earlier somatic tissue culture techniques (Ham and Puck, 1962). However, experimental analysis of oxygen concentrations within the oviduct and uterus of different mammalian species has revealed that oxygen concentration typically lies in the range of 2–8% (Fischer and Bavister, 1993, Maas et al., 1976, Mastroianni and Jones, 1965). Lower oxygen culture, at around 5–7%, has been shown to improve embryo development in several species including the sheep (Thompson et al., 1990), cow (Thompson et al., 1990), goat (Batt et al., 1991) and human (Catt and Henman, 2000, Ciray et al., 2009, Kovacic et al., 2009, Meintjes et al., 2009, Kovacic and Vlaisavljevi, 2008, Waldenström et al., 2009). Furthermore, increased blastocyst cell number and improved fetal development have been reported from mouse embryos cultured in reduced oxygen concentrations (Quinn and Harlow, 1978, Harlow and Quinn, 1979, Gardner and Lane, 1996, Karagenc et al., 2004, Quinn and Harlow, 1978).
Several studies have indicated that the preimplantation embryo exhibits temporal sensitivity to oxygen. Pabon et al. (1989) observed delays in development to the morula stage when embryos had been exposed to 20% oxygen as pronucleate oocytes, for as little as 1 h, followed by cultured in 5% oxygen. Similarly, Gardner and Lane (1996) determined that culture media equilibrated for 6 h in 20% oxygen prior to culture in 5% oxygen was detrimental to subsequent embryo development, while Karagenc et al. (2004) more recently reported that exposure of pronucleate oocytes to 20% oxygen for 23 h resulted in a significant decrease in blastocyst cell number. Of interest, several studies have reported no difference in blastocyst development rates when the preimplantation embryo was only exposed to atmospheric oxygen concentration at post-compaction stages (Karagenc et al., 2004, Kind et al., 2005, Feil et al., 2006). Collectively, such works suggest that the pronucleate oocyte has a greater sensitivity to atmospheric oxygen, the consequences of which may only be apparent with more accurate assessment over the complete period of preimplantation development.
Morphological assessment of the preimplantation embryo typically involves observations at discrete time points, usually once every 12–24 h, generally during work hours, in line with standard laboratory practices. Furthermore, the proportion of embryos that reach a specific stage at these discrete time points may be heavily influenced by the particular culture system employed and as a result the significance of specific time points will vary between laboratories (Bavister, 1995, Shoukir et al., 1997). A novel way to increase the sensitivity of morphological evaluation is through the use of high-frequency temporal assessment of embryo development with time-lapse microscopy. Using such an approach, this study aimed to determine the temporal effect of atmospheric oxygen on embryo development by assessing the embryos response to either a static or changing concentration of oxygen.
Section snippets
Animals and hormonal stimulation
Pronucleate oocytes were obtained from F1 hybrid (C57BL/6 × CBA/Ca) mice. Animals were housed in a 12 h light and 12 h dark photoperiod with food and water ad libidum. Six-week old females were superovulated with intraperitoneal injections of 5 IU pregnant mare’s serum gonadotrophin (Folligon; Intevet, UK) followed 48 h later by 5 IU human chorionic gonadotrophin (HCG, Chorulon; Intervet). Females were mated with F1 males overnight following the HCG injection. The presence of a vaginal plug the
Results
Embryos cultured in atmospheric oxygen for the first 48 h were delayed by 0.45 h (P < 0.05) at the first cleavage event and this delay was extended further to 0.84 h (P < 0.05) at the second cleavage event (Table 1). The length of the second cell cycle for embryos cultured in 20% oxygen was 20.79 ± 0.11 h compared with 20.40 ± 0.08 h for embryos cultured in 5% oxygen (P < 0.001) and the third cell cycle length was 14.30 ± 0.39 h for 20% and 11.95 ± 0.09 h for 5% (P < 0.001). At the third cleavage event, embryos
Discussion
This study has revealed that the detrimental effects of atmospheric oxygen on mouse embryos during in-vitro culture are evident from the first cleavage division. Continuous assessment of embryo development, using time-lapse microscopy, revealed that oxygen plays an important physiological role throughout the preimplantation period. Oxygen had a biphasic effect on embryo development with the greatest effect during the cleavage stages, plausibly reflecting a greater sensitivity of embryos to
Acknowledgements
The authors wish to thank Dr George Thouas for his comments on the manuscript.
Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos.
In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24–72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%–57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%).
In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.
