Flores et al.

Reproductive Biology and Endocrinology 2014, 12:38

http://www.rbej.com/content/12/1/38

RESEARCH

Open Access

Early detection and staging of spontaneous

embryo resorption by ultrasound biomicroscopy
in murine pregnancy
Luis E Flores1, Thomas B Hildebrandt1, Anja A Khl2 and Barbara Drews1*

Abstract
Background: Embryo resorption is a major problem in human medicine, agricultural animal production and in
conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse
model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to
reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect
embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound.
Methods: In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high
frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology
of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the
live ultrasound data with the respective histology.
Results: The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth
retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the
embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and
8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill
defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days,
the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled
caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and
lacunae filled with maternal blood were observed. In later stages (day 911) resorption prone embryos were one
day behind in their development compared to their normal siblings. The space between Reicherts membrane and
inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately
cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still
intact. In the subsequent resorption process first the embryo and then its membranes disappeared.
Conclusions: Our results provide a temporal time course of embryo resorption. With this method, animals
exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the
onset of total embryo disintegration.
Keywords: Embryonic failures, Yolk sac, Reicherts membrane, Embryo resorption, Ultrasound biomicroscopy,
Murine pregnancy, Placenta

* Correspondence: [email protected]

Equal contributors
1
Department Reproduction Management, Leibniz Institute for Zoo and
Wildlife Research (IZW), Alfred-Kowalke-Str. 17, 10315 Berlin, Germany
Full list of author information is available at the end of the article
2014 Flores et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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Background
Embryo resorption is not only a major problem in human
reproductive medicine [1,2] but also in agricultural animal
production [3] and in conservation breeding programs
(reviewed in Andrabi and Maxwell [4]). The underlying
mechanisms are manifold and include chromosomal anomalies [5], placental insufficiency [6] and disturbances in the
feto-maternal immune tolerance [7].
Studies on embryo resorption in humans are restricted
due to ethical reasons. Here, the mouse serves as a biomedical model. Its small size and its fast reproduction
mode enable large-scale studies. Moreover, knock-out
strains allow for a straight forward functional analysis of
genes involved in the establishment of pregnancy [8,9].
Up to date, studies on embryo resorption are performed
mainly post mortem [10-12] and pregnant animals are
sacrificed at certain days of pregnancy to determine the
resorption rate. The resorption rate is defined as the ratio between the number of resorptions and the number
of normal implantations [13]. The inaccuracy of this
method is rooted in the uncertain time point of embryonic death. Counting the number of resorptions on e.g.
day 12 post ovulation might very likely include embryos
that died considerably earlier in pregnancy. To yield a
reliable result on the actual rate of resorption in post
mortem studies, high animal numbers are needed to systematically evaluate every day of pregnancy. If animals
are sacrificed randomly, the identification of the cause of
embryo death and subsequent resorption is difficult due
to the rapid disaggregation of embryonic structures. In
studies on early embryo loss, among hundreds of implantation sites, only one conceptus was either not completely
normal, or completely destroyed but in a state of early resorption [10]. This fact is a major concern for the investigation of feto-maternal immune interactions, where cause
and effect are especially difficult to distinguish.
Ultra-high frequency ultrasound or so called Ultrasound
Biomicroscopy, (UBM) can depict structures smaller than
0.1 mm and enables in vivo monitoring of prenatal development in small animals. UBM has been used to highlighten the peculiarities of the long pregnancy of the
naked mole rat [14] and to describe embryo development, embryo resorption and corpus luteum regression
in the European brown hare [15,16]. In mouse development, UBM has been employed to establish growth
graphs for determination of gestational age [17] and to
describe the gross development of the mouse embryo
[18-20]. Phoon et al. have analyzed embryo cardiovascular function by UBM [21-23]. Most recently, UBM
has been used to evaluate the effect of defined quantitative trait loci on embryo lethality in a mouse model of
interspecific recombinant congenic strains [20,24]. In
these studies, the number of dead and living embryos
was assessed on defined days of gestation. To date,

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there is no in vivo description of the process of murine

embryo resorption.
In our study, we aimed to identify conceptuses undergoing spontaneous resorption at the earliest possible
stage by UBM. The dynamics between mother, placenta
and embryo of implantation sites under resorption were
compared to their normally developing littermates. Different stages of embryo resorption were exemplarily
evaluated by histology to verify UBM data. A time line
of the process of spontaneous murine embryo resorption
was established. On the basis of this study, growth retarded embryos and embryos that undergo resorption
can be identified in vivo.

Methods
Animals

All experimental work on live animals complied with our

institutional and governmental regulations (TierschutzVersuchstierordnung). The institutes committee for animal welfare and ethics and the State Office of Health and
Social Affairs Berlin approved the experimental design
(letter 03.11.2010) in accordance with 8a of the German
law of animal welfare (Tierschutzgesetz). Mice from the
inbred C57BL/6 strain were obtained from Harlan Laboratories, Rossdorf, Germany. A total of 30 females and 4
males were kept in open top-wire cages under a 12 h
lightdark regime with food and water ad libitum. A
microchip implant was used for individual identification
(Hong Teng Technology, Guangzhou, China). Mice were
mated for a period of 3 days in breeding groups comprised
of 4 females and one male.
Examination of pregnancies

Successful mating was confirmed by the presence of a

vaginal plug after establishment of breeding groups.
In some animals, a vaginal plug could not be detected
but pregnancy was confirmed by ultrasound. With the
exception of four animals (ID 4, 6, 7 and 13) ultrasound
examinations were performed on a daily basis starting
on day 4 after establishment of the breeding groups.
Pregnancy could be confirmed earliest by the ultrasonographic visualization of decidualized implantation sites
on day 5 after establishment of the breeding groups. If
implantation sites were detected one or two days later
(day 6 and 7 after establishment of breeding groups), we assumed that mating had occurred later, too. Consequently,
the day of the first visualization of the implantation sites
was always referred to as day 5 of pregnancy. If no pregnancy could be confirmed 8 days after establishment of
breeding groups, the animal was considered non-pregnant
and was mated again. For the ultrasound examination, an
Ultrasound Biomicroscope (Vevo 2100, Visual Sonics,
Toronto, Canada) equipped with a 30-70 MHz transducer
(MS700) was used. The ultrasound settings were standardized

Flores et al. Reproductive Biology and Endocrinology 2014, 12:38

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as follows: frequency50 MHz, power 100%, gain 30 db.

