# Sialoglycoproteins and sialyltransferases: Key regulators of blastocyst formation

Pélagie Douchez, Ingrid Fliniaux, Yoshiko Takeda-Uchimura, Matthieu Marin, Alain Martoriati, Anne Harduin-Lepers, Katia Cailliau

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## To cite this version:

Pélagie Douchez, Ingrid Fliniaux, Yoshiko Takeda-Uchimura, Matthieu Marin, Alain Martoriati, et al.. Sialoglycoproteins and sialyltransferases: Key regulators of blastocyst formation. Cells & Development, 2025, 10.1016/j.cdev.2025.204057. hal-05445943

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Submitted on 7 Jan 2026

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# Sialoglycoproteins and sialyltransferases: Key regulators of blastocyst formation

Pélagie Douchez<sup>a</sup>, Ingrid Fliniaux<sup>a</sup>, Yoshiko Takeda-Uchimura<sup>a</sup>, Matthieu Marin<sup>a</sup>, Alain Martoriati<sup>a</sup>, Anne Harduin-Lepers<sup>a</sup>, Katia Cailliau<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Sialic acid
Sialyltransferases
Glycoproteins
Blastulation
Implantation

## ABSTRACT

Following fertilization, there is an initial period of rapid cell division that leads to the formation of a multi-cellular structure known as the blastula, or blastocyst. Within this structure, sialic acids play a key role in influencing cellular processes such as signaling, cell-to-cell contact, and adhesion. In species that develop internally, the blastocyst undergoes implantation and placentation, which depend on maternal immunomodulation facilitated by sialylated proteins and enzymes involved in the biosynthesis of sialic acids. Although research has shown that the elimination of certain initial enzymes in the sialic acid synthetic pathway can lead to reduced embryonic viability, the precise role of these enzymes remains to be further investigated, particularly in the blastula of externally developing species, which have received limited attention. Recently developed blastoid models present promising prospects for future research in this field.

## 1. Introduction

Cell surfaces are coated by glycoconjugates with predominant terminal sialic acids (Sia) residues (Cohen and Varki, 2010; Sato and Kitajima, 2021; Varki, 2017). These glycoconjugates are highly complex, containing various core structures and terminals, and are processed by multiple enzymes. This structural diversity enables several cellular functions during early embryonic development. Different sets of structures, displayed on proteins with N- and O-linkages, are present in the zygote after fertilization and during the rapid phase of cell division, termed cleavage, which are addressed in this review.

Cleavage involves not only rapid cell divisions but also cell-to-cell interactions, and regulation of signaling and adhesion effectors that can potentially be modulated by sialic acid (Sia). Successful formation of multicellular structures, essential for the zygote to reach the blastula stage and initiate transcription, requires several sialoglycoconjugates and sialyltransferases, along with their fine-tuned regulation. Additionally, the use of embryonic stem cells derived from the blastocyst

inner cell mass and pluripotent induced stem cells have contributed to elucidate the functions of Sia regulatory enzymes and Sia glycoproteins.

Mammalian embryos that develop internally differ significantly from externally developing embryos, such as fish and amphibians, which are surrounded by large fertilization membranes containing Sia, or sea urchins that possess Sia polymers in their jelly coat, and chicken embryos that are protected by shells. In mammals, pregnancy begins with the formation of a blastocyst, which consists of an embryonic trophoblast containing an inner cell mass that subsequently implants into the uterine endometrium. At the trophoblast stage, successful implantation requires a transition from mesenchymal to epithelial cells (MET), accompanied by the regulation of sialylation (Zhang et al., 2024). Sia and polymers of Sia (PSA) are detected in the cytotrophoblast and the placenta formed from the blastocyst trophectoderm. Sia are involved in endometrial receptivity and implantation, trophoblast invasion, placentation, and maternal immunotolerance that requires T, NK, and mast cells, as well as Siglec receptor for Sia. The maternal aspects will be addressed from the blastula point of view. Furthermore, defects identified through knockout

<footnote><sup>a</sup> Univ. Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000, Lille, France</footnote>

<footnote>*Abbreviations:* B4Galnt2, β1,4-N-acetylgalactosaminyltransferase 2; CMAS or CSS, CMP-sialic acid synthetase; CMP, cytidine monophosphate; dpf, days post-fertilization; EMT, epithelial to mesenchymal transition; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglycosamine; GNE, glucosamine UDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase; hpf, hours post-fertilization; MET, mesenchymal to epithelial transition; Neu, neuraminic acid; Kdn, 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PSA, polysialic acids; Sia, sialic acid; sLe<sup>x</sup>, sialyl-Lewis x; sLe<sup>a</sup>, sialyl-Lewis a.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: katia.maggio@univ-lille.fr (K. Cailliau).</footnote>
studies and *in vitro* experiments have started to clarify essential and
dispensable sialylated proteins and enzymes involved in the biosynthesis
pathway of sialic acids (Lewis et al., 2022). However, externally
developing embryos at the blastula stage have received limited attention
before gastrulation. Many sialylated effectors and sialyltransferases still
have potential uncovered roles.

## 2. Sialylated glycoconjugates diversity in early dividing embryos

Sia derives from either Neu5Ac (*N*-Acetylneuraminic acid) or Kdn (2-keto-3-deoxynononic acid), with Neu5Ac and Neu5Gc (5-*N*-glycosylneuraminic acid) as the most abundant members. Sia are present in several phyla with internal and external early development, in Hemichordata, Cephalochorda, Vertebrata, Platyhelminthes, Cephalopoda, Crustacea, but also in viruses, bacteria, protozoa, and fungi (Ghosh, 2020). Sialylation occurs in *Drosophila* embryos (Aoki et al., 2007; Frappaolo et al., 2017), but no evidences are detected in other insects despite the detection of sialyltransferase homologs in their genome (Kajiura et al., 2015; Koles et al., 2004; Paschinger and Wilson, 2019). In contrast, C. elegans lacks Sia (Bacic et al., 1990). Notably, Neu5Gc is not present in humans and monotremes (also in platypus) and is only rarely present in Sauropsids (birds and reptiles) (Altman and Gagneux, 2019; Chen and Varki, 2010). Deaminated neuraminic acid, or Kdn, is abundantly present in lower vertebrates and bacteria (Ghosh, 2020).

