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In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media

Reproductive BioMedicine Online, Volume 34, Issue 5, May 2017, Pages 441 - 454

Editorial Comment

Many basic aspects of embryo culture are somewhat controversial. The increasing use of time-lapse monitoring systems led to more embryos being cultured individually in micro wells. In an elegantly designed set of experiments, Kelley and Gardner, explore the effects of group vs individual embryo culture, culture media volume, and whether possible adverse effects of individual culture can be negated using embryo-conditioned media. I believe this paper also provides a good overview of the subject for the clinicians, thus it is a must read both for embryologists and ART physician.


  • Individual culture of preimplantation mouse embryos is detrimental during both the precompaction and postcompaction stages, demonstrated by reduced hatching rate and blastocyst cell numbers.
  • The effect of embryo density on singly cultured embryos is dependent on the incubator oxygen concentration.
  • Culture in microwell dishes increased the percentage of inner cell mass per blastocyst, and altered timing of developmental events.
  • Addition of embryo-conditioned media to single embryos improves hatching rates and cell numbers.


Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture.

Keywords: Group culture, IVF, Osmolality, Single culture, Stress, Time-lapse.


Individual culture of in vitro fertilised embryos has become standard practice in many clinics, and will likely become more common with the application of new technologies for embryo monitoring and selection, such as time-lapse microscopy (Conaghan et al, 2013, Kirkegaard et al, 2014, and Rubio et al, 2014). Compared with embryos cultured in groups, individual culture in multiple species results in slower cleavage divisions, fewer embryos developing to the blastocyst stage, blastocysts with reduced cell number and altered cell allocation, and increased levels of apoptosis (Brison, Schultz, 1997, Gardner et al, 1994, Isobe et al, 2015, Keefer et al, 1994, Kelley, Gardner, 2016, Lane, Gardner, 1992, and Paria, Dey, 1990). Studies in humans have also reported lower cell numbers, blastocyst rates and pregnancy rates after single culture (Almagor et al, 1996, Ebner et al, 2010, Moessner, Dodson, 1995, and Rebollar-Lazaro, Matson, 2010), although some studies have found that single and group culture produce the same results (Rijnders, Jansen, 1999 and Spyropoulou et al, 1999). The reduced development rates and cell numbers in embryos cultured individually may be explained by deprivation from paracrine signalling molecules (Paria and Dey, 1990). It is estimated that about 55% of clinics worldwide routinely culture human embryos individually (Christianson et al., 2014); therefore, it is important to understand the consequences of this practice, and to determine if adjustments should be made to the culture system to improve outcomes for individually cultured embryos.

The embryo is more sensitive to stress in vitro during the precompaction stages than postcompaction (Gardner, Lane, 2005 and Lane, Gardner, 2005), but it is not known if this paradigm applies to the stress of single culture. It is not uncommon for clinics to culture embryos individually for part of the preimplantation period; for example, single culture to day 3, followed by group culture to day 5 and 6 (Christianson et al., 2014). Little evidence is available on the effects of this practice, but two studies suggest that single culture may be more detrimental in the precompaction stages of human embryo development than postcompaction (Rebollar-Lazaro, Matson, 2010 and Rijnders, Jansen, 1999). Stokes et al. (2005) made the same observation in the pig, but in the mouse, O'Neill (1998) found that any period of single culture was detrimental to blastocyst development. Further investigation is needed to determine if preimplantation embryos can be exposed to a period of single culture without compromising development.

The volume of culture medium per embryo (referred to as embryo density) is an important consideration in single embryo culture, but it is unclear how optimum embryo density may be influenced by other culture conditions, such as oxygen. Embryo density during single embryo culture can have a dramatic effect on cell numbers, as well as cleavage, blastocyst and hatching rates in the mouse (Lane, Gardner, 1992, Melin et al, 2009, O'Neill, 1997, and Paria, Dey, 1990). In humans, one study observed no effect of embryo density on blastocyst rate (Rijnders and Jansen, 1999), but evidence from two recent studies show that embryo density is an important factor in human blastocyst development, although their conclusions on optimal density were inconsistent (De Munck et al, 2015 and Minasi et al, 2015). The beneficial effect of increasing embryo density may be explained by the concentration of autocrine factors in the media. Media volume per embryo is currently inconsistent between clinics, and the practice of individual clinics may be a consequence of habit or convenience, rather than an optimised part of the embryo culture system (Bolton et al, 2014 and Reed, 2012).

Oxygen is a key element of embryo culture systems, and atmospheric (20%) oxygen causes multiple perturbations in embryo development, including increases in apoptosis (Van Soom et al., 2002), DNA damage (Takahashi et al., 2000), aneuploidy (Bean et al., 2002), and hydrogen peroxide levels (Goto et al., 1993), as well as changes to the transcriptome (Harvey et al., 2004), proteome (Katz-Jaffe et al., 2005), metabolome (Wale and Gardner, 2010), secretome (Kubisch and Johnson, 2007) and epigenome (Li et al., 2014). Despite this evidence, it is estimated that 20% oxygen is still used for human embryo culture in three-quarters of clinics worldwide (Christianson et al., 2014). Oxygen can change the embryo's response to other environmental stressors, demonstrated in the case of excess ammonium (Wale and Gardner, 2013), but it is not known if this applies to the stress of low embryo density. Evidence from the bovine suggests there may be an interaction between oxygen concentration and embryo density, but this has not yet been investigated in other species (Nagao et al., 2008).