2023, Handbook of Current and Novel Protocols for the Treatment of Infertility
The human in vitro fertilization (IVF) laboratory has evolved significantly since the birth of Louise Brown in 1978. Through an increase in our understanding of gamete and embryo biology, combined with the development of technologies specifically for human IVF, we have seen an increase in the success of assisted conception procedures. Further, increased awareness of in vitro sources of stress to both gametes and embryos, such as the use of atmospheric oxygen and media lacking amino acids, and inadequate lab construction and poor air quality, has led to the development of more suitable culture conditions combined with the building of laboratories specifically designed for the purpose, with dedicate air handling units to improve air quality. Maintaining a high-functioning laboratory requires a clear understanding of all procedures and the many interactions between individual components of the laboratory and treatment cycle. Hence with sufficient investment in trained staff, equipment, and laboratory build, it is feasible for clinics to attain successful and consistent outcomes. In this chapter, we present the essentials to facilitate the effective performance of an IVF laboratory. The content of such a chapter could comprise an entire book of its own, so here we provide a succinct overview with key references on specific topics.
Time lapse monitoring (TLM) is a commercial system of individual embryo culture based on the well-of-the-well system. It allows for collecting a panel of intrinsic parameters of special importance to non-invasive quality assessment. Besides, a combined analysis of TLM and metabolome data provides a deeper insight into the embryo quality. Two questions have been addressed by this study: (i) whether embryo culture in the Primo Vision affects embryo development and quality compared to the classical system and (ii) whether the first zygotic cleavage (FZC) dysmorphisms affect metabolomic profile of blastocysts. Presumptive zygotes from IVM/IVF oocytes were cultured either in classical drops (pools of 30 embryos, control group) or on Primo Vision plates (16 embryos in separate wells, sharing culture medium, Primo Vision group). The two categories of blastocysts were analysed for relative transcript abundance of five genes (real time PCR; FASN, Hsp70, PLIN2, Bax, Slc2a1) and for metabolite profile (mass spectrometry). Besides, basing on retrospective analysis of the TLM files, the Primo Vision blastocysts were allocated into one of two groups depending on the FZC pattern (normal NC or direct cleavage DC) and analysed for metabolite profiles.
The Primo Vision embryos showed higher cleavage rate (p < 0.01) but reduced blastocyst rate (p < 0.05) when compared to the control group. Although the culture system (Primo Vision vs classical) did not affect the relative transcript abundance, the metabolomic analysis revealed different activity of five pathways. Two of them, related to beta-oxidation of fatty acids were up-regulated, whereas the remaining three corresponding to ubiquinone synthesis, metabolism of phenyloalanine and tyrosine as well as vitamin B6 metabolism were down-regulated. The metabolite profiles were however particularly affected by the FZC pattern. The NC and DC blastocysts differed significantly in the activity of 16 metabolic pathways which mainly involved pyruvic acid. The DC embryos displayed a higher level of pyruvic acid (p < 0.05) which may imply disturbances in the switch from lipid to glucose metabolism. Besides, in our opinion, embryos cultured in separate wells in the Primo Vision may face increased ROS level which reduces their developmental potential.
Summarizing we underline application of metabolome analysis to embryo quality assessment due to providing a wider insight into embryo response to environmental factors.
To develop and validate a practical model for quality control monitoring of dichotomous in vitro fertilization (IVF) outcomes such as pregnancy resulting from the transfer of euploid blastocysts.
We designed and validated a model for quality control monitoring of dichotomous IVF outcomes. We demonstrate use of this model for assessment of euploid blastocyst transfer quality control based on fetal heartbeat rate per embryo. The model uses 3 weighted moving averages with window sizes of 21, 51, and 101 embryo transfers to detect short and long-term shifts in success rates. The quality warning limit was set to have a 2-sided type I error rate of 0.30 per 100 embryo transfers and the control limit was set to have a type I error rate of 0.05 per 100 embryo transfers. Simulation studies were performed to validate the model through assessment of type I and type II errors using custom computer programs.
Not applicable.
Patients undergoing IVF.
None.
Type I and type II error rates and statistical power analysis.
Validated quality warning and control limits are presented for a range of expected outcome rates. The power to detect a 20% decrease from an expected fetal heartbeat rate of 50%, when the decrease persisted for 50 embryo transfers, was 86% for the warning limit and 57% for the control limit.
This model can be used for continuous quality control assessment of dichotomous IVF outcomes such as pregnancy rates.
Diseño y validación de un modelo de monitorización de control de calidad de resultados dicotómicos en fecundación in vitro.
Desarrollar y validar un modelo práctico de control de calidad para la monitorización de resultados dicotómicos en fecundación in vitro (IVF) como es: gestación resultante de transferencias de blastocistos euploides.