Prior examination, mice were anesthetized using inhalation anesthesia via a mask. 5% isoflurane (CP-Pharma,
Burgdorf, Germany) was delivered for induction and
1.5%-2% for maintenance with an oxygen flow of 1 l/min.
To avoid hypothermia, animals were placed on an electric
heating mat and the ultrasound gel for the transducer
was warmed in a water bath before use. To ensure optimal image quality, the abdominal hair was removed
using commercial chemical hair removal gel (Veet, Kln,
Germany). In the course of the ultrasound examination
the number of conceptuses in each uterine horn was
determined. The viability and the staging of the conceptuses was evaluated according to biometric measurements
and morphologic parameters. Biometric measurements
included the size of the implantation site, the size of
the embryonic cavity (EC), the crown-rump-length (CRL)
the biparietal distance (BPD) and placental measurements.
The size of the implantation site was determined by averaging two perpendicular maximal diameters. The size of
the EC was measured in its maximal diameter. Morphologic parameters were the differentiation of decidua basalis (DB) and decidua capsularis (DC), formation of the
ectoplacental cone (EPC), the presence of embryonic
membranes and the presence and quality of heartbeat.
The duration of the scanning procedures ranged from 10
to 20 minutes per individual

Relevant ultrasound data was recorded for each conceptus. The resorption process was documented by UBM and
the respective animals were sacrificed at defined days after
the onset of embryo resorption for histological analysis.
Whole conceptus collection and processing

For the collection of normal conceptuses and conceptuses under resorption, the mouse was euthanized by
cervical dislocation and the reproductive tract was removed. The number of healthy embryos and resorption
sites was counted and photographed. The uterus was examined by UBM in a water bath with a 0.9% physiological saline solution (Braun, Melsungen, Germany) to
verify the in vivo ultrasound data. A solution of 4%
paraformaldehyde in 1x phosphate buffered saline with
a pH 7.4 was used for fixation. A standard protocol for
paraffin embedding and sectioning was followed. Sections had a set 35 m thickness. For morphological
analysis sections were stained with hematoxylin and
eosin (H&E) following standard protocol.

Results
In total, we followed 30 pregnancies in 30 different females by longitudinal UBM examinations. The mean
number of implantation sites per animal was 7.5 with
a range of 1 to 12 implantations per animal. Embryo

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resorptions were identified between day 7 and 13. In

total, 23 resorptions (R1-R23) were detected in 15 pregnancies. Taking all 30 pregnancies into account this resulted in a resorption rate of 10.22% (N = 225 normal
implantations versus 23 resorptions). To verify and supplement the ultrasound data, an exemplarily collection
of resorption sites for histological analysis was obtained.
The time points of detection and collection of resorption
are summarized in Table 1. In three animals, older resorption sites (R9, R10, R12 and R14) that had occurred
earlier in pregnancy were collected together with a fresh
resorption site that was identified later in pregnancy
(R11, R13 and R15).
The number and location of the conceptuses was
determined in every ultrasound examination

On day 5, we underestimated the total number of implantation sites by two in one animal, and by one in four
animals. On day 6, the number of conceptuses in these
animals was corrected and confirmed in subsequent examinations. On day 7, we counted one embryo twice in
one animal. The localization of the conceptuses in the
right and left uterine horn respectively was always correctly determined with the exception of one embryo on
day 6. This mistake was also corrected one day later during the next examination. Apart from these cases, the
number and position of embryos during in vivo examinations were consistent with the number and position of
the conceptuses and resorption sites as derived from the
exteriorized uterus and water bath examinations.
The central observations are life stream scans of embryos under resorption compared with their adjacent normal litter mates. Representative scans are documented in
the supplementary movies, which are much more informative than the single frames in the figure plates. Figure 1
shows an overview of the results. The major events of normal development are summarized on the abscissa and on
the ordinate the major observations in the respective embryos under resorption are shown.
Normal embryo development on days 5 to 8

By UBM, pregnancy could be diagnosed earliest on day

5. The implantation sites appeared as beadlike protrusions of the uterus measuring in average 1.95 mm in
diameter (SD = 0.25, N = 71), attributed to an extensive
decidualization of the endometrium (Figure 2A). An
additional movie file shows the normal development on
days 6 to 8 (see Additional file 1: Movie S1). On day 6,
the diameter of the implantation site had increased
to 2.28 mm (SD = 0.39; N = 71) (Additional file 1:
Movie S1, Figure 2B). The thick decidualized endometrium
appeared hyperechogenic compared to the surrounding
thin myometrium. Between implantation sites, endometrium and myometrium were difficult to differentiate. The

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Table 1 Ultrasonographic detection of embryo resorption and day of collection

Mouse ID

ID of 1st
resorption

Detection of 1st
resorption (day)

ID of 2nd
resorption

Detection of 2nd
resorption (day)

R1

d7

R2

d7

R3

d7

R4

d7

R5

d8

R7

R8

ID of 3rd
resorption

Detection of 3rd
resorption (day)

Number of
resorptions per
animal

Collection of
resorption sites
(day)

d8

d8

R6

d8

R9

d7

R10

d7

R12

d7

R13

d9

R11

d9

R14

d8

R15

d9

10

10

R16

d9

R17

d9

11

11

R18

d10

10

12

R19

d9

R20

d9

10

13

R21

d12

12

14

R22

d12

12

15

R23

d13

13

Figure 1 Timeline of embryo resorption. Ultrasonographic markers of normal development are outlined on the x-axis. The boxes on the y-axis
describe the different stages of resorption. The day of detection of the different resorption stages are given in brackets for each resorption site of
this study (R1-R23). The day of collection of the resorption sites is indicated by the cross symbol. Observations of follow up exams are aligned by
arrows. EPC ectoplacental cone; *embryo under resorption located outside the embryonic cavity.

