Challenges in studying preimplantation embryo-maternal interaction in cattle

Journal Pre-proof Challenges in studying preimplantation embryo-maternal interaction in cattle

Beatriz Rodríguez-Alonso, José María Sánchez, Encina González, Patrick Lonergan, Dimitrios Rizos PII:

S0093-691X(20)30025-X

DOI:

https://doi.org/10.1016/j.theriogenology.2020.01.019

Reference:

THE 15316

To appear in:

Theriogenology

Received Date:

09 January 2020

Accepted Date:

11 January 2020

Please cite this article as: Beatriz Rodríguez-Alonso, José María Sánchez, Encina González, Patrick Lonergan, Dimitrios Rizos, Challenges in studying preimplantation embryo-maternal interaction in cattle, Theriogenology (2020), https://doi.org/10.1016/j.theriogenology.2020.01.019

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Challenges in studying preimplantation embryo-maternal interaction in cattle Beatriz Rodríguez-Alonso1,2, José María Sánchez2, Encina González3, Patrick Lonergan2, Dimitrios Rizos1 1Department

of Animal Reproduction, INIA, Ctra. De la Coruña KM 5.9, 28040, Madrid, Spain. 2School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland. 3Department of Anatomy and Embryology, Veterinary Faculty, Complutense University of Madrid (UCM), Madrid, Spain. Highlights  Studies on physiological mechanisms in the maternal reproductive tract are essential  Embryo-maternal communication can be established through direct or indirect pathways  The crosstalk between the embryo and the reproductive tract is bidirectional  Novel strategies improving in vitro embryo conditions and reproductive outcomes in cattle Abstract A comprehensive understanding of the complex embryo-maternal interactions during the preimplantation period requires the analysis of the very early stages of pregnancy encompassing early embryonic development, maternal recognition and the events leading to implantation. Despite the fact that embryo development until blastocyst stage is somewhat autonomous (i.e., does not require contact with the maternal reproductive tract and can be successfully recapitulated in vitro), many studies on ruminant embryo production have focused on the fundamental question of why: (i) only 30% to 40% of immature oocytes develop to the blastocyst stage and (ii) the quality of such blastocysts continually lags behind that of blastocysts produced in vivo. Clear evidence indicates that in vitro culture conditions are far from optimal with deficiencies being manifested in short- and long-term effects on the embryo. Thus, enhanced knowledge of mechanisms controlling embryo−maternal interactions would allow the design of novel strategies to improve in vitro embryo conditions and reproductive outcomes in cattle. Keywords: Embryo-maternal interaction; oviduct; uterus; embryo; in vivo; in vitro.

Introduction Embryogenesis is a complex process that starts within the oviduct with zygote formation including fusion of the male and female pronuclei. The single-celled zygote starts to divide in a series of mitotic divisions or cleavages. Around the 8- to 16-cell stage, the bovine embryo undergoes a reprograming process termed embryonic genome activation (EGA), after which development is no longer dependent on oocyte-derived

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transcripts but is rather driven by de novo transcription [1]. In cattle, the embryo leaves the oviduct and enters the uterus around Day 4 after oestrus at approximately at the 16-cell stage [2]. Once in the uterus, the embryo continues its development forming a solid ball of cells, known as a morula, where individual blastomeres can no longer be distinguished accurately. The processes leading to blastocyst formation on Day 6 to 8 include: (i) the growth of the blastocoel due to an active sodium pump in the outer cells of the morula; and (ii) the differentiation of two cell populations (the trophoblast and the inner cell mass) [3]). On Days 9-11 post-fertilization, due to mechanical forces exerted by blastocyst expansion and the production of proteolytic enzymes by the trophoblast, the blastocyst hatches from the zona pellucida [4]. After hatching, the trophoblast undergoes a process of elongation that is initiated between Days 12 and 14. The uterine endometrium plays a key role in driving the elongation process via production and secretion of histotroph into the uterine lumen [5–7]. Spatiotemporal alterations in the endometrial transcriptome and histotroph composition are hypothesized to establish uterine receptivity to implantation and, in turn, are pivotal to the success of pregnancy [8]. These modifications are primarily regulated by progesterone (P4) and then influenced by conceptus-derived interferon tau (IFNT) in cattle, the signal for maternal recognition of pregnancy [5,9,10]. In cattle, even though fertilization occurs in over 85% of cases if insemination is carried out correctly [11], a significant proportion of the resulting embryos fail to develop to term [11,12]. Interestingly, much of this embryonic loss occurs before maternal recognition of pregnancy, encompassing the period between fertilization and Day 16 after insemination [11], and is greatest from fertilization to Day 7 in highproducing dairy cows [13]. Given the economic importance of the livestock industry, it is relevant to develop new strategies capable of improving the pregnancy rates by reducing early embryonic losses or improving assisted reproductive technologies (ARTs). The key to the development of such strategies is improving our understanding of the reproduction events. The aim of this review is to summarise key knowledge of the fine dialogue between the embryo and the maternal reproductive tract acquired through in vivo and in vitro studies. Moreover, we discuss the challenges currently faced when understanding and assessing these interactions. Embryo-maternal crosstalk Embryo-oviduct communication In spite of the fact that the oviduct is not essential for reproduction (i.e. successful pregnancies can be obtained by transferring in vitro produced embryos directly into the uterus), this organ has a key role in

