Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

in Reproduction

Correspondence should be addressed to P T Ruane; Email: peter.ruane@manchester.ac.uk

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

Abstract

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

Introduction

In vitro fertilisation (IVF) is widely used to treat infertility, however, establishment of pregnancy after transfer of embryos generated in vitro remains a significant hurdle (Calhaz-Jorge et al. 2017). Pregnancies arising from assisted reproductive technologies (ART) are associated with altered foetal growth, which continues into early childhood and may impact adult health (Ceelen et al. 2008, Hart & Norman 2013). Altered placental structure and function may underlie some of these effects (Haavaldsen et al. 2012, Feuer et al. 2014, Nelissen et al. 2014, Chen et al. 2015, Song et al. 2015). In vitro culture exposes embryos to environmental conditions, including non-physiological nutritive, oxidative, osmotic, temperature, pH and light/electromagnetic conditions, as well as toxic stress and shear forces involved in handling (Xie et al. 2007a). Altered nutrient availability in the preimplantation period can affect blastocyst growth in rodents, with knock-on effects on offspring weight and developmental progression (Kwong et al. 2000), and even a brief period in vitro at the blastocyst stage can affect placental development and function (de Waal et al. 2015). Conversely, some evidence suggests that exposure to sublethal stressors can improve developmental competence of gametes and embryos (Pribenszky et al. 2010). Effects of environmental stresses on intercellular interactions at implantation have not been examined, but a better understanding of this critical process has the potential to improve ART efficiency and efficacy.

Implantation involves attachment of the blastocyst trophectoderm (TE) to endometrial epithelial cells (EEC), followed by trophoblast invasion into the underlying endometrial stroma and eventual access to the maternal vasculature (Aplin & Ruane 2017). Dissecting implantation requires in vitro models, and primary EEC and cell lines have been used to investigate the mechanisms of human and mouse embryo attachment (Weimar et al. 2013). Recent studies have characterised the effects of pharmacologic agents on human embryo attachment in vitro (Petersen et al. 2005, Lalitkumar et al. 2007, 2013, Boggavarapu et al. 2016); however, studies of environmental impact on attachment have used only trophoblast cell spheroids as model embryos (Tsang et al. 2012, 2013). We recently described an in vitro model that allows the kinetics of mouse embryo-EEC attachment to be monitored and trophoblast breaching of EEC to be assessed; key parameters of the early stages of implantation which are difficult to investigate in vivo (Ruane et al. 2017).

Cells respond to environmental stressors by activating conserved signalling modules, including the mitogen-activated protein kinase (MAPK) superfamily member c-Jun N-terminal kinase (JNK). Active JNK phosphorylates transcription factors to coordinate the transcriptional stress response, leading to the regulation of cell growth, survival and differentiation (Weston & Davis 2007). In mouse embryos, there is some evidence of a role for JNK in blastocyst formation from the 8-cell stage (Maekawa et al. 2005), and additional work has identified a role specific to preimplantation development in sub-optimal medium (Xie et al. 2006). Ultimately, stress signalling pathways are thought to impinge on cell growth to divert energy to homeostatic processes that support short-term survival, with increased extent or duration of signalling leading to senescence and apoptosis (Puscheck et al. 2015).

Hyperosmolarity is a clinically relevant and experimentally tractable environmental variable that can be used to evaluate stress responses in mouse embryos and stem cells (Xie et al. 2007b). Here, we have characterised the effects of hyperosmotic stress on attachment and invasion parameters of mouse embryo implantation in an in vitro model.

Materials and methods

Cell culture

Ishikawa cells (ECACC 99040201) were cultured at 37°C, 95% air and 5% CO2 in growth medium (1:1 Dulbecco’s modified Eagle’s medium:Ham’s-F12 (Sigma, D5796 and N6658, respectively) containing 10% foetal bovine serum (Sigma) supplemented with 2 mM l-glutamine, 100 µg/mL streptomycin and 100 IU/mL penicillin (Sigma)). Ishikawa cells between passage 6 and 25 were used for experiments. Cells were grown to confluency in 24-well plates (Greiner) on 13 mm glass coverslips coated with 2% Matrigel (Sigma).

Mouse embryos

Experiments were licensed under the authority of a UK Home Office project license (PPL 70/07838) and were authorized by the Animal Welfare and Ethical Review Board of the University of Manchester, according to the Animal Act, 1986. CD1 mice were housed in the Biological Services Unit at the University of Manchester under standard environmental conditions of 12-h light and 12-h dark at 20–22°C and 40–60% humidity, with food and water provided ad libitum. Eight- to 10-week-old female CD1 mice (Charles River) were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotrophin (Intervet), followed by 5 IU human chorionic gonadotrophin (Intervet) 46 h later, and then kept overnight with ≤9-month-old CD1 stud males for mating. Midday the following day was designated E0.5. Embryos were gathered at E1.5 by flushing dissected oviducts with M2 medium (Millipore) containing 0.4% w/v BSA (Sigma). Embryo manipulation was performed using a Flexipet with 140 µm (E1.5 embryos) and 300 µm (E4.5–5.5 embryos) pipettes (Cook). E1.5 embryos were incubated for 72 h in drops of KSOM medium (Millipore) containing 0.4% BSA under oil (Ovoil, Vitrolife) at 37°C, 95% air and 5% CO2. E4.5 blastocysts were chemically hatched in acid Tyrode’s (pH 2.5) (Sigma) and washed in KSOM 0.4% w/v BSA.