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Figure 2 Normal development. (A) day 5. Note the uterine lumen between the two decidualized endometrial layers (arrows). (B) day 6. The
myometrium has a lower echogenicity (arrowheads) compared to the decidualized endometrium. The high echodensity spot indicates the embryo. (C)
Histological section of implantation site shown in (B). (D) Day 7. The embryonic cavity and the embryo proper are visible. Decidua capsularis and basalis
are differentiated. (E) Day 8. Embryo with amnion and allantois. (F) Day 9. The hyperechogenic decidua capsularis is stretched out at the antimesometrial
side and merges into the decidua basalis at the mesometrial side. The embryonic brain ventricles and the neural canal are visible. (G) Histological section
of implantation site shown in (F). Reicherts membrane and yolk sac membrane are only visible in the histologic section. In vivo, these membranes are
stretched out against the decidua and the placenta. (H) Day 10. The aortic arches and the mesencephalon are depicted. (I) Histology of the same embryo
as in (H). (J) Day 11. The placenta displays hyperechogenic calcification deposits (arrowheads) at the fetomaternal boundary. (K) Histological section of
placenta of same embryo as shown in (J). The giant trophoblast is disappearing. (L) Histology of same embryo as in (J and K) outlining the transition zone
between decidua capsularis, decidua basalis and new uterine lumen. AA - Aortic arches; Al Allantois; Am - Amnion; DB - Decidua basalis; DC - Decidua
capsularis; Dec Decidua; EC Embryonic cavity; ECC Excocoelomic cavity; Em- Embryo; EPC Ectoplacental cone; FE Fetal erythrocytes; He Heart;
La Labyrinth; Mes Mesencephalon; Ms Mesometrium; Myo Myometrium; NC Neural canal; nUL new uterine lumen; oUL old uterine lumen;
RM Reichert's membrane; Pc Pericardium; Pl Placenta; St Syncytiotrophoblast; UC Umbilical connection; UL Uterine lumen; UV Umbilical vessel;
VV Vitelline vessel; YS Yolk sac.

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uterine lumen was still visible. At that stage, the embryo

was located in its yolk sac cavity (Figure 2C). On day 7,
the embryo-maternal interface was characterized by a
bright echogenic ring and the wedge-shaped ectoplacental
cone protruded in the decidua basalis (Additional file 1:
Movie S1, Figure 2D). The embryonic cavity measured
0.54 mm in average (SD = 0.20; N = 80) and could be
subdivided into proamniotic, ectoplacental and exocoelom
cavity. The embryo proper could not yet be reliably identified (Figure 2D). On day 8, the S-shaped embryo started
to turn around its own axis. The allantois, which later gives
rise to the umbilical vessels, was visible. It transversed the
exocoelomic cavity and connected with the chorion to provide the embryonic vascular component for the chorioallantoic placenta (Additional file 1: Movie S1, Figure 2E).

disappeared. Only the Reichert's membrane was found in

the original embryonic cavity (Figure 3F). Between the
Reicherts membrane and the giant trophoblast layer in
the transition zone of the decidua capsularis, a massive influx of maternal blood was apparent. The central artery
was also filled with blood. Maternal erythrocytes, neutrophils and lymphocytes could be seen in the allantois, but
no embryonic erythrocytes were detected (Figure 3G).
In the resorptions R5, R7 and R14 no embryo or embryonic membranes were visible by ultrasound. The fluid
in the embryonic cavity was echodense. In the histologic
section (R10) only the Reichert's membrane was left (not
shown), as observed in R7. This resorption stage was
transformed into the typical echodense tissue with surrounding caverns within 24 h (R5, R8 and R14).

Embryo resorptions on day 7 and 8

Normal development on days 9 to 13

Identification of embryo resorption was first possible on

day 7 when the ectoplacental cone was visible. On day 7,
seven implantation sites were suspicious for resorption
(R1, R3, R2, R4, R9, R10, R12) because their embryonic
cavities were smaller than in their litter mates (EC =
0.30 mm; SD = 0.07; N = 7) and the ectoplacental cone
was not well defined. In R1 and R12 the fluid in the embryonic cavity, which could not be further differentiated,
was filled with clear fluid (Figure 3A) while on day 8, the
fluid was echodense (Figure 3B). In the histological section, the embryonic cavity was filled with proteinaceous
material and the placentation site consisted of condensed
trophoblast tissue and maternal haemorrhage (Figure 3C).
In four resorptions (R2, R4, R9, R10) the embryonic fluid
was already echodense on day 7. By day 8, the embryonic
cavities had disappeared and the implantation sites were
transformed into echodense tissue surrounded by fluid
filled caverns. A hyerechogenic spot was detected in the
periphery of the resorption sites. Histological analysis
showed that the caverns corresponded to maternal haemorrhage in the decidua basalis and the hyperechogenic
spot to fibrinoid tissue.
On day 8, four resorptions (R5, R6, R7 and R14) were
detected on the basis of their small cavities (EC = 0.70 mm;
SD = 0.34; N = 4) compared to 1.35 mm (SD = 0.35;
N = 86) in the normal developing conceptuses.
In one resorption (R6) the embryo proper was visible
but the embryonic cavities were smaller compared to the
normal siblings. This embryo was clearly growth retarded
one day later (CRL = 0.91 mm) compared to a mean CRL
of 1.72 mm (SD = 0.13; N = 6) in its litter mates. In another resorption (R7) the embryonic cavity was also
smaller and the shape of the embryo was ill defined
(Figure 3D). In that resorption (R7), only the embryonic
cavity was left in the follow up exam one day later
(Figure 3E). Histological analysis confirmed that the embryo proper and its inner membranes had completely

