Human embryo– conditioned medium stimulates

in vitro endometrial angiogenesis

Kitty Kapiteijn, M.D.,a,b Pieter Koolwijk, Ph.D.,a Robin M. F. van der Weiden, M.D.,c
Geerten van Nieuw Amerongen, Ph.D.,d Margreet Plaisier, M.D.,a,b
Victor W. M. van Hinsbergh, Ph.D.,a,d and Frans M. Helmerhorst, M.D.b
a
Division of Biomedical Research, TNO Quality of Life, Leiden; b Department of Gynecology, and Reproductive Medicine,
Leiden University Medical Center, Leiden; c Department of Obstetrics and Gynecology, St. Franciscus Gasthuis, Rotterdam;
and d Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam,
The Netherlands

Objective: Successful implantation and placentation depend on the interaction between the endometrium and the
embryo. Angiogenesis is crucial at this time. In this article we investigate the direct influence of the human
embryo on in vitro endometrial angiogenesis.
Design: In vitro study.
Setting: Human endometrial microvascular endothelial cells (hEMVEC) grown on an in vitro angiogenesis
model.
Intervention(s): Conditioned media (CM) of human embryos were used to stimulate in vitro angiogenesis.
Main Outcome Measure(s): In vitro angiogenesis of hEMVEC.
Result(s): Conditioned media of human embryos, containing significant amounts of vascular endothelial growth
factor (VEGF)–A, as determined by enzyme-linked immunosorbent assay (ELISA), caused an increase in
hEMVEC tube formation. This effect was prevented by soluble VEGF receptor 1, which quenches VEGF-A
activity. Recombinant EGF alone and leukemia inhibitory factor in combination with VEGF-A stimulated
hEMVEC tube formation. None of the other tested recombinant mediators, which have been described as
produced by the early embryo/trophoblast (interleukin (IL) 10, transforming growth factor (TGF) ␤, placental
growth factor, hCG, colony-stimulating factor 1, interferon-␥, insulin-like growth factor I and II, IL-6, platelet-
derived growth factor, and TGF␣), had an effect on tube formation by hEMVEC.
Conclusion(s): For the first time, it is shown that the human embryo is able to stimulate in vitro endometrial
angiogenesis at the time of implantation, a process that is mediated by VEGF-A. (Fertil Steril威 2006;85(Suppl
1):1232–9. ©2006 by American Society for Reproductive Medicine.)
Key Word: Endometrial angiogenesis

Successful implantation and subsequent placentation depend Inadequate angiogenesis in the peri-implantation phase may
on the interaction between a receptive decidualized endome- lead to a less receptive endometrium. This can result in
trium and an intrusive blastocyst. Angiogenesis plays a ma- implantation failure or aberrant placental formation, which
jor role in the formation of a receptive endometrium and an in turn may affect the pregnancy outcome, as demonstrated
adequate functioning of the placenta. in morphologic studies demonstrating poor placental vascu-
lar development in intrauterine growth restriction (2).
When the blastocyst enters the uterine cavity, its sur-
vival depends on endometrial secretion. After attachment Before the embryo and the endometrium can make phys-
and invasion, it is fed and oxygenated by the decidualized ical contact, an interaction by signaling molecules must have
endometrium. been established (3). The exact nature of this interaction is
not fully resolved yet. It is unknown whether the blastocyst
Maternal blood supply, via an extensive endometrial vas- is able to induce the angiogenic process in the endometrium
cular network, to the embryo is indispensable for further directly or indirectly via the epithelial or stromal cells (4 –7).
growth. In the peri-implantation period, local enhancement Following penetration of the epithelial lining, the embryo
of angiogenesis is necessary to support further differentia- has to establish a closer contact with endothelial cells. A
tion of the endometrium, ultimately leading to the formation direct regulation of angiogenesis by the blastocyst/early tro-
of the maternal part of the placenta. The stimulus for this phoblast might be possible during that phase of implantation.
process might very well come from the implanting blastocyst
itself, which in this way optimizes its implantation site (1). The production of cytokines and hormones varies at spe-
cific stages of embryonic differentiation. Human blastocysts
produce activin, colony stimulating factor (CSF)–1, epider-
Received August 5, 2005; revised and accepted November 3, 2005.
mal growth factor (EGF), interferon (IFN) ␥, insulin-like
Reprint requests: Pieter Koolwijk, Ph.D., TNO Quality of Life, Zernikedreef
9, 2333 CK Leiden, The Netherlands (FAX: ⫹31 71 5181901; E-mail: growth factor (IGF) I and II, interleukin (IL) 1␣ and -␤, IL-6,
[email protected]). IL-10, leukemia inhibitory factor (LIF), platelet-derived

