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Atmospheric oxygen and individual culture are standard embryo culture conditions, the effect of combining them is investigated here.
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Cleavage timings, hatching rates and blastocyst cell numbers were reduced by either individual culture or atmospheric oxygen.
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The combination of individual culture and atmospheric oxygen was more detrimental than either treatment alone.
Abstract
Embryos are routinely cultured individually, although this can reduce blastocyst development. Culture in atmospheric (20%) oxygen is also common, despite multiple detrimental effects on embryos. Although frequently occurring together, the consequences of this combination are unknown. Mouse embryos were cultured individually or grouped, under physiological (5%) or atmospheric (20%) oxygen. Embryos were assessed by time-lapse and blastocyst cell allocation. Compared with the control group (5% oxygen group culture), 5-cell cleavage (t5) was delayed in 5% oxygen individual culture and 20% oxygen group culture (59.91 ± 0.23, 60.70 ± 0.29, 63.06 ± 0.32 h post-HCG respectively, P < 0.05). Embryos in 20% oxygen individual culture were delayed earlier (3-cell cleavage), and at t5 cleaved later than embryos in other treatments (66.01 ± 0.40 h, P < 0.001), this delay persisting to blastocyst hatching. Compared with controls, hatching rate and cells per blastocyst were reduced in 5% oxygen single culture and 20% oxygen group culture (134.1 ± 3.4, 104.5 ± 3.2, 73.4 ± 2.2 cells, P < 0.001), and were further reduced in 20% oxygen individual culture (57.0 ± 2.8 cells, P < 0.001), as was percentage inner cell mass. These data indicate combining individual culture and 20% oxygen is detrimental to embryo development.
During culture in vitro, preimplantation embryos are exposed to various forms of stress, which influence the development of the embryo independently, and can also work in synergy to further compromise viability. These stressors may include culture media formulation, pH, temperature and oxygen, among others (
). While a single stressor may perturb embryo development to some degree, it also increases the embryo's vulnerability to a second stressor, as demonstrated in the case of light and temperature (
). Understanding the identity and nature of these stressors and their interactions has facilitated significant improvements in the efficacy and safety of embryo culture in vitro and the development of more suitable culture conditions. Consequently, embryo culture media have become more physiological over the past two decades, and are no longer simple salt solutions with carbohydrates (
). However, culture media still lack several components of the complex oviduct and uterine environments, including many paracrine signalling molecules (
). Embryos themselves secrete signalling molecules into the media in vitro, and these may partially compensate for the absence of signals from the reproductive tract.
The preimplantation embryo is unusual in that it does not have an absolute requirement for signalling from neighbouring cells in order to survive and proliferate (
Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development.
). Recent studies, including those with human embryos, demonstrate that advances in culture systems have not yet replicated the benefits of embryo group culture (
In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.
). In addition to negative effects on blastocyst development, cell numbers and allocation, individual culture has also been reported to increase apoptosis (
A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.
), and there have been no reports on birth weights or other pregnancy outcomes. The few animal studies that have investigated post-implantation outcomes following individual embryo culture have observed only a trend for decreased implantation rates or live birth rates (
In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.
Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial.
Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.
Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.
), both of which require embryos to be cultured individually (in the case of PGS this is not necessarily required if the biopsy is performed at the blastocyst stage). The widespread adoption of these technologies has therefore resulted in individual embryo culture becoming a standard procedure for many clinics. While some human IVF clinics culture embryos in groups, a recent web survey of 265 clinics from 71 countries indicated that 55% of clinics routinely culture human embryos individually during at least part of the culture period (
In addition to deprivation from paracrine factors, embryos cultured in vitro are also frequently exposed to atmospheric (20%) oxygen concentrations. Physiological oxygen concentrations are around 2–8% in the reproductive tract (
), and studies on multiple mammalian species have shown that culture in 20% oxygen is detrimental for embryo development, resulting in slower cleavage timings (
Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos.
Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos.
In vitro culture increases the frequency of stochastic epigenetic errors at imprinted genes in placental tissues from mouse concepti produced through assisted reproductive technologies.
