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Prostaglandin signaling regulates ciliogenesis by modulating intraflagellar transport

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signaling cascades that regulate cilia formation remain incompletely understood. Here we report that prostaglandin signaling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants display ciliogenesis defects, and lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme Cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates cAMP-mediated signaling cascade, are required for cilia formation and elongation. Importantly, PGE2 signaling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signaling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.


INTRODUCTION
In mammals, prostaglandins (PGs) regulate a wide variety of important physiological processes, including pain perception and body temperature, cardiovascular homeostasis, reproduction, and cancer progression 1,2 . The prostaglandin precursor PGH 2 is synthesized by COX-1 and COX-2 in the endoplasmic reticulum from arachidonic acid, a 20-carbon polyunsaturated fatty acid released from membrane phospholipids 1 . COX-1 serves a homeostatic function and is responsible for basal, constitutive prostaglandin synthesis, whereas COX-2 increases production of prostaglandins during inflammatory response and in cancer 1 . The PG precursor is metabolized by prostaglandin synthases to form structurally related, bioactive prostanoids in various tissues, including PGE 2 , PGD 2 , PGF2α PGI 2 and Thromboxane A 2 (TxA 2 ) 1 . PGE 2 functions through activation of G-protein-coupled receptors (GPCRs), including EP1 through EP4. Among them, EP2 and EP4 increase the intracellular cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA) signaling 1,3 . Although prostaglandins have important functions in a variety of physiological and pathological processes, their roles in ciliogenesis have not been previously investigated and remain virtually unknown.
Cilia are formed and extended by IFT, which transports cargo proteins along microtubules from the base to the tip of the cilium and back to the cell body. This process is mediated by kinesins in the anterograde direction and by cytoplasmic dynein motor in the retrograde direction 4,5 . Basal body proteins are also essential for cilia formation. They anchor the cilium at the cell surface, provide template for microtubules in the ciliary axoneme, and serve as a relay station for protein and lipid traffic from the Golgi complex to the ciliary membrane 6,7 . Ciliary dysfunction causes multisystemic genetic disorders commonly known as human ciliopathies 5,8 . Many developmental pathways have been shown to function in ciliogenesis 4,5 . Fibroblast growth factor (FGF) signalling regulates cilia length and function through ciliogenic transcription factor Foxj1 in diverse epithelia 9 . In zebrafish Kupffer's vesicle (KV), both Wnt/β-catenin and Notch pathway regulate Foxj1 expression and controls ciliogenesis 10,11 . Components of the phosphatidylinositol signaling cascade also regulate cilia formation in zebrafish. This conclusion is based on observations that knockdown of inositol-pentakisphosphate 2-kinase (Ipk1) reduced cilia length and decreased the cilia beating frequency 12 . Our understanding of ciliogenesis regulation is, however, incomplete. Using zebrafish genetics and cultured human epithelial cells we reveal for the first time the roles of prostaglandin signaling in vertebrate ciliogenesis.

leakytail mutants display defective ciliogenesis
In the course of a zebrafish genetic screen for mutations that affect organogenesis, we identified the leakytail (lkt) mutant that displayed randomized heart looping ( Fig. 1g-i), as well as cilia-associated phenotypes, including ventrally curved body axis (Fig. 1a, b), hydrocephalus (Fig. 1c, d), abnormal otolith number (Fig. 1e, f), and laterality defects of the brain and other organs (Supplementary Fig. S1a-d, g). Randomized expression of a nodalrelated gene southpaw (spaw) 13 and a nodal target gene pitx2 13 occurred in the lateral plate mesoderm (Fig. 1v, w; Supplementary Fig. 1h). To test roles of lkt in ciliogenesis, we visualized cilia formation in developing embryos. At 24 hour post-fertilization (hpf), zebrafish otic vesicles (OVs) contain two clusters of long tether cilia and many short cilia distributed throughout OVs (Fig. 1j). In contrast to wild-type OVs (Fig. 1j), mutant OVs lacked short cilia but had relatively normal tether cilia (Fig. 1k). At 96 hpf, cristae kinocilia in ear semicircular canals were lost in lkt mutants (Fig. 1l, m). In Kupffer's vesicle (KV), we observed cilia loss and length reduction in mutant embryos relative to wild-type (Fig. 1n, o; r, s). lkt mutants also exhibited a loss of ependymal cell cilia in the spinal canal (Fig. 1p, q). However, lkt mutants do not form kidney cysts (Fig. 1t, u), and the formation and growth of pronephric cilia are not affected in lkt mutants either ( Supplementary Fig. 1e, f).