1232 Fertility and Sterility姞 Vol. 85, Suppl 1, April 2006 0015-0282/06/$32.00
Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2005.11.029
growth factor (PDGF), transforming growth factor (TGF) ␣ and partment of Agriculture (Bethesda, MD). Recombinant hu-
␤, tumor necrosis factor (TNF) ␣, vascular endothelial growth man EGF, human CSF, IFN␥, IGF-I and -II, IL-1␣, -1␤, -6,
factor (VEGF)–A, and hCG, whereas human first trimester and -10, and TGF␣ and -␤ were commercially obtained from
trophoblasts produce EGF, IGF-2, placental growth factor PrepoTech (Rocky Hill, NJ). Human chorionic gonadotrophin
(PlGF), TGF␣ and ␤, TNF␣, and hCG. These molecules may (Pregnyl) was obtained from Organon (Oss, The Netherlands).
enable the implanting embryo to induce angiogenesis locally at Recombinant human LIF was purchased from Chemicon Inter-
the implantation site. Among these factors, VEGF-A is known national (Temecula, CA) and PDGF B/B from Roche (Mann-
to be a highly specific mitogen for endothelial cells (8). It heim, Germany).
induces angiogenesis and increases the permeability of blood
vessels (9).
Human Embryo Conditioned Medium
Here we have investigated the influence of the embryo on The study was conducted according to the guidelines of the
endometrial angiogenesis by evaluating the effect of condi- Institutional Review Board, and informed consent was ob-
tioned medium from human embryos (IVF culture medium) tained from each donor. The IVF culture media used in our
on isolated human endometrial microvascular endothelial experiments were obtained from the IVF Department of the
cells (hEMVEC) in an in vitro angiogenesis model, which Reinier de Graaf Group (Diaconessenhuis, Voorburg, The
we have previously characterized (10). Furthermore, indi- Netherlands) and the Infertility Centre from the Gent Uni-
vidual recombinant cytokines, known to be expressed by versity Hospital (Gent, Belgium). The media were collected
the human embryo and first trimester trophoblast, together during a period of 1 and 2 years. Early stage human embryos
with hCG were tested on hEMVEC to determine which obtained after oocyte pick-up and IVF were cultured in media
factors are involved in inducing angiogenesis at the time until embryo transfer. The culture media used were: GPO
of implantation. medium (for the exact compounds, see Rijnders et al. (12)),
Complete P-1 Irvine and Complete Blastocyst Irvine media
MATERIALS AND METHODS (Irvine Scientific, Santa Ana, CA), and Earle’s medium supple-
Materials mented with 0.08% (w/v) HSA, penicillin G (8 mg/L), sodium
Penicillin/streptomycin, L-glutamine, and medium 199 (M199) pyruvate (0.10 g/L), and sodium bicarbonate (2.1 g/L).
with and without phenol red and supplemented with 20 Early stage human embryos produce and accumulate me-
mmol/L HEPES was obtained from BioWhittaker (Verviers, diators in the medium in which they are cultured. Pool A, B,