On day 9, the originally concave embryo had assumed a

convex curvature and was enclosed in its amnion. Attributed to the inversion of germ layers, the exocoelomic
cavity is the main extraembryonic cavity and the yolk
sac cavity consists merely of a slim slit between exocoelom and Reicherts membrane. The yolk sac membrane
was therefore not visible by UBM. Details of the embryonic morphology such as the mesencephalon and the
neural tube became evident (Figure 2F) and the embryonic heartbeat could be detected (Additional file 2:
Movie S2). In corresponding histological images the exocoelomic cavity had collapsed and the space between the
folded visceral yolk sac membrane and the Reicherts
membrane was artificially enlarged (Figure 2G). In the
yolk sac membrane, numerous blood islands had developed. Nucleated erythrocytes were evident in the allantois. The originally antimesometrial decidua, the decidua
capsularis, consisted of densely packed cells and thinned
towards the mesmometrial pole, where it blended into
the richly vascularised mesmometrial decidua, the decidua basalis. Between the Reicherts membrane and the
decidua capsularis at the abembryonic pole, a layer of
giant trophoblast formed a ring and marked the border
between the placenta and the decidua basalis at the
mesometrial side.
Between days 10 and 13, the embryo considerably enlarged in size. The pericard and the heart with its atria
and ventricles could be clearly distinguished by ultrasound, as well as the aortic arches (Figure 2H and I). The
placenta exhibited a similar echogenicity as the decidua
basalis but could be differentiated by its pulsating blood
vessels and by a layer of higher echogenicity between
embryonic trophoblast and maternal decidua (Figure 2J,
Additional file 3: Movie S3). In the histological sections,
the placenta had differentiated in its placental labyrinth,
spongiotrophoblast and giant cell layer (Figure 2K). A transition zone between decidua capsularis, decidua basalis

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Figure 3 Embryo resorptions R1, R4, R7, R15 and R16. (A) R1, day 7. The resorption site with small embryonic cavity (arrow) lacking a well
defined ectoplacental cone. (B) R1, day 8. Embryonic fluid with increased echodensity. (C) R1, day 8. Maternal hemorrhage in the giant
trophoblast ring and spongious trophoblast in the transition zone. The former placental site is composed of fibrinous tissue infiltrated with
maternal granulocytes (Arrow). (D) R7, day 8. Embryo with ill defined morphology. (E) R7, day 9, scanned post mortem in the water bath. The
embryo has disappeared. (F) R7, day 9. The embryo and its membranes except for the Reichert's membrane have disappeared. The former
embryonic cavity is filled with denaturated proteins. There is a massive maternal hemorrhage between the giant trophoblast and the Reichert's
membrane. The central artery is filled with blood. (G) Placenta of R7. Embryonic erythrocytes are absent in the allantois but maternal
lyomphocytes, neutrophils and erythrocytes are present. (H) Growth retarded embryo R15, day 9. (I) R15, day 10. The embryo exhibited a reduced
heart rate. Pericardial effusion is evident. (J) R16, day 11. The heartbeat has ceased. Pericardium, amnion and yolk sac can be differentiated. (K)
R15, day 10. All embryonic membranes are visible, but the yolk sac is devoid of blood islands. The placental morphology is normal. (L) R15, day 10.
Magnification of areas outlined in (K). Umbilical connection filled with fetal erythrocytes (yellow rectangle). Maternal blood with high proportion
of immune cells between Reicherts membrane and giant trophoblast (green rectangle). Al Allantois; AC Amniotic cavity; CA Central artery;
DB - Decidua basalis; DC - Decidua capsularis; EC- Embryonic cavity; Em- Embryo; GT Giant trophoblasts; La Labyrinth; Ly Lymphocytes;
ME - Maternal erythrocytes; Ne Neutrophils; Pl Placenta; RM Reichert's membrane; UC Umbilical connection; YS Yolk sac.

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and new uterine lumen developed (Figure 2L). At this

stage, the decidua capsularis and giant trophoblast cells
were in the process of disappearing.
Embryo resorptions on days 9 to 13

On day 9, seven resorptions (R11, R13, R15, R16, R17,

R19, R20) were first identified on the basis of growth
retardation. R6, that had already exhibited a smaller
embryonic cavity on day 8 also showed growth retardation
on day 9. The crown rump length of the embryos (mean
CRL day 9 growth retarded embryos = 1.39 mm; N = 8)
was comparable to a developmental stage that was
reached one day earlier in the normal siblings (mean CRL
day 8 normal = 0.99 mm; SD = 0.30; N = 77) (Additional
file 2: Movie S2, Figure 3H). Placental size was also
smaller in the embryos under resorption (Table 2).
Within 24 hours, the growth retarded embryos exhibited bradycardia (92 bpm in resorption prone embryos
versus 130 bpm in normal siblings) and pericardial effusion (Additional file 3: Movie S3, Figure 3I). The size of
the heart corresponded to that of a normally developing
embryo and was therefore proportionally bigger to embryonic body size. Interestingly the resorption prone
embryos were still able to develop further, albeit at a reduced rate (Additional files 3, 4 and 5, Figure 3H and I).
Due to the loss of embryonic fluids, the formerly expanded exocoelomic cavity deflated and the folded yolk
sac membrane became visible by UBM (Additional file 4:
Movie S4, Figure 3J). One resorption prone embryo (R15)
that was first detected on day 9 (Figure 3H) showed a reduced heart beat on day 10 (Figure 3I). It was collected on
day 10 when it was still alive but its heartbeat was barely
detectable. In the corresponding histological sections all
membranes were identified (Figure 3K) though the yolk
sac membrane was lacking the typical blood islands. In
the transition zone, maternal blood had accumulated between the Reicherts membrane and the decidua capsularis, which was still delineated by giant trophoblast cells