). While the detrimental effects of 20% oxygen on human embryos have long been debated, clinical studies have reported that culture in 20% oxygen causes slower cleavage divisions (
) compared with embryos cultured in 5% oxygen. However, in spite of these animal and human data, the use of a reduced oxygen environment for human embryo culture is not common. In a web survey of 265 clinics by Christianson and colleagues (
) only 24% of surveyed clinics reported using reduced oxygen for all of their IVF and embryo culture, and 39% of clinics did not use physiological oxygen at all, meaning that 76% of human embryos are exposed to oxidative stress during some period of their culture in vitro culture.
While a single stressor may perturb embryo development to some degree, it also reduces the embryo's capacity to cope with a second stressor. The aim of this study was therefore to investigate the potential effect of combining two routinely employed clinical practices, individual embryo culture and atmospheric oxygen, in a mouse model. Time-lapse microscopy, which has previously been employed to investigate the effects of oxygen on the development of single embryos (
), was employed to assess cleavage event timings of grouped and singly cultured embryos.
Materials and methods
Animals
F1 hybrid (C57BL/6 × CBA) mice were maintained in a standard animal research facility in individually ventilated cages (Optimice, Animal Care Systems, Centennial, CO, USA) with a 12 h light–dark photoperiod (6.00 h–18.00 h) with controlled temperature and food and water available ad libitum.
Fertilized embryos were generated by superovulation of four week old F1 hybrid females with intraperitoneal injections of 5 international units (IU) pregnant mare serum gonadotrophin (PMSG, Folligon; Intervet, Bendigo East, Vic, Australia) at the mid-point of the light phase, followed 48 h later by 5 IU human chorionic gonadotrophin (HCG, Chorulon; Intervet), then mating with males of the same strain overnight. All mice were killed by cervical dislocation. All experiments were approved by The University of Melbourne Animal Ethics Committee on 2 May 2014 (reference number: 1413159).
Embryo culture
Pronucleate oocytes were collected 22 h after HCG injection in G-MOPS PLUS handling medium, containing 5 mg/ml human serum albumin (HSA) (Vitrolife, Göteborg, Sweden) as described previously (
). Embryos were incubated in G-MOPS PLUS containing 550 IU/ml hyaluronidase (bovine testes type IV-S; Sigma-Aldrich, St Louis, MO, USA) until cumulus cells were removed, then washed three times in G-MOPS PLUS and once in pre-incubated G1 culture medium. Embryos were pooled, then allocated to treatments. Embryos were cultured in G1 medium for 48 h, then transferred to G2 medium for a further 24 h or 48 h. All embryo manipulations were performed on a SMZ 1500 microscope with a heated stage (Nikon Instruments, Melville, NY, USA). Embryo development was assessed on the morning of days 3, 4 and 5 of culture (70, 94 and 118 h post-HCG). Developmental stages were defined as follows: ‘compacting’: the loss of membrane definition between blastomeres; ‘early blastocyst’: the presence of a blastocoel cavity less than half the volume of the embryo; ‘blastocyst’: the presence of a cavity occupying at least half the volume of the embryo; ‘expanded’: the increase in volume of the blastocyst and thinning of the zona; ‘hatching’: the appearance of cells outside the zona; and ‘fully hatched’: the complete evacuation of the embryo from the zona (
), except choline chloride, folic acid, inositol, nicotinomide and taurine were omitted from G2. Media were supplemented with hyaluronan (0.125 mg/ml; Vitrolife) and gentomycin (10 µg/ml; Sigma). Media were also supplemented with 2.5 mg/ml recombinant albumin (G-MM, Vitrolife), to eliminate the potential effects of contaminants inherent in serum albumin (
). All additional media chemicals were supplied by Sigma-Aldrich, except essential amino acids (MEM Cellgro; Corning Life Sciences, Tewksbury, MA, USA). All chemicals and plastics were tested in a mouse embryo assay prior to use (
Embryo cultures were performed in 35 mm or 60 mm Petri dishes (Falcon Easy-Grip; Corning Life Sciences, Tewksbury, MA, USA) under paraffin oil (Ovoil; Vitrolife) in a humidified multi-gas incubator at 37°C (MCO-5M; Sanyo Electric, Osaka, Japan). For reduced oxygen experiments the incubator atmosphere was 5% O2, 6% CO2 and 89% N2, for 20% oxygen experiments the incubator atmosphere was 6% CO2 in air. Drops of media were made under oil using an eVol positive displacement pipette (SGE Analytical Science, Ringwood, Vic, Australia) in order to accurately deliver the small volumes employed and prevent evaporation of media during dish preparation (accurate to ± 1.0%). Grouped embryos were cultured in drops of 20 µl (10 embryos per group) and single embryos in 2 µl, in order to maintain the embryo: media volume ratio. Embryos were cultured in groups or individually, in either physiologic (5%) or atmospheric (20%) oxygen, resulting in four treatment groups: 5% oxygen grouped (control group); 5% oxygen single; 20% oxygen grouped; and 20% oxygen single. The treatment of 5% oxygen group culture is referred to as the ‘control’ because it is considered the current best practise for mammalian embryo culture, but statistical comparisons have been performed between all groups.