Hyperosmolar treatment of blastocysts

E4.5 blastocysts were treated with KSOM containing 0.4% w/v BSA and 400 mM sorbitol (389 ± 2 mosmol increase in osmolarity). E5.5 blastocysts were treated without prior co-culture and were cultured in 1:1 DMEM:F12 supplemented with 2 mM l-glutamine, 100 µg/mL streptomycin, 100 IU/mL penicillin and 0.4% w/v BSA to allow comparison with blastocysts co-cultured from E4.5. E5.5 blastocysts were treated with 1:1 DMEM:F12, 2 mM l-glutamine, 100 µg/mL streptomycin, 100 IU/mL penicillin and 0.4% w/v BSA, containing 400 mM sorbitol (382 ± 4 mosmol increase in osmolarity). Repeated osmotic stress of blastocysts entailed four cycles of treatment, each of which consisted of a 30-min incubation in hyperosmotic medium containing 400 mM sorbitol followed by incubation in normosmotic medium for 30 min. Blastocysts were passed through three drops of treatment medium to prevent carry-over affecting osmolarity. Control blastocysts were treated as above but only normosmotic medium was used. All treatments were performed in drops under oil at 37°C, 95% air and 5% CO2. In some experiments, SP600125 (20 µM; Sigma) was added to medium to inhibit JNK during treatments. Embryos were imaged throughout treatment using an inverted phase contrast microscope (Evos XL Core) and embryo diameter was measured using ImageJ software.

In vitro implantation model

The in vitro implantation model was employed as described previously (Ruane et al. 2017). Briefly, confluent Ishikawa cells in 24-well plates were incubated with co-culture medium (1:1 DMEM:F12 containing 2 mM l-glutamine, 100 µg/mL streptomycin and 100 IU/mL penicillin) 24 h prior to co-culture with three hatched mouse blastocysts per well and incubation at 37°C, 95% air and 5% CO2. Blastocyst attachment stability was recorded at 4-h intervals from E5.5 to 6 and at E6.5, using an inverted phase-contrast microscope. Attachment stability was graded upon gentle and continuous agitation of the plate, and observation of blastocyst movements (not attached) or oscillations (weakly, intermediately or stably attached). After co-culture, samples were washed in PBS and fixed with 4% paraformaldehyde in PBS for 20 min.

Fluorescence staining and microscopy

Fixed attached embryo samples were washed with PBS, quenched with 50 mM ammonium chloride solution and permeabilised with 0.5% Triton-X100 PBS. Alexa568-phalloidin (Life Technologies) and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma), in PBS was added for 1 h before mounting in a chamber of 3% 1,4-diazabicyclo[2.2.2]octane (Sigma) in PBS. Fluorescence microscopy was performed with a Zeiss Axiophot microscope equipped with an Apotome module for optical sectioning. Images were analysed and processed using Zeiss Zen software.

Statistical analysis

2-Way ANOVA analysis with Dunnetts post hoc test was performed using SPSS and used to demonstrate significant differences from P < 0.05.

Results

Single-episode hyperosmotic treatment of mouse blastocysts does not affect implantation in an in vitro model

We challenged mouse embryos with hyperosmolar conditions for 2 h prior to implantation in our in vitro model system. Full activation of JNK in E3.5 early blastocysts has been shown to occur between 30-min and 2-h exposure to hyperosmotic conditions (Xie et al. 2007b). Placing E4+10 h blastocysts in medium containing 400 mM sorbitol caused immediate blastocoel collapse (Fig. 1A and B, diameter reduced by 17% to 74.7 µm). Recovery to expanded blastocyst morphology was apparent at the end of the 2-h treatment (E4.5) and subsequent replacement in normosmotic medium led to enhanced expansion (Fig. 1B, diameter increased by 7% to 99.8 µm). Blastocysts were then co-cultured with Ishikawa cells and their attachment was monitored from E5.5, at 24, 28, 32 and 48 h co-culture, as stable attachment occurs over this period (Ruane et al. 2017). The kinetics of stable attachment were not changed by hyperosmotic treatment (Fig. 2A).

Figure 1
Figure 1

(A) Brightfield images of E4.5 blastocysts in normosmotic medium, and immediately after placement in hyperosmotic medium. Scale bars 50 μm. (B) E4.5 blastocyst diameter was measured from brightfield images taken before incubation in treatment medium (normosmotic for control treatment and hyperosmotic for experimental treatment), immediately after the start of treatment, 2 h after incubation in treatment medium and immediately after transfer from treatment medium to normosmotic medium. Thirty-six embryos were measured per treatment group over three independent experiments, data expressed as mean diameter ± s.e.m. *P < 0.05, **P < 0.01 ANOVA compared to pre-treatment group.