Multiple combinations of Sia structures, substitutions, degree of polymerization (DP), linkages, and underlying glycans allow a high diversity (Ertunc et al., 2022; Schauer and Kamerling, 2018; Lewis et al., 2022). Sia possible substituents on the carbohydrate backbone generate around 50 different derivatives, with *O*-acetylation frequently occurring at C-9 (Angata and Varki, 2002). The glycosidic *α*-linkage attaching Sia moieties to other glycan units varies: either *α*2,3- and *α*2,6-linkages to galactose (Gal)-containing glycans or *α*2,6-linkage to *N*-acetylgalactosamine (GalNAc) and *N*-acetylglucosamine (GlcNAc) residues. In addition, *α*2,8-linkages form chains with different degrees of polymerization (Sato and Kitajima, 2013; Troy, 1992).

Derived from glucose metabolism, a multistep biosynthesis pathway,
occurring in several cellular compartments, forms sialylated proteins. As
a first compartment, the catalysis of Sia occurs in the cytoplasm with a
conversion by glucosamine UDP-GlcNAc-2-epimerase/*N*-ace-
tylmannosamine kinase (encoded by the *GNE* gene) of UDP-GlcNAc
(uridine diphosphate *N*-acetylglucosamine) to ManNAc-6-P (*N*-acetyl-
mannosamine 6-phosphate) and UDP (uridine diphosphate). ManNAc-6-
P is afterward condensed with phosphoenolpyruvate (PEP) by Neu5Ac-
9-P synthase (NANS) to form *N*-acetylneuraminate 9-phosphate
(Neu5Ac-9-P), with further dephosphorylation by *N*-acetylneur-
amine-9-phosphatase (NANP) to generate Neu5Ac. In a second
compartment, the nucleus, the sugar-nucleotide cytidine mono-
phosphate sialic acid (CMP-sialic acid) is catalyzed by the cytidine
monophosphate *N*-acetylneuraminic acid synthetase (CMAS). After the
transformation of Neu5Ac by hydroxylation of a methyl group in the *N*-
acetyl of activated CMP-Neu5Ac by cytidine monophosphate-*N*-ace-
tylneuraminic acid hydroxylase (CMAH), Neu5Gc is formed (Shaw and
Schauer, 1988). The CMAH gene is nonfunctional in humans (Altman
and Gagneux, 2019; Peri et al., 2018; Simakov et al., 2015). Sialylation
*per se* only occurs within a third compartment, the Golgi apparatus,
where activated sugar-nucleotide CMP-Sialic acid is transported by the
CMP-Sialic Acid Transporter (CMP-SAT) (Lepers et al., 1989; Lepers
et al., 1990; Petit et al., 2010). The *α*2,3-, *α*2,6-, or *α*2,8-sialyl-
transferases from four families of sialyltransferases, ST6Gal, ST6Gal-
Nac, ST3Gal, and ST8Sia (glycosyltransferase family 29 (GT29)) form
glycoside linkages (Harduin-Lepers et al., 2005; Harduin-Lepers et al.,
2008; Lombard et al., 2014; Teppa et al., 2016). Additionally, other
modifications occur in the Golgi apparatus. *O*-acetylation is performed
by the sialate *O*-acetyltransferase (SOAT) of CASD1 (Baumann et al.,
2015). Sialyl Lewis *x* (sLe*) and sialyl-Lewis *a* (sLeª) are generated by

fucosyltransferases (FUT) following the α2,3-linkage catalyzed by ST3Gal. In sLeª, the fucose group is linked to *N*-acetylglucosamine by an α1,3-linkage instead of an α1,4-linkage for sLeª, while the galactose is linked to *N*-acetylglucosamine *via* a β1,4-linkage for sLeª and a β1,3-linkage in sLeª. sLeª is synthesized in human mainly by fucosyltransferases FUT6 and FUT7 (CAzy GT10), to a lesser extent by FUT5 and FUT4, and FUT9 acts during the course of the development. sLeª is generated by FUT3 and to a lesser extent by FUT5 (Mondal et al., 2018). The Sda antigen is a sialylated trisaccharide (GalNAcβ1–4[Neu5Acα2–3]Galβ1) formed by the addition of an *N*-acetylgalactosamine residue through a β1,4-linkage to a sub-terminal galactose residue, which is substituted with an α2,3-linked sialic acid residue, by the β1,4-*N*-acetylgalactosaminyltransferase 2 (B4Galnt2) (Montiel et al., 2003; Dal’Olio et al., 2014; Groux-Degroote et al., 2021). Both Sda and sLeª have a common biosynthetic pathway that involves fucosyltransferases and B4Galnt2. Therefore, variations in the expression of B4GALNT2 can also impact the expression of sLeª (Groux-Degroote et al., 2021). Finally, a fourth lysosomal compartment acts when Sia are cleaved by neuraminidases/sialidases and released to be recycled (Henrissat and Bairout, 1996; Monti et al., 2010).

Fig. 1 illustrates the biosynthetic pathway of sialylation. It highlights the various enzymes involved in the biosynthesis of sialic acid, including sialyltransferases and other enzymes that contribute to the formation of specific patterns essential for the development of the blastula in both externally and internally developing species, as well as in embryonic stem cell models. The figure also emphasizes the major sialylated proteins and their functional contributions to the process of blastulation that will be discussed in the review.

## 3. Blastula and blastocyst formation

Following fertilization, the cleavage period begins, characterized by
the active division of blastomeres without overall growth of the embryo.
Cells composing the embryo, named blastomeres, assemble into a
compact mass called the morula, and cleavage culminates in the for-
mation of a blastula (or a blastocyst in mammals) characterized by the
appearance of an inner cavity called blastocoel (Fig. 2). At this stage, the
blastomere fate is prepared for gastrulation.

### 3.1. *External developing species*

Developing embryos that usually contain yolk are surrounded by
protective membranes, including a vitelline or pellucid membrane,
sometimes turning into a fertilization membrane upon fertilization, and
a jelly coat in some aquatic species. Enzymes from the Sia biosynthesis
pathway and Sia glycoproteins are present in some of those membranes
during the cleavage period; however, their specific roles remain
ambiguous and are subject to species specificity.