To improve the development of singly cultured embryos, Vajta et al. (2000) developed the ‘well-of-the-well’ (WOW) culture system, in which embryos are cultured in a microwell to concentrate the embryo-secreted autocrine factors. In addition, it may be beneficial to reduce the contact between media and oil, particularly in small volumes or for individually cultured embryos (Hughes et al, 2010 and Lane, Gardner, 1992). Embryo culture for time-lapse microscopy is typically carried out in commercially available microwell dishes, which may have multiple microwells under one drop of media (WOW), or each microwell may be under a separate drop of media (Figure 1). Culture of single bovine or porcine embryos in microwells increases cleavage rate and blastocyst rate compared with individual culture, equivalent to that observed in group culture (Hoelker et al, 2009, Kamiya et al, 2006, Pereira et al, 2005, Vajta et al, 2000, and Vajta et al, 2008). Single mouse embryos also benefit from culture in microwells, as blastocyst rates and cell numbers are reported to be equivalent to group culture (Chung et al, 2015 and Pribenszky et al, 2010) or higher than standard single culture (Dai et al, 2012 and Vajta et al, 2008), but these studies have all used 20% oxygen, which may influence the outcome. Two trials of human embryos cultured in custom-made microwell dishes increased blastocyst rates (Hashimoto et al, 2012 and Vajta et al, 2008), but most other human trials involving commercially available microwell dishes have not controlled for factors related to the incubator system. Although the use of microwells to improve single embryo culture is promising, further investigation is needed, in particular regarding the efficacy of commercially available dishes used in clinics.

Figure 1

Figure 1

Microwells in (A) well-of-the-well (Primo Vision) and (B) single microwell (EmbryoScope) format. Not to scale. The Primo Vision dish is shown here in 9-well format, but the 16-well format was also tested.


An alternative to increasing embryo density or culture in microwells may be the development of synthetic embryo-conditioned media. For this to be achieved, the composition and effects of embryo-conditioned media must be better characterised. Several studies have shown that embryos condition the media immediately surrounding them and that neighbouring embryos (<160 µm away) benefit from this microenvironment (Gopichandran, Leese, 2006, Matoba et al, 2010, Somfai et al, 2010, Spindler, Wildt, 2002, Spindler et al, 2006, and Stokes et al, 2005). Only two studies have added embryo-conditioned media to single embryos. These indicate that embryo-conditioned media improves single mouse and bovine blastocyst development, but these experiments were conducted from the two-cell or four-cell stages in suboptimal conditions, so it is unknown if embryo-conditioned media will benefit single embryo development from the pronucleate stage in complex media and 5% oxygen (Fujita et al, 2006 and Stoddart et al, 1996).

In the present study, we examine how culture conditions influence single mouse embryo development. First, we compare the development of mouse embryos in single culture for either the precompaction or postcompaction stages with group or single culture for the entire duration. Second, we investigate the combined effects of embryo density and oxygen on single embryo development. Third, we assess embryo development in 2 µl drops, Primo Vision WOW dishes, and EmbryoScope microwell dishes, using the same incubator. Finally, we investigate the effect of embryo-conditioned media supplementation on single embryo culture.

Materials and methods

All chemicals were supplied by Sigma-Aldrich (St Louis, MO, USA) unless otherwise specified.


C57BL/6 X CBA F1 hybrid mice were housed 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 am to 6 pm) and controlled temperature. Food and water were available ad libitum.

Four week old females were superovulated with 5 IU pregnant mare serum gonadotrophin (Folligon; Intervet, Bendigo East, Vic, Australia) administered intraperitoneally at the mid-point of the light phase, followed 48 h later by 5 IU human chorionic gonadotrophin (HCG) (Chorulon; Intervet) and followed by mating with males of the same strain overnight. Mice were killed by cervical dislocation. All experiments were approved by The University of Melbourne Animal Ethics Committee (reference number 1513740) on 21 January, 2016.

Embryo culture

Pronucleate oocytes were collected 22 h after HCG injection in G-MOPS PLUS handling medium, containing 5 mg/ml human serum albumin (Vitrolife, Göteborg, Sweden) as described previously (Gardner, Lane, 2007 and Gardner, Lane, 2014). Embryos were briefly incubated in G-MOPS PLUS containing 550 IU/ml hyaluronidase (bovine testes type IV-S) until cumulus cells were removed, then were washed three times in G-MOPS PLUS and once in pre-incubated G1 culture medium. Embryos were pooled and allocated randomly to treatments. Embryos were cultured in G1 medium for 48 h, and then in G2 medium for a further 48 h. When media were changed, embryos were washed once in G2 before they were moved to the culture drop. All embryo manipulations were carried out 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 after 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 one 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; ‘fully hatched’: the complete evacuation of the embryo from the zona (Gardner and Lane, 2014).

Culture media were prepared as described previously (Gardner, Lane, 2007 and Gardner, Lane, 2014), except that choline chloride, folic acid, inositol, nicotinamide and taurine were omitted from G2 to reflect the current formulation of G2 (Morbeck et al., 2014a). Media were supplemented with essential amino acids (MEM Cellgro; Corning Life Sciences, Tewksbury, MA, USA), hyaluronan (0.125 mg/ml; Vitrolife), gentomycin (10 ug/ml), and 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, 2014b). All chemicals and plastics were tested in a mouse pronucleate oocyte bioassay before use (Gardner, Lane, 2005 and Gardner et al, 2005).

Unless otherwise specified, embryo cultures were carried out 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) in a reduced oxygen environment (5% O2, 6% CO2 and 89% N2), unless specified otherwise. When 20% oxygen was required, the incubator atmosphere was 6% CO2 in air. All drops of media were made under oil using an eVol positive displacement pipette (accurate to ± 1.0%; SGE Analytical Science, Ringwood, Vic, Australia) to accurately deliver small volumes and prevent evaporation of media during dish preparation.