Citation: Reproduction 156, 5; 10.1530/REP-18-0154

Figure 2
Figure 2

(A) E4.5 blastocysts were incubated in normosmotic or hyperosmotic medium for 2 h before co-culture with Ishikawa cells. Stability of embryo attachment to Ishikawa cells was monitored from E5.5 every 4 h to E6, and at E6.5. A total of 64 blastocysts were used across 3 independent experiments. Mean stable attachment was plotted ±s.e.m. No significant differences in stable attachment were seen. (B) Attached embryos were fixed and labelled with actin (red) and nuclear (blue) stains (phalloidin and DAPI, respectively) before optical sectioning by fluorescence microscopy to reveal the embryo-Ishikawa interface. The top image shows an upper section of the embryo above the Ishikawa cell plane, while the bottom image shows breaching of the Ishikawa cell layer by embryonic cells (area within dotted line) in a lower section. Scale bars 20 µm. (C) E4.5 normosmotic and hyperosmotic-exposed embryos that had attached to Ishikawa cells by E6.5 were analysed by fluorescence microscopy. Embryo breaching of the Ishikawa cell layer was scored for 52 embryos from 3 independent experiments and the mean ± s.e.m. percentage of imaged embryos to have breached the Ishikawa cells was plotted. No significant difference in breaching was seen (NS). (D) E5.5 blastocysts were cultured in normosmotic or hyperosmotic medium for 2 h before incubation with Ishikawa cells. Stability of attachment to Ishikawa cells was monitored every 4 h to E6, and at E6.5. A total of 60 blastocysts were used in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were observed. (E) E5.5 normosmotic and hyperosmotic-exposed embryos attached to Ishikawa cells at E7.5 were subjected to fluorescence microscopic analysis. Mean ± s.e.m. percentage of 30 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. No significant difference in breaching was seen (NS).

Citation: Reproduction 156, 5; 10.1530/REP-18-0154

We previously showed that blastocyst apposition to Ishikawa cells from E4.5 to 5.5 activates them to breach Ishikawa cell layers during the following 24 h, which resembles progression from attachment to invasion at implantation (Ruane et al. 2017). This process was not affected by hyperosmotic treatment (Fig. 2B and C).

Attachment kinetics do not differ between embryos beginning co-culture with Ishikawa cells at E4.5 and those beginning co-culture at E5.5 (Ruane et al. 2017). E5.5 blastocysts, not previously co-cultured, were exposed to hyperosmolar conditions to establish whether concurrence of such stressors with the onset of attachment to Ishikawa cells affects attachment and breaching. The profile of stable attachment over time was not affected by hyperosmotic treatment from E5+10 h to E5.5 (Fig. 2D). Additionally, the limited embryonic breaching seen when co-culture was initiated at E5.5 was not affected by hyperosmotic treatment (Fig. 2E).

Inhibition of JNK signalling during hyperosmotic treatment does not affect embryo attachment or breaching

To establish whether stress-activated JNK signalling is required for in vitro implantation after exposure to stress, we exposed blastocysts to hyperosmotic conditions for 2 h at E4.5 and E5.5 in the presence of JNK inhibitor SP600125 prior to co-culture with Ishikawa cells. SP600125 did not affect E4.5 blastocyst collapse and re-expansion in hyperosmotic medium (data not shown). Moreover, SP600125 treatment during normosmotic or hyperosmotic treatment did not affect blastocyst attachment or breaching (Fig. 3A, B, C and D).

Figure 3
Figure 3

(A) E4.5 blastocysts were incubated in normosmotic or hyperosmotic medium for 2 h in the presence or absence of 20 µM JNK inhibitor SP600125 before co-culture with Ishikawa cells. Stability of embryo attachment was monitored from E5.5 to E6.5. A total of 134 blastocysts were used in 3 separate experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were detected. (B) Embryos from A that had attached to Ishikawa cells by E6.5 were stained with phalloidin and DAPI to detect embryos breaching the Ishikawa cell layer. Mean ± s.e.m. percentage of 134 imaged embryos from 3 independent experiments to have breached was plotted. No significant differences in breaching were observed (NS). (C) E5.5 blastocysts were cultured in normosmotic or hyperosmotic medium for 2 h in the presence or absence of 20 µM JNK inhibitor SP600125 before incubation with Ishikawa cells. Stability of attachment was monitored to E6.5. A total of 147 blastocysts were used in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted and no significant differences were found. (D) E7.5 attached embryos from C were labelled with phalloidin and DAPI and imaged to analyse embryo breaching. Mean ± s.e.m. percentage of 96 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. No significant differences in breaching were seen (NS).

Citation: Reproduction 156, 5; 10.1530/REP-18-0154

Repeated osmotic stress at E5.5 promotes breaching of Ishikawa cells through JNK signalling

The re-expansion of mouse blastocysts within 2 h after initial collapse in hyperosmotic medium revealed a remarkable ability to acclimatise to hyperosmolarity (Fig. 1B). We hypothesised that repeated changes in osmolarity may result in stress responses, which impact upon attachment and breaching. We therefore moved blastocysts between normosmotic and hyperosmotic media at 30-min periods over 4 h in the absence or presence of SP600125. Alongside this treatment blastocysts were moved between normosmotic media to control for repeated manipulation. Blastocysts were therefore exposed to a total of 2 h hyperosmolarity, mirroring single episode exposures, and 30 min exposure has been shown to activate JNK (Xie et al. 2007b).

E4+8 h and E5+8 h blastocysts subjected to repeated osmotic stress exhibited initial collapse in hyperosmotic medium that did not recover within 30 min or during the subsequent 30 min in normosmotic medium. This decrease in size was exceeded upon the second exposure to hyperosmolarity; however, the embryos then recovered, in both hyperosmotic and normomostic media, to the size seen after the initial hyperosmotic treatment. This latter collapse and re-expansion was observed in the following two cycles of hyperosmotic and normosmotic treatment and was not affected by SP600125 (Fig. 4A, data shown for E5.5 embryos only). The manipulation control blastocysts were significantly decreased in size after two cycles through normosmotic medium and maintained this size, which was similar to that of blastocysts after two cycles of hyperosmotic shock, during subsequent manipulations (Fig. 4A).