In marine species, such as *sea urchins*, embryos undergo a radial
holoblastic cleavage characterized by an arrangement of each upper
blastomere directly over those of the next lower ones. The sea urchin
blastula lines up along the animal-vegetal *axis* established in the egg
cytoplasm before fertilization. Tight junctions connect blastomeres
assembled into a seamless epithelial sheet that encircles the blastocoel.
Blastomeres adhere to an external layer of hyaline and are submitted to a
water influx that expands the blastocoel. Sea urchin eggs' plasma
membrane and their large surrounding protective jelly coat are char-
acterized by a high content of PSA (Kitazume et al., 1994; Kitazume
et al., 1996). A *poly α2,8 sialosylsialyltransferase (ST8Sia)* is highly
expressed in the blastula, with a level increasing until the onset of
gastrulation (Cho et al., 1996; Harduin-Lepers et al., 2008). In egg, jelly
glycoconjugates with α2,9-linked PSA chains identified as (5-*O*-glycolyl-
Neu5Gcα2)ₙ contain n ranging from 4 to over 40 Neu5Gc residues, and
Neu5Gc9HSO₃, a nonreducing terminal residue of oligo/poly(5-*O*-gly-
colyl-Neu5Gcα2)ₙ group is present (Kitazume et al., 1994; Kitazume
et al., 1996; Kitazume-Kawaguchi, 1998). PSA linear homopolymers are
negatively charged and restrict homophilic and heterophilic interactions due to charge repulsion and intercellular steric hindrance. PSA also absorbs considerable amounts of water, leading to an increase in size and volume and playing a protective role (Varki and Gagneux, 2012).

In freshwater vertebrate species, particularly in *Xenopus*, the dorsal-ventral axis is set up upon fertilization with a parallel network of microtubules, dynein, and kinesin, allowing a massive translocation of the cytoplasm of 30 degrees in the vegetative cortex for the future dorsal pole, a phenomenon called the “cortical rotation”. Then, a radial holoblastic cleavage places two organizing centers (Nieuwkoop and Speemann) to define intercellular signaling and axis later at the onset of gastrulation. In the *Xenopus* blastula, half of the acidic glycans are sialylated N-glycans. A decrease in *LacdiNAc*-containing sialylated glycans starts at the cleavage stage and continues through later stages (Qu et al.,

2020).

In *zebrafish*, a dorsalizing activity is translocated to the future dorsal side at fertilization before the discoidal meroblastic cleavage takes place in a thin yolk-free region of the cytoplasm at the animal cap, and only divides a portion of the egg. At mid-blastula, three distinct cell populations are formed: 1/ the yolk syncytial layer, located at the vegetal margin of the blastoderm, fused with the underlying yolk cell essential for gastrulation; 2/the enveloping layer, made up of the outermost cells of the blastoderm, forming a single-cell layer that acts as a protective extraembryonic envelope. 3/between these two layers are the deep cells that give rise to the embryo. Zebrafish development exhibits extensive and structurally diverse sialylation at all stages, starting from fertilized eggs to hatched embryos. Complex and oligomannose N-glycans feature a wide range of mono- and oligo-sialylated termini with

<figure><img src="image_3.png" /></figure>

D Plasma membrane

<table><tr><td>Mice (16)</td><td>α 2,6 sialylated PECAM</td><td>Antiapoptotic, cell adhesion</td></tr><tr><td>* Human</td><td>α 2,6 sialylated TNFR1</td><td>Potential protection against apoptosis</td></tr><tr><td>ESC (8,17)</td><td>α 2,3 sialylated CD44</td><td>Pluripotency</td></tr><tr><td>ESC human (9)</td><td>Sialomucin Podocalyxin</td><td>Blastocoe formation</td></tr><tr><td>* EMT Mice Human</td><td>α 2,6 sialylated Glycans</td><td>Facilitation of undifferentiated state</td></tr><tr><td></td><td>Sialylated integrin β4</td><td>Potential facilitation of pluripotency</td></tr></table>

(caption on next page)
**Fig. 1.** Enzymes involved in the biosynthesis pathway of sialic acid, sialic motifs, and sialylated proteins present during cleavage stages in the blastula and blastocyst formation, in embryonic stem cells (ESCs), and during the epithelial-to-mesenchymal transition (EMT) across various species. The production of sialylated proteins follows a multistep pathway derived from the glucose metabolism and occurs across three cellular compartments, ultimately leading to the placement of sialylated proteins in the plasma membrane. (A) In the cytoplasm, UDP-GlcNAc is converted to ManNAc-6-P by the enzyme glucosamine UDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase (GNE). This reaction is followed by the condensation of ManNAc-6-P with phosphoenolpyruvate (PEP) forming N-acetylneuraminate 9-phosphate (Neu5Ac-9-P), a process catalyzed by the N-acetylneuraminate 9-phosphate synthase (NANS). Neu5Ac-9-P is then dephosphorylated to produce Neu5Ac by the N-acetylneuraminate-9-phosphatase (NANP). (B) In the nucleus, cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) converts Neu5Ac into CMP-sialic acid. Sialylation occurs in the Golgi apparatus, where CMP-sialic acid is transported. Both GNE ((1) Schwarzkopf et al., 2002) and CMAS ((2) Abeln et al., 2019) knockdowns affect differently the mouse blastula development. (C) In the Golgi apparatus, several families of sialyltransferases (ST6Gal, ST6GalNAc, ST3Gal, and ST8Sia) are responsible for creating glycosidic linkages, α2,3, α2,6, α2,8, and α2,9. The sialyltransferases particularly relevant to blastulation include ST8Sia in sea urchin ((3) Kitazume et al., 1994; Kitazume et al., 1996), which is necessary for synthetizing the polysialic acid chains, sialyltransferases responsible for α2,3 and α2,6 linkages (ST3, ST6) in Xenopus (44) Hanzawa et al., 2017), zebrafish (5) Flanagan-Steet and Steet, 2013), and chicken (6) Nakano et al., 1994); α2,3 linkage (ST3) in mice (7) (Kimber et al., 1993) and ESC ((8) Walker et al., 2022); the ST6Gal1 in human ESC ((9) Hasehira et al., 2012; Saito et al., 2011; Wang et al., 2015); the expression of fucosyltransferases FUT3, FUT6 or ST6Gal1 during EMT ((10) Pucci et al., 2021). Additionally, the β1,4-N-acetylgalactosaminyltransferase 2 (B4Galnt2) in mice interacts with the Sda antigen (11) Li et al., 2011 and 2012). Further modifications lead to the generation of sialylated structures such as the sialyl-Lewis x (sLe^x) in zebrafish (5) Flanagan-Steet and Steet, 2013), mice (12) Zhang et al., 2009), and human (13) Carson, 2002; Ma et al., 2021; Margarit et al., 2009), as well as the sialyl lactotetra motif in mice and human ESC ((14) Barone et al., 2014 and 2020), and O-acetylation provided by sialate O-acetyltransferase (SOAT) in mice ((15) Varki et al., 1991). (D) Lastly, sialylated proteins reported to play a role in blastulation across different models include: α 2,6 sialylated PECAM (Platelet endothelial cell adhesion molecule) in mice ((16) Redick and Bautch, 1999; Robson et al., 2001); potential α 2,6 sialylated TNFR1 (Tumor necrosis factor receptor 1 in human); α2,3 sialylated CD44 (8) Walker et al., 2022) and sialomucin podocalyxin (17) Shahbazi et al., 2017; Nishihara, 2018; Kim et al., 2021) in ESC; α2,6 sialylated Glycans in human ESC ((9) Hasehira et al., 2012; Saito et al., 2011; Wang et al., 2015); and potential sialylated integrin β4 in EMT. An asterisk indicates potential action.