Diseñamos y validamos un modelo para la monitorización del control de calidad de los resultados dicotómicos de IVF. Demostramos el uso de este modelo para evaluar la transferencia de blastocistos euploides basado en la tasa de latido cardíaco fetal por embrión. El modelo utiliza 3 medias móviles ponderadas con tamaños de ventana de 21, 51 y 101 transferencias de embriones, para detectar desplazamientos de las tasas de éxito a corto y largo plazo. El límite de alarma de calidad se puso para tener una tasa de error tipo I de 2 colas de 0.30 por cada 100 transferencias de embriones. Se realizaron estudios de simulación para validar el modelo a través de la valoración de los errores tipo I y tipo II utilizando programas de ordenador a medida.
No aplica.
Pacientes en tratamiento de IVF.
Ninguna.
tasas de errores tipo I y tipo II y análisis de la potencia estadística.
Se presentan unos límites validados de calidad, de alarma y de control, para un rango de tasa de resultados esperado. La potencia para detectar una disminución de un 20% de una tasa esperada de latido cardiaco fetal del 50%, cuando la disminución persistió más allá de 50 transferencias embrionarias, fue del 86% para el límite de alarma y del 57% para el límite de control.
Este modelo puede utilizarse para la evaluación del control de calidad continua de resultados dicotómicos de IVF tales como la tasa de gestación.
Recently, infertility has become one of the most important endemic conditions, affecting approximately 15–20 % of couples worldwide. Among others, the careerist lifestyle, the increasing maternal age and the parallel increment in the aneuploidy rate of embryos play a crucial role in this phenomenon. In this study, embryological parameters and pregnancy outcomes were investigated in IVF cycles using either sequential embryo culture or a single step culture system. By sequential media, oocytes/embryos are needlessly exposed to the potentially negative effects of light exposure, temperature decrement and altered oxygen tension. In comparison with sequential media, single step media induced 1.28, 1.21 and 1.21-fold increments in implantation, biochemical pregnancy and clinical pregnancy rates, respectively. Pregnancy outcomes showed strong maternal age-dependency, so the difference between the two investigated culture systems was equalized by the increasing maternal ages (35–44 years) and the supposed incidence of embryo aneuploidy. Nevertheless, the significant enlargements in the outcomes of the younger ages (25–34) induced by the single step cultures suggest that, beside the resultant maternal aneuploidy, aneuploidy (reduced pregnancy rates) may evolve from exposure to the mentioned environmental stress factors.
Mammalian oocyte maturation and early embryo development are highly sensitive to the in vitro culture environment, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effects of different oxygen concentrations (20%, 10%, 5% or 1% O2) on yak oocyte maturation, in vitro fertilization (IVF), and embryo development competence, as well as its effects on the oxidative response, metabolism, and apoptosis in cumulus-oocyte complexes (COCs) and the embryo. The results revealed that the maturation rate of oocytes, blastocysts rate and hatched blastocysts rate in the group with 5% oxygen concentration were significantly higher (P < 0.05) than other groups, but the cleavage rate with 5% oxygen concentration was significantly lower (P < 0.05) than the 20% and 10% oxygen concentrations. The maturation rate of oocytes, the cleavage rate, blastocysts rate and hatched blastocysts rate with the 1% oxygen concentration were the lowest. The blastocyst cultured with 5% oxygen concentration had significantly greater (P < 0.05) numbers of total cells, inner cell mass (ICM) cells and trophectoderm (TE) cells compared to the other groups. Analysis of the apoptosis index of oocytes and blastocyst cells by transferase dUTP nick end labeling (TUNEL) showed that the number of apoptotic cells significantly reduced (P < 0.05) with 5% oxygen concentration, but increased significantly (P < 0.05) in the 1% oxygen concentration group. Also, the qRT-PCR and western immunoblotting analysis confirmed that the transcription levels of the metabolism genes, antioxidant response genes, apoptosis genes, oocyte competence genes and embryonic developmental markers showed significant differences (P < 0.05) in the COCs or blastocysts matured in 5% oxygen concentration group compared to the other groups. In summary, our findings demonstrate that 5% oxygen concentration improves oocyte maturation and blastocyst development in the yak, increases blastocyst cell numbers, reduces apoptosis rate in the oocyte and blastocyst as well as reduces embryo cleavage rate.
Petra Wale has worked in clinical IVF for over a decade both in Australia, at Melbourne IVF, and in the USA, at the Colorado Center for Reproductive Medicine. She is currently undertaking her PhD with Professor David Gardner at the University of Melbourne, Australia. Her main interests include embryo culture technologies and the development of embryo viability assays.