Differential stain

Blastocyst cell allocation to the inner cell mass (ICM) or trophectoderm was determined using a differential staining protocol (Hardy et al, 1989 and Thouas et al, 2001). This technique identifies the inner and outer cells of the blastocyst, rather than specific cell lineage markers. All procedures were carried out at 37°C, and blastocysts were washed in G-MOPS PLUS between all steps except the last. Simple G1 medium without non-essential amino acids, alanyl glutamine, taurine or human serum albumin, containing 4 mg/ml polyvinylpyrrolidone (Simga-Aldrich) was used to dilute 2,4,6-trinitrobenzenesulfonic acid and anti-dinitrophenyl. Blastocysts were incubated in 0.5% pronase until the zona was no longer visible, then in 0.5% 2,4,6-trinitrobenzenesulfonic acid for 10 min. Blastocysts were then transferred to 10% anti-dinitrophenyl antibody (produced in rabbit) for 10 min, and then into guinea pig serum (IMVS, Adelaide, SA, Australia) diluted 50% in 0.02 mg/ml propidium iodide in G-MOPS, until blebbing of the cell membranes was observed. The final step was incubation in 0.1 mg/ml bisBenzimide (Hoescht 33258) in G-MOPS and 10% ethanol for 20 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 (Rasband WS, US National Institutes of Health, Bethesda, Maryland, USA,, 1997–2016) using the ‘Cell Counter’ plugin. Percent ICM was calculated as the number of ICM cells divided by the total number of cells per blastocyst.

Experiment 1: effect of single culture during precompaction or postcompaction stages

Embryos were cultured in groups of 10 in 20 µl or individually in 2 µl (to maintain embryo density). When the embryos were moved from G1 to G2 on day 3 of culture, one half of the group-cultured embryos were changed to single culture, and one half of the singly cultured embryos were moved to group culture. This resulted in four treatments: single, single/group, group/single, and group. On day 5, ICM and trophectoderm were stained at 118 h after HCG as outlined above.

Experiment 2: effect of embryo density and oxygen on single embryo development

Single embryos were cultured in 20 µl or 2 µl drops of media, under 5% or 20% oxygen, resulting in four treatments: 5% 20 µl, 5% 2 µl, 20% 20 µl, and 20% 2 µl. On day 5, ICM and trophectoderm were stained at 118 h after HCG as outlined above.

To ensure that a reduced drop volume of 2 µl did not affect osmolality compared with 20 µl drops, the osmolality in both drop sizes was measured after incubation. Drops of G1 and G2 media (Vitrolife) were made under oil, and the dishes were incubated without embryos in either 5% or 20% oxygen for 48 h. Osmolality was measured on a Model 3320 Osmometer (Advanced Instruments Inc, Norwood, MA, USA). Because of the volume required by the osmometer, drops were pooled to make the required 40 µl before measuring.

Experiment 3: effect of culture in microwell dishes

Embryos were cultured in the same incubator in four different dish types. Control embryos were cultured individually in 2 µl drops in 35 mm dishes as described above. Their development was compared with embryos in three commercially available microwell dishes designed for the Primo Vision and EmbryoScope time-lapse systems (Figure 1). Microwell dishes were prepared according to the manufacturer's instructions. When the medium was changed after 48 h culture, the manufacturers recommend aspirating 90% of the media and replacing it with fresh pre-incubated media, but to maintain consistency with the 2 µl drop controls the entire dish was changed. The microwell dishes are made from polystyrene, tested for embryo toxicity, and have conical-shaped microwells. The Primo Vision culture dishes (Vitrolife) are available in a 9-well or 16-well format. The microwells in the 9-well dish are 0.4 mm diameter, 0.27 mm deep, with a volume of 0.11 µl. The microwells in the 16-well dish are 0.35 mm diameter, 0.27 mm deep, with 0.09 µl volume. A single drop of media covers all the microwells: 40 µl in the 9-well dish and 70 µl in the 16-well dish (4.4 µl per embryo). An additional 20 µl wash drop was also made. One half of the media drop was laid down first, then covered with 3.5 ml oil. The dish was tapped on the bench to remove air bubbles, and media was then added to the final volume. All the microwells of each dish were used for embryo culture (i.e. nine or 16 embryos per dish). The EmbryoSlide culture dish (Vitrolife), designed for the EmbryoScope time-lapse system, has 12 separate wells. At the bottom of each well is a microwell, 0.2 mm in diameter with a volume of about 0.02 µl. 25 µl media was placed in each well, plus the extra wells for washing, and overlaid with 1.5 ml oil.

Embryos in all dish types were monitored using a multi-gas cell imaging incubator fitted with an inverted microscope (MCOK-5M[RC]; Sanyo Electric), as described previously (Kelley, Gardner, 2016, Lee et al, 2015, and Wale, Gardner, 2010). The embryo culture dishes rest on a motorised stage, and images are taken through the fixed Olympus microscope with a 10X 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. At 124 h after HCG, ICM and trophectoderm were stained as outlined above. Cleavage times were defined as the first time point when new blastomeres were separated by cell membranes. The notation system is based on that described by 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, and 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, and s3 is the duration of the third synchrony, i.e. the interval between t5 and t8. Notation of postcompaction events are as described in the embryo culture section. Cleavage times are expressed as hours post-HCG injection or as hours post-t2 to account for variations in time of fertilisation. Because of incompatibility between the design of the dishes and the time-lapse system, not all embryos cultured and stained were able to be monitored by time-lapse.

Experiment 4: effect of embryo-conditioned media on individual embryos

Embryo-conditioned media was produced by culturing 10 embryos in 20 µl media. After 48 h culture, grouped embryos were moved from G1 to G2 media, and the 20 µl drop of conditioned G1 was used to culture a single pronucleate oocyte. After a further 48 h, the grouped embryos were removed from G2 and the nuclei stained with bisBenzimide. The single embryos were then moved from conditioned G1 to conditioned G2. Control embryos were cultured individually in G1 that had been incubated without embryos. Embryos were stained at 118 h post-HCG in 0.1 mg/ml bisBenzamide in G-MOPS and 10% ethanol for 50 min, then mounted on a microscope slide and analysed as described above.

Statistical analysis

For all tests, differences were considered statistically significant when P < 0.05. Percentage data, i.e. the number of embryos to reach each stage of development on each day, were compared by Generalised Linear Model with binomial distribution and logit link function, using JMP 13.0.0 (SAS, Cary, NC, USA). Comparison of means, i.e. the number of cells per embryo, were compared by General Linear Model Univariate analysis with replicate as a random factor in SPSS Statistics v22 (IBM, Armonk, NY, USA). Multiple comparisons were conducted with Bonferroni post-hoc analysis, except for the microwell experiments, where the least significant difference test was used to compare each treatment with the control. 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.