Neu5Ac/Neu5Gc mono-sLe^x. From midcleavage to the pharyngula stage, α2,6-sialyl LacNAc antenna structures increase, while α2,3-sialyl LacNAc antenna, core α1,6-Fuc, and bisecting GlcNAc are gradually modified (Flanagan-Steet and Steet, 2013). This increase in sialylated N-glycans with LacNAc antenna structures is accompanied by a decrease in LacdiNAc-containing sialylated glycans at midcleavage, as observed in Xenopus (Hanzawa et al., 2017). An unusually abundant oligosialylated O-glycosylated mucin-type is present in the pharyngula stage before 24 h post-fertilization (hpf) (Flanagan-Steet and Steet, 2013; Guérardel et al., 2006). The amount of oligoSia on glycoproteins gradually decreases throughout later stages (Chang et al., 2009). The yolk predominantly contains complex-type glycans with sLe^x (Hanzawa et al., 2017).

The chicken terrestrial vertebrate model undergoes a discoidal meroblastic cleavage. The developing embryo is surrounded by a superimposed shell above the protective membranes. The vitelline membrane, closest to the embryo, is rich in N-glycoproteins (Xiao et al., 2020) and exhibits the highest Sia content (Nakano et al., 1994), and no sialyltransferases α2,3 and α2,6 in the oviduct magnum portion and the egg white (Kojima et al., 2015). Recently, it was determined that the downregulation of the vitelline membrane tension and stiffness in the early blastoderm disk, necessary for the body axes formation, are linked to glycoprotein fibers (Kunz et al., 2023). The involvement of their Sia is raised.

3.2. Placental species

Mammalian embryo cleavage is rotational holoblastic, characterized
by a slow rate of division without synchrony and a unique cleavage
orientation. An initial cellular differentiation event occurs in the em-
bryos before preimplantation, when the morula transforms into a blas-
tocyst after the blastomeres undergo compaction, forming an inner mass
composed of pluripotent cells that give rise to embryonic and extra-
embryonic tissues. The outer cells develop into an epithelium, the tro-
phectoderm responsible for the formation of a fluid-filled cavity, the
blastocoel. Epithelial cells form a lumen at their apical pole, via polar-
ized transport of fluid and proteins containing negatively charged Sia, to
start the formation of the proamniotic cavity (Kim et al., 2021). They
repel the apical membranes and tight junctions prevent fluid from
escaping by establishing cell-cell contacts, ensuring the cavity's imper-
meability. By that time, the embryo also constructs one of its main body
polarities with the dorsal to ventral axis, with blastomeres facing the
maternal side being dorsal. Importantly, Sia motifs and Sia receptors
seem to have considerable significance in cell-cell and cell-matrix

adhesion molecules and antiapoptotic signaling to define and maintain
the properties of the blastocyst as follows.

In mice, sLe^x (sialyl SSEA1, sialyl CD15), known to play a pivotal role in cell adhesion, is present concomitantly with morula compaction during preimplantation at the 8-16 cell stage (Kimber et al., 1993).

In terms of cell adhesion, PECAM1/CD31, is expressed in the inner cell mass of mice blastocyst and on embryonic stem cells (Redick and Bautch, 1999; Robson et al., 2001), where it could establish heterophilic interactions with sialylated glycans. ST6Gal1 regulates PECAM anti-apoptotic signaling (Kitazume et al., 2010) and could protect the early embryo against death. α2,6 sialylated TNFR1 death receptor could prevent maternal apoptosis (Boeddeker and Hess, 2015) by blocking ligand-induced receptor internalization in mice inner mass, and human trophectoderm as shown in an in vitro model (Holdbrooks et al., 2018).

In mice, the selective destruction of 9-O-acetyl groups of Sia, achieved through the 9-O-acetyl-sialic acid specific esterase expression in fertilized eggs, arrests the embryonic development at the two-cell stage (Varki et al., 1991).

3.3. Embryonic stem cells, blastocoel and amniotic cavity

Embryonic stem cells (ESC) derived from the inner cell mass of
blastocysts, specifically from 3.5 to 4.5 days post-fertilization (dpf) in
mice, have contributed to understand cellular aspects of differentiation
and morphogenesis (Abeln et al., 2017). In mice blastocysts, epiblast
pluripotent cells undergo epithelialization and create an apical surface
in contact with a cavity (Bedzhov and Zernicka-Goetz, 2014). During the
peri-implantation stages, the embryo transforms from a hollow-shaped
blastocyst to an egg cylinder. The epiblast reorganizes from a simple
ball of cells to a cup-shaped epithelial monolayer enclosing the
proamniotic cavity. As a result, a lumen forms through charge repulsion
among the cell apical membranes coated by negatively charged sialo-
mucin podocalyxin, a CD34 family of transmembrane antiadhesins, at
around 5 to 5.25 dpf in mice (Kim et al., 2021). Notably, in mice or
human ESC with a knockout of antiadhesive podocalyxin, the lumeno-
genesis does not occur, and the proamniotic cavity is not formed
(Shahbazi et al., 2017; Nishihara, 2018). Interestingly, sialylated E-
cadherin, integrin, and catenin are also present in ESC (Melo-Braga
et al., 2014).