To ensure that using two drop volumes did not affect osmolality, the osmolality was measured in both the 2 µl and 20 µl drops after 48 h incubation. There was no difference in osmolality between the two drop sizes, and both were well within normal range for embryo culture media (data not shown). Furthermore, single embryos were cultured in 2 µl or 20 µl drops and the study found that culture in 2 µl did not impair development (data not shown). Consequently, any differences detected in this study cannot be attributed to compromised conditions in the 2 µl drops.
Differential stain
Allocation of cells in the blastocyst to the inner cell mass (ICM) or trophectoderm was assessed using a differential staining protocol (
). All procedures were performed at 37°C, and blastocysts were washed in G-MOPS PLUS between all steps except the last. Simple G1 (sG1) medium without non-essential amino acids, alanyl glutamine, taurine or HSA, containing 4 mg/ml polyvinylpyrrolidone (PVP; Sigma-Aldrich) (sG1 + PVP) was used to dilute trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) and anti-DNP produced in rabbit (Sigma Aldrich).
Blastocysts were incubated in 0.5% pronase (Sigma-Aldrich) until the zona was no longer visible, then in 0.5% 2,4,6-TNBS for 10 min. Blastocysts were then transferred to 10% anti-DNP antibody for 10 min, and then into guinea pig serum (IMVS, Adelaide, SA, Australia) diluted 50% in 0.02 mg/ml propidium iodide (Sigma-Aldrich) in G-MOPS until blebbing of the cell membranes was observed. The final step was incubation in 0.1 mg/ml bisBenzimide (Hoescht 33258, Sigma) in G-MOPS and 10% ethanol for 30 min. Blastocysts were then washed briefly and mounted in glycerol on a glass microscope slide. Images were captured on an Eclipse TS100 inverted fluorescent microscope with DS-Fi1 camera and Digital Sight control unit (Nikon Instruments). Cells were counted using ImageJ (1.49v, National Institutes of Health, USA).
Morphokinetics
Embryos were monitored using a multi-gas cell imaging incubator fitted with an inverted microscope (MCOK-5M[RC]; Sanyo Electric), as described previously (
Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.
). The embryo culture dishes (which were the same as described in the embryo culture section) rest on a motorised stage, while images are taken through the fixed Olympus microscope with a ×10 objective. The light source is a 0.1 W white LED. Images were obtained every 15 min during culture and the time of cleavage events recorded. Cleavage times were defined as the first time point when new blastomeres were separated by cell membranes. To date, numerous systems for notation of cleavage events have been used, and while there have been proposals for standardization (
): cleavage time to two cells is represented as t2, and so on for three to eight cells. Syn-t2 is the duration from syngamy to two cells; cc2 is the duration of the second cell cycle of the embryo, i.e. the interval between t2 and t3, likewise cc3 is the third cell cycle, i.e. the interval between t4 and t5; s2 is the duration of the second synchrony, i.e. the interval between t3 and t4, likewise s3 is the duration of the third synchrony, i.e. the interval between t5 and t8. Notation of post-compaction events are as described in the embryo culture section. Cleavage times are expressed as h post-HCG injection, or as h post-t2 (to reduce the potential influence of variation in mating and fertilization times). Time-lapse experiments (but not earlier experiments) in either 5% oxygen or 20% oxygen were conducted in series to compare individual and group culture at each oxygen concentration.