leakytail locus encodes the ABCC4 transporter
To determine the molecular underpinnings of lkt ciliopathy, we mapped lkt to chromosome 6 of the zebrafish genome using whole genome bulk segregant analyses (Fig. 2a) 14 . Subsequent fine mapping refined lkt genetic interval to a single bacterial artificial chromosomal clone (BAC) (Fig. 2a). One of four transcribed regions on this BAC encodes ABCC4 (Mrp4), a subfamily 'C' member of the ATP-binding cassette (ABC) superfamily of transport proteins (Fig. 2a, b) [15][16][17] . At the amino acid level, zebrafish Lkt/ABCC4 is 68.5% identical to the human ABCC4 transporter ( Supplementary Fig. 2), with a high degree of conservation in transmembrane (TM) domains and nucleotide binding domains (NBD) 18 critical for normal ABC transporter function ( Fig. 2b; Supplementary Fig. 2). Sequencing of abcc4 from lkt mutants identified a threonine to methionine substitution at position 804 (T804M) (Fig. 2b, c). This threonine localizes to the third cytoplasmic loop of the lkt polypeptide and is evolutionarily conserved from Drosophila to human (Fig. 2b, d). We next performed abcc4 knockdown and rescue experiments to verify that abcc4 was indeed the gene affected in lkt mutants. Injection of antisense abcc4 morpholinos (abcc4-MO) caused ciliogenesis defects and phenocopied lkt mutants ( Supplementary Fig. 3a-h). abcc4 mRNA injection also rescued lkt mutant phenotypes ( Supplementary Fig. 4a-i). Together, these findings lead us to conclude that the lkt locus encodes ABCC4 and that lkt T804M is a loss of function allele.
Organ laterality in zebrafish is established by cilia-driven fluid flow in KV 19,20 . Since lkt/ abcc4 deficiency causes a loss of KV cilia (Fig. 1n, o, r, s; Supplementary Fig. 3c), we assessed the status of KV fluid flow in lkt/abcc4-deficient embryos. Fluorescent beads were injected into KV of lkt mutants and abcc4 morphants. While both wild-type and control embryos displayed strong directional flow in KV (Fig. 3h (Fig. 3n, q). In comparison, normally asymmetric expression of spaw was observed in uninjected wild-type embryos, as well as in embryos injected with control-MO (DFC ctrl-MO embryos), or embryos injected with abcc4-MO when yolk-DFC bridges have closed, restricting the morpholinos to the yolk cell (Yolk abcc4-MO ) (Fig. 3l, m, o, q). These findings indicate that Lkt/ABCC4 functions autonomously in DFC/KV cells to regulate ciliogenesis and organ laterality.