Belgium); Newborn calf serum (NBCS) was obtained from and C consisted of medium in which 12, 40, and 79 embryos
Life Technologies (Grand Island, NY). Human serum (HS) (2– 8 cell stage) were cultured originating from 7, 10, and 17
was obtained from a local blood bank and was prepared from patients, respectively. Pool D consisted of medium from 90
fresh blood from 10 –20 healthy donors, pooled, and stored blastocysts originating from an unknown number of patients.
at 4°C; it was heat inactivated before use. Human serum
albumin (HSA) was obtained from Sanquin (Amsterdam, Earle’s medium or GPO medium (pool A, B, and C) were
The Netherlands). Tissue culture plastics and microtiter refreshed after 1 or 3 days, respectively. The Irvine me-
plates came from Costar/Corning (Cambridge, MA) and dium (pool D) was changed on day 3 from Complete P-1
Falcon (Becton Dickinson, Bedford, MA). A crude prepara- Irvine medium to Complete Blastocyst Irvine medium. No
tion of endothelial cell growth factor (ECGF) was prepared data are available on the success rate of implantation of
from bovine hypothalamus as described by Maciag et al. these embryos.
(11). Heparin and thrombin were obtained from Leo Phar- Other materials used have been specified in the methods
maceutics Products (Weesp, the Netherlands). Human fibrin- described or in the related references mentioned.
ogen was purchased from Chromogenics (Mölndal, Swe-
den). Dr. H. Metzner and Dr. G. Seeman (Aventis Behring,
Marburg, Germany) generously provided factor XIII. Fi- Cell Culture
bronectin was a gift from Dr. J. van Mourik (CLB, Amster- Human endometrial microvascular endothelial cells (hEMVEC)
dam, The Netherlands). Human recombinant VEGF-A and were isolated from endometrial tissue (collected according to
PlGF were purchased from ReliaTech (Braunschweig, Ger- the guidelines of the Institutional Review Board and with
many); soluble VEGF receptor 1 (sVEGFR-1) was a gener- informed consent was obtained from each donor) as previ-
ous gift from Dr. H.A. Weich (GBF, Braunschweig, Ger- ously described (10) and maintained in indicator-free M199
many). Tumor necrosis factor ␣ was a gift from Dr. J. supplemented with 20 mmol/L HEPES (pH 7.3), 20% hu-
Travernier (Gent, Belgium). Recombinant human basic fi- man serum, 10% heat-inactivated NBCS, 150 ␮g/mL ECGF,
broblast growth factor (bFGF) was purchased from Pepro- 5 ng/mL VEGF-A, 5 U/mL heparin, 100 IU/mL penicillin,
Tech (Rocky Hill, NJ). Recombinant human activin was and 100 mg/mL streptomycin to constitute the hEMVEC
obtained from Dr. Pawson via the National Hormone and culture medium. hEMVEC were cultured on fibronectin-
Pituitary Program, The National Institute of Diabetes and coated wells at 5% CO2/95% air until confluence was
Digestive and Kidney disease, The National Institute of reached and were subsequently detached with 0.05% trypsin/
Child Health and Human Development, and the U.S. De- 0.025% EDTA and transfered into coated dishes at a split