Statistical analysis
For all tests, differences were considered statistically significant when P < 0.05. Percentage data (e.g. the number of embryos to reach each stage of development on each day) were compared by Fisher's Exact Test in Prism v5.04 (GraphPad, La Jolla, CA, USA). Comparison of means (e.g. the number of cells per embryo) were compared by General Linear Model Univariate analysis with ‘oxygen’ and ‘grouping’ as factors and ‘replicate’ as a random factor in SPSS Statistics v22 (IBM, Armonk, NY, USA). This was followed by the same test with ‘treatment’ as a factor and ‘replicate’ as a random factor. Multiple comparisons were performed with Bonferroni post-hoc analysis. The homogeneity of variance was assessed by visualisation of the residuals on a scatterplot, and if the data were considered skewed then it was transformed by either a square root or logarithmic transformation, depending on which was most appropriate for the data.
Results
Development rates and cell allocation
Single culture in 5% oxygen had no effect on the number of embryos that were compacting on day 3 compared with the control group (5% oxygen group culture) (Figure 1), but did reduce the proportion of embryos hatching on day 4 (Supplementary Table S1). On day 5, in 5% oxygen, fewer individually cultured embryos were hatching than those in groups, and more were expanded. Atmospheric oxygen did not alter the number of embryos in groups compacting on day 3 compared with controls, but on days 4 and 5 fewer embryos were hatching. While there was no effect of oxygen or individual culture on the proportion of embryos that had developed to the compacting stage on day 3 compared with controls, the combination of both 20% oxygen and individual culture did reduce the number of compacting embryos compared with controls. This pattern continued on days 4 and 5, when the combination of both 20% oxygen and single culture resulted in fewer embryos forming a blastocyst or hatching than embryos cultured in either 20% oxygen in groups, or in 5% oxygen individually.
Figure 1Development of embryos in 5% or 20% oxygen, in groups of 10 or individually. Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h post-HCG). Stages: day 3 compacting, day 4 blastocyst, day 5 hatching. n = at least 180 embryos per treatment, 11 replicates, half of the embryos were removed from culture on day 4 for staining. Different letters indicate significant differences between treatments; day 3 P < 0.05; day 4 P < 0.01, day 5 P < 0.001. HCG = human chorionic gonadotrophin.
The general linear model (GLM) univariate analysis showed that there was a significant overall effect of both oxygen (P < 0.001) and grouping (P < 0.001) on the total number of cells per blastocyst on day 4, and the interaction between the two factors was significant (P < 0.05). A Bonferroni post-hoc analysis showed that the mean number of cells per embryo in 5% oxygen was the same in single culture or group culture, but in 20% oxygen embryos cultured individually had fewer cells than those in groups (P < 0.001) (Figure 2A). All embryos in 20% oxygen had fewer cells than those in 5% oxygen (P < 0.001). The same pattern was observed in the trophectoderm cells (overall effect of oxygen P < 0.001, grouping P < 0.01, interaction P < 0.01), and the post-hoc showed differences between all treatments except the 5% oxygen group and single (P < 0.001). The number of ICM cells was affected by both oxygen (P < 0.001) and grouping (P < 0.001), but there was no significant interaction. The post-hoc analysis showed all treatments were significantly different to each other (P < 0.001). Consequently, the proportion of ICM per embryo was affected by both oxygen (P < 0.05) and grouping (P < 0.001), with a significant interaction (P < 0.05). A Bonferroni post-hoc analysis found the percentage ICM was reduced by single culture compared with group culture when embryos were cultured in 5% oxygen (P < 0.001) but not 20% oxygen (or, conversely, the percentage ICM was reduced by 20% oxygen compared with 5% oxygen when embryos were in groups [P < 0.01] but not when they were cultured individually). All treatments had a lower proportion of ICM compared with the control (P < 0.01).