Figure 4
Figure 4

(A) E5+8 h blastocysts were incubated in hyperosmotic medium for 30 min followed immediately by incubation in normosmotic medium for 30 min. This regimen was repeated four times in the presence or absence of SP600125. Manipulation control blastocysts were passed through normosmotic medium only. Brightfield images of embryos were taken after 1min and 30 min in each culture condition and embryo diameter measured. Mean ± s.e.m. diameter of 106 embryos over 3 independent experiments was plotted. (B) 104 E5.5 blastocysts exposed to repeated osmotic stress in the presence or absence of SP600125, or control conditions, were co-cultured with Ishikawa cells from E5.5 and the stability of attachment monitored to E6.5 in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were observed (NS). (C) Embryos attached to Ishikawa cells by E7.5 from B were stained with phalloidin and DAPI to image the embryo-Ishikawa interface. Mean ± s.e.m. percentage of 62 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. *P < 0.05 ANOVA.

Citation: Reproduction 156, 5; 10.1530/REP-18-0154

E4+8 h blastocysts cycled through hyperosmotic and normosmotic medium until E4.5 collapsed and expanded similarly to E5+8 h blastocysts; however, there was no effect on subsequent embryo attachment and breaching on Ishikawa cells (data not shown). E5.5 blastocysts exposed to repeated osmotic stress in the presence or absence of SP600125 also exhibited undisturbed attachment to Ishikawa cells (Fig. 4B); however, embryos subjected to repeated osmotic stress went on to breach the Ishikawa cell layer at a significantly higher rate than the manipulation control embryos (Fig. 4C). Notably, this effect was dependent on JNK signalling since the presence of SP600125 during repeated osmotic stress abolished the increase in breaching.

Discussion

Despite concerted efforts to optimise the in vitro culture environment, exposure to stressors is inevitable during IVF and embryo culture. Using hyperosmolarity as a well-defined experimental stressor with some clinical relevance, we show that single hyperosmotic events do not compromise mouse embryo attachment and invasion of Ishikawa cells in vitro. Repeated changes from normosmotic to hyperosmotic conditions also do not affect TE function at attachment; however, embryonic breaching of Ishikawa cells is increased in a JNK signalling-dependent manner.

Trophoblast responses to stress are coupled to development and invasion in interstitially implanting species, with hypoxic and nutritive stress promoting proliferation and migration and thus driving implantation and embryonic survival through access to maternal nutrients (Rosario et al. 2008, Watkins et al. 2015). Nutritive stress restricted to the preimplantation stage in vivo leads to more invasive trophoblast, suggesting that stress during cleavage and first lineage allocation stages can affect subsequent trophoblast development at implantation. Effects on epigenetic reprogramming may underlie these outcomes (Choux et al. 2015), though signalling through the nutrient-responsive mTOR complex partially mediates enhanced trophoblast formation from affected blastocysts (Eckert et al. 2012). To investigate the impact of stresses relevant to embryo culture and blastocyst transfer in ART on implantation, we used a defined stressor and a characterised in vitro model based on mouse embryos and the Ishikawa EEC line. This enabled the analysis of blastocyst attachment to EEC and trophoblast penetration of the EEC layer as key early steps in implantation (Ruane et al. 2017). To assess stress effects on human implantation in this model, careful powering would be required to account for variability of human embryo quality. Further work is also merited with primary human endometrial epithelial and stromal cells, especially in light of evidence that decidualised stromal cells respond differentially to embryos of high and low quality (Brosens et al. 2014). Though it is difficult to generate polarised primary human EEC layers in vitro (Campbell et al. 2000), recent developments in epithelial organoid culture promise sophisticated models of human implantation (Boretto et al. 2017, Turco et al. 2017).

In vitro studies using rodent embryos and TE-derived trophoblast stem cells (TSC) have investigated acute and chronic stress effects on differentiation to early extraembryonic lineages, especially trophoblast giant cells (TGCs) (Puscheck et al. 2015). The activity of the stress-responsive protein kinases, JNK, p38 MAPK and adenosine monophosphate-activated protein kinase (AMPK), increases in embryos and TSC subjected to sub-optimal culture medium, shear stress, hyperosmolarity, hypoxia and microgravity (Bell et al. 2009, Wang et al. 2005, 2009, Xie et al. 2007a,b, 2013). Moreover, hyperosmolarity leads to downregulation of the TE and TSC progenitor marker Cdx2 and upregulation of trophoblast differentiation markers Eomes, Hand1, Stra13 and Prl3d1 in a JNK- and AMPK-dependent manner (Awonuga et al. 2011). These studies strongly evidence stress signalling as a mechanism for promoting trophoblast differentiation, and this has been suggested as a response to the developmental hurdle of implantation when the embryo signals to establish maternal recognition of pregnancy and strives to secure resources for growth. Conversely, excessive stress-driven differentiation at the expense of TSC renewal has been proposed to negatively affect placental homeostasis and adaptation due to a reduction in the TSC pool (Puscheck et al. 2015). The data presented here provide the first evidence of preimplantation embryo exposure to an environmental stressor promoting subsequent invasion into maternal cells. In addition, we evidence JNK signalling as a mediator of stress-induced embryonic invasion at implantation because pharmacological inhibition of JNK signalling during embryo exposure to stress blocked the stimulation of trophoblast breaching of Ishikawa cell layers. We previously showed that blastocyst apposition to Ishikawa cells during E4.5–5.5 induced gene expression changes characteristic of TGC differentiation, and in turn, TGCs were seen to mediate breaching of the Ishikawa cell layer at E6.5 (Ruane et al. 2017). We therefore speculate that stress signalling through JNK in the TE at E5.5 induces the expression of genes underpinning TGC differentiation, such as Hand1.