Experiment 1: effect of single culture during precompaction or postcompaction stages

Single embryos were cultured under 5% oxygen in 2 µl, or in groups of 10 in 20 µl. After 48 h, embryos were either maintained in the same treatment or switched to the other, resulting in four treatment groups (group, group/single, single/group, and single; n = 90–120 embryos per treatment). On day 3, most embryos had reached the compacting stage with no significant effect of single culture (Figure 2A and Supplementary Table S1). On day 4, fewer embryos reached the expanded blastocyst stage or were hatching when cultured individually for the first half of culture, even if they had been moved into groups on day 3 (Figure 2A and Supplementary Table S1), although this difference was not statistically significant. On day 5, there was no effect of single culture on the number of hatching blastocysts, and although there were more fully hatched blastocysts when the second half of culture was grouped, this difference was not statistically significant (Supplementary Table S1).

Figure 2

Figure 2

Effect of single culture during precompaction or postcompaction stages. Between 90 and 120 embryos were cultured per treatment, from five independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). No significant differences were found between treatments. (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments in total cell number (Group versus Single/Group: P < 0.05; others: P < 0.01); trophectoderm cells (P < 0.05); inner cell mass (Group versus Single: P < 0.05; others P < 0.01). ICM, inner cell mass; TE, trophectoderm.


Total cell numbers on day 5 were lower in embryos cultured individually for any period, regardless of whether it was precompaction, postcompaction, or the entire culture period, compared with group-cultured embryos (P < 0.05) (Figure 2B). The number of trophectoderm cells was significantly lower after continuous single culture (P < 0.05) (Figure 2B), and there were fewer ICM cells after any period of single culture (P < 0.05) (Figure 2B). The percent ICM was not significantly altered by individual culture.

Experiment 2: effect of embryo density and oxygen on single embryo development

Single embryos were cultured in either 5% or 20% oxygen, in 2 µl or 20 µl drops of media (n = 92–101 embryos per treatment). No difference was observed in the proportion of embryos that reached the compacting or morula stage on day 3 (Figure 3A). On day 4, however, fewer embryos cultured in 20% oxygen in 2 µl had reached the blastocyst stage or beyond than embryos in other treatments (P < 0.01) (Figure 3A). Also, fewer embryos in 20% oxygen in both drop sizes were hatching than embryos in 5% oxygen in both drop sizes (P < 0.001) (Supplementary Table S2). Similarly, on day 5 fewer embryos in 20% oxygen were hatching or fully hatched compared with embryos cultured in 5% oxygen in both drop sizes (P < 0.001) (Figure 3A).

Figure 3

Figure 3

Effect of embryo density and oxygen on single embryo development. Between 92 and 101 embryos were cultured per treatment, individually, from four independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). Different letters indicate significant differences between treatments within each time point (day 4: oxygen: P < 0.01; density: P > 0.05; interaction between oxygen and density: P < 0.05. Day 5: oxygen: P < 0.001; density: P > 0.05; interaction: P > 0.05). (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments in total cell number (a–b: P < 0.05; ab–c: P < 0.001), trophectoderm cells (d–e: P < 0.01; de–f: P < 0.001) or inner cell mass (g–h: P < 0.01). No significant differences in percent inner cell mass. ICM, inner cell mass; TE, trophectoderm.


A general linear model univariate analysis of blastocyst total cell numbers showed an overall effect of both drop size (P < 0.05) and oxygen (P < 0.001), and that the interaction between the two factors was significant (P < 0.01). A Bonferroni post-hoc showed that total cell number was lower after culture in 20 µl than 2 µl drops in 5% oxygen (P < 0.05) (Figure 3B), but at 20% oxygen there was no effect of drop size on cell numbers. Also, embryos cultured in 20% oxygen had fewer cells than those cultured in 5% oxygen in both drop sizes (P < 0.01). The number of trophectoderm cells per blastocyst followed the same pattern as total cells (Figure 3B), but the general linear model univariate analysis showed that ICM numbers were affected only by oxygen (P < 0.001) and not drop size. Post-hoc analysis found that embryos cultured in 20% oxygen had fewer ICM cells than embryos in 5% oxygen, in both drop sizes (P < 0.01) (Figure 3B). Percent ICM was not significantly different between any treatments.

Osmolality in 2 µl and 20 µl culture drops

Drops of media incubated under oil for 48 h increased in osmolality compared with media from the bottle (P < 0.001), but there was no effect of drop size on this increase. The starting osmolality of the media was 286.8 ± 1.0 mOsm/kg for G1 and 287.6 ± 0.6 mOsm/kg for G2 (n = 6 each). After 48 h incubation, the osmolality of the 2 µl drops was 291.5 ± 0.8 mOsm/kg for G1 and 292.3 ± 0.5 mOsm/kg for G2 (n = 12 each). The osmolality of the 20 µl drops was 291.9 ± 1.3 mOsm/kg for G1 and 291.8 ± 0.9 mOsm/kg for G2 (n = 12 each).

No significant effect of oxygen (5% or 20%), media (G1 or G2), or drop size (2 µl or 20 µl) was observed on the change in osmolality during incubation. The increase in osmolality during incubation was 5.6 ± 0.7 mOsm/kg in 20 µl drops, and 5.2 ± 0.4 mOsm/kg in 2 µl drops.

