1. Update TRENDS in Endocrinology and Metabolism Vol.14 No.5 July 2003 197
|Research Focus
The role of estrogen in uterine receptivity and
blastocyst implantation
Carlos Simon1,2, Francisco Domı´nguez1, Diana Valbuena1 and Antonio Pellicer1,2
1Foundation of the Instituto Valenciano de Infertilidad Foundation (FIVI), Plaza de la Policia Local 3, 46015, Valencia, Spain
2Department of Pediatrics, Obstetrics & Gynecology, School of Medicine, Valencia University, Valencia, Spain
The endometrium is a specialized, hormonally regu-lated
organ that is non adhesive for embryos through-out
most of the reproductive cycle in mammals. Thus,
the window of implantation is a self-limited period in
which the endometrial epithelium (EE) acquires a func-tional
and transient ovarian steroid-dependent status.
The luminal EE initiates the adhesion of the developing
blastocyst during this period, owing mainly to the pre-sence
of progesterone (P) after appropriate 17b-estra-diol
(E2) priming in humans or because of the addition
of E2 after appropriate P priming in rodents. Wen-ge
et al. have now demonstrated in mice that low levels of
exogenous E2 can maintain the window of receptivity
for an extended period of time, whereas high doses of
E2 can rapidly initiate a refractory state. In summary,
levels of E2 within a very narrow range determine the
duration of the window of implantation in uterine
receptivity in mice. These outstanding results demon-strate
the possibility of manipulating the receptivity
window with the use of different doses of E2.
Endometrial receptivity is a self-limited period in which
the endometrial epithelium permits blastocyst adhesion
[1]. In humans, this period, termed the ‘window of
implantation’, initiates four to five days after progesterone
(P) production or administration and ends nine to ten days
later. The ‘open’ window is thus limited to days 19–24 of
the menstrual cycle in humans [2] and to days eight to ten
postovulation in other primates [3]. Indeed, the adminis-tration
of P antagonist [4] or 17b-estradiol (E2) antiserum
[5] during the preimplantation period disrupts endo-metrial
receptivity in primates. P administration following
E2 priming is routine practice in ovum donation programs
for inducing a clinical endometrial receptive window,
thereby making it possible to synchronize the timing of
embryo transfer [6]. In mice, the uterus becomes receptive
on day four of pregnancy or pseudopregnancy and proceeds
to the refractory state on day five [7].
Ovarian steroids, acting through their endometrial
nuclear receptors, effect an alteration of expression
patterns in the uterus that provoke the receptive status.
Gene-knockout strategies reveal that several genes are
crucial for embryonic implantation in mice. For example
Lif (leukemia inhibitor factor), Dtr (diptheria toxin
receptor), Ptgs2 (prostaglandin-endoperoxide synthase 2)
and Il11a (interleukin 11 receptor) [7] promote both
embryo attachment and stromal decidualization [8].
However, global gene expression analysis suggests that
the regulated expression of a wide range of genes is
required for endometrial receptivity both in humans
[9–12].
Estrogens as crucial determinants of the window of
implantation in mice
Wen-ge et al. [13] investigated the role of estrogen in
determining the window of implantation in mice with the
use of the P-treated delayed-implantation model. In a first
set of experiments, different doses of E2 (1.5, 3,10 or 25 ng
per mouse) were injected at day seven of pseudopregnancy
immediately before blastocyst transfer. Implantation sites
were examined 48 h later (Fig. 1a) and stablished that the
optimal range of exogenous E2 for inducing implantation
was between 3 and 25 ng. The authors then determined
the effects of different levels of estrogens on the duration of
the window of endometrial receptivity by repeating the
experiment, this time administering a second E2 injection
(3 ng) on day eight (Fig. 1b). The results obtained showed
that mice treated with 1.5 or 3 ng as the first dose achieved
implantation but, the uterus became refractory in those
that received high doses of E2 (10 or 25 ng). Increasing the
second dose to 10 or 25 ng did not improve implantation
rates.