3.4. Cell pluripotency

Sialylation is important for stem cell pluripotency maintenance (Li
and Ding, 2019). In human, ESC, ST6Gal1 knockdown leads to
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Blastula from various species. The coeloblastula surrounds an inner fluid-filled blastocoel, whereas the stereoblastula shows no cavity. In sea urchin, the blastomeres build a monolayer of cells encasing a central blastocoel. The animal pole secretes enzymes to degrade the fertilization membrane, and the epithelial cells of the vegetal pole become thicker before a free-swimming primary mesenchyme blastula hatches (*Paracentrotus lividus* 6.5 h post-fertilization (hpf) until 13 hpf at 18 °C, (Formery et al., 2022). In *Xenopus*, the blastula roof is formed by pigmented micromeres which form an outer layer of epithelial cell type. The vegetal half consists of large macromeres containing endodermal cells, while a blastocoel occupies the animal half (*Xenopus laevis* 3.5 hpf or stage 6.5 until 7 hpf or stage 9 at 23 °C, (Nieuwkoop and Faber, 1994). In Zebrafish, following an incomplete undercut, the blastodisc appears oblong and lacks a blastocoel. The epiboly begins with the blastodisc becoming thinner and spreading over the yolk (*Danio rerio* from 2.25 hpf at 128 cells to 4.75 hpf with epiboly 30 % at 28,5 °C, (Kimmel et al., 1995). In chicken, the blastoderm disc, which consists of several layers, sits on top of the blastocoel, above a hypoblast layer and a subgerminal cavity. The peripheral disc cells form a yolk syncytial layer. This blastulation runs until 23 hpf (stage 7) at 38 °C (Hamburger and Hamilton, 1992). Notably, externally developing species are illustrated without protective surrounding membranes and shells. In Mice, the blastomeres in the morula undergo compaction, arranging into a compact cluster of cells before the process is followed at 3.5 days post-fertilization (dpf) by the genesis of an outer trophectoderm, an internal blastocoel, and an inner cell mass at one pole, which subsequently becomes the blastocyst (Dyban et al., 1991). In humans, embryos compaction starts at 4 dpf, with the blastocyst formation at 5 dpf (Gauster et al., 2022). In both mice and humans, the formed blastocysts subsequently implant into the inner lining of the female uterine wall (endometrium).</figcaption></figure>

hyposialylation and altered cellular pluripotency. In pluripotent stem cells (PSC), α2,6 sialylated glycans maintain an undifferentiated state by regulating the expression of oct4 and sox2 (Hasehira et al., 2012; Saito et al., 2011; Wang et al., 2015). In ESC and induced pluripotent stem cells, a **sialyl-lactotetra carbohydrate** sequence (S-Lc4; Neu5-Aα3Galβ3GlcNAcβ) is specifically expressed on a 250 Kda glycoprotein in addition to glycosphingolipids and decreases upon differentiation (Barone et al., 2014; Barone et al., 2020). Stem cell pluripotent properties are facilitated by α2,3 sialylated **O-glycans on CD44** that promote an interaction with hyaluronan (HA), a glycosaminoglycan component of the extracellular matrix (such as in breast cancer cells (Walker et al., 2022).

Interference with **9-O-acetylation of Sia** halts embryonic development at the two-cell stage (Varki et al., 1991). While the blocking of Sia de novo biosynthesis in GNE knockout mice proves lethal around midgestation (Schwarzkopf et al., 2002), the hyposialylation resulting from **loss of GNE** activity in mice ESC can be rescued through the Sia

lysosomal salvage pathway, indicating a potential role for sialylation in the regulation of early embryonic differentiation *in vitro* (Weidemann et al., 2010). To the contrary, mice ESCs knocked out for **CMAS** exit from pluripotency and differentiate without sialylation (Abeln et al., 2017). The discrepancy observed between GNE and CMAS experiments may reflect different effects of hypo- and asialylation, possibly mediated by various adhesive sialylated proteins (D'Addio et al., 2020).

### 3.5. Epithelial to mesenchymal and mesenchymal to epithelial transitions

Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) occur during several stages of early development, including the cleavage stage when the blastocyst forms, as well as during implantation and placentation. In the blastocyst, the trophectoderm cells, precursors of the cytotrophoblast, undergo MET, adopt an epithelial morphology necessary for efficient interaction with the uterine endometrium, and facilitate invasion for successful
implantation and the subsequent placenta formation (Kalluri and Weinberg, 2009). The invasion of the placental extravillous cytotrophoblast requires an EMT. Villous cytotrophoblast differentiates into extravillous cytotrophoblast and gains the capacity to migrate and invade the maternal endometrium.

During an EMT, epithelial cells lose their intercellular contacts (through the downregulation of E-cadherin), extend filopodia, and migrate (Hay, 2005). The process is reversible for a MET. During EMT in several mouse and human cell lines and models, in the first instance, sialylation is down-regulated and then upregulated by the time of differentiation (Du et al., 2015). The upregulation of ST6Gal1 and the expression of fucosyltransferases FUT3/6 (for the synthesis of sLeX) or FUT3 (for sLeA), favor EMT (Pucci et al., 2021). The expression of sialylated Integrin β4 (Du et al., 2015) and ST6Gal1 could possibly be promoted by a decrease in Sox2 (as in cancer cell models, Dorsett et al., 2019), considering Sox2 is necessary for EMT (Avilion et al., 2003; Goolam et al., 2016).

## 4. Placental species and embryo implantation

Pregnancy presents several maternal aspects, adhesion/attachment, implantation, and placentation, and an immunological paradox. They also include regulation of apoptosis, no rejection by the mother's immune system, from which the embryo is distinct, and a favorable environment through angiogenesis, vascular remodeling, and trophoblast invasion (De Kleer et al., 2014; Polese et al., 2014; Schauer, 2009). Sia modulates these phenomena to various degrees through sialylated glycoproteins, and Sia receptors Siglecs (Sialic acid-binding immunoglobulin-type lectin) and selectins (Li and Ding, 2019). Table 1 summarizes enzymes from the sialic acid biosynthesis pathway, sialic motifs, sialylated proteins, and sialic acid receptors found in the maternal environment of internally developing species necessary for the blastocyst's successful implantation.

### 4.1. Endometrial contact, attachment and implantation

Trophoblast adhesion to the uterine wall is essential for implantation. This process allows the embryo to attach to the uterine endometrial epithelium, penetrate the luminal epithelium, and invade the decidual/stromal cells to form the placenta.