Figure 2Cell numbers on day 4 (A) and 5 (B) of culture in 5% or 20% oxygen, in groups of 10 or individually. Bars represent mean ± sem; n = at least 80 embryos per treatment, day 4 = 5 replicates, day 5 = 6 replicates. Embryos were stained at 94 and 118 h post-HCG, respectively. Different letters represent significant differences between treatments; (A) Total, trophectoderm P < 0.001; ICM, percentage ICM P < 0.01; (B) Total, trophectoderm, ICM <0.001, percentage ICM <0.05. HCG = human chorionic gonadotrophin; ICM = inner cell mass; TE = trophectoderm.
The total number of cells per day 5 blastocyst, the number of cells in the trophectoderm and the number of cells in the ICM (Figure 2B) were analysed by GLM univariate and there was a negative overall effect of both single culture (P < 0.001) and 20% oxygen (P < 0.001), but the interaction was not significant, implying that single culture had a detrimental effect at both 5% or 20% oxygen (or conversely, that 20% oxygen had a detrimental effect whether embryos were cultured in groups or singly). Post-hoc analysis showed that all treatments were significantly different to each other (P < 0.001). The proportion of ICM per embryo was affected by single culture (P < 0.05), but not oxygen, and there was no significant interaction.
Morphokinetics
To determine the point at which delayed development was observable, embryos were monitored in a time-lapse system. In 20% oxygen, the timing of syngamy and t2 were not different between individual and group cultured embryos, but mean cc2 was 38 min slower in individually cultured embryos (Table 1). As a result, t3 was delayed in these embryos by nearly 1 h compared with group culture in the same oxygen concentration. cc3 was also slower by 1 h 45 min, resulting in t5 delayed by nearly 3 h. By the time cavitation was reached, single embryos were delayed by 5 h 45 min, and time at hatching was similarly delayed by 4 h 45 min. Although 92% of embryos in single culture in 20% oxygen formed a cavity, only 51% initiated hatching. This was significantly fewer than the control group (95%), single embryos in 5% oxygen (90%) or embryos in groups in 20% oxygen (98%) (P < 0.001). Similarly, significantly fewer embryos had completely escaped the zona after individual culture in 20% oxygen (7%) compared with the controls (31%), single culture in 5% oxygen (18%) or group culture in 20% oxygen (20%).
Table 1Time of cleavage events of embryos cultured in 5% or 20%, in groups of 10 or individually.
Values are mean±sem of cleavage events (h post-HCG), or time between cleavage events (h). Notation for cleavage events are described in the Methods section. n = at least 60 embryos per treatment, 4 replicates at 5% oxygen, 3 replicates at 20% oxygen. cc2 = the duration of the second cell cycle of the embryo; cc3 = the third cell cycle; HCG = human chorionic gonadotrophin; syn-t2 = the duration from syngamy to two cells; s2 = the duration of the second synchrony; s3 = the duration of the third synchrony.
* indicates difference compared with group culture in the same oxygen concentration, *P < 0.05, ***P < 0.001.
When embryos were cultured in 5% oxygen, no difference was observed between group cultured and single embryos until cc3, when individually cultured embryos were delayed by 43 min. This resulted in a delay of t5 by 47 min. There was no significant difference in the time taken for embryos to complete the third synchrony, however t8 was delayed by 1 h 17 min. There was no significant difference in timing of post-compaction events, despite observations in the previous section that fewer individually cultured embryos were hatching on day 5.
When cleavage times were expressed relative to the time each embryo reached t2, the differences between the treatments remained the same as when cleavage times were expressed relative to HCG injection (Table 2). As such, the delays in development remained evident irrespective of the normalisation time point used.
Table 2Time of cleavage events of embryos cultured in 5% or 20% oxygen, in groups or individually.