The observation that breaching was promoted only by repeated osmotic stress may reflect a stress signalling threshold that must be reached to advance trophoblast differentiation faster than in unstressed conditions (Puscheck et al. 2015). The observation that blastocyst expansion was fully rescued 2 h after initial collapse in hyperosmotic medium highlights the homeostatic resilience of pre-implantation mouse embryos and suggests that stress signals may have rapidly abated after initial exposure to hyperosmolarity. Our data suggest that a single episode of collapse and re-expansion may cause stress equivalent to simple embryo manipulation by pipetting (Xie et al. 2007a). Blastocysts undergo repeated cycles of partial collapse and re-expansion during normal development, likely due to transient losses of epithelial integrity during cytokinesis in the TE layer. Once the blastocoel fluid has equilibrated with the external environment and epithelial integrity has been restored, directional ion pumping rapidly restores osmotic pressure allowing the blastocyst to re-expand (Biggers et al. 1988). Repeated osmotic stress caused repeated blastocyst collapse-expansion cycles, perhaps indicating prolonged or higher magnitude stress signalling, which reached a threshold that led to altered gene expression. One possibility is that reduced actomyosin tension upon repeated blastocoel collapse acts upstream of JNK through Rho GTPases (Coso et al. 1995). Other mechanisms of osmotic sensing, which persist through repeated osmotic stress, such as ion channel activity, may also act upon JNK (Furst et al. 2002).

There is a lack of understanding regarding the role of stress signalling in human embryos, especially at the implantation stage, despite the exposure to sub-optimal environmental conditions that is inherent in in vitro culture. Environmental stressors have been shown to impact on human trophoblast development and function (Burton et al. 2009), and if TE differentiation to trophoblast at implantation is positively regulated by stress signalling in human as in mouse, it follows that stress invoked during ART procedures may not impede implantation. Whether this response is capable of rescuing a failing conceptus, with possible implications for foetal development and long-term health or whether stress-affected embryos may implant only to fail at a later stage of pregnancy, will require investigation in vivo.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for S C B from the Anatomical Society.

Acknowledgments

The authors acknowledge staff at the Biological Services Unit, University of Manchester, for their support with animal care.

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  • de WaalEVroomanLAFischerEOrdTMainigiMACoutifarisCSchultzRMBartolomeiMS 2015 The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model. Human Molecular Genetics 24 69756985. (https://doi.org/10.1093/hmg/ddv400)

    • Search Google Scholar
    • Export Citation
  • EckertJJPorterRWatkinsAJBurtEBrooksSLeeseHJHumphersonPGCameronITFlemingTP 2012 Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health. PLoS ONE 7 e52791. (https://doi.org/10.1371/journal.pone.0052791)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • FeuerSKLiuXDonjacourALinWSimbulanRKGiritharanGPianeLDKolahiKAmeriKMaltepeE 2014 Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis. Endocrinology 155 19561969. (https://doi.org/10.1210/en.2013-2081)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • FurstJGschwentnerMRitterMBottaGJakabMMayerMGaravagliaLBazziniCRodighieroSMeyerG 2002 Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflugers Archiv: European Journal of Physiology 444 125. (https://doi.org/10.1007/s00424-002-0805-1)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • HaavaldsenCTanboTEskildA 2012 Placental weight in singleton pregnancies with and without assisted reproductive technology: a population study of 536,567 pregnancies. Human Reproduction 27 576582. (https://doi.org/10.1093/humrep/der428)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • HartRNormanRJ 2013 The longer-term health outcomes for children born as a result of IVF treatment: Part I – general health outcomes. Human Reproduction Update 19 232243. (https://doi.org/10.1093/humupd/dms062)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KwongWYWildAERobertsPWillisACFlemingTP 2000 Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127 41954202.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LalitkumarPGLalitkumarSMengCXStavreus-EversAHambilikiFBentin-LeyUGemzell-DanielssonK 2007 Mifepristone, but not levonorgestrel, inhibits human blastocyst attachment to an in vitro endometrial three-dimensional cell culture model. Human Reproduction 22 30313037. (https://doi.org/10.1093/humrep/dem297)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • LalitkumarSBoggavarapuNRMenezesJDimitriadisEZhangJGNicolaNAGemzell-DanielssonKLalitkumarLP 2013 Polyethylene glycated leukemia inhibitory factor antagonist inhibits human blastocyst implantation and triggers apoptosis by down-regulating embryonic AKT. Fertility and Sterility 100 11601169. (https://doi.org/10.1016/j.fertnstert.2013.06.023)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MaekawaMYamamotoTTanoueTYuasaYChisakaONishidaE 2005 Requirement of the MAP kinase signaling pathways for mouse preimplantation development. Development 132 17731783. (https://doi.org/10.1242/dev.01729)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • NelissenECDumoulinJCBusatoFPongerLEijssenLMEversJLTostJvan MontfoortAP 2014 Altered gene expression in human placentas after IVF/ICSI. Human Reproduction 29 28212831. (https://doi.org/10.1093/humrep/deu241)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • PetersenABentin-LeyURavnVQvortrupKSorensenSIslinHSjogrenAMosselmannSHambergerL 2005 The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro. Fertility and Sterility 83 (Supplement 1) 12551263. (https://doi.org/10.1016/j.fertnstert.2004.08.040)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • PribenszkyCVajtaGMolnarMDuYLinLBolundLYovichJ 2010 Stress for stress tolerance? A fundamentally new approach in mammalian embryology. Biology of Reproduction 83 690697. (https://doi.org/10.1095/biolreprod.110.083386)