Fertility and Sterility姞 1233

ratio of 1:3. Fresh medium was given three times a week RESULTS
with twice a two-day interval and once a three-day interval Early Stage Embryo Conditioned Medium Enhances
(weekend). hEMVEC Proliferation and Tube Formation
We used conditioned IVF culture medium of pool A to
evaluate the effect of cytokines and other mediators pro-
Incorporation of 3H-Thymidine duced by early stage human embryos on hEMVEC prolifer-
Incorporation of 3H-thymidine into DNA was determined as ation and the media of pools B, C, and the blastocyst-derived
the measurement of endothelial cell proliferation. Confluent D on in vitro angiogenesis.
cultures of endothelial cells (passages 5 to 9 of three differ-
An increase in hEMVEC proliferation was observed when
ent donors) were detached by trypsin/EDTA solution and hEMVEC were stimulated with pool A. When 5% of the
allowed to adhere and spread at a density of 104 cells per cm2 conditioned media was added, a maximum stimulation was
on fibronectin-coated dishes in indicator-free M199-HEPES observed, although this increase was not significant as com-
supplemented with 10% heat-inactivated and charcoal- pared with the control condition (with 0.75 ng/mL VEGF-A
treated NBCS, penicillin/streptomycin, and 0.75 ng/mL as maintenance factor; data not shown). When 5% of non-
VEGF-A for 18 h. The 0.75 ng/mL VEGF-A was added as conditioned culture media was added, no effect on hEMVEC
a maintenance factor to prevent hEMVEC death under these proliferation was seen.
control culture conditions. Then the cells were stimulated
with conditioned medium, increasing concentrations of cy- Pools B, C, and D independently induced an increase in
tube formation by hEMVEC when these cells were stimu-
tokines or hCG in the presence or absence of extra 6.25
lated with 2.5%–10% of the conditioned media (Fig. 1). The
ng/mL VEGF-A, as indicated in the preceding text. After a
results of the three pools were taken together for statistical
total incubation period of 42 h, 3H-thymidine was added and
analysis. Quantification of the tube formation revealed that
the cells were incubated for another 6-h period. Subse-
the effect was significant when 2.5%–10% of the condi-
quently, the 3H-labeled DNA was precipitated and counted
tioned medium was used and showed an increase of 150%,
in a liquid scintillation counter, and the stimulation index
151%, and 135% when 2.5%, 5%, and 10%, respectively, of
was calculated as previously described (10).
the conditioned medium was used (control set at 100%) (Fig.
2A). At higher concentrations (20%) the ingrowth of vascu-
In Vitro Angiogenesis Model lar structures declined. The nonconditioned IVF culture me-
dium (control) had no effect on tube formation (Fig. 2A).
Human fibrin matrices were prepared as described by Kool-
wijk et al. (10). Confluent hEMVEC (passages 6 to11 of two To test whether the enhancement of tube formation was
different donors) were detached and seeded in a split ratio of due to expression of VEGF-A by the early stage embryos,
2:1 on the surface of the fibrin matrices and cultured for 24 h sVEGFR-1 was added to the pools. Soluble VEGFR-1 cap-
in indicator-free M199 medium supplemented with 20% tures VEGF-A and prevents its binding to cellular receptors.
human serum, 10% NBCS, and penicillin/streptomycin. The presence of sVEGFR-1 completely prevented VEGF-A–
Then the endothelial cells were cultured with the mediators induced tube formation under our standard conditions. Addi-
indicated for 2–5 days. Invading cells and the formation of tion of sVEGFR-1 inhibited the tube formation that was
capillary-like structures of endothelial cells in the three- enhanced by pool C (Fig. 2B). Similar results were obtained
dimensional fibrin matrix were analyzed by phase-contrast with pools B and D (data not shown). This suggests that
early stage embryos are able to express VEGF-A to such a
microscopy; the total length of the tube-like structures was
level that it was involved in the stimulation of tube formation
measured as described by Kroon et al. (13).
by hEMVEC.
To confirm the presence of VEGF-A in the conditioned
Enzyme-Linked Immunosorbent Assays (ELISA) medium of day 2–3 and blastocyst stage embryos, VEGF-A
The VEGF-A antigen determinations were performed by the concentrations in the media of pools B, C, and D were
commercially available DuoSet ELISA Development Kit for assayed by ELISA. In the pooled media B, C, and D,
human VEGF-A (R&D Systems, Minneapolis, MN), which respectively, 10,700 pg/mL, 5200 pg/mL, and 14 pg/mL
recognizes VEGF-A165 and VEGF-A121. Human recombi- VEGF-A antigen was detected. The control medium of the
nant VEGF-A165 (R&D Systems) was used as a standard. pooled media B and C, in which no embryos were grown,
contained 70 pg/mL VEGF-A, whereas no VEGF-A antigen
was detectable in the control medium of pool D.
Statistics
The data are expressed as the mean ⫾ SD/SEM or range. Effect of Cytokines and hCG Produced
Statistical evaluations of the data were performed using the by the Early Embryo and First Trimester
paired t test and Wilcoxon rank test after the control condi- Trophoblast on hEMVEC Proliferation
tions were set at 100%. P⬍0.05 was considered statistically Because conditioned medium of early embryos was able to
significant. induce hEMVEC proliferation and tube formation, the ques-

1234 Kapiteijn et al. Embryo-conditioned medium and angiogenesis Vol. 85, Suppl 1, April 2006
 FIGURE 1
Conditioned medium of early stage embryos stimulate in vitro human endometrial microvascular endothelial
cells (hEMVEC) tube formation. Phase-contrast pictures were taken of hEMVEC cultured on top of a three-
dimensional fibrin matrix under control conditions (A), after stimulation with vascular endothelial growth
factor (VEGF)-A (10 ng/mL, B), 5% (v/v) control IVF medium (C), or 5% (v/v) pooled conditioned medium (D).
The number of tubular structures (examples indicated by arrows) increased after stimulation with VEGF-A or
5% pooled conditioned medium. Bar ⫽ 500 ␮m.