Values are mean±sem of cleavage events (h post-t2). Notation for cleavage events are as described in the Methods section. n = at least 60 embryos per treatment, 4 replicates at 5% oxygen, 3 replicates at 20% oxygen.
* indicates difference compared with group culture in the same oxygen concentration, *P < 0.05, **P < 0.01, ***P < 0.001.
The 5% oxygen and 20% oxygen time-lapse experiments were done in series, and so the cleavage times for embryos cultured under the two oxygen concentrations could not be directly compared. It was observed however, that when embryos were cultured in groups, syngamy, t2 and t3 occurred at the same time in 5% and 20% oxygen (Figure 3). Then cc3 was approximately 3 h longer for those embryos cultured in groups in 20% oxygen, resulting in a delay of t5 compared with controls. While there appeared to be no difference in cavitation or hatching times, fewer embryos hatched fully in 20% oxygen group culture than controls (20% vs 31%), and it occurred around 3 h later than in 5% oxygen. When embryos were cultured individually, the delay caused by 20% oxygen compared with single embryos in 5% oxygen was observable earlier and was more exaggerated than the delay caused by 20% oxygen when embryos were cultured in groups (1 h delay at t3, 6 h delay at t5). There was also an observable delay in cavitation (6 h) and hatching (4 h), and fewer single embryos reached the hatching stage in 20% oxygen than 5% oxygen (51% vs 91%).
Figure 3Time of cleavage events relative to group culture at 5% oxygen (control). Data from Table 1 is represented here as (mean cleavage time) − (mean control cleavage time) ± sem. Notation of cleavage events are described in the Methods section. * indicates difference compared with group culture in the same oxygen concentration, *P < 0.05, ***P < 0.001. n = at least 60 embryos per treatment, 4 replicates at 5% oxygen, 3 replicates at 20% oxygen. Not all embryos reached each developmental stage.
This study has demonstrated that two common practices of embryo culture, atmospheric oxygen and individual culture, are not only detrimental independently, but that combining these two conditions perturbs embryo development further.
The adverse effects of single culture and 20% oxygen were observed early in culture by time-lapse microscopy. In 5% oxygen, individually cultured embryos were slower to complete the third cell cycle than embryos cultured in groups, which resulted in later cleavage to the 5-cell stage. This delay in cleavage timings persisted to the 8-cell stage, but was not observed later in post-compaction events, although blastocyst cell number was significantly affected. In 20% oxygen, single culture induced a delay compared to group culture that occurred earlier than in 5% oxygen and was more exaggerated. Single embryos at 20% oxygen took longer than embryos in groups at 20% oxygen to complete the second cell cycle, resulting in a delay of cleavage to 3-cells, and this delay increased during the cleavage stages and was maintained post-compaction. These results are in agreement with previous studies that have demonstrated 20% oxygen induces a delay in cleavage timings of individually cultured embryos compared with those in physiological oxygen (
), but this study is the first to compare morphokinetics of group cultured and individually cultured embryos. Cleavage timings are predictive of embryo viability in multiple species, although debate remains over which morphokinetic parameters are most appropriate to use to select the best embryo for transfer (
Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial.
Relationship between the length of cell cycles, cleavage pattern and developmental competence in bovine embryos generated by in vitro fertilization or parthenogenesis.
). In the mouse, faster cleaving embryos are more likely to form a blastocyst, have more ICM cells and different mRNA and metabolic profiles at the blastocyst stage, as well as improved fetal development compared with slow-cleaving embryos (
Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.