Lkt/ABCC4 transporter regulates ciliogenesis through PGE2 secretion
Mammalian ABCC4 has been shown to transport several physiological substrates such as prostaglandins, cyclic nucleotides, steroid conjugates and folates 15 . Among related ABCC subfamily members, only ABCC4 is known to directly transport PGE 2 22 . We hypothesized that defective ciliogenesis in lkt/abcc4 mutants could be caused by diminished PGE 2 export resulting in PGE 2 signaling defects. To test this idea, we assessed whether addition of exogenous PGE 2 could rescue cilia-associated phenotypes in lkt mutants. Mutant embryos were incubated in media supplemented with PGE 2 or PGF2α. lkt mutants treated with PGE 2 exhibited straight body axis, normal heart looping and otolith biogenesis (Fig. 4a, b; Supplementary Fig. 5a), while all mutants treated with PGF2α showed cilia-associated phenotypes ( Supplementary Fig. 5a). We then assessed whether PGE 2 treatment could reverse defective ciliogenesis in lkt mutants. In PGE 2 -treated lkt mutants, reduction of KV cilia number and length was partially reversed (Fig. 4c, d, i, j); restoration of kinocilia or spinal canal cilia was also evident ( Fig. 4e-h). Together, these findings are consistent with the idea that PGE 2 deficiency is responsible, at least in part, for ciliogenesis defects in lkt mutants.
To assess whether Lkt/ABCC4 exports PGE 2 , we performed PGE 2 efflux assays using abcc4-deficient murine embryonic fibroblasts (MEF abcc4−/− ) 23 expressing human ABCC4, zebrafish Lkt/ABCC4 or mutant Lkt/ABCC4 T804M . Transfection of human or zebrafish ABCC4 increased extracellular PGE 2 and reduced intracellular PGE 2 , relative to vehicletransfected cells (Fig. 4k). In contrast, cells expressing the Lkt/ABCC4 T804M mutant were defective in PGE 2 export (Fig. 4k). Addition of MK571, an inhibitor of ABCC4 transporter 15,22 , blocked ABCC4-mediated PGE 2 export, mimicking the effect of the Lkt/ ABCC4 T804M mutation (Fig. 4k). These data reveal the molecular bases of Lkt/ABCC4 function, linking PGE 2 export to ciliogenesis. To determine whether zfABCC4 directly exports PGE 2 across the plasma membrane, we performed vesicular transport assays using inside-out membrane vesicles 22,24 . The ATP-dependent PGE 2 uptake levels were 3-fold greater in vesicles expressing ABCC4 than vesicles derived from untransfected controls (Fig. 4l). We next assessed the sub-cellular localization of mammalian ABCC4 and zebrafish Lkt/ABCC4. Although ABCC4 was reported to localize to the plasma cell membrane, its sub-cellular localization in ciliated cells was not examined 15 . We observed that murine ABCC4 localizes to the plasma cell membrane but not to the cilium in inner medullary collecting duct 3 (IMCD3), ciliated cells ( Fig. 4m-o). We next transfected IMCD3 cells with constructs encoding zebrafish lkt/abcc4 or lkt/abcc4 T804M mutant gene fused with EGFP. Similar to its mammalian counterpart, zebrafish Lkt/ABCC4 protein (EGFP-zfABCC4) was detected on the plasma cell membrane but not in the cilium (Fig. 4pr). In contrast, Lkt/ABCC4 T804M mutant protein (EGFP-zfABCC4 T804M ) was mis-localized in the cytoplasm in ciliated cells ( Fig. 4s-u). Collectively, these findings demonstrate that zebrafish Lkt/ABCC4 has a strong activity in exporting PGE 2 , whereas the T804M mutation, likely due to its loss of normal membrane localization, is incapable of PGE 2 export.

COX-EP4 signaling regulates Lkt/ABCC4-mediated ciliogenesis
PGE 2 is synthesized by COX-1 and COX-2, and after secretion from the cell, it binds and signals through G-protein-coupled receptors EP1-4 1, 3 . Knowing that cox1, cox2 and ep4 are expressed during zebrafish development [25][26][27][28] , where cox1 is expressed in the gastrula and cox2 is detected in the anterior neuroectoderm 25 , we assessed whether embryos deficient in PGE 2 signaling exhibit ciliogenesis defects. Microinjection of cox1-MO 25, 27 into wild-type embryos at low doses caused cilia-associated phenotypes, including hydrocephalus, randomized heart looping, abnormal otolith number and curved body axis ( Fig. 5a-c, g-h; Supplementary Fig. 5h, i), whereas cox2-MO 25,27 injection resulted in hydrocephalus, abnormal otoliths and curved body axis, but did not affect heart looping ( In contrast to that, administration of Cox2 inhibitor NS398 only caused hydrocephalus and three otoliths but not randomized heart laterality and KV cilia reduction ( Fig. 6a-d; h, i), thus phenocopying cox-2 morphants. Moreover, Indomethacin treatment in lkt mutants exacerbated ciliogenesis defects in KV and ears ( Fig. 6j-q). These findings complement our antisense knockdown studies and refine our understanding of time frames when PGE 2 singling functions in ciliogenesis.