(Figure 3L). The umbilical vessel was filled with embryonic erythrocytes (Figure 3L), which underlined the previous ultrasonographic observation of a faint heartbeat
(Additional file 5: Movie S5). Surprisingly, the cells of the
embryo proper showed signs of necrosis to a great extent.
In the next phase of the resorption process, the heartbeat finally ceased. Histological analysis showed that the
resorption process continued with the necrosis of the
embryo proper, which was still surrounded by its membranes. In two cases (R13, R16), the embryo was located
in the uterine lumen due to a rupture of the decidua
capsularis (Figure 4A and B, Additional file 4: Movie S4).
In the histological section of R13, yolk sac and Reichert's
membrane were found inside the embryonic cavity. The
exteriorized embryo was surrounded by its amniotic
membrane. The morphology of its placenta was unsuspicious (Figure 4C). In our study, there were no indications for a first detection of the resorption process on
day 11.
On day 12, two embryos that were unsuspicious on
the previous day were found dead (R21, R22). One embryo (R21) showed reduced embryonic fluids and condensed embryonic tissue (Figure 4D). On day 13 an
embryo that presented a normal morphology had no visible heart beat (R23).
In conclusion, the process of embryo resorption is
characterized by four distinct phases: in the first phase,
growth retardation is observed which manifests in the
reduced size of the embryonic cavities and the delayed
developmental stage of the embryo itself. In the second
phase, the embryo exhibits a reduced, sometimes irregular heartbeat, reduced placental blood flow, detachment
of the yolk sac membrane from the outer Reicherts
membrane and pericardial effusion which subsequently
results in stalling of the heartbeat. In the third phase,
first the embryo disintegrates, then its inner membranes
disappear and finally the placental integrity is lost. In
the final stage of resorption, the implantation site is

Table 2 Crown rump-length (CRL) and placental size of normal embryos and embryo resorptions
day 9

day 10

day 11

day 12

day 13

Placental width (normal embryo)

1.66 mm; SD =
0.32; N = 57

3.14 mm; SD =
0.50; N = 38

4.71 mm; SD =
0.63; N = 21

5.83 mm; SD =
0.14; N = 13

8.03 mm; SD =
1.36; N = 2

Placental height (normal embryo)

0.54 mm; SD =
0.09; N = 57

0.83 mm; SD =
0.15; N = 38

1.17 mm; SD =
0.20; N = 21

1.37 mm; SD =
0.14; N = 13

2.11 mm; SD =
0.07; N = 2

Placental width (resorption)

1.22 mm; SD =
0.38; N = 12

2.40 mm; SD =
0.58; N = 8

3.78 mm; SD =
0.77; N = 4

4.81 mm; SD =
0.98; N = 3

4.22 mm; N = 1

Placental height (resorption)

0.42 mm; SD =
0.09; N = 12

0.60 mm; SD =
0.10; N = 8

1.04 mm; SD =
0.21; N = 4

0.84 mm; SD =
0.21; N = 3

1.18 mm; N = 1

CRL (normal embryo)

2.11 mm; SD =
0.46; N = 72

4.01 mm; SD =
0.54; N = 50

5.71 mm; SD =
0.85; N = 29

7.38 mm; SD =
0.81; N = 18

9.31 mm; SD =
0.70; N = 5

CRL (resorption)

1.39 mm; SD =
0.43; N = 8

2.41 mm; SD =
0.75; N = 6

1.47 mm; SD =
0.44; N = 2

6.54 mm; SD =
1.48; N = 2

5.60 mm; N = 1

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Figure 4 Embryo resorptions R12, R13 and R21. (A) R12 and R13, day 9. In R12, the embryo is no longer visible. In R13, the embryo is still
present but it is located in the uterine lumen. (B) R13, day 9. The decidua capsularis is ruptured. The embryo is encased in its amnion but outside
the yolk sac membrane and Reicherts membrane, which are still located in the original embryonic cavity. The yolk sac membrane exhibits blood
islands and is folded due to fluid loss. Between Reichert's membrane and yolk sac membrane, there is maternal hemorrhage. (C) The placental barrier
is intact. Blood spaces filled with maternal blood are evident as well as embryonic blood vessels containing nucleated erythrocyte. (D) R21, day 12.
There was no visible heartbeat and less fluid in the embryonic cavity. Al Allantois; Am Amnion; DC Decidua capsularis; EC Embryonic cavity;
Em Embryo; GT Giant trophoblasts; MH - Maternal hemorrhage; nUL New uterine lumen; PE Pericardial effusion; Pl Placenta; RM Reichert's
membrane; UC Umbilical connection; YS Yolk sac.

characterized by hypoechogenic caverns and a central,

hyperechogenic spot which correspond to maternal
haemorrhage and fibrinoid tissue, respectively. The time
course of the outlined resorption stages and the respective morphological characteristics vary according to gestational age as outlined above.

Discussion
In our study we described the in vivo process of murine
embryo resorption using UBM and correlated our ultrasound data to histology. The normal development served
as a reference for the successful in vivo detection of embryo resorption.
The resorption followed a specific pattern, independent
from the time in gestation when the resorption process
started. The first sign to presage resorption prone embryos was delayed development or growth retardation. On

the basis of our study growth retardation can be strongly

associated with the resorption process, even though a considerable variability in developmental stage within one litter has been demonstrated in the post mortem study of
Thiel et al. [25]. The developmental difference observed in
the study of Thiel et al. varied by almost one day. However, since these observations rely on post mortem findings, the subsequent development of the smaller embryos
was not documented. We assume that some of embryos
which exhibited the least development were in fact
prone for resorption. Measurements of CRL on day 9
from our study support this possibility since the gestational age difference between embryos of the same litter
was not greater than half a day if we excluded the resorption prone embryos.
In early pregnancy stages, growth retardation manifested
in smaller embryonic cavities and an undifferentiated