In the present study, these delays in cell cleavage timings resulted in a reduction in blastocyst cell numbers on days 4 and 5. On day 4, single culture in 5% oxygen reduced the number of ICM cells and the proportion of ICM per embryo compared with group culture in 5% oxygen. Atmospheric oxygen in groups reduced the total number of cells, trophectoderm, ICM and percentage ICM compared with group culture in 5% oxygen. Combining single culture and 20% oxygen resulted in a further reduction in total cell numbers, trophectoderm and ICM in day 4 blastocysts than either single culture in 5% oxygen or group culture in 20% oxygen. These blastocysts had roughly half the number of cells compared with blastocysts cultured at 5% oxygen in groups. Cell numbers on day 5 followed a similar pattern, except that the total number of cells per embryo was reduced by single culture in 5% oxygen. Also, percentage ICM was reduced by single culture but not 20% oxygen compared with group culture at 5% oxygen. Again, the blastocysts cultured individually in 20% oxygen had roughly half the number of cells compared with blastocysts cultured in groups in 5% oxygen. This reduction in cell numbers is in agreement with the majority of studies on single culture (
Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro.
Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro.
), which indicates that the most viable embryos were those cultured in groups in 5% oxygen, and the least viable were those cultured individually in 20% oxygen.
The observed higher rate of hatching following group culture is consistent with the majority of previous reports (
Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development.
). Although hatching is necessary for implantation and ispredictive of improved pregnancy rates and live birth in human assisted reproductive technologies (
Blastocyst expansion score and trophectoderm morphology strongly predict successful clinical pregnancy and live birth following elective single embryo blastocyst transfer (eSET): a national study.
). Blastocyst hatching in vitro is brought about by increasing pressure from the expanding blastocyst combined with zona-thinning embryo-secreted proteases, where implantation serine protease 1 (ISP1, also known as strypsin) is thought to play a vital role (
). An increase in hatching rate may be due to an overall improvement in embryo health, but during culture proteases are secreted into the culture media (
Regulation of proteinases during mouseperi-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9.
), and could assist in the zona degradation and hatching of neighbouring embryos. Also, it has been suggested that protease production for hatching may be stimulated by growth factors and cytokines (
). If this were the case, it may partially explain the increased hatching rates reported when embryos are cultured in groups, as embryos secrete many growth factors (
), which may elicit protease production. The authors are not aware of any direct evidence of a link between growth factors or cytokines and ISP1 production in hatching blastocysts, but leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) upregulate matrix metalloproteinase (MMP) and urokinase-type plasminogen activator (uPA) activity in blastocyst outgrowths (
). The connection between proteases and growth factors can also operate in reverse, as proteases are involved in protein processing and secretion and thereby can regulate growth factor activity (
). An alternative theory is that group culture results in increased hatching due to secretion and local concentration of lactate, which may reduce the pH in the immediate microenvironment (
). A slightly acidic pH can improve the stability of some proteases and prolong their activity, or can increase their secretion, thereby possibly enhancing zona-degrading activity (
Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated protein kinase signaling.
Although the benefits of group culture have been recognized since the 1990s, the exact mechanisms responsible remain unclear. Embryos both secrete and deplete substances in the culture media, and it is likely that combinations of multiple changes to their microenvironment are responsible for the observed improvements in development during group culture (
Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization.
), the embryo secretome remains only partially defined, and the role of each secreted or depleted substance in contributing to the beneficial effect of group culture is largely unknown. To date, most attention has been given to the potential role of embryo-secreted growth factors and cytokines in promoting growth and/or survival (
A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization.
). Yet there are other embryo-secreted molecules that alter the microenvironment which have not yet been investigated for their role in the beneficial effect of group culture (
). One example is lactate, often considered a waste product with no physiological consequence, it can lower pH and act as a signalling molecule, both of which can alter cell behaviour (
). This study observed a delay in cleavage timings from the 3-cell stage in individually cultured embryos, indicating that embryo-mediated alterations to the microenvironment are important from early in culture. This is in keeping with previous evidence in the mouse that the earliest period of development is sensitive to the stress of individual culture (
Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro.
In this study, culture in 20% oxygen resulted in slower cleavage divisions, reduced blastocyst and hatching rates, and fewer cells per blastocyst compared with culture in 5% oxygen (in groups or individually), all of which is in agreement with previous studies in the mouse (
). Embryos cultured at 20% oxygen have also been shown to display changes in gene and protein expression, metabolism and reduced post-implantation viability, among other effects (
), but the mechanism by which oxygen perturbs embryo development is not fully understood. Excess oxygen is proposed to induce an increase in intracellular reactive oxygen species (ROS), possibly by changing the activity of NADPH and xanthine oxidases (
). In support of this hypothesis, one form of ROS, hydrogen peroxide (H2O2), has been found at higher concentrations in embryos cultured in 20% oxygen compared with 5% oxygen in the mouse (
Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos.