    • Crossref
    • PubMed
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    • Export Citation
  • PuscheckEEAwonugaAOYangYJiangZRappoleeDA 2015 Molecular biology of the stress response in the early embryo and its stem cells. Advances in Experimental Medicine and Biology 843 77128. (https://doi.org/10.1007/978-1-4939-2480-6_4)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RosarioGXKonnoTSoaresMJ 2008 Maternal hypoxia activates endovascular trophoblast cell invasion. Developments in Biologicals 314 362375. (https://doi.org/10.1016/j.ydbio.2007.12.007)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • RuanePTBerneauSCKoeckRWattsJKimberSJBrisonDRWestwoodMAplinJD 2017 Apposition to endometrial epithelial cells activates mouse blastocysts for implantation. Molecular Human Reproduction 23 617627. (https://doi.org/10.1093/molehr/gax043)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • SongSGhoshJMainigiMTuranNWeinermanRTruongcaoMCoutifarisCSapienzaC 2015 DNA methylation differences between in vitro- and in vivo-conceived children are associated with ART procedures rather than infertility. Clinical Epigenetics 7 41. (https://doi.org/10.1186/s13148-015-0071-7)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • TsangHCheungTYKodithuwakkuSPChaiJYeungWSWongCKLeeKF 2012 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses spheroids attachment on endometrial epithelial cells through the down-regulation of the Wnt-signaling pathway. Reproductive Toxicology 33 6066. (https://doi.org/10.1016/j.reprotox.2011.11.002)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • TsangHCheungTYKodithuwakkuSPChaiJYeungWSWongCKLeeKF 2013 Perfluorooctanoate suppresses spheroid attachment on endometrial epithelial cells through peroxisome proliferator-activated receptor alpha and down-regulation of Wnt signaling. Reproductive Toxicology 42 164171. (https://doi.org/10.1016/j.reprotox.2013.08.001)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • TurcoMYGardnerLHughesJCindrova-DaviesTGomezMJFarrellLHollinsheadMMarshSGEBrosensJJCritchleyHO 2017 Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nature Cell Biology 19 568577. (https://doi.org/10.1038/ncb3516)

    • Crossref
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    • Export Citation
  • WangYPuscheckEELewisJJTrostinskaiaABWangFRappoleeDA 2005 Increases in phosphorylation of SAPK/JNK and p38MAPK correlate negatively with mouse embryo development after culture in different media. Fertility and Sterility 83 (Supplement 1) 11441154. (https://doi.org/10.1016/j.fertnstert.2004.08.038)

    • Crossref
    • PubMed
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    • Export Citation
  • WangYXieYWygleDShenHHPuscheckEERappoleeDA 2009 A major effect of simulated microgravity on several stages of preimplantation mouse development is lethality associated with elevated phosphorylated SAPK/JNK. Reproductive Sciences 16 947959. (https://doi.org/10.1177/1933719109337544)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • WatkinsAJLucasESMarfy-SmithSBatesNKimberSJFlemingTP 2015 Maternal nutrition modifies trophoblast giant cell phenotype and fetal growth in mice. Reproduction 149 563575. (https://doi.org/10.1530/REP-14-0667)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • WeimarCHPost UiterweerEDTeklenburgGHeijnenCJMacklonNS 2013 In-vitro model systems for the study of human embryo-endometrium interactions. Reproductive Biomedicine Online 27 461476. (https://doi.org/10.1016/j.rbmo.2013.08.002)

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  • WestonCRDavisRJ 2007 The JNK signal transduction pathway. Current Opinion in Cell Biology 19 142149. (https://doi.org/10.1016/j.ceb.2007.02.001)

  • XieYPuscheckEERappoleeDA 2006 Effects of SAPK/JNK inhibitors on preimplantation mouse embryo development are influenced greatly by the amount of stress induced by the media. Molecular Human Reproduction 12 217224. (https://doi.org/10.1093/molehr/gal021)

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  • XieYWangFPuscheckEERappoleeDA 2007a Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos. Molecular Reproduction and Development 74 12871294. (https://doi.org/10.1002/mrd.20563)

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  • XieYZhongWWangYTrostinskaiaAWangFPuscheckEERappoleeDA 2007b Using hyperosmolar stress to measure biologic and stress-activated protein kinase responses in preimplantation embryos. Molecular Human Reproduction 13 473481. (https://doi.org/10.1093/molehr/gam027)

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  • XieYAwonugaALiuJRingsEPuscheckEERappoleeDA 2013 Stress induces AMPK-dependent loss of potency factors Id2 and Cdx2 in early embryos and stem cells (corrected) Stem Cells and Development 22 15641575. (https://doi.org/10.1089/scd.2012.0352)

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    (A) Brightfield images of E4.5 blastocysts in normosmotic medium, and immediately after placement in hyperosmotic medium. Scale bars 50 μm. (B) E4.5 blastocyst diameter was measured from brightfield images taken before incubation in treatment medium (normosmotic for control treatment and hyperosmotic for experimental treatment), immediately after the start of treatment, 2 h after incubation in treatment medium and immediately after transfer from treatment medium to normosmotic medium. Thirty-six embryos were measured per treatment group over three independent experiments, data expressed as mean diameter ± s.e.m. *P < 0.05, **P < 0.01 ANOVA compared to pre-treatment group.