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determining the quality of the embryo [14]. Therefore, improved knowledge of embryo-oviduct interaction during the few days that the embryo spends in the oviduct are important to achieve better embryo survival. More specifically, the relevance of the oviduct resides, on the one hand, in its ability to produce high quality embryos (compared with those produced in vitro [14–18]), and on the other hand, its contribution to one of the major causes of infertility, early embryo mortality [11]. Taking into account the complexity of the physiological environment, different approaches could help in the ultimate goal of understanding the mechanisms of the oviduct´s contribution to the early reproductive events (i.e. sperm capacitation, oocyte fertilization and early embryo development) - Figure 1. Despite the challenge of studying embryo-oviduct interactions, knowledge acquired to date suggests that: (i) the communication can be established through direct or indirect pathways; (ii) the crosstalk is bidirectional; (iii) the interactions can be affected by external conditions; and (iv) the crosstalk elicits a feedback in the recipient of the message. With respect to the communication establishment through direct or indirect pathways, traditionally, indirect intercellular communication was thought to occur due to the transfer of secreted molecules. These molecules could be a range of biochemical messengers known as embryotropins including proteins, saccharides, lipids, neurotransmitters, microRNAs, and can be secreted in several ways such as active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles (EVs) [19]. Besides the traditional concept of indirect cell-tocell communication, in recent years, membrane-limited vesicles found in the extracellular environment known as EVs have attracted interest in relation to communication between the embryo and the oviduct [20]. EVs mediate cell-to-cell communication by transferring biomolecules (i.e. mRNAs, miRNAs, proteins) that can modulate the activities of recipient cells. The assessment of bovine oviductal EVs protein content and their role during oviduct-embryo crosstalk has revealed key proteins with different roles such as exosome biogenesis, intracellular vesicle trafficking and release, tetraspanins and GTPases, exosome sorting, annexin proteins involved in membrane trafficking and fusion events, and heat-shock proteins (HSP) [20,21]. Additionally, Alimiñana et al. [22] identified proteins characteristic of the oviduct such as OVGP1, ANXA2, HSPA8, HSP90, HSP70, gelsolin and ezrin. Proteins in EVs could exert an effect on embryos when they are taken up. For example, proteins of the HSP family are crucial for fertilization and early embryo development and can exert anti-apoptotic effects and cryoprotection [20]. Moreover, RNAsequencing of EVs transcripts revealed functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins [21]. Among the identified mRNAs, protein-coding RNAs encoding ribosomal proteins and translation elongation factors were the most abundant in oviductal EVs. Furthermore, functional analysis of oviductal EVs content revealed genes 3

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involved in chromatin modification, which suggests that chromatin modification in the early embryo could be in part under maternal control via oviductal EVs transcripts (reviewed by Almiñana and Bauersachs, 2019). Furthermore, the crosstalk is bidirectional; the fact that EVs can be both secreted and received by the bovine embryo [22–24], and secreted by the oviduct [25,22,21], suggests that embryo-oviductal communication is a two-way (or bidirectional) process. Furthermore, other molecules potentially involved in the embryo-maternal communication such as embryotropins [19] or embryokines [26] are also secreted by the embryo and the reproductive tract, respectively. It has also been shown that the interactions can be affected by external conditions. For example, it has been shown that the metabolic status of postpartum dairy cows (a disorder linked with suboptimal follicle development, oocyte quality, sperm transport and fertilization and an altered reproductive tract environment) can impact fertility. Assessment of the blastocyst yield after in vitro produced zygotes were transferred to the oviducts of postpartum lactating dairy cows vs. dry cows (i.e. never milked) [27] and nulliparous heifers [28], indicated that blastocyst development in lactating cows was significantly reduced, indicating that the reproductive tract of postpartum dairy cows contributes to poor fertility. Among the factors that can affect these interactions, is the timing (i.e. synchrony between the developing embryo and the maternal reproductive tract environment) at which they occur. While the importance of synchrony between the embryo and the uterus has been well described in embryo transfer (ET) studies in sheep and cattle [29–33], little is known about the relevance of synchrony between the oviduct and the early embryo. To address this, we recently endoscopically transferred Day 1 in vitro produced bovine zygotes to the oviducts of heifers either synchronous or asynchronous (2 days in advance) with the embryos; embryo recovery was performed at different developmental stages (Day 4, 7 or 15). The main findings revealed that a 48 h difference between the embryo age and the reproductive tract stage of the cycle resulted in decreased embryo development and lower embryo survival. Results suggest that the development of the majority of early embryos from the asynchrony group in an altered environment (in an advanced oviduct, in the uterus instead of the oviduct or a combination of both) may explain the detrimental effect. On Day 15, despite the negative impact observed on those embryos exposed to an advanced maternal environment, those conceptuses that survived to Day 15 were longer in comparison with those cultured in synchrony. The increased size of the asynchronous conceptuses, likely driven by effects of P4 on the uterus [33], did not lead to higher recovery rates of Day 15 conceptuses (only 50% of the asynchronous heifers yielded conceptuses vs. 100% in the synchronous group). These findings highlight the importance of synchrony between the embryo and the oviduct in achieving a successful pregnancy and suggest that an asynchronous environment could be among the factors influencing early embryo mortality. Moreover, this could explain inadequacies in in

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vitro culture (IVC), where the media composition is not “in synchrony” with the embryo’s needs, and thus does not fulfil the changing needs of the developing embryo. Finally, the crosstalk established between the embryo and the oviduct elicits a feedback in the recipient of the message: o