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shape of the embryo itself. In our study, a smaller embryonic cavity suggestive for resorption could be first observed on day 7.
On day 8, resorption prone conceptuses could be identified by their smaller size. In some cases, the embryo itself first developed but its morphology was ill defined
and it then disappeared within 24 hours. The detection
of embryos under resorption that early in gestation is
therefore highly dependent on the level of experience of
the sonographer, considering that the morphological
changes are delicate and occur at a fast rate. It seems
that with advancing development, the time period between the first signs of growth retardation and death of
the embryo is elongated, making it more likely to identify living but resorption prone embryos. Beginning on
day 9, when the heart beat was reliably detectable, the
second stage of the resorption process could be visualized. Here, the dysfunction of the embryonic circulation
manifested in bradycardia, reduced placental perfusion,
and pericardial edema.
Another finding, the visualization of the shrivelled
yolk sac membrane by UBM which is not evident in
normal embryos might account for the fact that the
limited and finally ceased production of embryonic
fluids reduces the physiological turgor of the exocoelomic cavity. This eventually leads to an artificial increase
of the yolk sac cavity.
The third phase of the resorption process implies the
death of the embryo. In other studies, the death of the
embryo is defined by a ceasing heartbeat. The heart of
the mouse starts to beat between day 8 and 9 [26,27].
Diagnosis of embryonic death on the basis of heart
action is therefore not possible prior this day. Theoretically, the determination of the exact time point of embryonic death based on the absence of a heartbeat would
require permanent live ultrasound scanning. In our
study, bradycardia, arrhythmia and pericardial edema in
growth retarded conceptuses preceded the final cessation
of heart function. After observation of these markers,
ultrasound examinations can be performed twice daily
to delineate the time window of death. However, one
has to consider that ceasing of the heartbeat might in
fact not be the appropriate marker for embryonic
death. We showed that one embryo under resorption,
which still exhibited a faint heartbeat, already showed
severe necrosis of the cells of the embryo proper. This
finding shows that the border between the end of life
of an individual is equally fluent and difficult to define
as its beginning. In early gestation, it is even harder to
narrow down the period of death since the embryo
proper only begins to develop and its heart is not yet
beating. Death in that developmental stage occurs at
the cellular level only and is reflected in arrested development of the conceptus, accompanied by an increased

Page 10 of 12

echogenicity of embryonic fluids as seen by high frequency ultrasound.

In the fourth stage the conceptus dissolves and is
subject to haemorrhage and necrosis. The final resorption stage consists of fibrinoid, condensed scar tissue
which persists for an extended period of time. In post
mortem studies, this is the stage where the resorption
site is identified macroscopically in the exteriorized
uterus [11,28-30]. By ultrasound, this final stage of resorption observed in early pregnancy was characterised by a
high echodensity spot. We documented similar highechodensity spots along the ectoplacental cone and in the
placenta at the embryo-maternal border in normal conceptuses. In a study of Akirav et al., these high density foci
have been identified as calcium deposits [31]. The concretions most likely originate from dystrophic calcification
processes in dysfunctional cells where the active calcium
transport is impaired. They can therefore be considered as
a marker for the last stages of apoptosis and necrosis.
This process seems to originate from the embryo itself,
since we always observed first the death of the embryo,
then its dissolution, and then the disappearance of its
inner membranes. These observations have also been
made in an ultrasound study on embryo resorption in
the European brown hare [32]. In human reproductive
medicine, an anembryonic gestational sac is considered
as an ultrasonographic marker for embryonic demise
[33]. High levels of alpha-fetoprotein of yolk sac origin
in the maternal circulation are indicative for an early
death of the embryo which was resorbed before it became ultrasonographically detectable [34].
In our study, the Reicherts membrane which is unique
to rodents and acts as a filter between embryonic and
maternal tissue [35], was the last membrane to disappear. Together with the finding that the placenta was
morphologically unsuspicious these observations support
the hypothesis that death is triggered within the embryo
itself. By means of cell competition, embryonic cells can
compare their fitness with that of neighbouring cells
resulting in apoptosis of the less fitter cells [36]. This
mechanism has been demonstrated to play a crucial
role in the selection of mouse embryonic epiblast [37].
If that cell competition becomes unbalanced, it could
result in embryonic death. There seems to be a higher
selection pressure on the long lived epiblast cells than
on the short lived cells of the extraembryonic membranes [37], reflecting our finding that the membranes
undergo resorption only after the embryo has already
disappeared.
In knockout mice, the depletion of specific genes may
result in a characteristic embryonic phenotype [20,24]
which can be further examined with additional methods
such as hybridization and immunohistochemistry. Longitudinal UBM examinations will enable to determine the

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exact stage at which certain genes need to be expressed to

ensure healthy embryo development and survival.
The monitoring of embryo development by UBM
will also be useful in the field of epigenetics, where
certain environmental factors acting on the adult individual influence the gene expression and intra-uterine
development of their offspring [38,39]. The use of UBM to
detect embryo resorptions will significantly reduce the
number of experimental animals in studies investigating embryo failure for two reasons: first, pregnant animals can be reliably detected from day 5 onward and
non-pregnant animals can be saved. Second, embryo
resorptions can be identified in vivo at an early stage enabling the sacrifice of the experimental animal at the
appropriate time.

Conclusions
In our study we have shown that UBM is a useful tool to
detect resorption prone embryos and to follow their involution process over time. With this method, resorption
prone embryos can be specifically targeted and harvested
before the onset of decomposition. This is particularly
important for the study of embryo-maternal immune reactions, where the specific maternal immune response
towards the dying embryo must be differentiated from a
general immune reaction necessary to clear the uterus
from apoptotic tissue. Furthermore, the morphology of
the placenta and extraembryonic membranes can be
evaluated in vivo over a period of time before its integrity is compromised by dissection.
Additional files
Additional file 1: Movie S1. Normal development day 68. On day 6,
the implantation site is visible as a bulge of the uterus. The endometrial
layers are seen as a hyperechogenic, white line. On day 7, the ectoplacental
cone and the proamniotic cavity become evident. On day 8, the embryo is
encased in its amnion and the allantois traverses the exocoelom. The
ectoplacental cone invades the mesometrial decidua basalis.
Additional file 2: Movie S2. R16 and R17 day 9. In the normal embryo,
head and rump can be differentiated and the heartbeat is evident. The
umbilical cord attaches to the placenta. The resorption prone embryos
R16 and R17 display the same morphological features but are visibly
smaller compared to the normal embryo.
Additional file 3: Movie S3. R16 and R17 day 10. The normal embryo
displays a pulsating heart with atria and ventricles enclosed in the
pericardium. The blood flow from the umbilical cord to the placenta is
visible. The embryo-maternal interface is characterized by calcifications
between the trophoblast and the placenta. The resorption prone embryo
R16 is visibly smaller than its normal sibling. However, its heart rate is not
yet reduced. The resorption prone embryo R17 shows growth retardation,
pericardial effusion and a reduced heartbeat.
Additional file 4: Movie S4. R16 and R17 day 11, first and second scan.
The normal embryo has increased in size. In contrast, the R16 is now
clearly in the process of resorption: the heart is compressed by
pericardial effusion and a heartbeat is barely detectable. The yolk sac is
visible as a shrivelled membrane after having lost its close connection to
the Reichert's membrane and underlying maternal mucosa. R17 has died.
The amniotic cavity is small and the yolk sac has also lost its balloon like

