Theriogenology xxx (xxxx) xxx

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

Challenges in studying preimplantation embryo-maternal interaction

in cattle

María Sa
Beatriz Rodríguez-Alonso a, b, Jose
nchez b, Encina Gonza

lez c, Patrick Lonergan b,
Dimitrios Rizos a, *
a
Department of Animal Reproduction, INIA, Ctra. De la Corun ~ a KM 5.9, 28040, Madrid, Spain
b
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
c
Department of Anatomy and Embryology, Veterinary Faculty, Complutense University of Madrid (UCM), Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: A comprehensive understanding of the complex embryo-maternal interactions during the preimplan-
Received 9 January 2020 tation period requires the analysis of the very early stages of pregnancy encompassing early embryonic
Accepted 11 January 2020 development, maternal recognition and the events leading to implantation. Despite the fact that embryo
Available online xxx
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
Keywords:
embryo production have focused on the fundamental question of why: (i) only 30%e40% of immature
Embryo-maternal interaction
oocytes develop to the blastocyst stage and (ii) the quality of such blastocysts continually lags behind
Oviduct
Uterus
that of blastocysts produced in vivo. Clear evidence indicates that in vitro culture conditions are far from
Embryo optimal with deficiencies being manifested in short- and long-term effects on the embryo. Thus,
In vivo enhanced knowledge of mechanisms controlling embryo
maternal interactions would allow the design
In vitro of novel strategies to improve in vitro embryo conditions and reproductive outcomes in cattle.
© 2020 Elsevier Inc. All rights reserved.

1. 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 se-
ries 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 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 for-
mation on Day 6e8 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
* Corresponding author.
E-mail address: drizos@inia.es (D. Rizos).
inner cell mass) [3]). On Days 9e11 post-fertilization, due to me-
chanical 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 un-
dergoes 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 [5e7]. Spatiotemporal alterations in the endo-
metrial transcriptome and histotroph composition are hypothe-
sized 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 high-producing dairy cows [13]. Given the economic importance
of the livestock industry, it is relevant to develop new strategies

https://doi.org/10.1016/j.theriogenology.2020.01.019
0093-691X/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
2 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx

capable of improving the pregnancy rates by reducing early em-
bryonic 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 re-
view 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.
2. Embryo-maternal crosstalk
2.1. Embryo-oviduct communication
In spite of the fact that the oviduct is not essential for repro-
duction (i.e. successful pregnancies can be obtained by transferring
in vitro produced embryos directly into the uterus), this organ has a
key role in 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 ach-
ieve 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 [14e18]),
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 envi-
ronment, 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 fertil-
ization and early embryo development) - Fig. 1. Despite the chal-
lenge 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 inter-
cellular 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 pro-
teins, 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-to-cell 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, Ali-
min~ ana 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, RNA-
sequencing of EVs transcripts revealed functions related to exo-
some/vesicles, cilia expression, embryo development and many