Experiment 3: effect of culture in microwell dishes

Embryos were cultured in either 2 µl control drops in 35 mm dishes or in one of three types of microwell dishes, all in the same time-lapse incubator (n > 71 per treatment). In the Primo Vision 16-well dishes, t3 occurred later than in control 2 µl drops, due to cc2 taking approximately 1.6 h longer (P < 0.001) (Table 1). This delay continued through to t8, with no difference in s2, s3 or cc3. After compaction, a 3 h delay in cavitation was observed compared with the control (P < 0.01). In contrast, embryos in the Primo Vision 9-well dishes had a slightly shorter cc2 and cc3 than control embryos (P < 0.05), and t5 and t6 occurred around 1 h earlier. After compaction, no differences were observed compared with controls. Embryos in the EmbryoSlide cleaved at the same time as control embryos during the precompaction stages, but they cavitated around 3 h earlier (P < 0.05) and hatched around 7 h earlier than controls (P < 0.01).

Table 1

Effect of culture in microwell dishes on morphokinetics.


Time of event (h after HCG) Duration between eventsa (h) 2 µl drop Primo Vision 9-well Primo Vision 16-well EmbryoSlide
Syngamy 28.66 ± 0.32 28.73 ± 0.25 28.91 ± 0.24 28.24 ± 0.24
syn-t2 1.70 ± 0.06 1.66 ± 0.05 1.80 ± 0.05 1.82 ± 0.07
First cleavage (t2) 30.13 ± 0.29 30.32 ± 0.24 30.76 ± 0.23 29.95 ± 0.33
cc2 (t3–t2) 19.96 ± 0.21 19.27 ± 0.17b 20.91 ± 0.16e 19.42 ± 0.24
Second cleavage (t3) 50.08 ± 0.31 49.6 ± 0.26 51.67 ± 0.25e 49.36 ± 0.31
s2 (t4–t3) 0.89 ± 0.16 0.84 ± 0.13 0.85 ± 0.12 0.63 ± 0.18
t4 50.82 ± 0.35 50.43 ± 0.28 52.52 ± 0.28e 50.00 ± 0.40
cc3 (t5–t4) 10.38 ± 0.27 9.64 ± 0.22b 10.62 ± 0.21 10.16 ± 0.31
Third cleavage (t5) 61.20 ± 0.42 60.08 ± 0.34c 63.14 ± 0.33e 60.15 ± 0.47
t6 61.71 ± 0.43 60.52 ± 0.35b 63.68 ± 0.34d 60.47 ± 0.48
Fourth cleavage (t7) 62.36 ± 0.47 61.08 ± 0.37 64.37 ± 0.36d 61.15 ± 0.52
t8 63.00 ± 0.53 61.66 ± 0.42 65.00 ± 0.42d 61.57 ± 0.59
s3 (t8–t5) 1.89 ± 0.23 1.59 ± 0.18 2.15 ± 0.18 1.41 ± 0.26
Morula (Cavitation–t8) 23.64 ± 0.69 23.77 ± 0.55 24.66 ± 0.56 22.26 ± 0.75
Cavitation 86.56 ± 0.79 85.39 ± 0.65 89.47 ± 0.66d 83.78 ± 0.87b
Hatching 98.69 ± 1.73 97.90 ± 1.36 102.63 ± 1.19 91.02 ± 1.82d
n 30 45 48 23

a Notation for cleavage events are as described in the Materials and methods section. Three independent biological replicates. All cultures were conducted in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric).

c P = 0.052.

d P < 0.01.

e P < 0.001.

Values are mean ± SEM of cleavage events (h after HCG), or time between cleavage events (h).

Significantly different to the control (2 µl drop); bP < 0.05.

When cleavage times were normalised to the two-cell cleavage time for each embryo to account for variation in time of fertilisation, embryos in the Primo Vision 9-well dishes reached t3, t5, t7 and t8 significantly earlier than embryos in 2 µl control drops (P < 0.05) (Figure 4 and Table 2). Differences between the control embryos and embryos in the 16-well dishes or EmbryoSlide remained the same.

Figure  4

Figure  4

Effect of culture in microwell dishes on morphokinetics, relative to single culture in a 2 µl drop. Data from Table 2 are represented here as (mean cleavage time) – (mean control cleavage time) ± SEM. This is for graphical representation only, and statistics have are described in the Materials and methods section. Notation of cleavage events are described in the Materials and methods section. All cultures were performed in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric). Between 88 and 96% of embryos developed to the hatching blastocyst stage in all treatments. Between 23 and 48 embryos were cultured per treatment, from three independent biological replicates. *indicates significantly different to control 2 µl drop; *P < 0.05, **P < 0.01, ***P < 0.001.


Table 2

Effect of culture in microwell dishes on morphokinetics according to cleavage times normalised to the two-cell cleavage time.


Time of eventa (h after t2) 2 µl drop Primo Vision 9-well Primo Vision 16-well EmbryoSlide
Syngamy −1.70 ± 0.06 −1.66 ± 0.05 −1.80 ± 0.05 −1.82 ± 0.07
First cleavage (t2) n/a n/a n/a n/a
Second cleavage (t3) 19.96 ± 0.21 19.27 ± 0.17b 20.91 ± 0.16d 19.42 ± 0.24
t4 20.69 ± 0.29 20.11 ± 0.24 21.76 ± 0.23c 21.76 ± 0.23
Third cleavage (t5) 31.06 ± 0.38 29.75 ± 0.31b 32.38 ± 0.30c 30.21 ± 0.43
t6 31.57 ± 0.40 30.20 ± 0.32b 32.94 ± 0.31c 30.52 ± 0.45
Fourth cleavage (t7) 32.16 ± 0.43 30.76 ± 0.35b 33.62 ± 0.33c 31.20 ± 0.48
t8 32.81 ± 0.48 31.34 ± 0.38b 34.27 ± 0.38b 31.66 ± 0.54
Cavitation 56.61 ± 0.76 55.19 ± 0.63 58.94 ± 0.63b 53.83 ± 0.84b
Hatching 68.75 ± 1.76 67.38 ± 1.38 72.02 ± 1.21 61.25 ± 1.85c

a Notation for cleavage events are as described in the Materials and methods section. Three independent biological replicates were used. All cultures were carried out in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric).

c P < 0.01.

d P < 0.001.

Values are mean ± SEM of cleavage events (h after t2).