Oviduct effects on the embryo

The relevance of understanding the oviduct effects on the embryo (and thus its response) resides in the fact that the post-fertilization culture environment has a major influence on embryo quality [34]. As a consequence of this influence, embryos cultured in the suboptimal conditions provided by the in vitro systems are of a lower quality compared with those cultured in vivo, evidenced by different embryonic features such as modifications at the ultrastructural level [35] or limited compaction at the morula stage [36]. The lower cryotolerance of in vitro cultured embryos is further evidence of the negative impact of the in vitro environment on embryo quality [37]. Furthermore, ‘omics’ studies have found that IVC results in embryos with altered transcriptomes [38,39] or epigenomic changes [40] such as DNA methylation dysregulation [41] or imprinting disorders [42]. Moreover, it has been reported that the impact on the embryonic transcriptome is particularly critical during embryonic genome activation [38]. Ultimately, all of these short-term consequences result in lower pregnancy rates of IVP embryos compared to in vivo derived embryos [43]. Consequences of IVC conditions on embryo development and quality have been extensively reviewed [44]. Besides the positive effect in terms of quality of embryos cultured in vivo (in the oviduct), the fact that the quality of in vitro produced embryos is improved in culture systems which use different oviductal components such as oviductal fluid (OF), bovine oviductal epithelial cells (BOECs) or their respective EVs is evidence of the ability of the oviduct to exert a positive effect on the embryo [45]. o

Embryo effect on the oviduct

Most of the published work in relation to embryo-oviduct communication relates to the effect on the embryo; meanwhile there are only a few studies that have reported the converse effect of the embryo on the oviduct. The earliest evidence in the literature was published more than five decades ago and reported that mare´s oviducts had the ability to allow (or not) the passage of the oocyte depending on its fertilization status [46]. Similarly, experiments in hamsters [47] and rats [48] demonstrated how the embryo was differentially transported in time depending on the developmental stage. It seems that other oviductal features such as the cilia beat frequency, vascularization or the formation of secretory cells are also subjected to change in response to the presence of the embryo [49].

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In recent years, few studies have been published showing that the presence of the embryo has an effect on oviductal gene expression. In general terms, the reported effect of the embryo on the oviduct consists of a regulation of the immune system response [50,51]. Under physiological conditions, the oviduct has to fight the presence of pathogens while allowing the presence of the immunologically semi-allogeneic embryos [52]. The embryo’s ability to regulate the expression of genes related to the immune system suggests that it is a key player in avoiding its own rejection. The first results came from litter-bearing species such as mice [53] and swine [54], where the presence of multiple embryos might amplify the effect on the oviduct. With respect to monoovulatory species, where the effect on the oviduct is exerted by a single embryo, there are relatively few studies. An experiment performed in mares reported a local influence of the equine embryo on the oviductal epithelium transcriptome in the ampullary-isthmic junction, where the equine embryo stays during its early development [51]. They described the effect of the early embryo on the immune response-related genes, where up- and down-regulated genes were found. Thus, they proposed that a delicate balance exists between stimulating and inhibiting factors in these processes [51]. In cattle, no differences in the oviduct epithelium transcriptome were found in the presence of a single embryo in vivo [50]. However, a response to a putative embryo signal in terms of differential gene expression of the oviductal isthmic cells was detected after the oviductal transfer of multiple embryos (up to 50) [50]. Given the size of the bovine embryo (120m in diameter), it is very likely that any effects on the oviductal epithelium induced by a single embryo are very local in nature. In an effort to detect any local embryo-induced changes in the bovine oviduct epithelium gene expression, we took the approach of dissecting and sequentially flushing small sections of the bovine oviduct (2 cm long) on Day 2.5 after AI in order to recover the epithelium in direct contact with the embryo [55]. The expression pattern of 8 differentially expressed genes (DEG) between the isthmus of pregnant (with multiple embryos, [50]) and cyclic heifers was assessed by qRT-PCR. Comparisons between (i) the section in which the embryo was found (ipsilateral oviduct) vs the corresponding section from the contralateral isthmus, or (ii) along the ipsilateral oviduct (i.e. oviductal section through which the embryo had passed, section in which the embryo was found and a last section on the uterine side of the embryo), resulted in no statistical differences. Of the 8 candidate genes, VAT1L was the only one showing a statistical trend when comparisons between the embryo section (ipsilateral oviduct) and the corresponding section from the contralateral oviduct were carried out, exhibiting lower relative mRNA abundance in the presence of the embryo. One of the molecular functions of VAT1L is related with zinc ion binding. Interestingly, the presence of a single equine embryo in the mare oviduct regulates a gene, SLC39A2, involved with zinc ion homeostasis [51]. Although this study failed to detect embryo-induced changes in the abundance of