The regulation of specific glycan structures influences the outcome of embryo implantation. sLeX, a primary ligand for L-selectin, reaches its peak levels during implantation in healthy women (Carson, 2002; Ma et al., 2021; Margarit et al., 2009). The upregulation of sLeX via the transfection of the fucosyltransferase FUT7 enhances embryo adhesion and implantation in both mice and in vitro (Zhang et al., 2009). Conversely, the downregulation of sLeX and sLeA in human patients experiencing miscarriage is associated with alterations of trophoblast adhesion and invasion (Ma et al., 2021). Additionally, the deregulation of sLeA, FUT1/3/4, and ST3GAL3/4 has been observed in the decidua of unexplained recurrent miscarriage patients (Ma et al., 2022). In mice, ST3Gal3, which is responsible for sLeX production, facilitates uterus endometrium receptivity, while its downregulation impairs it (Yu et al., 2018). Moreover, the interaction between sLeX and L-selectin is critical to mediate the adhesion of the human blastocyst to the endometrium. L-selectin is present on the maternal side in the endometrium (Genbacev et al., 2003; Lai et al., 2005), where it is up-regulated during receptivity (Genbacev et al., 2003) and altered in infertile women with unexplained infertility (Margarit et al., 2009). The interaction between sLeX on the human trophoblast/L-selectin mediates the adhesion of blastocyst to the endometrium in an in vitro implementation model (Liu et al., 2011). However, L-selectin is also present on the blastocyst (Fukuda and Sugihara, 2008; Yucha et al., 2014). Moreover, the interaction between sLeX mucins (MUC1 and MUC16/CA125) localized on the endometrial epithelial cells (Dharmaraj et al., 2014; Gipson et al., 2014) and the embryos' trophoblast L-selectin (Dharmaraj et al., 2014; Francis et al.,

<caption>**Table 1**</caption>

Enzymes from the sialic acid biosynthesis pathway (BioSynPath), sialic motifs (SiaMotif), sialylated proteins (Siaprotein), and sialic acid receptors (SiaR-ceptor) found in the maternal environment of internally developing species and crucial for the blastocyst adhesion, implantation, and inhibition the maternal immune response. E: equine; H: human; M: Mouse; P: pig. An asterisk indicates possible involvement. ST3Gal: ST3 beta-galactoside alpha-2,3-sialyltransferase; B4Galnt2: β1,4-N-acetylgalactosaminyltransferase 2; ST6GalNAc1: alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; ST6Gal1: beta-galactoside alpha-2,6 sialyltransferase 1.

<table><thead><tr><th></th><th>Species</th><th>Uterine environment</th><th>Function</th><th>References</th></tr></thead><tbody><tr><td>Implantation<br>BioSynPath</td><td>M</td><td>ST3Gal3</td><td>Endometrium receptivity</td><td>Yu et al., 2018</td></tr><tr><td>BioSynPath<br>+<br>SiaMotif</td><td>M</td><td>B4Galnt2 and Sda antigen</td><td>Implantation</td><td>Li et al., 2011 and 2012</td></tr><tr><td>SiaMotif</td><td>H, M</td><td>Sialyl-Lewis x</td><td>Morula compaction and epithelial-mesenchymal transition favored</td><td>Pucci et al., 2021; Draper et al., 2022</td></tr><tr><td rowspan="2">Siaprotein</td><td>H</td><td>Sialyl-Lewis x and sialyl-Lewis a</td><td>Adhesion to L-selectin and implantation</td><td>Zhang et al., 2009; Liu et al., 2011</td></tr><tr><td>H, M</td><td>Sialyl-Lewis x, mucin MUC1</td><td>Implantation</td><td>Dharmaraj et al., 2014; Gipson et al., 2014; Francis et al., 2021</td></tr><tr><td rowspan="3">SiaReceptor</td><td>E</td><td>Sialylated Mucin</td><td>Antiadhesive properties</td><td>Vichera et al., 2018</td></tr><tr><td>H, M</td><td>ST6GalNac1-regulated Sialyl-Tn antigen</td><td>Interaction with Siglec-6 and Siglec-G</td><td>Dong et al., 2024</td></tr><tr><td>p</td><td>ST6Gal1-regulated E-cadherin</td><td>Uterine lumen closure</td><td>Han et al., 2022</td></tr><tr><td>Placentation<br>SiaMotif</td><td>H</td><td>Polysialylation</td><td>Cytotrophoblast migration</td><td>Genbacev et al., 2003; Liu et al., 2011</td></tr><tr><td>Siaprotein</td><td>H</td><td>Sialylated Glycodelin-A</td><td>Limit trophoblast invasion through Siglec-6</td><td>Hromatka et al., 2013<br>Brinkman-Van der Linden et al., 2007; Lam et al., 2011</td></tr><tr><td rowspan="2">Immunomodulation<br>BioSynPath</td><td>H</td><td>Sialylated human chorionic gonadotropin</td><td>Favor hemochorial placentation</td><td>Cole, 2012; Ibeto et al., 2020</td></tr><tr><td>M</td><td>* Sialylated Lymphatic endothelial hyaluronan receptor-1 (LYVE1)</td><td>Vasculature development</td><td>Gu et al., 2006; Gordon et al., 2008; Nightingale et al., 2009</td></tr><tr><td rowspan="2">SiaMotif</td><td>M</td><td>CMP-sialic acid synthetase (CMAS)</td><td>Avoid maternal complement attack</td><td>Abeln et al., 2019</td></tr><tr><td>H</td><td>Sialyl-Lewis x</td><td>Avoid Natural Killer cells action</td><td>King and Loke, 1990; Yoshimura et al., 1996; Chen et al., 2016</td></tr><tr><td rowspan="2">Siaprotein</td><td>M</td><td>α2,6 Sialylation</td><td>B cell suppression through Siglec-2</td><td>Rizzuto et al., 2022</td></tr><tr><td>H</td><td>LacdiNac Glycodelin-A</td><td>Immune cell inhibition</td><td>Rachmilewitz et al., 2003;</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Species</th><th>Uterine environment</th><th>Function</th><th>References</th></tr></thead><tbody><tr><td>H, M</td><td>Mucin MUC16/ CA125</td><td>Natural Killer cells inhibition through Siglec-9</td><td>Poornima and Karande, 2007<br/>Tyler et al., 2012</td></tr></tbody></table>