Indicates significantly different to the control (2 µl drop); bP < 0.05.

Of the embryos monitored by time-lapse, most reached the hatching blastocyst stage before the end of culture: 2 µl drop 90%, Primo Vision 9-well 96%, Primo Vision 16-well 88%, EmbryoSlide 91%.

No significant differences were observed in total cell numbers on day 5 after culture in microwells (Figure 5). An increase in ICM cells was, however, observed in blastocysts cultured in microwells compared with the control 2 µl drop (P < 0.05 for 9-well and 16-well Primo Vision dishes and P < 0.01 for EmbryoSlide) (Figure 5). This caused an increase in the percent ICM in embryos cultured in the EmbryoSlide or 9-well Primo Vision dishes compared with the 2 µl drop (P < 0.01), but the increase in percent ICM in the Primo Vision 16-well dish was not significant (Figure 5).

Figure  5

Figure  5

Effect of culture in microwell dishes on cell numbers. Bars represent cells per blastocyst (mean ± SEM); n = 71–116 embryos per treatment, from five independent biological replicates. Embryos were stained at 124 h after HCG. All cultures took place in a MCOK-5M[RC] time-lapse incubator (Sanyo Electric). *indicates significantly different to control 2 µl drop; *P < 0.05; **P < 0.01. ICM, inner cell mass; TE, trophectoderm.


Experiment 4: effect of embryo-conditioned media on individual embryos

Embryo-conditioned media was produced by culture of groups of 10 embryos in 20 µl drops (n = 56 groups, total 560 embryos). Single embryos were then cultured in these 20 µl drops of embryo-conditioned media (n = 56). No effect of conditioned media was observed compared with control media on the numbers of embryos at each stage of development on days 4 or 5 (Supplementary Table S3); however, more embryos in conditioned media were either hatching or hatched on day 5 than those cultured individually in control media (P < 0.05) (Figure 6A). Embryos cultured in conditioned media also had more total cells (P < 0.01) (Figure 6B) than embryos cultured individually in control media. No correlation was observed between the mean number of cells in the group of embryos that produced the conditioned media, and the single embryo that was cultured in it (Supplementary Figure S1).

Figure 6

Figure 6

Effect of embryo-conditioned media on individual embryos. Embryos were cultured individually in conditioned media (obtained from the culture of grouped embryos; n = 10 per group; total = 560) or individually or grouped in control media (media incubated without embryos); n = 56–57 per treatment, from three independent biological replicates. (A) Bars represent the proportion of embryos that reached the nominated developmental stage or beyond on each day of culture (70, 94 and 118 h after HCG). *indicates significantly different to other treatments (P < 0.05). (B) Bars represent cells per blastocyst (mean ± SEM). Embryos were stained at 118 h after HCG. Different letters represent significant differences between treatments (P < 0.01).



Single culture during precompaction or postcompaction stage is detrimental

Embryos that were cultured individually for the entire culture or any portion thereof had reduced cell numbers at the blastocyst stage compared with those cultured in groups, mostly attributable to reduced ICM cell numbers. No significant decrease in percent ICM, however, occurred after single culture. Higher cell numbers are predictive of viability in the mouse, as is a larger ICM and higher percent ICM (Lane and Gardner, 1997). These results show that single culture during either the precompaction or postcompaction stage is detrimental; however, there may be some benefit in culturing in groups for one-half of the culture period compared with continuous single culture, as the number of trophectoderm cells in group/single or single/group embryos was not significantly different to group culture.

These findings are in agreement with a study by O'Neill (1998), who found that transfer of mouse embryos from group culture to single culture or vice versa after 48 h lowered blastocyst rates compared with group culture, but were higher than continuous single culture. Taken together, this experiment and others suggest that there may be some benefit of group culture for one half of the culture period compared with single culture, although it will likely still be inferior to continuous group culture (Larson, Kubisch, 1999, O'Neill, 1998, Rebollar-Lazaro, Matson, 2010, Rijnders, Jansen, 1999, Stokes et al, 2005, and Wright et al, 1978).

No evidence that embryos were more sensitive to the stress of single culture during the precompaction stage was found, unlike a previous study using porcine embryos (Stokes et al., 2005), and examples of other in vitro stresses (Wale, Gardner, 2010 and Zander et al, 2006). Also, unlike exposure to other stresses, in the present study single embryos seemed to benefit somewhat from transfer to group culture in the postcompaction stages, as trophectoderm cell numbers of single or group embryos were not significantly different to group culture.

These data are also in agreement with studies on grouped and single mouse embryos, which determined that single culture results in fewer blastocysts, lower cell numbers, resulting mostly from a smaller ICM (Brison, Schultz, 1997, Dai et al, 2012, Jin, O'Neill, 2014, Kato, Tsunoda, 1994, Kelley, Gardner, 2016, Lane, Gardner, 1992, O'Neill, 1997, O'Neill, 1998, Paria, Dey, 1990, Salahuddin et al, 1995, Stoddart et al, 1996, and Vutyavanich et al, 2011). Group culture results in faster cell divisions (Kelley and Gardner, 2016), and reduction of apoptosis (Brison and Schultz, 1997), indicating both an increase in proliferation and a reduction in cell death.

The effect of embryo density on single embryo development is dependent on oxygen concentration

Embryo density and oxygen both affect in vitro embryo development, but the potential interaction between these treatments has not been investigated in the mouse. Culture of individual embryos in 5% oxygen increased development rates and cell numbers compared with culture in 20% oxygen in both 2 µl and 20 µl drops. Higher embryo density reduced the proportion of embryos that developed to the blastocyst stage on day 4 compared with low density, but only in 20% oxygen, not 5% oxygen (Figure 3A). Conversely, higher embryo density increased cell numbers compared with low density at 5% oxygen, but not 20% oxygen, demonstrating an interaction between oxygen concentration and optimal embryo density (Figure 3B). Higher embryo density in 5% oxygen increased cells in the trophectoderm rather than the ICM (Figure 3B), in contrast to group culture, which typically results in more ICM cells than single culture (Figure 2B) (Kelley and Gardner, 2016). Also, culture in 5% oxygen in high density resulted in the most cells per embryo of these four treatments (Figure 3B), but these embryos still have fewer cells than group-cultured embryos (Figure 2B) (Kelley and Gardner, 2016).