Kapiteijn. Embryo-conditioned medium and angiogenesis. Fertil Steril 2006.

tion arose of which factors produced by the human embryo TGF␣, TGF␤, PlGF, and hCG) did not have a significant
could be held responsible for these effects. As such, recom- effect. Only incubation with increasing concentrations of
binant cytokines and hCG, known to be expressed by the IL-1␣ had a significant inhibitory effect on hEMVEC
human embryo and first trimester trophoblast, were tested on proliferation induced by 6.25 ng/mL VEGF-A (data not
hEMVEC proliferation and tube formation, both in the ab- shown).
sence or presence of 6.25 ng/mL VEGF-A.
Cell death in hEMVEC cultures was caused by TNF␣, at
The VEGF-A was a potent stimulator of hEMVEC pro- concentrations of 1 and 2.5 ng/mL, but addition of 6.25
liferation, as measured by 3H-thymidine incorporation (10) ng/mL VEGF-A prevented the TNF␣-induced cell death
and confirmed by an increase in cell number (determined at (data not shown).
48 h; data not shown).
Under control conditions (with 0.75 ng/mL VEGF-A as Effect of Cytokines and hCG Produced by the Early
maintenance factor), IL-1␣ and activin (only the highest Embryo and First Trimester Trophoblast on in Vitro
concentration) significantly inhibited hEMVEC prolifera- Angiogenesis by hEMVEC
tion, whereas all the other tested cytokines (EGF, LIF, Subsequently, the effect of factors expressed by the human
CSF-1, IFN␥, IGF-I, IGF-II, IL-1␤, IL-6, IL-10, PDGF, embryo and first trimester trophoblast on in vitro angiogen-

Fertility and Sterility姞 1235

 FIGURE 2
Conditioned medium of embryos stimulate in vitro human endometrial microvascular endothelial cells
(hEMVEC) tube formation, an effect which is vascular endothelial growth factor (VEGF)-A mediated. (A)
hEMVEC, from passage 5–10 of one donor, were cultured on top of a three-dimensional fibrin matrix and
stimulated with 2.5%, 5%, 10%, and 20% (v/v) of pooled medium (pool B, C, or D; solid triangles with
solid line). As control, the cells were stimulated with 2.5%, 5%, and 10%, and 20% (v/v) of the IVF
culture medium in which no embryos were grown (open circles with dotted line) or 10 ng/mL VEGF
(solid bar). After 3–5 days of culturing, mean tube length was measured. The data are expressed as a
percentage of the control and represent mean ⫾ SEM of four independent experiments performed in
duplicate wells. *P⬍.05 compared to control condition. The mean tube length of the controls was 63.4
mm/cm2. (B) Addition of 0.5 ␮g/mL sVEGFR-1 to control (M199 supplemented with 10% newborn calf
serum (NBCS) and 20% human serum (HS)) and VEGF-A–stimulated conditions, inhibited the amount of
capillary-like structures formed. The enhanced formation of capillary-like structures by hEMVEC after
stimulation with 10% (v/v) embryo culture medium was also reduced by sVEGFR-1. Mean tube length
was measured and expressed as a percentage of the control range. Hatched bars: without sVEGFR-1;
solid bars: with sVEGFR-1. The mean tube length of the controls was 77.4 mm/cm2. The experiments
were performed in duplicate wells.

A B
300

tube formation (% of control)

300
tube formation (% of control)

Pool C

200 200
* *
*

100 100

0
0 2.5 5 10 20 VEGF-A control VEGF-A Pooled
medium
medium added (%)
Kapiteijn. Embryo-conditioned medium and angiogenesis. Fertil Steril 2006.

esis by hEMVEC was studied in the absence or presence of concentrations had no effect on tube formation (data not
VEGF-A. shown).
In the absence of VEGF-A, EGF significantly stimulated
tube formation concentration dependently. However, in DISCUSSION
combination with VEGF-A no additive effect of EGF was
observed (Fig. 3C). Similarly to their inhibitory effect on The data presented here demonstrate that conditioned media
hEMVEC proliferation, high concentrations of activin (Fig. of human embryos contained VEGF-A and stimulated in
3B), IL-1␣ (Fig. 3A) and IL-1␤ (not shown) significantly vitro endometrial angiogenesis, an effect counteracted by
inhibited the amount of tubes formed. Interestingly, LIF (1 sVEGFR-1. The VEGF-A was the most potent mediator in
and 10 ng/mL) significantly stimulated the VEGF-A– stimulating hEMVEC proliferation and tube formation
enhanced tube formation but did not alter basal tube forma- among the known mediators expressed by the human em-
tion (Fig. 3D). Interleukin 10, TGF␤, PlGF, hCG, CSF-1, bryo and first trimester trophoblast, of which LIF could
IFN␥, IGF-I/-II, IL-6, PDGF, and TGF␣ in the indicated increase the VEGF-mediated tube formation.