Lkt/ABCC4 function in ciliogenesis is conserved in mammalian cells
To assess roles of Lkt/ABCC4-mediated PGE 2 signaling in ciliogenesis in mammalian cells, we depleted ABCC4 or EP4 in human retinal pigment epithelial 1 (hRPE1) cells using shortinterfering RNA (siRNA) 32 . Immunoblotting confirmed that both ABCC4 and EP4 protein levels were significantly reduced in cells transfected with ABCC4-siRNA or EP4-siRNA ( Fig. 7e, f). We observed that depletion of ABCC4 or EP4 in hRPE1 cells caused a reduction of ciliaformation and elongation when compared with use of the control siRNA ( Fig. 7a, b, c). Bothcilia length and percentages of ciliated cells were significantly reduced in ABCC4 or EP4 depleted cells ( Fig. 7g. h). Addition of exogenous PGE 2 increased both cilia length and percentages of ciliated cells in control cells but not in EP4-depleted cells (Fig. 7a Fig. 8a-h). We next assessed the sub-cellular localization of human EP4. EP4 staining in the plasma cell membrane has been described previously 33 . We observed that EP4 localizes to the cilium and is enriched in the perciliary cell membranę in hRPE1 cells (Fig. 7i-k). Similar ciliary localization of EP4 was also seen in IMCD3 cells (Fig. 7l). Consistent with the plasma membrane localization of murine and zebrafish Lkt/ ABCC4 transporters (Fig. 4m-r), human ABCC4 also localizes to the plasma membrane but not to the cilium (Fig. 7m, n), whereas EP4 is detected on the cilum in the same hRPE cell (Fig. 7m, n). Collectively, these findings suggest that PGE 2 released by ABCC4 binds and signals through the EP4 receptor in the cilium.

PGE 2 signaling affects anterograde velocity of IFT during ciliogenesis
cAMP signaling is known to promote ciliogenesis by increasing anterograde IFT 29 . Given that PGE 2 acts upstream of cAMP signaling, we assessed the possibility that Lkt/ABCC4mediated PGE 2 signaling regulates ciliogenesis via the control of IFT. To test this idea, we imaged the movement of IFT particles in IMCD3 cells stably expressing IFT88-EYFP using time-lapse microscopy 34 . As expected, we observed that IFT particles are transported along axonemal microtubules from the ciliary base to the tip of the cilium (Fig. 8a). To determine velocities of individual IFT88 particles, the time-lapse image sequences were converted into kymographs ( Fig. 8b-d). We observed that similar to FSK treatment 29 , PGE 2 increased anterograde velocities of IFT88 particles but had no obvious effects on retrograde velocities ( Fig. 8g; Supplementary Fig. 7). Moreover, PGE 2 -treated cells displayed more intense fluorescence at the cilium tip revealing accumulation of IFT88 particles (Fig. 8e, f). Furthermore, we found that PGE 2 treatment partially rescues cilia defect in oval/ift88 mutants (Fig. 8h, i, l, n). A rescue is not observed however for elipsa/ift54 at this stage ( Fig.  8j, k, m, n). This is most likely due to the fact that elipsa/ift54 phenotype is more severe, compared to that seen in oval/ift88 7,35 . These observations are consistent with the increased IFT velocity and efficiency in PGE 2 -treated cells. Hence, PGE 2 signaling increases the anterograde speed of IFT, thereby accelerating the movement of cargos into the axoneme and resulting in enhanced ciliogenesis.