2021; Margarit et al., 2010) modulates implantation by an increase of adherence and recognition/adhesion in human. Successful embryo apposition at the endometrial surface requires a balance between adhesion and antiadhesion events; a mucin-rich endometrium acts as a natural barrier against embryo attachment except at specific sites (Achache and Revel, 2006; Aplin and Aplin and Ruane, 2017; Meseguer et al., 2001). In transgenic mice, the loss of MUC1 at the implantation site correlated with embryo attachment, whereas in human, MUC1 expression is high during implantation (Dharmaraj et al., 2009; Meseguer et al., 2001). MUC1 in humans promotes recognition/adhesion and guidance for the embryo to the implantation site. MUC1 expression is altered in infertile endometrium (Margarit et al., 2010). The expression of another mucin, MUC16/CA125, modifies trophoblast adherence in humans (Gipson et al., 2008). The endometrial MUC16/CA125 transcript only increases during the receptive phase in fertile women, but not in those experiencing fertilization failure (Bhagwat et al., 2013). However, the regulation of its sialylation needs clarification. In equine embryos, a specific capsule composed of mucin-like glycoproteins produced by the trophectoderm contains a high quantity of Sia, which confers anti-adhesive properties essential for the intrauterine migration of the embryo before implantation (Vichera et al., 2018). The intricate interplay of Sia glycan structures, selectins, and mucins in the endometrium facilitates the process of embryo implantation. Understanding these molecular mechanisms is vital for unraveling the complexities of reproductive physiology and may have implications for addressing infertility and pregnancy complications.

ST6GalNac1-regulated sTn in the endometrium supports embryo attachment by interacting with trophoblastic Siglecs. The inhibition of endometrial ST6GalNac1/sTn pathway impairs embryo implantation in vivo. The adhesion molecule CD44 carries sTn in human endometrial cells and mouse endometrial epithelial organoids. In humans, Siglec-6 and Siglec-G, expressed in the embryonic trophectoderm, facilitate embryo attachment binding with sTn-modified CD44 (Dong et al., 2024).

In pigs, an increase in the activity of maternal ST6Gal1 governs the structure and functionality of membrane receptors. This activity influences collective luminal epithelial migration through α2,6-sialylation of E-cadherin, contributes to the extension of the endometrial fold, and enables closure of the uterine lumen (Han et al., 2022).

During mice pregnancy, B4Galnt2 contributes to normal embryo implantation but not to embryo development (Li et al., 2011). B4Galnt2 is expressed in blastocysts and interacts via Sda antigen on the endometrial surface to permit implantation. However, B4Galnt2 is also expressed in the uterine endometrium, suggesting a dual function depending on its localization (Li et al., 2012).

## 4.2. Placentation

After implantation, endometrial stromal cells differentiate into decidual cells, undergoing a mesenchymal-epithelial transition (MET) to support trophoblast invasion and placenta formation. Inadequate decidualization is a major factor implicated in implantation failure, spontaneous miscarriage, preeclampsia (incomplete transformation of spiral arteries), and infertility (Zhang et al., 2013).

Eutherian species exhibit distinct patterns of placentation. In humans, primates, and some rodents, hemochorial placental trophoblast

cells establish direct contact with maternal cells at the villous surface (syncytiotrophoblast) and just beneath within the maternal decidua and myometrium (cytotrophoblast). Some placental stem cells penetrate the anchoring villi and differentiate into invasive trophoblast, the extra-villous cytotrophoblast, which remodels arteries to support increased blood flow and reduced pressure compared to the maternal circulation. In contrast, other species experience no direct contact between maternal blood vessels and the embryo (Bornhöft et al., 2018).

In human placental trophoblast, the expression of PSA may enhance the migration and invasion of cytotrophoblast cells through steric hindrance. PSA promote cytotrophoblast migration in a villous chorion model and is often overexpressed in patients with gestational trophoblastic diseases. The removal of PSA reduces cytotrophoblast penetration and may serve as a stop signal for cytotrophoblast invasion (Hromatka et al., 2013).

Human sialylated Glycodelin-A (GdA), a dimeric lipocalin, is synthesized by the glandular epithelium of the endometrium, secreted into the amniotic fluid, and concentrated in the placenta. When the cytotrophoblast differentiates into villous and extravillous cytotrophoblast and invades the decidualized endometrium, the decidual GdA inhibits trophoblast invasion by the downregulation of proteinase expression and its interaction with the trophoblastic Siglec-6 (Brinkman-Van der Linden et al., 2007; Lam et al., 2011). Abnormally elevated levels of placental Siglec-6 are associated with trophoblast abnormalities such as modified downstream signaling capabilities and preeclampsia (Stefanski et al., 2022).

Human chorionic gonadotropin (hCG), which is highly enriched in Sia, is secreted by the syncytiotrophoblast and linked to key processes, including successful implantation into the uterus, formation of villous trophoblast, relaxation of smooth muscle that maintains quiescence during pregnancy, and facilitation of placental vasodilatation (Cole, 2012; Islami et al., 2003; Keay et al., 2004; Slattery et al., 2001). The glycosylation of hCG features complex-type N-glycans, with terminal Sia with or without fucose content (Ibeto et al., 2020; Kessler et al., 1979; Valmu et al., 2006), and core-1 O-glycan structures, with two Sia bound to each glycan (Kessler et al., 1979). A high number of sialylated glycoforms of hCG, along with alterations in sialyltransferases and sialidase, is implicated in several pregnancy diseases (Ibeto et al., 2020; Lee et al., 2011).

Another potential effector is LYVE1 (lymphatic endothelial hyaluronan receptor-1) observed in embryonic mouse blood vessels at 9.5 dpf (Gordon E.J., Gale N.W., Harvey N.L., 2008), and in human placenta (Gu et al., 2006). In endothelial cell lines, LYVE1 is functionally silenced by α2,3 or α2,6 Sia linkages on its O-glycans (Nightingale et al., 2009) and could act on cell adhesion and vasculature formation.

Canine trophoblasts, even when not in direct contact with maternal blood, also express Neu5Ac (Zanuzzi et al., 2024). However, the content of Sia in the placenta of wild and domestic animals has only been minimally studied.

## 4.3. Immunoregulation of maternal complement attack

The placenta nourishes the fetus and negotiates a peaceful coexistence between the mother and her histoincompatible embryo. Within the placenta, epithelial cytotrophoblast directly engages contact with maternal immune cells at two interfaces. The syncytiotrophoblast and cytotrophoblast from the fetal compartment are juxtaposed to maternal macrophages within the chorion at the villous interface. The extravillous cytotrophoblast and its derivatives encounter maternal natural killer (NK) cells, macrophages, and T cells as they invade the decidua, myometrium, and spiral arteries within the uterine wall.