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specific transcripts in the bovine oviduct in vivo, the methodology used represents the foundation for further assessment of the early embryo-maternal dialogue [55]. Overall, the studies that have succeeded in the detecting an oviductal response in terms of changes in its epithelial gene expression, support the idea that the embryo might be able to regulate specific genes in order to avoid its own rejection by enhancing the maternal immune tolerance, a process which is still not fully elucidated [56]. With respect to the way in which embryos could modify the expression of immune response-related genes, the IFNT protein could be an intermediate candidate for induction of the anti-inflammatory response. IFNT is a pregnancy recognition signal in ruminants that indirectly inhibits prostaglandin F2 alpha, thereby preventing CL regression and maintaining pregnancy [57,58]. IFNT is also an immunosuppressive molecule that inhibits proliferation of lymphocytes, and thus may protect the fetus from maternal immune attack [59]. It has been shown that the endometrium recognizes the embryo around Day 16 in cattle, when the trophoblast is elongated and the embryo secretes increasing amounts of IFNT [60]. With respect to the early embryo in the stages corresponding to the first 4 days of development (undergone in the oviduct), it has been shown that IFNT mRNA is expressed in the 8 to 16-cell bovine embryo in vitro [17,61]. However, in vitro studies with endometrial explants [62] or BOECs [63] indicate that at this stage the IFNT secreted by the early embryo does not induce changes in the interferon-stimulated genes (ISGs). Although Talukder et al., [63] did not find this effect on the ISGs from oviductal cells, they provided results on how BOECs stimulate Day 4 bovine embryos at the 16-cell stage to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct. As described, the study of embryo-oviductal crosstalk remains one of the most challenging subjects in reproductive biology. With the aim of describing the widely unknown oviductal response to the presence of an embryo in terms of changes in the proteomic, amino acid and carbohydrate content in the OF, a pilot study was recently conducted (Rodriguez-Alonso et al., unpublished data). Proteomic and metabolomic assessments were carried out using in vivo OF samples collected from bovine oviducts on Day 3 of the oestrous cycle from pregnant (8-cell embryo) and cyclic unstimulated heifers. In addition to the evaluation of the effect of the embryo, the influence of the oviduct region on OF composition was assessed. Results revealed that, on Day 3 post-oestrus, OF composition varied based on (i) anatomical region, where isthmic metabolites were present in lower (i.e. lactate, glycine, and alanine) or higher (i.e. arginine) concentrations compared to the ampulla; and (ii) embryo presence, which was correlated with greater arginine, Phosphoglycerate kinase 1, Serum albumin, Alpha-1-antiproteinase, IGL@ protein concentrations (Rodriguez-Alonso et al., unpublished data).

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Embryo-uterine communication Once the embryo enters the uterus, an appropriate communication between the developing conceptus and the maternal endometrium is essential for the establishment and maintenance of pregnancy. In this regard, and despite the efforts to further our knowledge, many questions remain unresolved in relation to the processes leading to successful establishment of pregnancy. Unlike communication between the embryo and the oviduct, much has been demonstrated with respect to the embryo-uterus communication [64]. Thus, in the present review we will focus on addressing questions that remains a challenge within the field such as (i) what are the key players determining the success of the embryo development?, (ii) when and how does the embryo-uterus communication happen?, and (iii) is the endometrium able to detect differences in embryo quality that may account for early pregnancy loss? o

Oocyte quality vs. endometrium as key player for embryo development

The use of ARTs such as ovum pick up (OPU) coupled with IVF (in which the donor cow’s uterus is bypassed), artificial insemination (AI) and ET (in which the recipient cow’s oocyte is bypassed), has allowed the comparison of the relative importance of oocyte quality and uterine capacity in pregnancy outcomes. In terms of the importance of oocyte quality for embryo development, several studies have reported lower pregnancy rates in lactating dairy cows after AI compared with ET [65–68], which suggests that failure to establish pregnancy may be due to a poor quality oocyte and/or resulting embryo. In fact, according to Sartori et al., [13], a significant proportion of embryos degenerate before the blastocyst stage in unstimulated high-producing dairy cows. Moreover, studies using multiple ET have reported a significant variation in size between age-matched conceptuses regardless of the embryo source (in vivo or in vitro) or P4 concentrations [69–72]. The fact that such embryos were exposed to a common uterine environment would suggest that part of the ability to elongate is intrinsic to the embryo and is probably related to the oocyte/embryo quality, consistent with the hypothesis that the quality of the oocyte regulates developmental competence [18]. In fact, many other studies have shown that any disturbance in the oocyte’s environment may affect the oocyte’s developmental quality through an altered gene expression pattern in the oocyte and the cumulus investment [73], highlighting that oocyte quality is a key contributor to subfertility. Conversely, it has been reported that many of the reproductive failures observed in natural or assisted pregnancies during the preimplantation period may be attributed to other maternal factors, such as the inability of the uterus to support conceptus growth and implantation [74]. Indeed, elongation has not been recapitulated in vitro (in vivo and in vitro produced bovine blastocysts must be transferred to a

