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Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro

Reproductive BioMedicine Online, Volume 33, Issue 5, November 2016, Pages 537–549

Highlights

  • Atmospheric oxygen and individual culture are standard embryo culture conditions, the effect of combining them is investigated here.
  • Cleavage timings, hatching rates and blastocyst cell numbers were reduced by either individual culture or atmospheric oxygen.
  • 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.

Keywords: group culture, IVF, oxidative stress, paracrine factors, single culture, time-lapse.

Introduction

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 (Wale and Gardner, 2016). 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 (Fischer et al., 1988), single culture and peroxides (Hughes et al., 2010) or oxygen and ammonium (Wale and Gardner, 2013). 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 (Bavister, 1995, Gardner, Lane, 1997, and Lane, Gardner, 2007). However, culture media still lack several components of the complex oviduct and uterine environments, including many paracrine signalling molecules (Aviles et al, 2010, Coy, Yanagimachi, 2015, Hannan et al, 2011, Robertson et al, 2015, and Salamonsen et al, 2016). 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 (Raff, 1992). Nonetheless, blastocyst development rates and cell numbers in vitro are improved by group culture with other embryos (Gardner et al, 1994, Keefer et al, 1994, Lane, Gardner, 1992, Moessner, Dodson, 1995, and Paria, Dey, 1990). Recent studies, including those with human embryos, demonstrate that advances in culture systems have not yet replicated the benefits of embryo group culture (Ebner et al, 2010, Isobe et al, 2015, Sun et al, 2014, and Vutyavanich et al, 2011). In addition to negative effects on blastocyst development, cell numbers and allocation, individual culture has also been reported to increase apoptosis (Brison and Schultz, 1997), and affect the embryo secretome (Contramaestre et al, 2008 and Larson, Kubisch, 1999). It remains unclear whether pregnancy rate in humans is affected by individual culture (Almagor et al, 1996, Ebner et al, 2010, and Spyropoulou et al, 1999), 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 (Isobe et al, 2015, Kato, Tsunoda, 1994, and Lane, Gardner, 1992), and the long term development and health of such offspring has not been monitored.

In recent years, improved technologies for embryo monitoring and diagnosis have become available, such as time-lapse microscopy (Conaghan et al, 2013, Kirkegaard et al, 2014, Pribenszky et al, 2010b, and Rubio et al, 2014) and advanced molecular techniques for preimplantation genetic screening (PGS) (Schoolcraft et al, 2010, Scott et al, 2013, and Yang et al, 2012), 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 (Christianson et al., 2014). Some of these single embryos will be cultured in microwells, which can improve embryo development compared with single culture in a drop (Chung et al, 2015, Dai et al, 2012, and Vajta et al, 2008), but many single embryos are cultured in conventional drops with a wide variety of volumes (Bolton et al., 2014).