Sialylation is essential to maintain the immune homeostasis between the embryo and the mother and to protect the embryo from attacks by the maternal innate immune system. In CMAS knockout mice, a deficiency of sialylated glycans is lethal at 9.5 dpf. Maternal complement activation occurs in trophoblasts, leading to the infiltration of
neutrophils at the fetal-maternal interface, which results in impaired intrauterine growth and defective placentation. As secondary effects, embryos lacking cell surface sialic acid suffer from malnutrition due to inadequate placentation and perish (Abeln et al., 2019).

Human trophoblast expresses Sia N-glycans, predominantly ending with NeuAcaα2,3Gal sequences, while a minor fraction carries sLeₓ (Chen et al., 2016). These sequences are known to resist NK cell cytotoxicity in assay systems (Yoshimura et al., 1996), and trophoblast cells demonstrate resistance to NK cell-mediated cytotoxicity in vitro (King and Loke, 1990), indicating that these Sia N-glycans play a protective role against NK.

Glycodelin A (GdA), which is the most heavily sialylated glycodelin isoform, possesses three N-glycosylation sites and LacciNac structures with terminal sialic acid residues that bind to T cell receptors, contributing to T cell apoptosis (Poornima and Karande, 2007). GdA contributes to the apoptosis-inducing action on T-cells by binding CD45 receptors on T-cells (Rachmilewitz et al., 2003). In humans, GdA also facilitates immune tolerance by blocking T cell proliferation, reducing IgM release and MHC class II from lymphocytes, inhibiting chemoattractant-stimulated migration of monocytes, blocking E-selectin-mediated adhesion of neutrophils, and inducing IL-6 release from monocytes and macrophages by L-selectin binding (Clark et al., 1996; Clark, 2014; Clark and Schust, 2013). The removal of Sia from GdA abrogates these immunosuppressive activities (Poornima and Karande, 2007), through lymphocytic cell death (Lee et al., 2009). In cases of gestational and pregnancy disorders, the Sia content of GdA decreases, and sialidase activity increases in the placenta (Lee et al., 2011).

In mice, a mechanism of B cell tolerance to trophoblast antigens has been identified, whereby ligand α2,6-Sia glycans from the trophoblast mediates B cell immunosuppression through Siglec-2 (CD22) inhibitory signaling. The B cells' activity, in turn, leads to the suppression of CD4+ T-cells (Rizzuto et al., 2022).

The mucin MUC16/CA125 is shed into the extracellular milieu following proteolytic cleavage (Clark, 2014) and binds to immune NK cells (Tyler et al., 2012) via Siglec-9, using α2–3 α2–6 Sia linkages. This binding inhibits immune cell receptors in humans (Angata et al., 2000; Belisle et al., 2010) and alters immune response (Clark, 2014). In mice, during the pre-placental period, depletion of NK cells correlates with an increase in O- and N-linked sialylation in the decidual vascular zone, leading to defects in the placental cells and spongiotrophoblasts within the junctional zone (Borowski et al., 2020).

## 5. Conclusion

Sialylation is an important aspect of blastula formation and is essential for interactions with the maternal structures in species that develop internally. However, many questions remain regarding the precise regulation of sialylation in the blastula and in the establishment of its connectivity to the maternal uterine environment for embryotrophic support.

Recently, small non-coding RNAs associated with sialylated glycans, referred to as “glycoRNAs,” have been identified in various cell types and mammalian species, both in cultured cells and *in vivo*. The formation of these glycoRNAs is dependent on the N-glycan biosynthetic machinery, resulting in structures that are rich in sialic acid (Flynn et al., 2021). Notably, they have also been found in the embryos of *Xenopus tropicalis*, underscoring their significance in early external development. Most glycoRNAs are present on the cell surface, where they can interact with members of the Siglec receptor family (Flynn et al., 2021). Additionally, glycoRNAs are emerging as important players in immune regulation and evasion (Graziano et al., 2025; Montag et al., 2025) and could play a role in blastocyst endometrium interaction in internal species.

Blastoids, or blastocyst-like structures, are composed of cavitating and multilayered clusters of differentiating embryonic stem cells. These structures resemble the cell types and tissue organization found in a blastocyst and hold promising potential for further research (Luijkgx

et al., 2022; Simunovic and Brivanlou, 2017). Their trophoblast and embryonic stem cells cooperate to create structures that morphologically and transcriptionally resemble blastocysts at embryonic day 3.5 (Rivron et al., 2018). Uncoupling physically and genetically the human trophectoderm from the embryonic compartment revealed that trophoblast stemness (proliferation, self-renewal) and epithelial morphogenesis are maintained by the embryonic cells (Kagawa et al., 2022). Furthermore, blastoids generate inductive signals from their inner embryonic cells to promote the development of the outer trophectoderm, robustly implant, and trigger decidualization *in utero*, thereby facilitating the construction and deconstruction of our knowledge on early development (Kagawa et al., 2022; Shahbazi and Shahbazi and Zernicka-Goetz, 2018). Blastoids could provide more precise insights into the role of Sia in the embryonic inductions necessary to maintain trophoblasts in a state conducive to implantation. Endometrial organoids and assembloids are also being developed as potential models to study implantation and future placenta generation (Kleinová et al., 2024). Additionally, blastoids were obtained from reprogrammed aged somatic cells from monkey, enhancing the study of embryonic development for regenerative medicine (Liu et al., 2024). The animal cap part of *Xenopus* blastula that was used to generate intriguing kinematically replicating organisms (Xenobots), capable of moving through their environment, and these structures raise interesting questions about their potential to deepen our understanding of the early stages and phases of development and differentiation (Kriegman et al., 2021). Overall, all blastula-derived structures could yield more precise information regarding the involvement of Sia in embryonic inductions and their requirements for maintaining trophoblast in a state that implants *in utero*, and certainly information well beyond.

## CRediT authorship contribution statement

**Katia Cailliau:** Conceptualization, Writing – review & editing, Writing – original draft. **Ingrid Fliniaux:** Writing – review & editing, Writing – original draft. **Yoshiko Takeda-Uchimura:** Writing – review & editing. **Pélagie Douchez:** Writing – review & editing, Writing – original draft. **Anne Harduin Lepers:** Writing – review & editing. **Matthieu Marin:** Writing – review & editing. **Alain Martoriati:** Writing – review & editing.

## Funding

This work was supported by the CNRS, and the University of Lille.

## Declaration of competing interest

The authors declare no conflict of interest.

## Data availability

No data was used for the research described in the article.

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