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    (A) E4.5 blastocysts were incubated in normosmotic or hyperosmotic medium for 2 h before co-culture with Ishikawa cells. Stability of embryo attachment to Ishikawa cells was monitored from E5.5 every 4 h to E6, and at E6.5. A total of 64 blastocysts were used across 3 independent experiments. Mean stable attachment was plotted ±s.e.m. No significant differences in stable attachment were seen. (B) Attached embryos were fixed and labelled with actin (red) and nuclear (blue) stains (phalloidin and DAPI, respectively) before optical sectioning by fluorescence microscopy to reveal the embryo-Ishikawa interface. The top image shows an upper section of the embryo above the Ishikawa cell plane, while the bottom image shows breaching of the Ishikawa cell layer by embryonic cells (area within dotted line) in a lower section. Scale bars 20 µm. (C) E4.5 normosmotic and hyperosmotic-exposed embryos that had attached to Ishikawa cells by E6.5 were analysed by fluorescence microscopy. Embryo breaching of the Ishikawa cell layer was scored for 52 embryos from 3 independent experiments and the mean ± s.e.m. percentage of imaged embryos to have breached the Ishikawa cells was plotted. No significant difference in breaching was seen (NS). (D) E5.5 blastocysts were cultured in normosmotic or hyperosmotic medium for 2 h before incubation with Ishikawa cells. Stability of attachment to Ishikawa cells was monitored every 4 h to E6, and at E6.5. A total of 60 blastocysts were used in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were observed. (E) E5.5 normosmotic and hyperosmotic-exposed embryos attached to Ishikawa cells at E7.5 were subjected to fluorescence microscopic analysis. Mean ± s.e.m. percentage of 30 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. No significant difference in breaching was seen (NS).

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    (A) E4.5 blastocysts were incubated in normosmotic or hyperosmotic medium for 2 h in the presence or absence of 20 µM JNK inhibitor SP600125 before co-culture with Ishikawa cells. Stability of embryo attachment was monitored from E5.5 to E6.5. A total of 134 blastocysts were used in 3 separate experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were detected. (B) Embryos from A that had attached to Ishikawa cells by E6.5 were stained with phalloidin and DAPI to detect embryos breaching the Ishikawa cell layer. Mean ± s.e.m. percentage of 134 imaged embryos from 3 independent experiments to have breached was plotted. No significant differences in breaching were observed (NS). (C) E5.5 blastocysts were cultured in normosmotic or hyperosmotic medium for 2 h in the presence or absence of 20 µM JNK inhibitor SP600125 before incubation with Ishikawa cells. Stability of attachment was monitored to E6.5. A total of 147 blastocysts were used in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted and no significant differences were found. (D) E7.5 attached embryos from C were labelled with phalloidin and DAPI and imaged to analyse embryo breaching. Mean ± s.e.m. percentage of 96 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. No significant differences in breaching were seen (NS).

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    (A) E5+8 h blastocysts were incubated in hyperosmotic medium for 30 min followed immediately by incubation in normosmotic medium for 30 min. This regimen was repeated four times in the presence or absence of SP600125. Manipulation control blastocysts were passed through normosmotic medium only. Brightfield images of embryos were taken after 1min and 30 min in each culture condition and embryo diameter measured. Mean ± s.e.m. diameter of 106 embryos over 3 independent experiments was plotted. (B) 104 E5.5 blastocysts exposed to repeated osmotic stress in the presence or absence of SP600125, or control conditions, were co-cultured with Ishikawa cells from E5.5 and the stability of attachment monitored to E6.5 in 3 independent experiments. Mean ± s.e.m. stable attachment was plotted. No significant differences in stable attachment were observed (NS). (C) Embryos attached to Ishikawa cells by E7.5 from B were stained with phalloidin and DAPI to image the embryo-Ishikawa interface. Mean ± s.e.m. percentage of 62 imaged embryos from 3 independent experiments to have breached the Ishikawa cells was plotted. *P < 0.05 ANOVA.

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  • de WaalEVroomanLAFischerEOrdTMainigiMACoutifarisCSchultzRMBartolomeiMS 2015 The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model. Human Molecular Genetics 24 69756985. (https://doi.org/10.1093/hmg/ddv400)