DISCUSSION
In this study, we demonstrate that normal cilia formation and elongation require the COX-Lkt/ABCC4-EP4 signaling cascade. While ABCC4 has the ability to transport a variety of physiological metabolites 15 , the current study reveals a crucial and unexpected role for Lkt/ ABCC4-mediated PGE 2 export in ciliogenesis. cAMP-dependent kinase signaling is known to increase anterograde IFT during ciliogenesis 29 . Our data suggest that Lkt/ABCC4mediated PGE 2 signaling affects cAMP level and promotes ciliogenesis via an increase in the anterograde velocity of IFT. These results reveal a conserved ciliogenic cascade. A model is proposed where PGE 2 is exported from cells via Lkt/ABCC4 on the plasma cell membrane and signals through the EP4 receptor on the cilium or at the base of the cilium, thereby activating Gs and cAMP signaling to promote the anterograde IFT (Fig. 8o). In support of this model, we found that PGE 2 treatment causes an increase of intracellular cAMP but not Ca 2+ during ciliogenesis in IMCD3 cells ( Supplementary Fig. 8i, j), which is consistent with PGE 2 studies in other cells and tissues 36,37 . Furthermore, adenylate cyclase is detected in the cilium 38 . Cilia formation requires coordinated regulation of the bidirectional IFT particle traffic, including its frequency and speed 39,40 . The dynamic modulation of IFT velocity by COX-Lkt/ABCC4-EP4 signaling represents a key regulatory step in axoneme formation and elongation. PGE 2 is derived from the prostaglandin precursor, PGH 2 , which is biosynthesized by COX1 and COX2 enzymes from arachidonic acid, a 20-carbon polyunsaturated fatty acid 1,41 . We propose that after being exported from cells, PGE 2 acts in an autocrine and/or paracrine manner, as it is known that cells can respond to PGE 2 released by either themselves or by their neighbors 42 .
In human cancer cells, interaction of PGE 2 with EP4 receptor induces Wnt/β-catenin signaling resulting in COX2 expression, thereby setting up a positive feedback loop leading to further PGE 2 synthesis 42 . Such positive feedback loop in PGE 2 autocrine signaling mechanism may enable cells in the epithelial field of the KV or the otic vesicle to rapidly upregulate ciliogenesis 43 . This effect may be further enhanced by paracrine PGE 2 signaling between neighboring cells. The interplay between PGE 2 autocrine and paracrine signaling may create a community effect, whereby all cells in the epithelial field are coherently activated to generate cilia and ciliary motility. Although PGE 2 is implicated in influencing the ciliary beat frequency of cultured nasal mucosa cells 44 , the role of PGE 2 signaling in cilia formation was not reported in previous murine genetic studies [45][46][47] . This is most likely due to the presence of maternal contribution of prostaglandins in the placenta, which allows PGE 2 -deficient mouse embryos to develop normally 48 . The use of externally developing zebrafish embryos allowed us to reveal the ciliogenenic roles of PGE 2 signaling. In agreement with animal model studies, cultured mammalian cells deficient in PGE 2 signaling also display defective ciliogenesis. Given the role of cilia dysfunction in human disorders 49,50 , our findings suggest the existence of a previously unexplored aspect of human ciliopathies arising from abnormal prostagladin signaling.

Zebrafish maintenance
Zebrafish (Danio rerio, AB line) care and breeding were described previously 1 . Embryos were staged according to their morphology 51 . lkt mutants were isolated from a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis (Boston screen 2000) 52 . Fudan Animal Care Committee advises animal care and research.

Video microscopy analysis of KV fluid flow and cilia motility
Fluorescent beads (Polysciences, Inc.) were injected into KV and fluid flow was analyzed as previously described 19 . Details of videomicroscopy analysis of KV flow and cilia motility were described 19 . Flow was imaged using a Zeiss Axio Imager M1 microscope with a 63x Plan Apochromat water-dipping objective. Movies were generated using a Zeiss Axiocam high speed monochromatic digital camera and Axiovision (Zeiss) and Quicktime (Apple) software.