). Although production of ROS is part of normal cell metabolism, elevated ROS can be deleterious, resulting in dysfunction of signalling and other processes (
). Furthermore, oxygen is a metabolite, and as such changes in its concentration can influence the relative activities of metabolic pathways within the embryo (
). In addition to regulating ATP generation and cell growth, changes to cellular metabolism can also influence signalling pathways, including long term alterations of the epigenome (
The synergistic effect of combining 20% oxygen and single culture, both of which can be considered stressors, is consistent with previous studies that have treated preimplantation embryos with two stressors (
). Individually cultured embryos may be more sensitive to the additional stress of 20% oxygen, but there may also be an interaction between oxidative stress and the secretome. Changes to oxygen concentration can alter secretions of interferon tau (IFN-τ) from bovine embryos (
), resulting in an altered microenvironment through consumption and production of carbohydrates and amino acids. As metabolites also act as signalling molecules, oxygen has the potential to alter paracrine signalling through regulating metabolism. Addition of antioxidants (
In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.
) during culture has been demonstrated to improve single embryo development more than when embryos are cultured in groups, indicating that culturing embryos in groups may reduce oxidative stress.
Although mammalian embryos exhibit many species-specific traits, there are also many commonalities in their physiologies and requirements, and hence the mouse has proved to be a good model for human embryo culture (
). A possible reservation regarding the use of mouse embryos for experiments investigating group culture is that mice are polyovulatory. We would suggest that the embryotrophic effect of group culture may not necessarily be embryo-specific, but neighbouring embryos may substitute for the lack of paracrine signals from the reproductive tract. This is supported by the observation that embryos from bovine, which is a monovulatory species, also benefit from group culture (
In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.
). A second issue regarding the use of mouse embryos, particularly hybrid mice, is that the embryos are perceived to be more robust than human embryos. Additionally, mouse embryos are less heterogeneous and obtained from young, fertile donors, in contrast to the heterogeneous, older, infertile human population that undergo IVF treatment, and consequently the mouse embryos used may be of higher quality than many cultured human embryos. The presence of poor quality embryos in a group may reduce the benefits of group culture (
The potential combined effects of single culture and oxygen have not been examined in the human; the studies that have compared group and single culture have mostly used 20% oxygen (
A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.
), where 5% oxygen was used (personal communications). The range of clinical study designs and culture techniques do not allow for comparison across studies to discern the role of oxygen in determining the outcome of single culture.
Optimisation of each element of the culture system is essential to prevent damage to the embryo from known and unknown stressors, since exposure to one stressor can predispose the embryo to be less resilient to additional stressors. While it is necessary to culture embryos individually in order to facilitate analyses for embryo selection, there is no situation that necessitates the use of atmospheric oxygen for embryo culture. If these results from the mouse are replicated in humans, the combination of atmospheric oxygen and single culture will decrease the number of good quality blastocysts available for transfer. To date, no study has compared the effects of reduced and atmospheric oxygen in individually cultured and group cultured human embryos, but results of this study suggest such an investigation is warranted.
Acknowledgement
This work was funded through the University of Melbourne. The authors thank Drs Alexandra Harvey and Lisa Lee for their comments on the manuscript.
Appendix. Supplementary material
The following is the supplementary data to this article:
Culture of embryos in atmospheric or reduced oxygen, in groups of 10 or individually. Numbers in columns represent the % of embryos at each developmental stage. N = 180–250 embryos per treatment, 11 replicates. Approximately half of the embryos were removed from culture on day 4 for staining, resulting in fewer embryos on day 5. Grouped 5% oxygen (5G), Single 5% oxygen (5S), Grouped 20% oxygen (20G), Single 20% oxygen (20S).
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