    • Search Google Scholar
    • Export Citation
  • EckertJJPorterRWatkinsAJBurtEBrooksSLeeseHJHumphersonPGCameronITFlemingTP 2012 Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health. PLoS ONE 7 e52791. (https://doi.org/10.1371/journal.pone.0052791)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • FeuerSKLiuXDonjacourALinWSimbulanRKGiritharanGPianeLDKolahiKAmeriKMaltepeE 2014 Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis. Endocrinology 155 19561969. (https://doi.org/10.1210/en.2013-2081)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • FurstJGschwentnerMRitterMBottaGJakabMMayerMGaravagliaLBazziniCRodighieroSMeyerG 2002 Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflugers Archiv: European Journal of Physiology 444 125. (https://doi.org/10.1007/s00424-002-0805-1)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • HaavaldsenCTanboTEskildA 2012 Placental weight in singleton pregnancies with and without assisted reproductive technology: a population study of 536,567 pregnancies. Human Reproduction 27 576582. (https://doi.org/10.1093/humrep/der428)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • HartRNormanRJ 2013 The longer-term health outcomes for children born as a result of IVF treatment: Part I – general health outcomes. Human Reproduction Update 19 232243. (https://doi.org/10.1093/humupd/dms062)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KwongWYWildAERobertsPWillisACFlemingTP 2000 Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127 41954202.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LalitkumarPGLalitkumarSMengCXStavreus-EversAHambilikiFBentin-LeyUGemzell-DanielssonK 2007 Mifepristone, but not levonorgestrel, inhibits human blastocyst attachment to an in vitro endometrial three-dimensional cell culture model. Human Reproduction 22 30313037. (https://doi.org/10.1093/humrep/dem297)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • LalitkumarSBoggavarapuNRMenezesJDimitriadisEZhangJGNicolaNAGemzell-DanielssonKLalitkumarLP 2013 Polyethylene glycated leukemia inhibitory factor antagonist inhibits human blastocyst implantation and triggers apoptosis by down-regulating embryonic AKT. Fertility and Sterility 100 11601169. (https://doi.org/10.1016/j.fertnstert.2013.06.023)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MaekawaMYamamotoTTanoueTYuasaYChisakaONishidaE 2005 Requirement of the MAP kinase signaling pathways for mouse preimplantation development. Development 132 17731783. (https://doi.org/10.1242/dev.01729)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • NelissenECDumoulinJCBusatoFPongerLEijssenLMEversJLTostJvan MontfoortAP 2014 Altered gene expression in human placentas after IVF/ICSI. Human Reproduction 29 28212831. (https://doi.org/10.1093/humrep/deu241)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • PetersenABentin-LeyURavnVQvortrupKSorensenSIslinHSjogrenAMosselmannSHambergerL 2005 The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro. Fertility and Sterility 83 (Supplement 1) 12551263. (https://doi.org/10.1016/j.fertnstert.2004.08.040)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • PribenszkyCVajtaGMolnarMDuYLinLBolundLYovichJ 2010 Stress for stress tolerance? A fundamentally new approach in mammalian embryology. Biology of Reproduction 83 690697. (https://doi.org/10.1095/biolreprod.110.083386)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • PuscheckEEAwonugaAOYangYJiangZRappoleeDA 2015 Molecular biology of the stress response in the early embryo and its stem cells. Advances in Experimental Medicine and Biology 843 77128. (https://doi.org/10.1007/978-1-4939-2480-6_4)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RosarioGXKonnoTSoaresMJ 2008 Maternal hypoxia activates endovascular trophoblast cell invasion. Developments in Biologicals 314 362375. (https://doi.org/10.1016/j.ydbio.2007.12.007)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • RuanePTBerneauSCKoeckRWattsJKimberSJBrisonDRWestwoodMAplinJD 2017 Apposition to endometrial epithelial cells activates mouse blastocysts for implantation. Molecular Human Reproduction 23 617627. (https://doi.org/10.1093/molehr/gax043)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • SongSGhoshJMainigiMTuranNWeinermanRTruongcaoMCoutifarisCSapienzaC 2015 DNA methylation differences between in vitro- and in vivo-conceived children are associated with ART procedures rather than infertility. Clinical Epigenetics 7 41. (https://doi.org/10.1186/s13148-015-0071-7)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • TsangHCheungTYKodithuwakkuSPChaiJYeungWSWongCKLeeKF 2012 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses spheroids attachment on endometrial epithelial cells through the down-regulation of the Wnt-signaling pathway. Reproductive Toxicology 33 6066. (https://doi.org/10.1016/j.reprotox.2011.11.002)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • TsangHCheungTYKodithuwakkuSPChaiJYeungWSWongCKLeeKF 2013 Perfluorooctanoate suppresses spheroid attachment on endometrial epithelial cells through peroxisome proliferator-activated receptor alpha and down-regulation of Wnt signaling. Reproductive Toxicology 42 164171. (https://doi.org/10.1016/j.reprotox.2013.08.001)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • TurcoMYGardnerLHughesJCindrova-DaviesTGomezMJFarrellLHollinsheadMMarshSGEBrosensJJCritchleyHO 2017 Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nature Cell Biology 19 568577. (https://doi.org/10.1038/ncb3516)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • WangYPuscheckEELewisJJTrostinskaiaABWangFRappoleeDA 2005 Increases in phosphorylation of SAPK/JNK and p38MAPK correlate negatively with mouse embryo development after culture in different media. Fertility and Sterility 83 (Supplement 1) 11441154. (https://doi.org/10.1016/j.fertnstert.2004.08.038)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • WangYXieYWygleDShenHHPuscheckEERappoleeDA 2009 A major effect of simulated microgravity on several stages of preimplantation mouse development is lethality associated with elevated phosphorylated SAPK/JNK. Reproductive Sciences 16 947959. (https://doi.org/10.1177/1933719109337544)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • WatkinsAJLucasESMarfy-SmithSBatesNKimberSJFlemingTP 2015 Maternal nutrition modifies trophoblast giant cell phenotype and fetal growth in mice. Reproduction 149 563575. (https://doi.org/10.1530/REP-14-0667)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • WeimarCHPost UiterweerEDTeklenburgGHeijnenCJMacklonNS 2013 In-vitro model systems for the study of human embryo-endometrium interactions. Reproductive Biomedicine Online 27 461476. (https://doi.org/10.1016/j.rbmo.2013.08.002)

    • Crossref
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