PGE2 cell export assay
Extracellular and intracellular PGE 2 were extracted and measured using PGE 2 EIA kit (Enzo Life sciences). MEF abcc4−/− Cells transfected with pAcGFP-huABCC4, pAcGFP-zfABCC4 or pAcGFP-zfABCC T804M were seeded into six-well plates at 2×10 5 cells/well in 1 ml of complete medium. 24hr after the transfection 1ml fresh DMEM 10% FBS media was fed to the transiently transfected cells. Supernatant was collected and processed for measurement of extracellular PGE 2 levels after 12h incubation. Purification of extracellular PGE 2 was accomplished using C-18 mini columns. Cell monolayer was rinsed twice with ice-cold PBS and cells were collected by scraping into 1 ml of ice-cold PBS. Cells were pelleted and stored at 80°C before the purification of intracellular PGE 2 . Frozen cell pellets were taken up in 1 ml of homogenization buffer and then sonicated briefly three times in 3-second bursts. Duplicate sets of samples were saved for determination of cellular protein concentrations using western analysis. An aliquot of each sample was spiked with PGE 2 (20 pg) before purification for monitoring the recovery rate. After the addition of 1 ml of acetone, samples were vortexed, allowed to stand for 20 min and centrifuged at 25°C for 10 min; the acetone was then evaporated under a gentle stream of nitrogen. The pH of samples was adjusted to 4.0 using 1N HCl and the C-18 columns were activated before addition of sample with 10 ml of ethanol and 10 ml of water. After the sample addition, columns were washed with 10 ml of 15% ethanol followed by 10 ml of hexanes and PGE 2 was eluted with 10 ml of ethyl acetate. Samples were subsequently dried under a gentle stream of nitrogen for the ELISA PGE 2 determinations.

PGE2 vesicular transport assay
Membrane vesicles were prepared from both the SV40-transformed HEK293T cells and those cells transiently expressing pAcGFP-zfABCC4. pAcGFP-zfABCC4 was transfected into HEK293T cells using PolyJet (FroggaBio) after 48 h, inside-out membrane vesicles were prepared and zfAbcc4 levels in the membrane vesicles were confirmed by immunoblotting using Living Colors® EGFP(Clonetech). ATP-dependent uptake of 3 Hlabeled prostaglandin E 2 (PGE 2 ) (180 Ci/mmol) by the membrane vesicles was measured using a rapid filtration technique. Briefly, 30 μl reaction mixtures containing 5 μM [ 3 H]PGE 2 (100 nCi), 4 mM AMP or ATP, 10 mM MgCl 2 , and membrane vesicle protein (10 μg) were incubated at 37°C for 10 min. Uptake was stopped by rapid dilution of the reaction mixtures in ice-cold buffer and vesicles were immediately collected by filtration through a Unifilter-96 GF/B plate (Perkin Elmer) using a Packard Filtermate harvester. Tritium associated with the vesicles was counted using a Top Count NXT microplate scintillation counter (Perkin Elmer). Uptake in the presence of AMP was subtracted from uptake in the absence of ATP to determine ATP-dependent uptake values. Experiments were carried out in duplicate and repeated twice.

Live cell imaging of intraflagellar transport
M368-4 cells (clonal line of IMCD3 cells stably expressing IFT88-EYFP, a gift from Jagesh V. Shah, Harvard Medical School, Boston, USA; ref. 34) were cultured and treated with 10 μM PGE2 (Sigma) for 12 hours and DMSO, respectively. Cells were plated on Poly-L-Lysine-coated glass coverslips and were cultured at 37°C, 5% CO 2 in DMEM medium containing 10% FBS until reaching 100% confluency. Cells were examined for IFT88 movement at 37°C on an OLYMPUS ZX81 inverted microscope system equipped with a spinning disk confocal head (ANDOR) using a 150 x oil immersion objective (1.45 NA). Time-lapse images (200 frames) were captured continuously with exposure 150 ms and 7 frames per second. Time-lapse image sequences were assembled into kymographs and the velocities of IFT88-EYFP particles was calculated using the ImageJ software package (http://rsbweb.nih.gov/ij) as described (ref. 29,34).

Statistics Methods
For comparing two independent groups on a given variable, independent sample t-tests are justified when the dependent variable is normally distributed. When the normality assumption is violated, non-parametric Mann-Whitney U Tests are justified to be appropriate. For determining independent association between two categorical variables, non-parametric Chi-square tests are justified. For Student t-test, the minimal sample size for embryo was determined to be 10 and the minimal sample size for cell was determined to be 50. This sample size is able to detect a standardized effect size 0.2 and to reject the null hypothesis that the two population means are equal with probability (power) 0.85. For continuity-corrected chi-square test, the minimal sample size for embryo was determined to be 20. This gives power 0.80 to detect true difference 0.4 with default Type I error probability 0.05. Each subject (embryo, cell or image field) was randomly chosen and allocated to their respective group, and we were not blinded to the group allocation.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.