Further experiments were conduced by delaying the
second dose of E2 (3 ng) until days nine, ten, eleven and
twelve of pseudopregnancy, immediately after blastocyst
transfer (Fig. 1c). Again, the closing of the window of
receptivity was postponed for at least four days in most
mice if the first E2 dose was low (3 ng). By contrast, when
the first injection was 25 ng, the uterus became refractory
within 24 h and remained refractory for the next 72 h.
What was responsible for this interesting outcome? The
authors investigated the possibility that aberrant gene
expression induced by high doses of E2 had caused this
effect. They examined the temporal expression of Lif,
Hoxa10, Dtr, Areg, Ptsg1 and Ptsg2 by in situ hybridization
in the uterus of animals previously investigated. The
expression of these genes at the implantation site
remained normal when only one dose of E2 (3–25 ng)
was administered at day seven or when the first injection
was 3 ng. However, when the first injection was 25 ng, the
uterus showed aberrant expression of several genes,
suggesting that the uterus becomes refractory at higher
Corresponding author: C. Simon (csimon@interbook.net). E2 levels owing to the altered expression of these genes.
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2. 198 Update TRENDS in Endocrinology and Metabolism Vol.14 No.5 July 2003
3–25 ng
1.5 ng
10–25 ng
1.5 ng
24 48 72 96 120 hours
4 5 6 7 8 9 10 11 12 13
Blastocyst
transfer
3 ng
Implantation
No implantation
Refractory
Receptive
Pre-receptive
TRENDS in Endocrinology & Metabolism
Uterine sensitivity
to E2
Uterine sensitivity
to E2
Days of pseudopreganancy
Ovariectamized mice E2 injection
Fig. 1. The experiments performed by Wen-ge et al. [13] (a) Different doses of E2 (1.5, 3, 10 or 25 ng per mouse) were injected at day seven of pseudopregnancy immediately
before blastocyst transfer. Implantation sites were examined 48 h later (Fig. 1a) and it was established that the optimal range of exogenous E2 for inducing implantation
was 3–25 ng. (b). To determine the effects of different levels of estrogens on the duration of the window of endometrial receptivity, a second E2 injection was administered
(3 ng) on day eight. The results obtained showed that mice treated with 1.5 or 3 ng as the first dose achieved implantation but, the uterus became refractory in those that
received high doses of E2 (10 or 25 ng). Increasing the second dose to 10 or 25 ng did not improve implantation rates. (c) The second dose of E2 (3 ng) was delayed until
days nine, ten, 11 and 12 of pseudopregnancy, immediately after blastocyst transfer. Again, the closing of the window of receptivity was postponed for at least four days in
most mice if the first E2 dose was low (3 ng). By contrast, when the first injection was 25 ng, the uterus became refractory within 24 h and remained refractory for the
next 72h.
Application to humans
The mouse has become an indispensable model for the
study of endometrial receptivity and implantation, yet
differences between species mean that we must be
cautious about applying the results obtained to a human
context. First, the hormonal regulation that leads to
endometrial receptivity is not identical in humans and
mice. In humans, the implantation window opens owing to
the presence of P after appropriate E2 priming, whereas in
rodents it is the addition of E2 after appropriate P priming
that induces the receptive phenotype. Second, there are
important differences in the genomic of endometrial
receptivity between the two species. Genes that are
functionally crucial for implantation in mice, such as Lif
[8] or Ptgs2 [7], were not detected as regulated genes in
global gene expression analysis in humans [10–12].
However, global gene expression in mice compared the
expression profiles of the implantation versus interim-plantation
sites, whereas in humans, similar analysis
compared the expression profiles of the receptive with the
non-receptive endometrium in the absence of implanting
embryo. Thus, comparing the results obtained in mice with
those of humans in the absence of embryo–uterine cross-talk
might provide limited information.