These data are consistent with previous studies on the mouse. Oxygen was demonstrated to be detrimental to mouse embryo development as early as the 1970s (Quinn, Harlow, 1978 and Whitten, 1971), and embryo density has been known to effect mouse embryo culture since the 1980s (Brison, Schultz, 1997, Kato, Tsunoda, 1994, Lane, Gardner, 1992, O'Neill, 1997, and Wiley et al, 1986). Nagao et al. (2008) found that the optimum oxygen concentration for bovine embryo culture depended on embryo density; when the media volume was kept constant, single embryos had higher blastocyst rates at 1% oxygen than higher oxygen concentrations, whereas groups of five embryos had highest blastocyst rates at 2.5% oxygen, and groups of 25 embryos had highest blastocyst rates at 5% oxygen. In the present study, we have used the opposite approach, keeping the number of embryos constant and changing the media volume, and have found that optimum embryo density is dependent on oxygen concentration.

The reasons for the interaction between embryo density and oxygen are unclear. The benefits of reduced media volume are potentially derived from the concentration of embryo-secreted factors in the medium, resulting in greater embryotrophic autocrine signalling. Culture in 20% oxygen can change the secretion of cytokines into the media (Kubisch, Johnson, 2007 and Rodina et al, 2009), which may influence the optimum media volume for enhanced autocrine–paracrine signalling. Embryos cultured in 20% oxygen are also less viable than those at 5% oxygen, and therefore they may secrete fewer embryotrophic factors into the microenvironment, or more harmful factors (Salahuddin et al, 1995, Spindler et al, 2006, and Tao et al, 2013). Oxygen alters embryo metabolism (Khurana, Wales, 1989 and Wale, Gardner, 2012), and therefore the uptake and production of nutrients and metabolites, thereby changing their concentration in the media. Metabolites and nutrients can alter the activity of metabolic pathways, act as autocrine signalling molecules and can change the pH, all of which have dramatic consequences for the embryo (reviewed in Gardner, 2015). All of these differences between embryos cultured at 20% and 5% oxygen may result in different microenvironments and thereby influence the optimal embryo density.

These data emphasise the need to optimise embryo density as a key element of embryo culture systems. Two recent studies have investigated the role of embryo density in human single embryo culture, and produced contrasting results. Minasi et al. (2015) found that reducing the culture volume from 35 µl to 7 µl resulted in a higher blastocyst rate. Conversely, De Munck et al. (2015) observed a lower blastocyst rate in 7 µl drops compared with 25 µl drops. Key differences were observed between the two studies, including experimental design, day of transfer, number of patients, media formulation and protein supplement, and oxygen, and these conflicting results support the hypothesis that optimum embryo density is dependent on other culture conditions (Gardner et al, 1994, Kato, Tsunoda, 1994, Nagao et al, 2008, and Wiley et al, 1986).

Osmolality is not affected by culture drop volume

Optimum osmolality for in vitro mammalian embryo development is around 255–295 mOsm/kg, and above this range embryo development is compromised (Brinster, 1965, Hay-Schmidt, 1993, and Swain et al, 2012). A study by Swain et al. (2012) highlighted the importance of drop size and dish preparation method in maintaining osmolality of culture media (Swain et al., 2012). In our study, drops were made under oil, and no difference between the osmolality of the two drop sizes was determined, indicating that changes in osmolality are not responsible for the differences observed between embryos in 20 µl and 2 µl drops. A small increase in osmolality, however, occurred after 48 h of incubation compared with the media in the bottle in all culture conditions. This increase may be caused by absorption of water by the oil overlay and possibly the handling required to collect the media after incubation (Heo et al., 2007).

Culture in microwells increases ICM and alters timing of developmental events

Culture in the three microwell dishes in the same incubator produced different timings of developmental events. Embryos in 16-well Primo Vision dishes were delayed compared with controls from cc2 to cavitation, whereas embryos in 9-well Primo Vision dishes cleaved faster than controls during the precompaction stages, and embryos in EmbryoSlides were faster than controls in the postcompaction stages. Timing of early cleavage events is predictive of blastocyst formation and postimplantation viability in the mouse (Chung et al, 2015, Lee et al, 2015, Pribenszky et al, 2010, and Weinerman et al, 2016), although it is unknown if the delay observed in the 16-well dishes (3 h maximum) is long enough to be indicative of reduced viability. Unlike early cleavage events, hatching time may not be a reliable marker of viability for mouse in vitro cultured embryos (Lane and Gardner, 1997), but hatching earlier is typical of group-cultured embryos (Figure 2A) (Contramaestre et al, 2008, Dai et al, 2012, Kelley, Gardner, 2016, Paria, Dey, 1990, and Salahuddin et al, 1995), so earlier hatching supports the hypothesis that embryos in EmbryoSlide microwells create a microenvironment similar to group culture by concentrating embryo-secreted factors.

Despite variation in cleavage timings, no difference was observed in overall development rates or blastocyst total cell numbers between the control and the microwell dishes. An increase was, however, observed in ICM cell numbers in all microwells, and an increase in percent ICM in 9-well and EmbryoSlide dishes. Increased allocation of cells to the ICM indicates that embryos cultured microwells are more viable than those in a 2 µl drop (Lane and Gardner, 1997). In addition, a larger ICM is more similar to group-cultured embryos, which supports the hypothesis that microwells concentrate autocrine factors (Figure 2B) (Brison, Schultz, 1997, Dai et al, 2012, Kelley, Gardner, 2016, and Vutyavanich et al, 2011). These data are contrary to previous studies in mice that found microwells increased total blastocyst cell number (Dai et al, 2012 and Vajta et al, 2008). This may be due to our use of small volume control drops and 5% oxygen. Also, the previous two studies used a single medium without renewal, which may affect the outcome.

