We conducted experiments to more closely replicate the mechanical environment that embryos encounter in vivo during their journey from the fallopian tube to the uterus. Our results indicate that embryos culture on a substrate with a stiffness approximating that of the fallopian tube and uterus is associated with more rapid preimplantation development and larger placentas at mid gestation. These findings have important implications and we believe that serious consideration should be given to developing technologies that allow culturing embryos in a physical environment that more closely mirrors the physiologic features of the genital epithelia.
First, we found that initial development to both 2-cell and blastocyst stage was dependent on the substrate stiffness, as the frequency of development from zygote to the 2-cell stage and from 2-cell to blastocyst were significantly different for embryos cultured on soft collagen gels (Col-1k) versus stiffer substrates (Col-1G or PD).
It is very likely that the zona pellucida functions as a shock absorber to dampen the effects of different mechanical environments when forces have profound effects in shaping future development 
. Therefore, the blastomeres in contact with the ZP will experience only the mechanical forces generated by the embryo weight (FBZ
, Figure S3
). Our assessment suggests that the embryo weight generates a force of approximately 1 nN, which is sufficient to affect developmental fate 
. In fact, we calculated the wet weight of preimplantation embryos to be approximately 100 ng (), a value similar to the weight measured by others. Turner et al, in fact calculated that the dry weight of a blastocyst is 33 ng 
. Considering that the embryo is composed for 80% water, these values are comparable.
Experiments using zona free embryos () confirm that the constant mechanical stress shielding provided by the zona is beneficial. In fact, zona free embryos cultured on polystyrene have a dramatically lower blastocyst frequency than that of zona-intact embryos (ZFP: 22% and PD
78%, p<0.05). In addition, experiments on zona-free embryos indicate that the stiffness of the substrate has a greater effect; culture of zona free embryos on collagen improved not only the blastocyst development frequency (ZFC: 53%), but also the number of TE cells (ZFP: 30 cells and PD
49 cells, p<0.05) and ICM cells (ZFP: 9 cells and PD
12 cells, p<0.05) compared to zona-intact embryos. The beneficial effect of collagen served to blunt the negative effect of zona removal on the ICM (ZFC: n
12 ICM cells). Culturing on soft collagen also increases the number of TE cells, but it does not completely restore TE cell number to that seen in control, zona-intact embryos (ZFC: n
44; Col-1k: n
65 TE cells).
A second finding was that softer environments appear more permissive for blastocyst hatching; furthermore, embryos cultured on softer environments had greater numbers of TE cell. These effects were consistent across different substrate types. On PDMS the effect was graded: culture on 200 KPa resulted in greater blastocyst development and hatching frequency than culture on 1.8 MPa or PD (109
Pa). It is unclear why culture on 50 KPa PDMS resulted in lower hatching frequency and lower TE cell number compared to 200 KPa. It is however known that there is often an optimal substrate stiffness that is unique for every material
It is unclear how the softness of the substrate affects hatching. An exhaustive explanation of the hatching process in different species is surprisingly missing 
. Importantly, the ZP stiffness decreases by an order of magnitude from zygote to the morula and blastocyst stage, potentially facilitating the exit from the ZP 
. The hatching process appears to be regulated by a variety of autocrine and paracrine molecules and by the presence of trophectodermal projections (TEPs). TEPs are cytoplasmic extensions of trophectodermal cells that protrude through the ZP into the extra-embryonic environment
. During a 4-hour time window prior to hatching, TEPs reach a length of 17 microns and protrude and retract beyond the ZP
. It is possible that the TEP detect physical and biochemical cues from the environment, specifically in this case its stiffness. The softer surface environment appears to be a pro-hatching signal.
While TEP could provide an explanation for the increase in hatching following culture in softer environment, it is unclear how to explain the increase in TE cell number noted in softer environment. Overall, it is believed that increases in TE and total cell number may give rise to better-quality in vitro-derived embryos: an Increase in TE cell number is observed following addition of folate, while culture in vitro or in stressful condition is associated with a decrease in TE cells
To further explore the mechanism of increase in TE cell number we tested a series of candidate genes. Surprisingly the gene expression data were not helpful, as expression of genes involved in trophectodermal differentiation (cdx2, eomes
or responsive to mechanical cues (ctgf
was not different in zona intact embryo cultured in Col-1k or PD. Notwithstanding that embryos cultured in Col-1k have increased in TE cells number, these embryos had a paradoxical decreased expression of cycd
, a marker of proliferation. It is unclear how to explain this last finding, although it is possible that measuring cycd
level in isolated TE cells would provide different results.
Zona free embryos cultured on collagen have more TE cells and showed an increased expression of cdx2.
Overall the expression studies suggest that the selected genes are not reflective of the biochemical events that results from culturing cells on different substrate. Analysis of RhoA phosphorylation, a marker of activation of mechano-sensitive pathways, could provide an answer 
. Unfortunately, the paucity of material does not permit embryonic testing.
Third, we found that in vitro
embryos growing in softer (col-1K) and harder (PD) environments had equal frequencies of implantation and survival to E12.5 and equal fetal weights. However they had significantly larger placentas at E12.5 compared to embryos cultured on PD or FB controls. The lower fetal weight of in vitro
embryos compared to in vivo
embryos supports our prior data and confirms that in vitro
generated embryo have a different in utero growth pattern compared to in vivo embryos
. The larger placenta in the Col-1k is likely to be a result of the initial greater TE cell number. Overall the larger placentae following culture in soft environment might facilitate a compensatory growth in utero 
. Indeed placental weight is positively correlated to bodyweight at term in a wide range of species and overall placental size directly affects the capacity for nutrient transfer via changes in the surface area for transport 
In this study we employed a newly devised method to apply protein based extracellular matrices, like collagen I, on hydrophobic material like polystyrene. This method included the fabrication of PDMS based wells that were activated via reactive ion etching and poly-d-lysine coating to form precisely sized 3D gels in wells for oil-immersed droplet culture. The method used here is highly modifiable to accommodate virtually any gel or well size.
One limitation of our study is that we tested few materials and stiffness in comparison with the myriad of those available. In addition, recapitulating the in vivo mechanical conditions goes beyond just modifying the surface on which the embryos rest. Ideally culture conditions should replicate all the in vivo mechanical conditions, including other externally applied forces, such as fluid shear, oviduct and uterine contractions, and electrostatic forces.
In summary, we found that embryos are mechano-sensitive and develop differently according to the specific stiffness of the environment. Our results suggest that culture on softer environments like collagen alters both pre-implantation and post-implantation development; current culture settings in an oil-immersed culture droplet on a polystyrene dish, are a far cry from the in vivo condition where the embryo is either suspended in a viscous tubal fluid or in contact with a uterine epithelium roughly six orders of magnitude softer than polystyrene. Future studies should include attempts to closely model the uterine environment, but should also better define the mechanical properties of the fallopian and uterine environment.