Page 11 of 12

shape, resulting in a shriveled yolk sac membrane. In the second scan,

3 hours later, the morphology of the normal embryo is not altered. In
R16, a heartbeat cannot be detected. The embryo of R17 has completely
lost its original morphology and its tissue looks condensed. The embryo
seems to be outside its original cavity in the uterine lumen. During
collection of the resorption site, the embryo was lost. Only the Reichert's
membrane was found in the uterine lumen.
Additional file 5: Movie S5. R15 day 9 and 10. On day 9 the resorption
prone embryo R15 shows visible growth retardation. Compared to its
normal sibling, the morphology is poorly defined. On day 10, the
resorption prone embryo has increased in size and its heart can be
differentiated. The pericard is filled with excess fluid and its heart rate is
greatly reduced as illustrated by color doppler flow.

Competing interests
The authors have no competing interests.
Authors contributions
TBH, LEF and BD developed the study design. LEF and BD performed
ultrasound imaging and retrospective analysis. LEF, AAK and BD evaluated
the histological sections. LEF and BD wrote the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
We would like to express our very great appreciation to Prof. Dr. Katarina
Jewgenow for her support and for granting us access to laboratory
equipment. We are also especially thankful for the excellent technical
assistance of Sigrid Holz.
Author details
1
Department Reproduction Management, Leibniz Institute for Zoo and
Wildlife Research (IZW), Alfred-Kowalke-Str. 17, 10315 Berlin, Germany.
2
Charit Department of Medicine I for Gastroenterology, Infectious Disease
and Rheumatology, Research Center ImmunoSciences / Universittsmedizin
Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin,
Germany.
Received: 19 February 2014 Accepted: 26 April 2014
Published: 10 May 2014
References
1. Macklon NS, Geraedts JP, Fauser BC: Conception to ongoing pregnancy:
the black box of early pregnancy loss. Hum Reprod Update 2002,
8(4):333343.
2. Bulletti C, Flamigni C, Giacomucci E: Reproductive failure due to
spontaneous abortion and recurrent miscarriage. Hum Reprod Update
1996, 2(2):118136.
3. Diskin MG, Parr MH, Morris DG: Embryo death in cattle: an update. Reprod
Fertil Dev 2011, 24(1):244251.
4. Andrabi SM, Maxwell WM: A review on reproductive biotechnologies for
conservation of endangered mammalian species. Anim Reprod Sci 2007,
99(34):223243.
5. Simpson J, Bombard A: Chromosomal abnormalities in spontaneous
abortion: frequency, pahtology and genetic counseling. In Spontaneous
Abortion. London: Blackwell; 1991:51.
6. Reynolds LP, Caton JS, Redmer DA, Grazul-Bilska AT, Vonnahme KA, Borowicz PP,
Luther JS, Wallace JM, Wu G, Spencer TE: Evidence for altered placental
blood flow and vascularity in compromised pregnancies. J Physiol 2006,
572(1):5158.
7. Zenclussen AC, Gerlof K, Zenclussen ML, Sollwedel A, Bertoja AZ, Ritter T,
Kotsch K, Leber J, Volk HD: Abnormal T-cell reactivity against paternal
antigens in spontaneous abortion - Adoptive transfer of pregnancyinduced CD4 (+) CD25 (+) T regulatory cells prevents fetal rejection in
a murine abortion model. Am J Pathol 2005, 166(3):811822.
8. Farese RV Jr, Ruland SL, Flynn LM, Stokowski RP, Young SG: Knockout of
the mouse apolipoprotein B gene results in embryonic lethality in
homozygotes and protection against diet-induced hypercholesterolemia
in heterozygotes. Proc Natl Acad Sci U S A 1995, 92(5):17741778.

Flores et al. Reproductive Biology and Endocrinology 2014, 12:38

http://www.rbej.com/content/12/1/38

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.
22.

23.

24.

25.

26.

27.

28.

29.

30.

Chang H, Huylebroeck D, Verschueren K, Guo Q, Matzuk MM, Zwijsen A:

Smad5 knockout mice die at mid-gestation due to multiple embryonic
and extraembryonic defects. Development 1999, 126(8):16311642.
Passey RJ, Williams E, Lichanska AM, Wells C, Hu S, Geczy CL, Little MH,
Hume DA: A null mutation in the inflammation-associated S100 protein
S100A8 causes early resorption of the mouse embryo. J Immunol 1999,
163(4):22092216.
Murphy SP, Fast LD, Hanna NN, Sharma S: Uterine NK cells mediate
inflammation-induced fetal demise in IL-10-null mice. J Immunol 2005,
175(6):40844090.
Lin Y, Nakashima A, Shima T, Zhou X, Saito S: Toll-like receptor signaling in
uterine natural killer cellsrole in embryonic loss. J Reprod Immunol 2009,
83(12):95100.
Clark DA, Petitbarat M, Chaouat G: How should data on murine spontaneous
abortion rates be expressed and analyzed? Am J Reprod Immunol 2008,
60(3):192196.
Roellig K, Drews B, Goeritz F, Hildebrandt TB: The long gestation of the
small naked mole-rat (Heterocephalus glaber Ruppell, 1842) studied
with ultrasound biomicroscopy and 3D-ultrasonography. PLoS One 2011,
6(3):e17744.
Schroeder K, Drews B, Roellig K, Menzies BR, Goeritz F, Hildebrandt TB:
In vivo tissue sampling of embryonic resorption sites using ultrasound
guided biopsy. Theriogenology 2011, 76(4):778784.
Drews B, Ringleb J, Waurich R, Hildebrandt TB, Schroder K, Roellig K:
Free blastocyst and implantation stages in the European brown hare:
correlation between ultrasound and histological data. Reprod Fertil
Develop 2012, 25(6):866878.
Mu J, Slevin JC, Qu D, McCormick S, Adamson SL: In vivo quantification
of embryonic and placental growth during gestation in mice using
micro-ultrasound. Reprod Biol Endocrinol 2008, 6:34.
Zhou YQ, Foster FS, Qu DW, Zhang M, Harasiewicz KA, Adamson SL:
Applications for multifrequency ultrasound biomicroscopy in mice from
implantation to adulthood. Physiol Genomics 2002, 10(2):113126.
Pallares P, Gonzalez-Bulnes A: Use of ultrasound imaging for early diagnosis
of pregnancy and determination of litter size in the mouse. Lab Anim 2009,
43(1):9195.
Laissue P, Burgio G, L'Hote D, Renault G, Marchiol-Fournigault C, Fradelizi D,
Fellous M, Serres C, Montagutelli X, Monget P, Vaiman D: Identification of
Quantitative Trait Loci responsible for embryonic lethality in mice
assessed by ultrasonography. Int J Dev Biol 2009, 53(4):623629.
Phoon CK, Turnbull DH: Ultrasound biomicroscopy-Doppler in mouse
cardiovascular development. Physiol Genomics 2003, 14(1):315.
Phoon CK, Ji RP, Aristizabal O, Worrad DM, Zhou B, Baldwin HS, Turnbull DH:
Embryonic heart failure in NFATc1/ mice: novel mechanistic insights
from in utero ultrasound biomicroscopy. Circ Res 2004, 95(1):9299.
Phoon CK, Aristizabal O, Turnbull DH: 40 MHz Doppler characterization of
umbilical and dorsal aortic blood flow in the early mouse embryo.
Ultrasound Med Biol 2000, 26(8):12751283.
Vatin M, Burgio G, Renault G, Laissue P, Firlej V, Mondon F, Montagutelli X,
Vaiman D, Serres C, Ziyyat A: Refined mapping of a quantitative trait locus
on chromosome 1 responsible for mouse embryonic death. PLoS One
2012, 7(8):e43356.
Thiel R, Chahoud I, Jurgens M, Neubert D: Time-dependent differences in
the development of somites of four different mouse strains. Teratog
Carcinog Mutagen 1993, 13(6):247257.
Srinivasan S, Baldwin HS, Aristizabal O, Kwee L, Labow M, Artman M,
Turnbull DH: Noninvasive, in utero imaging of mouse embryonic heart
development with 40-MHz echocardiography. Circulation 1998,
98(9):912918.
Ji RP, Phoon CK, Aristizabal O, McGrath KE, Palis J, Turnbull DH: Onset of
cardiac function during early mouse embryogenesis coincides with
entry of primitive erythroblasts into the embryo proper. Circ Res 2003,
92(2):133135.
Kusakabe K, Naka M, Ito Y, Eid N, Otsuki Y: Regulation of natural-killer cell
cytotoxicity and enhancement of complement factors in the spontaneously
aborted mouse placenta. Fertil Steril 2008, 90(4 Suppl):14511459.
Duclos AJ, Haddad EK, Baines MG: Presence of activated macrophages
in a murine model of early embryo loss. Am J Reprod Immunol 1995,
33(5):354366.
Clark DA, Foerster K, Fung L, He W, Lee L, Mendicino M, Markert UR, Gorczynski
RM, Marsden PA, Levy GA: The fgl2 prothrombinase/fibroleukin gene is

Page 12 of 12

31.

32.

33.

34.

35.

36.
37.
38.
39.

required for lipopolysaccharide-triggered abortions and for normal mouse

reproduction. Mol Hum Reprod 2004, 10(2):99108.
Akirav C, Lu Y, Mu J, Qu DW, Zhou YQ, Slevin J, Holmyard D, Foster FS,
Adamson SL: Ultrasonic detection and developmental changes in
calcification of the placenta during normal pregnancy in mice. Placenta
2005, 26(23):129137.
Schroeder K, Drews B, Roellig K, Goeritz F, Hildebrandt TB: Embryonic
resorption in context to intragestational corpus luteum regression:
A longitudinal ultrasonographic study in the European brown hare
(Lepus europaeus PALLAS, 1778). Theriogenology 2013, 76(4):778784.
Odeh M, Tendler R, Kais M, Grinin V, Ophir E, Bornstein J: Gestational sac
volume in missed abortion and anembryonic pregnancy compared to
normal pregnancy. J Clin Ultrasound 2010, 38(7):367371.
Jauniaux E, Gulbis B, Jurkovic D, Gavriil P, Campbell S: The origin of
alpha-fetoprotein in first-trimester anembryonic pregnancies.
Am J Obstet Gynecol 1995, 173(6):17491753.
Salamat M, Gotz W, Horster A, Janotte B, Herken R: Ultrastructural
localization of carbohydrates in Reichert's membrane of the mouse.
Cell Tissue Res 1993, 272(2):375381.
de la Cova C, Abril M, Bellosta P, Gallant P, Johnston LA: Drosophila myc
regulates organ size by inducing cell competition. Cell 2004, 117(1):107116.
Claveria C, Giovinazzo G, Sierra R, Torres M: Myc-driven endogenous cell
competition in the early mammalian embryo. Nature 2013, 500(7460):3944.
Simmons RA: Developmental origins of adult disease. Pediatr Clin North
Am 2009, 56(3):449466.
Jammes H, Junien C, Chavatte-Palmer P: Epigenetic control of development
and expression of quantitative traits. Reprod Fertil Develop 2012, 23(1):6474.

doi:10.1186/1477-7827-12-38
Cite this article as: Flores et al.: Early detection and staging of
spontaneous embryo resorption by ultrasound biomicroscopy in murine
pregnancy. Reproductive Biology and Endocrinology 2014 12:38.

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