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receptive uterus in order to grow and develop into an elongated filamentous conceptus) [75]. Furthermore, elongation does not occur in vivo in the absence of uterine glands [76], thus, demonstrating that conceptus elongation is a maternally driven process. We and others have made significant contributions in determining that the main driver of elongation is the steroid hormone P4, the effects of which are mediated through effects on the endometrium rather than directly affecting the developing embryo [70,77]. Global gene expression profiling studies have identified temporal changes that occur in endometrial gene expression in both cyclic [78] and pregnant [79] heifers following an elevation or reduction of postovulatory P4 during early dioestrus that promotes or delays conceptus elongation, respectively [70,78–80]. In addition to the temporal changes in P4 concentrations, differences in endometrial tissue P4 concentrations between different regions of the same uterine horn, and between the horns ipsilateral and contralateral to the ovary bearing the corpus luteum (CL), have also been reported [81–85]. In line with this, early ET studies established that the incidence of embryo loss is higher following transfer to the uterine horn contralateral to the ovary containing the CL compared to transfer to the ipsilateral horn [86– 88]. Based on this evidence, in a recent study from our group [89] we hypothesized that the knowledge of differences in gene expression between the uterine horns during the oestrous cycle could further enhance our understanding of uterine receptivity and the process of conceptus elongation. In that approach, we found that there were many more altered genes between the uterine horns ipsilateral and contralateral to the CL in the early (Day 5 and 7) as compared to late (Day 13 and 16) luteal phase in cyclic heifers. Signalling pathways regulating pluripotency of stem cells were highly dysregulated when both uterine horns were compared, regardless of the day of luteal phase. In agreement with this, greater expression of pluripotent markers in the ipsilateral than in the contralateral horn have been reported [90], suggesting that ovarian hormones influence these characteristics. Interesting findings showing that a decrease in the expression of stem cell markers, or in the actual number of stem cells, are related to factors affecting pregnancy rate (uterine horn, cow age, and uterine health status) strongly support the hypothesis that uterine stem cells may play a key role in many physiological and pathological reproductive events [89–92]. In a separate experiment within the same study [89], no differences in conceptus survival or development/elongation were observed when ten Day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral or contralateral to the CL and recovered on Day 14. Interestingly, in a follow-on study (Bagés-Arnal et al., unpublished data), we found that the local response to a Day 14 bovine conceptus was different between the ipsilateral and contralateral uterine horns (32 DEG). Gene Ontology analysis of these 32 genes revealed 10 enriched biological processes, mainly related to immune response, 9

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response to an external stimulus, and homeostasis. These results suggest that the environment in the contralateral uterine horn may not be favourable for embryo development, but these differences in embryo development cannot be detectable before maternal recognition of pregnancy. Other approaches have used serial ET (up to 6 separate attempts) to establish a model of uterine insufficiency where heifers were classified as high fertile (HF), subfertile (SF) or infertile (IF) based on pregnancy success beyond Day 28 [93–95]. In agreement with our ipsilateral vs. contralateral model, embryo recovery rate and conceptus length did not differ among groups on Day 14 [94]. Moreover, the differences in the endometrial transcriptome between fertility-classified heifers were minimal (26 DEG HF vs. SF; 12 DEG SF vs. IF; 3 DEG HF vs. IF). The results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. However, elongating conceptuses were approximately two-fold longer in HF than SF heifers when advancing in gestation to Day 17 [95]. According to these findings, substantial differences in the transcriptional profiling of the endometrium to pregnancy between HF and SF heifers were also found. From the embryo site, conceptuses from HF and SF animals showed distinct gene expression pattern, with many of the genes downregulated in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Remarkably, analyses of biological pathways, key players and ligand-receptor interactions based on transcriptome data produced substantial evidence for dysregulation of conceptus–endometrial interactions in SF animals. As with the oocyte, metabolic factors also affect the endometrium [96,97] and the conceptus [98]. To avoid the confounding metabolic effect on oocyte quality, Leane et al., [98] evaluated the impact of exogenous glucose infusion on early embryonic development in lactating dairy cows following the transfer of Day 7 blastocysts. Glucose infusion from Day 7 to Day 14 post-oestrus had an adverse impact on early embryonic development. With regard to the endometrium, few analyses have addressed the impact of intensive milk production at the initiation of implantation, a critical milestone ensuring a successful pregnancy. It has been reported that maternal metabolism (lactating vs. dry cows) had a higher impact on gene expression in endometrial intercaruncular areas compared with caruncular areas, affecting endometrial expression of oxidative stress and forkhead box L2 (FOXL2) genes [97]. In addition, using RNAsequencing on intercaruncular endometrial samples from pregnant animals on Day 19 (n = 5 lactating and nonlactating pregnant cows by ET ; n = 4 pregnant heifers by AI), Bauersachs et al., [96] concluded that metabolic status of the animal (lactating vs. dry and heifer vs. cow) modulates the response of the endometrium to the developing conceptus.

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Altogether, the studies cited above support the notion that both oocyte quality and uterus receptivity impact embryo survival and program its development, and dysregulated conceptus-endometrial interactions elicit loss of the post-elongation conceptus in cattle. o

How early does the embryo-maternal communication in the uterus begin? Is this a systemic or local dialogue?

Optimal dialogue between the developing embryo and mother during the peri-implantation period is essential for pregnancy recognition and uterine receptivity, ultimately setting the stage for implantation and placentation [9]. However, up to the blastocyst stage, development is independent of oviductal or uterine signalling, as illustrated by the relative ease with which blastocysts can be produced in vitro. Data from in vivo studies have shown that temporal changes in uterine transcriptome occur irrespective of whether the cow is pregnant or not and it is only during maternal recognition of pregnancy, around Day 16, when major changes in gene expression between cyclic and pregnant endometrium become apparent [99,100]. During pregnancy recognition, conceptus-derived IFNT exerts its antiluteolytic effect via inhibition of transcription of oxytocin receptor gene (OXTR) in the endometrium of cattle [101]. Further, this paracrine action of IFNT on the endometrium also stimulates expression of ISGs that are hypothesized to regulate uterine functions important for conceptus elongation, implantation and establishment of pregnancy [5,74]. Interestingly, there is evidence to support the idea of an endocrine action of IFNT, which navigates the endometrium, enters uterine vein blood and is delivered in amounts sufficient to induce ISGs in peripheral tissues such as peripheral blood mononuclear cells, CL, and liver [10]. Although we and others did not find any response of the endometrium to pregnancy prior to Day 16, it has been recently reported that the presence of a single Day 7 blastocyst altered the abundance of transcripts (ISGs and prostaglandin biosynthesis, channel water, and solute carrier genes) in the cranial part of the ipsilateral uterine horn to the CL in cattle [102]. Therefore, factors secreted by the preelongating embryo enhancing changes in the transcriptome of the endometrium are most likely to be local in nature. Due to the small size of the early pre-elongating embryo relative to the volume of the uterine lumen, collection of endometrium adjacent to the developing conceptus is difficult by conventional uterine flushing methods. Using an ex vivo model of intact bovine endometrium to amplify any potential embryoderived signals (co-culture of endometrial explants in the absence or presence of embryos or medium conditioned by blastocysts), we recently investigated potential local effects of blastocyst stage embryos on endometrial gene expression and demonstrated that: (i) the ability to detect a response of the