Embryos developed differently in each type of microwell dish. In the two Primo Vision dishes, the media volume per embryo was the same, so differences may be because the microwells in the 16-well dishes are smaller and more closely spaced than the microwells in the 9-well dishes (see Materials and methods section), and this slight difference may be enough to affect embryo development (Hoelker et al., 2010). In addition, differences between the EmbryoSlide and Primo Vision dishes could be a result of communication between embryos in the Primo Vision dishes. It is debatable whether embryo-secreted factors can diffuse between embryos in WOW dishes (Dai et al, 2012, Kang et al, 2015, Pereira et al, 2005, Sugimura et al, 2013, and Wydooghe et al, 2014), this would depend on the shape and depth of the microwells, and the distance between them (Hoelker et al, 2010 and Matsuura, 2014). If communication occurred between embryos in the Primo Vision dishes, it was not observable in this study; however, group culture also allows for the possibility of detrimental communication between embryos, and this cannot be excluded as the cause of the delayed cleavage times observed in the 16-well dish.

Human embryos are more metabolically active than mouse embryos, and consequently the microwells best suited to mouse embryo culture may not be optimal for human embryos, as they will consume more nutrients and produce more waste products. In addition, much of human embryo culture in microwells is performed using a single medium without renewal, rather than the sequential media used in these experiments. Undisturbed culture may affect singly cultured embryos, and although evidence to date shows no effect of media renewal or changeover (Biggers et al, 2005, Costa-Borges et al, 2016, Lane, Gardner, 1994, and Macklon et al, 2002), the use of a single-step medium could potentially influence the outcome of these experiments comparing microwells with standard drops.

Mouse preimplantation embryos are generally a useful model for human embryos, owing to many similarities in their physiologies and requirements in vitro (Lane and Gardner, 2007). For experiments comparing group and individual culture, however, mouse embryos may benefit more from group culture than human embryos. Mouse embryos are sourced from young fertile donors, in contrast to the older infertile human population undergoing IVF treatment, and therefore mouse embryos used for these experiments are generally of higher quality than many cultured human embryos. The importance of embryo quality in determining the embryotrophic effect of group culture has not yet been determined in humans, but in animal models poor quality embryos can have no beneficial effect on other embryos, or their presence may even be detrimental (Salahuddin et al, 1995 and Spindler, Wildt, 2002). Conversely, it is possible that a lower quality human embryo could be more sensitive to the stress of individual culture, and therefore benefit from culture in a group with higher quality embryos.

Culture in embryo-conditioned media improves single embryo development

Culture of individual embryos in embryo-conditioned media increased hatching rates and cell numbers compared with single control embryos. This is in agreement with previous studies that added embryo-conditioned media to singly cultured bovine or mouse embryos later in culture under 20% oxygen (Fujita et al, 2006 and Stoddart et al, 1996).

Evidence shows that embryo quality has a role in regulating the development of neighbouring embryos (Salahuddin et al, 1995, Spindler, Wildt, 2002, and Tao et al, 2013). With this in mind, we examined the data for a correlation between the cell number of single embryos cultured in conditioned media, and the cell numbers of the grouped embryos that conditioned the media. No correlation was found, which may be because the conditioning embryos were all of high quality with little variation in cell number.

Embryo-conditioned media may stimulate growth for a variety of reasons, as embryos make many changes to media during culture, including consumption of nutrients, production of metabolites, and secretion of growth factors, cytokines, hormones, nucleic acids, and many other molecules, reviewed in detail elsewhere (Thouas et al, 2015 and Wydooghe et al, 2015). All of these changes to the media can influence preimplantation development independently and in combination. Further analysis of embryo-conditioned media will help elucidate the molecular basis for the embryotrophic effect of group culture, and perhaps lead to the development of improved media specifically for single embryo culture; however, it is likely that the interactions between these factors are highly complex.

In conclusion, although single embryo culture reduces development compared with group culture, it is necessary for embryo tracking and identification. Therefore, efforts should be made to improve the conditions used. The easiest and most obvious action is to reduce the oxygen concentration in the incubator, as we have shown here and previously that 20% oxygen is highly detrimental to individually cultured embryos (Kelley and Gardner, 2016). Second, embryo density is an important consideration, and given the influence of other culture conditions, the optimal embryo density will likely need to be determined for each culture system. Microwells may be the solution to this, but our data suggest that the outcome is variable between commercially available dishes. Some clinics currently culture embryos in groups for part of the culture period only, but the data presented here suggest that, although there may some benefit from this practice compared with single culture, it is still inferior to group culture throughout. The marked improvement of single embryo development with the addition of conditioned media suggests that efforts should be made to further analyse the embryo secretome to identify embryotrophic factors that can be used to supplement media. Taken together, these data demonstrate that changes can be made to culture systems to improve single embryo development, and that the formulation of a better media for single embryo culture is possible, if a synthetic version of embryo-conditioned media can be created.


This work was funded through the University of Melbourne. The authors thank Dr Alexandra Harvey and Mai Truong for their constructive comments on the manuscript and Professor Michael Keough for his advice on statistical analyses.

Appendix. Supplementary material

The following is the supplementary data to this article:

Download file

Figure S1

Correlation between cell number of group cultured embryos used to make conditioned media, and cell number of single embryos cultured in embryo-conditioned media (n = 56–57; three independent biological replicates).


Download file

Table S1

The effect of precompaction or postcompaction single embryo culture. Development assessed on days 3, 4 and 5 of culture (70, 94 and 118 h after HCG). Five independent biological replicates. No significant differences between treatments.



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


Key message


Single embryo culture is typical for time-lapse systems but, at least in the mouse, it reduces development compared with group culture. Embryo development during individual culture was improved by using reduced oxygen, reduced media volume or microwell dishes, and conditioned medium. The latter could possibly lead to modified media formulations.

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

* Corresponding author.