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 (Fischer, Bavister, 1993 and Mastroianni, Jones, 1965), and studies on multiple mammalian species have shown that culture in 20% oxygen is detrimental for embryo development, resulting in slower cleavage timings (Wale, Gardner, 2010 and Weinerman et al, 2016), lower blastocyst rates and cell numbers (Batt et al, 1991, Quinn, Harlow, 1978, Tervit et al, 1972, Thompson et al, 1990, and Whitten, 1971), increased apoptosis (Van Soom et al, 2002 and Yuan et al, 2003), more frequent aneuploidy (Bean et al., 2002), more DNA damage (Kitagawa et al, 2004 and Takahashi et al, 2000) and higher hydrogen peroxide concentrations (Goto et al, 1993, Kitagawa et al, 2004, and Kwon et al, 1999). Atmospheric oxygen in culture is also associated with differential preimplantation gene expression (Harvey et al, 2004, Kind et al, 2005, Meuter et al, 2014, and Rinaudo et al, 2006), histone remodelling and global methylation (Gaspar et al, 2015 and Li et al, 2014), alterations of the proteome (Katz-Jaffe et al., 2005), secretome (Kubisch, Johnson, 2007 and Rodina et al, 2009) and metabolism (Khurana, Wales, 1989 and Wale, Gardner, 2012) compared with 5% oxygen. These changes result in perturbed post-implantation development following culture in 20% oxygen (de Waal et al, 2014, Fischer-Brown et al, 2005, and Karagenc et al, 2004). 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 (Kirkegaard et al., 2013), reduced blastocyst rates and blastocyst cell number (Dumoulin et al, 1999 and Waldenstrom et al, 2009) and altered gene expression (Mantikou et al., 2016). When embryos cultured in 20% oxygen are transferred to patients, they result in decreased implantation and pregnancy rates (Catt, Henman, 2000, Gomes Sobrinho et al, 2011, and Meintjes et al, 2009) and decreased live birth rates (Bontekoe et al, 2012, Meintjes et al, 2009, and Waldenstrom et al, 2009) 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 (Christianson et al., 2014) 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 (Wale and Gardner, 2010), 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 (Gardner, Lane, 2007 and Gardner, Lane, 2014). 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 (Gardner and Lane, 2014).

G1/G2 media were prepared in the laboratory as described previously (Gardner, Lane, 2007 and Gardner, Lane, 2014), 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 (Bar-Or et al, 2005, Dyrlund et al, 2014, and Morbeck et al, 2014). 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 (Gardner et al., 2005).

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 (Hardy et al., 1989). 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 (Binder et al, 2012, Lee et al, 2015, and Wale, Gardner, 2010). 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 (Ciray et al, 2014 and Kaser, Racowsky, 2014), there is still no consensus within the field (Lundin and Ahlstrom, 2015). Ths study has used a common notation system based on that used by Meseguer and colleagues (Meseguer et al., 2011): 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 1

Figure 1

Development 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 2

Figure 2

Cell 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 1

Time of cleavage events of embryos cultured in 5% or 20%, in groups of 10 or individually.

 

Parameter 5% Oxygen 20% Oxygen
Time of event (h post-HCG) Duration between events (h) Grouped Single Grouped Single
Syngamy 29.69±0.19 30.04±0.24 29.44±0.21 29.63±0.25
syn − t2 1.60±0.05 1.57±0.06 1.63±0.06 1.61±0.07
1st cleavage (t2) 30.88±0.17 30.93±0.22 30.62±0.18 31.15±0.22
cc2 (t3 − t2) 19.62±0.16 19.43±0.19 19.94±0.20 20.58±0.24*
2nd cleavage (t3) 50.28±0.18 50.26±0.25 50.28±0.25 51.21±0.31*
s2 (t4 − t3) 0.90±0.06 0.71±0.08* 0.89±0.10 0.96±0.12
t4 51.17±0.20 50.96±0.28 51.17±0.27 52.17±0.33*
cc3 (t5 − t4) 9.26±0.13 9.97±0.17*** 12.23±0.21 14.01±0.26***
3rd cleavage (t5) 59.91±0.23 60.70±0.29* 63.06±0.32 66.01±0.40***
t6 60.18±0.21 61.25±0.27*** 63.80±0.37 66.39±0.49***
4th cleavage (t7) 60.79±0.24 61.66±0.30* 64.37±0.43 67.30±0.58***
t8 61.17±0.24 62.46±0.30*** 65.03±0.44 68.26±0.60***
s3 (t8 − t5) 1.42±0.09 1.46±0.11 2.01±0.13 2.39±0.18
Cavitation 85.38±0.31 85.62±0.43 85.87±0.69 91.65±0.83***
Hatching 94.08±0.71 93.94±0.968 93.20±0.87 97.96±1.12***
Fully hatched 119.78±1.02 119.58±1.60 122.56±0.50 122.57±1.05

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 2

Time of cleavage events of embryos cultured in 5% or 20% oxygen, in groups or individually.