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endometrium to the embryo is dependent on the number of embryos present (minimum of 5 blastocysts); (ii) the response of the endometrium to the early embryo is stage specific (only blastocysts but not earlier stages induce gene expression changes); (iii) direct contact between the embryo and the endometrium is not required to induce expression of candidate ISGs; (iv) diffusible factors present in blastocystconditioned medium alter the expression of ISGs in the endometrium; and (v) all of the DEG (n = 40) induced in the endometrium by blastocyst-stage embryos are IFNT-stimulated [62,103]. Together, embryo-maternal communication in the uterus seems to begin as early as the blastocyst stage when it appears to be due solely to IFNT, but the factors released by pre-elongating embryos alter the transcriptome of the endometrium locally. However, whether such changes play any role in subsequent pregnancy recognition remains to be established. In contrast, around the pregnancy recognition, the dialogue between the embryo and the mother can be detectable systemically, which is promising for the development of early pregnancy diagnosis test. o

Could the endometrium detect differences in embryo quality that may account for early pregnancy loss?

Conception rates are lower following the transfer of in vitro produced embryos (30-40%) compared with AI or embryos recovered nonsurgically from donor animals (50-70%) [104]. Furthermore, in a large scale ET study under field conditions, Hasler et al., [105] reported that the highest pregnancy rate was achieved with grade 1, fresh in vivo embryos. Significantly, lower pregnancy rates were achieved, in order, by in vivo frozen, fresh Day-7 in vitro produced embryo, fresh Day-8 and frozen Day-7 in vitro, and, lastly, by frozen Day-8 in vitro produced embryos. The results obtained in this study would suggest that not only embryo origin but also the cryopreservation state and the age of the embryo at the time of the transfer influence in the reproductive outcomes. An interesting study comparing Day 18 conceptuses produced by AI, in vitro production and different types of somatic cell nuclear transfer (SCNT) found that, although most of the conceptuses exhibited normal elongation (46/50), the likelihood of observing normal gastrulation was low (36/50) [106]. A severe uncoupling of embryonic and extra-embryonic differentiation observed in all groups of SCNT conceptuses was highly correlated with embryo loss at implantation. It has also been demonstrated different capabilities of secreting IFNT for in vivo derived and in vitro produced embryos compared to cloned and demi-embryos, suggesting that it may, at least in part, be responsible for those differences in pregnancy rate following the ET [107]. However, can these differences in embryo quality (development) be sensed by the mother and, in turn, activate the mechanism to disrupt or, in contrast, to maintain the pregnancy?

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Recent data have indicated that the endometrium can act as a sensor of embryo quality and that it responds differently in terms of its gene expression signature to such embryos of varying quality which may also reflect their subsequent viability [108–110]. To further understand this phenomenon, we combined in vitro and in vivo production of bovine blastocysts, multiple ET techniques, a conceptusendometrial explant co-cultured system and RNA-sequencing technology [111]. Approximately two-thirds (67%) of all genes differentially expressed in response to in vivo-derived conceptuses were IFNTdependent. The top 10 most up-regulated genes in response to in vivo- and in vitro-derived conceptuses were classical ISGs. In addition, 240 DEG were uniquely altered by conceptuses (in vivo- and in vitroderived) but not IFNT. Of these transcripts, 46 were shared between both groups, while 133 and 61 were specific to in vivo- and in vitro-derived conceptuses, respectively. Biological processes associated with genes up-regulated uniquely by in vivo-derived conceptuses, both dependent and independent of IFNT, were related to inflammation and tumour necrosis factor (TNF) and nuclear factor kappa-light-chainenhancer of activated B cells (NFKB) signalling. In addition, the transport of micro- and macromolecules across cell membranes within the endometrium by the solute carrier transporters seems to be altered by conceptus origin. These data support the hypothesis that conceptus regulation of gene expression in the endometrium involves factors other than IFNT that may have a biological role in pregnancy establishment. Among others, these IFNT-independent factors may include prostaglandin, cortisol, growth factors, and proteins associated with gestation produced and/or induced by such conceptuses. As stated above, there is wide variation observed in length among conceptuses recovered on the same day, even from the same uterine environment [69,70,72]. In terms of embryo size, bigger Day 7 blastocysts (more cell number) are positively correlated with conceptus length on Day 14 [112]. Interestingly, longer conceptuses produce higher amount of the IFNT [113]. As IFNT is the signal for the maternal recognition of pregnancy, conceptus length on a given day in the period around pregnancy recognition is thought to be indicative of its quality and the likelihood of establishing and maintaining a pregnancy [114], although this has yet to be definitively established. While significant differences in the transcriptomes of long and short Day 15 conceptuses have been reported [114], where ovoid conceptuses appeared to have reduced viability based on gene expression profiles, the interaction between such divergent conceptuses and the endometrium had not been described. Our group has recently studied the transcriptome profile of endometrial explants (ex vivo model) exposed to 100 ng/mL IFNT, Day 15 long conceptuses (25.4 ± 5.7 mm) or Day 15 short conceptuses (1.8 ± 0.3 mm) [72]. We concluded that bovine endometrium responds differently in terms of its gene expression signature to age-matched long and short conceptuses in an IFNT-dependent and independent manner, which may be critical for embryo survival. In particular, short 13