Assisted reproductive technologies (ART) have
provided much insight into human reproductive processes,
but lower implantation rates are a major problem. In
ovarian hyperstimulation (COH) protocols, the impact of
supraphysiological levels of E2 on the day of human
coriogonadotropin (hCG) administration on human endo-metrial
receptivity is a matter of debate, because different
clinical studies have produced different results. It was
previously demonstrated that, in high responder patients,
high serum E2 levels (.3,000 pg ml21) on the day of hCG
administration are detrimental to uterine receptivity [14],
regardless of the number of oocytes retrieved or serum P
levels. In addition, an increase in serum E2 levels during
the preimplantation period in high responders was
documented, which was not observed in normal responder
patients, suggesting that this abnormal endocrine milieu
is responsible for an impaired implantation [14]. Moreover,
decreasing E2 levels during the preimplantation period by
a step-down protocol increases implantation and preg-nancy
rates in high responder patients [14]. Finally, an
established in vitro model for embryonic adhesion has used
in which E2 dose–response and time-course experiments
are performed to establish whether the effect of E2 targets
the embryo and/or the endometrium and to test whether
late embryo transfer is a possible approach for minimizing
the unwanted effects of high E2 on embryo implantation.
(a)
(b)
(c)
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3. Update TRENDS in Endocrinology and Metabolism Vol.14 No.5 July 2003 199
With the use of this in vitro model, it was corroborated that
E2 concentrations of $1026
M reduce embryo adhesion [15].
However, although E2 reduces the receptivity of the endome-trium,
it is sensible to also consider the embryo a target [15].
Wen-ge et al. [13] demonstrate in mice that the uterus
can be maintained in a receptive status with low doses of
E2, but that the uterus becomes refractory in response to
high doses of E2. Further results from human clinical
studies indicate that in patients displaying a high
response to gonadotrophins, supraphysiological levels of
E2 on the day of hCG are deleterious to embryonic
implantation [14,15]. Regardless of differences between
the species, this research provides valuable information
for this field, and should be an important reference in the
pursuit for improved endometrial receptivity in ART.
References
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mid-luteal (receptive phase) transition in human endometrium
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1043-2760/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S1043-2760(03)00084-5
Prolactin-induced neurogenesis in the maternal brain
Robert S. Bridges1 and David R. Grattan2
1Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, MA 01536, USA
2Centre for Neuroendocrinology, Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand
New and exciting findings reported by Shingo and
colleagues indicate that the hormone prolactin stimu-lates
neurogenesis in adult female mice. New neurons
produced in the forebrain during pregnancy and lacta-tion
migrate to the olfactory bulb where they likely
participate in processing olfactory cues received by the
new mother as she adapts to the needs and challenges
of raising young.
Using a mouse model, Shingo and colleagues [1] found that
both during early pregnancy and lactation, new neurons
originated in the subventricular zone (SVZ) of the mouse
forebrain and migrated to the periglomerular and granule
layers of the olfactory bulb, where these new neurons
differentiated and established functional connections.
This neurogenesis was evident on day 7 of pregnancy as
well as day 7 of lactation. It was also apparent in
pseudopregnant females, showing that embryo implan-tation
was not required, and that the response could
be generated solely by changes associated with mating
and early pregnancy. Finally, they demonstrated that
neurogenesis was inducible by either systemic or
central administration of prolactin (PRL), a hormone
significantly elevated during these reproductive states,
acting via neural PRL receptors. This is a particularly
exciting finding, because it is the first demonstration
that a hormone can stimulate the genesis, migration
and differentiation of neurons in the adult mammalian
brain.
Mechanism of the action of PRL
This elegant set of studies convincingly demonstrated
that the actions of PRL appear to be mediated by PRL
receptors in the SVZ. PRL receptor immunoreactivity for
the short form of the PRL receptor measured by
fluorescence microscopy was present in the dorsolateral
corner of the SVZ and in the choroid plexus. Furthermore,
the enhanced neurogenesis on day 7 of pregnancy, as
measured by the number of bromodeoxyuridine (BrdU)-
labeled cells in the SVZ, was attenuated in mice
heterozygous for the PRL receptor (PRLrþ/2) when
Corresponding author: R.S. Bridges (robert.bridges@tufts.edu). compared with the number of BrdU-labeled SVZ cells in
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