 

Parameter 5% Oxygen 20% Oxygen
Time of event (h post-t2) Grouped Single Grouped Single
Syngamy −1.59±0.05 −1.52±0.07 −1.63±0.06 −1.61±0.07
1st cleavage (t2) n/a n/a n/a n/a
2nd cleavage (t3) 19.60±0.16 19.43±0.19 19.94±0.20 20.58±0.24*
t4 20.55±0.18 20.15±0.22 20.87±0.22 21.61±0.26*
3rd cleavage (t5) 29.27±0.23 29.91±0.25* 32.78±0.31 35.27±0.40***
t6 29.51±0.20 30.46±0.22** 33.57±0.38 35.74±0.51**
4th cleavage (t7) 30.15±0.25 30.87±0.26* 34.20±0.47 36.92±0.63**
t8 30.27±0.28 31.68±0.29** 34.97±0.48 38.02±0.67***

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 3

Figure 3

Time 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.

 

Discussion

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 (Kirkegaard et al, 2013, Wale, Gardner, 2010, and Weinerman et al, 2016), 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 (Conaghan et al, 2013, Hashimoto et al, 2012, Meseguer et al, 2011, Petersen et al, 2016, Somfai et al, 2010, Sugimura et al, 2012, and Wong et al, 2010). 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 (Arav et al, 2008, Chung et al, 2015, Lee et al, 2015, Pribenszky et al, 2010a, and Weinerman et al, 2016).

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 (Dai et al, 2012, Jin, O'Neill, 2014, Kato, Tsunoda, 1994, Lane, Gardner, 1992, O'Neill, 1998, Paria, Dey, 1990, Salahuddin et al, 1995, Stoddart et al, 1996, and Vutyavanich et al, 2011) and 20% oxygen (Gardner, Lane, 1996, Karagenc et al, 2004, Quinn, Harlow, 1978, Rinaudo et al, 2006, and Wale, Gardner, 2010), and may be caused by an increase in apoptosis that has been observed following single culture (Brison, Schultz, 1997 and O'Neill, 1998) and culture in 20% oxygen (Van Soom et al, 2002 and Yuan et al, 2003). Alternatively, 20% oxygen can also induce cellular senescence, which would slow the rate of cell divisions, resulting in fewer cells (Meuter et al., 2014). Higher numbers of cells per blastocyst, and particularly a larger ICM, is associated with improved fetal development in the mouse (Lane and Gardner, 1997), 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 (Contramaestre et al, 2008, Dai et al, 2012, Gardner et al, 1994, Larson, Kubisch, 1999, Lee et al, 1996, Paria, Dey, 1990, Salahuddin et al, 1995, and Stoddart et al, 1996). Although hatching is necessary for implantation and ispredictive of improved pregnancy rates and live birth in human assisted reproductive technologies (Thompson et al, 2013 and Yoon et al, 2001), in-vitro hatching may not be an indicator of post-transfer viability in the mouse (Lane and Gardner, 1997). 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 (Ichikawa et al, 1985, O'Sullivan et al, 2001, Perona, Wassarman, 1986, Sawada et al, 1990, and Sharma et al, 2006). 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 (Hwang et al, 2000, Martinez-Hernandez et al, 2011, Sawada et al, 1990, Whiteside et al, 2001, and Yu et al, 2014), 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 (Seshagiri et al., 2016). 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 (Thouas et al, 2015 and Wydooghe et al, 2015), 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 (Harvey et al., 1995), and many other examples exist of proteinase regulation by growth factors and cytokines in other cell types (Lemaire et al, 1997 and Zhang et al, 2013). 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 (Westermarck and Kahari, 1999). 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 (Gardner, 2015 and Griffiths, 1991). 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 (Brömme, Wilson, 2011 and Kato et al, 2005).