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conceptuses failed to alter the expression of a large number of ISGs that were altered by both IFNT and long conceptuses, suggesting that insufficient IFNT production is a major contributory factor to lower survival of such conceptuses. Furthermore, the alteration of >100 endometrial transcripts uniquely by long conceptuses suggests that other aspects of maternal–embryo communication at this critical time are IFNT-independent. Understanding embryo-maternal communication through in vitro models Based on the need to bypass the difficulties of studying embryo-maternal communication in vivo, the development of adequate in vitro systems is necessary. One of the main challenges to generate such models is to recreate the complex and dynamic maternal environment. In the last decade, numerous studies have been carried out aiming to understand the role of oviductal and uterine components (i.e. epithelial cells and fluids) using in vitro models. As a result of these studies, the dialogue between the embryo and the genital tract has been furtherer elucidated [60,115,116]. One of the approaches used to study early local embryo-maternal paracrine and autocrine interactions, which are difficult to investigate in vivo, is to co-culture embryos with oviduct epithelial cells. This system has been especially developed in the study of monovulatory species such as cattle [117-121]. BOECs can be cultured in vitro in many systems such as monolayers, in perfusion chambers, in suspension, and in polarized or three-dimensional (3D) systems. The co-culture of embryos with an oviductal epithelial cells (OEC) monolayer is a simple and well-established model since the early days of in vitro production [122], and have provided a good starting point to study embryo signals. Even though aspects of this system are still suboptimal - the BOECs monolayer culture has been associated with a dedifferentiation process [115] and a decrease in the expression of some genes [119,123] such as the oviduct-specific oestrus-associated glycoprotein gene [115] – it has been used successfully as an in vitro model to improve the quality of the produced embryos [120]. Moreover, Schmaltz-Panneau et al., [124] demonstrated that BOECs monolayer modify their transcription in the presence of blastocysts, concluding that BOECs co-culture may be a suitable model to study the complex embryo-maternal cross talk in cattle. Besides the use of BOECs monolayer to address the challenging question of the oviduct response to the embryo presence, other studies have provided evidence which supports different hypotheses related to this crosstalk. Our group has demonstrated that the embryo is able to communicate with the oviduct in different ways, given that the BOECs transcriptome is affected by the embryo presence via direct contact or through embryo secretions and this effect is embryo stage-specific [121]. Furthermore, new knowledge with regards to possible specific signaling components such as bone morphogenetic proteins (BMP) has been described.

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García et al. [125] demonstrated that oviduct-embryo interactions in vitro induce specific changes in the transcriptional levels of BMP signaling, suggesting that the BMP signaling pathway could be involved in the crosstalk between the bovine embryo and the oviduct during the first stages of development. Other IVC systems involve the use of oviductal cells obtained by mechanical means which can be maintained in suspension culture dishes as explants. In these systems, the cells have their apical surface directed outwards, and constitute an adequate well-defined short-term culture for functional genomics and proteomics studies [126]. These cell clusters preserve oviduct morphological features and gene markers present in the oviduct epithelial cells in vivo throughout a 24 h culture period [126] but they are not able to adhere and undergo mitosis [127]. Overall, primary cultures have two limitations: lack of reproducibility due to the variability of the cells employed, and risk of diseases transmission. To overcome these issues, established frozen-thawed BOEC lines that maintain primary culture attributes can be used [120,128] or even BOEC conditioned media which is able to support embryo development to the blastocyst stage and improve embryo quality [120]. Additionally, BOEC cultured in polarized system have been used as a model to elucidate the mechanism and physiology employed by the oviduct to face situations of metabolic stress produced by elevated nonesterified fatty acids [129,130]. Besides, air-liquid interface cultures have been used to study the early embryonic environment and interactions with the maternal environment [131]. More specific, Chen et al., (2017)[132] reported that polarized culture using an air-liquid interface system supports embryo development in vitro without culture medium supply, in porcine, mouse and bovine species. However, the obtained blastocyst rates could not yet match the outcome of optimized standard IVP procedures, suggesting further improvement of the model by: a) simulation of the hormonal changes taking place during the periconceptional period and b) development of a sequential culture system using oviductal as well as uterine epithelial cell [132]. More recently, Ferraz et al., [133,134] used a three-dimensional (3D) printing technology in combination with microfluidics ‘oviduct-on-a-chip platform’, in which oviductal epithelial cells could culture and maintain their morphological and functional structure, similar to the in vivo oviduct. They demonstrated that, although, the “oviduct-on-a-chip” supported fertilization and early embryo development, the production rates were lower compared to normal in vitro procedures, suggesting the need of further improvements [134]. Regarding the study of the communication between the embryo and the uterus, the establishment of in vitro models is highly complex due to the cyclic changes experienced by the endometrium under the hormonal control of estrogen and P4, the presence of both epithelial and stromal cells, and the initiation