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

Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
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 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
Almin ~ ana and Bauersachs, 2019). Furthermore, the crosstalk is
bidirectional; the fact that EVs can be both secreted and received by
the bovine embryo [22e24], and secreted by the oviduct [21,22,25],
suggests that embryo-oviductal communication is a two-way (or
bidirectional) process. Furthermore, other molecules potentially
involved in the embryo-maternal communication such as embry-
otropins [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 post-
partum 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. syn-
chrony between the developing embryo and the maternal repro-
ductive 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 [29e33], 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 asyn-
chronous (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. Re-
sults 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 com-
bination 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 sur-
vived 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 syn-
chronous group). These findings highlight the importance of syn-
chrony between the embryo and the oviduct in achieving a
successful pregnancy and suggest that an asynchronous environ-
ment could be among the factors influencing early embryo mor-
tality. Moreover, this could explain inadequacies in in 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:
2.1.1. Oviduct effects on the embryo
The relevance of understanding the oviduct effects on the em-
bryo (and thus its response) resides in the fact that the post-
Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
3
fertilization culture environment has a major influence on em-
bryo 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 com-
ponents 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].
2.1.2. 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].
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 immu-
nologically 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 re-
ported 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]. How-
ever, 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
4 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
size of the bovine embryo (z120 mm 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 em-
bryos, [50]) and cyclic heifers was assessed by qRT-PCR. Compari-
sons between (i) the section in which the embryo was found
(ipsilateral oviduct) vs the corresponding section from the contra-
lateral 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 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 devel-
opment (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 em-
bryo 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 unstimu-
lated heifers. In addition to the evaluation of the effect of the
Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
embryo, the influence of the oviduct region on OF composition was
assessed. Results revealed that, on Day 3 post-oestrus, OF compo-
sition varied based on (i) anatomical region, where isthmic me-
tabolites 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).
2.2. Embryo-uterine communication
Once the embryo enters the uterus, an appropriate communi-
cation between the developing conceptus and the maternal endo-
metrium 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 communi-
cation [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 devel-
opment?, (ii) when and how does the embryo-uterus communi-
cation happen?, and (iii) is the endometrium able to detect
differences in embryo quality that may account for early pregnancy
loss?
2.2.1. 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 [65e68], 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 signifi-
cant proportion of embryos degenerate before the blastocyst stage
in unstimulated high-producing dairy cows. Moreover, studies us-
ing multiple ET have reported a significant variation in size be-
tween age-matched conceptuses regardless of the embryo source
(in vivo or in vitro) or P4 concentrations [69e72]. 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 fac-
tors, such as the inability of the uterus to support conceptus growth
and implantation [74]. Indeed, elongation has not been recapitu-
lated in vitro (in vivo and in vitro produced bovine blastocysts must
be transferred to a 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
 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
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 tem-
poral 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 [70,78e80]. In addi-
tion 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 [81e85]. In line with this, early ET studies estab-
lished 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 [86e88]. 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 contralat-
eral 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 regu-
lating 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 [89e92]. 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 (Bage
s-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 im-
mune response, 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 at-
tempts) 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 [93e95]. 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 be-
tween fertility-classified heifers were minimal (26 DEG HF vs. SF;
12 DEG SF vs. IF; 3 DEG HF vs. IF). The results indicate that preim-
plantation 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
Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
5
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 conceptuseendometrial
interactions in SF animals.
As with the oocyte, metabolic factors also affect the endome-
trium [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 devel-
opment 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 RNA-sequencing on intercaruncular endometrial
samples from pregnant animals on Day 19 (n ¼ 5 lactating and
nonlactating pregnant cows by ET; n ¼ 4 pregnant heifers by AI),
Bauersachs et al., [96], concluded that metabolic status of the ani-
mal (lactating vs. dry and heifer vs. cow) modulates the response of
the endometrium to the developing conceptus.
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.
2.2.2. 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 signal-
ling, 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 evi-
dence 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 endo-
metrium 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 pre-
6 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
elongating 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 embryo-derived 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 endometrium to the embryo is depen-
dent on the number of embryos present (minimum of 5 blasto-
cysts); (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 blastocyst-conditioned me-
dium alter the expression of ISGs in the endometrium; and (v) all of
the DEG (n ¼ 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.
2.2.3. 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 (30e40%) compared with AI or embryos
recovered nonsurgically from donor animals (50e70%) [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 pro-
duction 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 implanta-
tion. 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, acti-
vate the mechanism to disrupt or, in contrast, to maintain the
pregnancy?
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 [108e110]. To
further understand this phenomenon, we combined in vitro and
Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
in vivo production of bovine blastocysts, multiple ET techniques, a
conceptus-endometrial 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 IFNT-dependent. 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 vitro-derived) 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, respec-
tively. Biological processes associated with genes up-regulated
uniquely by in vivo-derived conceptuses, both dependent and in-
dependent of IFNT, were related to inflammation and tumour ne-
crosis factor (TNF) and nuclear factor kappa-light-chain-enhancer
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 in-
volves 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 defini-
tively established. While significant differences in the tran-
scriptomes 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 con-
ceptuses (1.8 ± 0.3 mm) [72]. We concluded that bovine endome-
trium 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 sur-
vival. In particular, short 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
maternaleembryo communication at this critical time are IFNT-
independent.
3. 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 car-
ried out aiming to understand the role of oviductal and uterine
components (i.e. epithelial cells and fluids) using in vitro models. As
 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
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 [117e121]. 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 dedifferenti-
ation process [115] and a decrease in the expression of some genes
[119,123] such as the oviduct-specific oestrus-associated glyco-
protein gene [115] e 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 pro-
vided 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 morphoge-
netic proteins (BMP) has been described. 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 sys-
tems, the cells have their apical surface directed outwards, and
constitute an adequate well-defined short-term culture for func-
tional 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 non-esterified 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 pro-
cedures, suggesting further improvement of the model by: a)
Please cite this article as: Rodríguez-Alonso B et al., Challenges in studying preimplantation embryo-maternal interaction in cattle,
Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.019
7
simulation of the hormonal changes taking place during the peri-
conceptional 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 endome-
trium under the hormonal control of estrogen and P4, the presence
of both epithelial and stromal cells, and the initiation 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 anti-inflammatory
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 ex-
plants [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 self-organization, exhibiting similar
organ functionality as the tissue of origin [139]. Recently, endo-
metrial 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 [141,142] or uterine fluids (UF) [143] during embryo culture.
Our group has demonstrated that the use of low concentrations 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 [25,145,146].
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 con-
centration of EVs secreted from day 7 to day 9 varies depending on
embryo competence and origin. Furthermore, we observed that EVs
8 B. Rodríguez-Alonso et al. / Theriogenology xxx (xxxx) xxx
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 communi-
cations 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 endo-
metrium, 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], high-
lighted 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.
4. 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 pro-
duction 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.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Spanish Ministry of Science,
Innovation and Universities (AGL2015-70140-R); Science Founda-
tion Ireland (13/IA/1983); and European Union H2020 Marie Sklo-
dowska-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)”.
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