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 (Bavister, 1995). Although embryo-conditioned media has been analysed using advanced technologies such as proteomics and metabolomics (Katz-Jaffe et al, 2006 and Seli et al, 2007), 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 (Hardy, Spanos, 2002, Spanos et al, 2000, and Ziebe et al, 2013). Paf, an embryo-secreted ester phospholipid, is also beneficial to singly cultured embryos (O'Neill, 2005). 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 (Thouas et al, 2015 and Wydooghe et al, 2015). 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 (Gardner, 2015). In addition, as the embryo develops the secretome also changes (Austgulen et al, 1995, Fishel et al, 1984, and Hemmings et al, 1992), as can the embryo's response to embryo-secreted factors due to stage-specific expression of some receptors (Sharkey et al, 1995 and Thouas et al, 2015). 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 (O'Neill, 1998). It is also in keeping with observations that the receptors of some growth factors and cytokines are expressed from the 2-cell stage or earlier (Dadi et al, 2004 and Sharkey et al, 1995).

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 (Quinn, Harlow, 1978, Rinaudo et al, 2006, Wale, Gardner, 2010, and Weinerman et al, 2016). 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 (Karagenc et al, 2004, Katz-Jaffe et al, 2005, Khurana, Wales, 1989, Kind et al, 2005, Rinaudo et al, 2006, and Wale, Gardner, 2012), 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 (Guerin et al., 2001). 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 (Goto et al, 1993 and Kwon et al, 1999), cow (Favetta et al., 2007) and pig (Kitagawa et al., 2004). Although production of ROS is part of normal cell metabolism, elevated ROS can be deleterious, resulting in dysfunction of signalling and other processes (Zhang et al., 2016), and elevated intracellular ROS is associated with poor embryo viability (Esfandiari et al, 2005 and Yang et al, 1998). Furthermore, oxygen is a metabolite, and as such changes in its concentration can influence the relative activities of metabolic pathways within the embryo (Khurana, Wales, 1989, Lane, Gardner, 2005, Wale, Gardner, 2012, and Wale, Gardner, 2013). 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 (Harvey et al., 2016).

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 (Fischer et al, 1988, Hughes et al, 2010, and Wale, Gardner, 2013). 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 (Kubisch, Johnson, 2007 and Rodina et al, 2009). Oxygen is also known to affect embryo metabolism (Khurana, Wales, 1989 and Wale, Gardner, 2012), 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 (Truong et al., 2016) or ROS-reducing protein (Isobe et al., 2015) 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 (Lane and Gardner, 2007). 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 (Carolan et al, 1996, Doherty et al, 1997, Donnay et al, 1997, Ferry et al, 1994, Fujita et al, 2006, Ikeda et al, 2000, Isobe et al, 2015, Keefer et al, 1994, Larson, Kubisch, 1999, Nagao et al, 2008, and Senatore et al, 2010). Interestingly, this effect also occurs between species; mouse embryos can stimulate the development of both cat and cow embryos (Spindler et al, 2006 and Stilley et al, 2003). 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 (Spindler and Wildt, 2002) or even have a negative effect on other embryos (Salahuddin et al., 1995).

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 (Almagor et al, 1996, Moessner, Dodson, 1995, Rijnders, Jansen, 1999, and Spyropoulou et al, 1999), except Ebner and colleagues (Ebner et al., 2010) and Rebollar-Lazaro and Matson (Rebollar-Lazaro and Matson, 2010), 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:

Table S1

Table S1

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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rbmo1588-fig-5001

Rebecca Kelley is currently undertaking her PhD with Professor David Gardner at the University of Melbourne, Australia. She has over 10 years experience in embryology research at both the University of Melbourne and the University of Adelaide, and in haematology research at the University of Cambridge. Her main interest is in finding ways to improve embryo culture in vitro by better understanding mammalian embryo development.

Footnotes

School of BioSciences, The University of Melbourne, Parkville, Victoria 3010, Australia

* Corresponding authors.