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of embryo implantation. So far, different in vitro models have been developed to assess these interactions: (i) co-culture of primary bovine endometrial epithelial cells (BEEC) and trophoblast cells from CT-1 cell line [135] to study the attachment of trophoblast cells to uterine epithelium [136]; (ii) co-culture of primary BEEC with in vitro produced morulae for four days until the blastocyst hatched stage to explore early embryo-maternal uterine communication (showing that the embryo can produce an antiinflammatory response in BEEC via IFNT signaling) [137], (iii) co-culture of a single embryo using matrigel coating inserts with separate basal and apical compartments that allow independent growth of BEEC in the upper region and stromal uterine cells in the lower compartment [138] demonstrating that at very early stages the epithelial layer of the endometrium is responsible for recognition of the early embryo; and, finally, (iv) the above discussed ex vivo model in which embryos are co-cultured with intact uterine explants [62]. In any case, none of the systems above mentioned recreate the important changes suffered by the endometrium during the oestrous cycle. However, a model that is acquiring special relevance in this respect is the organoid [139]. An organoid is an in vitro 3D cellular cluster derived exclusively from primary tissue, embryonic stem cells or induced pluripotent stem cells, capable of self-renewal and selforganization, exhibiting similar organ functionality as the tissue of origin [139]. Recently, endometrial organoids that can mimic epithelium physiology have been developed in human and in mouse models [140]. An alternative to embryo co-culture with BOEC and BEEC is the use of OF [141,142] or uterine fluids (UF) [143] during embryo culture. Our group has demonstrated that the use of low concentrations of OF and/or UF in serum-free culture medium during IVC supports early embryo development and improves blastocyst quality by increasing their cryotolerance [141], altering the abundance of developmentally related genes including imprinted genes [142] and providing better antioxidant activity [143]. OF and UF contain simple and complex carbohydrates, ions, lipids, phospholipids and proteins [144], as well as an important content of EVs [145,146,25]. As described above, EVs have been recognized in recent years as a new way of intercellular communication [147]. A study by de Avila et al., [148] demonstrated that EV miRNA contents are distinct in follicles at different estrous cycle stages and that supplementation of oocyte maturation in vitro with EVs impacts gene expression and biological processes in cumulus cells. Furthermore, it was shown that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from heat stress [149]. Beyond this, Melliso et al., [150] evidenced that bovine blastocysts secrete EVs into the culture media and that the concentration of EVs secreted from day 7 to day 9 varies

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depending on embryo competence and origin. Furthermore, we observed that EVs from conditioned media of an extended culture of BOEC monolayer can be isolated, characterized and successfully used for in vitro embryo culture, improving the quality of the produced blastocysts with the objective of trying to mimic the intercellular communications between oviductal tissue and the embryo in vitro [120]. In addition, it was evidenced that supplementation of EVs from bovine OF in in vitro embryo culture enhance development and increase blastocysts quality in terms of cryotolerance and the expression patterns of development-related genes [22,25]. In addition, it is well accepted that successful implantation is dependent on coordination between the embryo and the endometrium, and EVs may participate in this required cross-talk. Thus, it has been suggested that the endometrial epithelium releases EVs that are involved in the transfer of signalling miRNAs and adhesion molecules either to the blastocyst or to the adjacent endometrium into the uterine cavity, which in turn can affect endometrial receptivity and implantation [151,152]. Furthermore, by comparing miRNAs contained in exosomes isolated from plasma during early, middle and late pregnancy in dairy cows, Zhao et al., [153] highlighted several specific miRNAs for each stage of pregnancy, providing new insight into maternal-foetal communication during pregnancy. While, Qiao et al., [154], indicated that the early luteal phase uterus secretes exosomes and their supplementation in vitro improved the developmental capacity of SCNT embryos. Thus, these evidences suggest that EVs effect on early embryonic development may be fundamental, and in vitro culture systems containing EVs can provide new insight into gametes maturation, fertilization and early embryo-maternal interactions and their involvement in the reproductive accomplishment. Conclusion Studies on physiological mechanisms and interactions in the maternal reproductive tract (oviduct and uterus) are essential. Such studies will help advance our knowledge of mechanisms related to early embryo development and will support the improvement of assisted reproductive technologies including in vitro embryo production that seek to mimic physiological conditions and generate good quality embryos. Moreover, these advances could help improve fertility treatments in humans and domestic animals to increase the efficiency of production and breeding schemes. Acknowledgments This work was supported by the Spanish Ministry of Science, Innovation and Universities (AGL2015-70140-R); Science Foundation Ireland (13/IA/1983); and European Union H2020 Marie

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Sklodowska-Curie (MSCA) Innovative Training Network (ITN), REP-BIOTECH - 675526. The authors are members of the COST Action 16119 “In vitro 3D total cell guidance and fitness (Cellfit)”. Conflict of interest The authors declare no conflict of interest. References [1] [2] [3] [4] [5] [6] [7] [8]

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Figure 1. Schematic representation of early embryo development and embryo-oviduct communication in cattle. Following fertilization in the oviduct, the developing embryo undergoes the first cleavage divisions incorporating embryo genome activation, entering the uterus at around

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Day 4 after oestrus at approximately at the 16-cell stage. Bi-directional communication between the oviduct and embryo is essential for the development of a high quality embryo capable of subsequently establishing and maintaining pregnancy.

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