1236 Kapiteijn et al. Embryo-conditioned medium and angiogenesis Vol. 85, Suppl 1, April 2006
 FIGURE 3
The influence of recombinant cytokines and human chorionic gonadotropin (hCG) on the formation of
capillary-like structures by human endometrial microvascular endothelial cells (hEMVEC). hEMVEC were
cultured on top of a three-dimensional fibrin matrix in M199 supplemented with 20% Human serum and
10% newborn calf serum and stimulated with increasing amounts of different cytokines and hCG in the
absence (solid line) or presence (dotted line) of vascular endothelial growth factor (VEGF)-A (10 ng/mL). After
2–5 days of culturing, mean tube length was measured by image analysis as described and expressed as a
percentage of the control ⫾ range of two independent experiments performed in duplicate wells. *P⬍.05
compared to control condition; **P⬍.05 compared to VEGF-stimulated condition.

A B
300 300

tube formation (% of control)

tube formation (% of control)

200 200

100
*
100
*
0 0
0 1 10 50 0 1 10 50
IL-1 (ng/mL) Activin (ng/mL)

C D
300 300
** **
tube formation (% of control)

*
tube formation (% of control)

200
* 200

*
100 100

0 0
0 1 10 50 0 1 10 50
EGF (ng/mL) LIF (ng/mL)
Kapiteijn. Embryo-conditioned medium and angiogenesis. Fertil Steril 2006.

Fertility and Sterility姞 1237

 Adequate interaction between embryo and endometrium is between angiogenesis-stimulating and -inhibiting factors
essential for successful implantation and placentation. The which strictly controls angiogenesis.
embryo locally prepares the endometrium for its nidation by
producing various mediators (3, 14 –17). Previously, Sakkas Heterozygous and homozygous deletion of the VEGF-A
et al. (18) described that the human blastocyst directly in- gene in mouse embryos resulted in embryonic mortality at
duces changes in endometrial epithelial cells. Therefore it midgestation and in impaired placental development due to
was suggested that the human embryo might also directly abnormal formation of intra- and extraembryonic vessels
affect the endometrial endothelium, thus regulating endome- (26 –28). This further underlines the importance of VEGF-A.
trial angiogenesis, an important factor in the preparation However, the embryo might also produce other mediators
process (19). Studies in rats support the hypothesis that which are able to induce or enhance angiogenesis at the site
angiogenesis at the implantation site is a localized process of implantation. Such cytokines derived from the human
controlled by the embryo, whereas angiogenesis, which oc- embryo may act directly on angiogenesis or may affect
curs in the entire endometrium, is maternally controlled (20, angiogenesis indirectly by inducing VEGF-A (e.g., IL1␤,
21). Our data indicate that the human embryo is able to hCG (29, 30)), or its receptors in endometrial cells.
produce detectable concentrations of active VEGF-A and One other candidate mediator that, in the presence of
thus to stimulate local angiogenesis in the endometrium VEGF, might be involved in angiogenesis at the site of
during the peri-implantation phase. implantation is LIF. In our studies LIF had an indirect effect
Krüssel et al. (22,23) previously demonstrated that human on angiogenesis. It increased the VEGF-mediated tube for-
embryos from the 10 cell up to the blastocyst stage were able mation, whereas LIF by itself did not have an effect.
to express mRNA, encoding for four different isoforms of In vivo, it has been shown that LIF was able to induce
VEGF-A (121, 145, 165, and 189). Relatively highly ex- angiogenesis in the rabbit cornea (31) and that female mice
pressed were isoforms 121 and 165, which are both secreted lacking a functional LIF gene are fertile but their blastocysts
VEGF-A isoforms. However, they were not able to detect fail to implant and their uteri were found to be poorly
VEGF-A protein in the embryo culture medium, presumably vascularized (32, 33). However, in vitro, inconsistent effects
because it was below the detection limit of the ELISA, which of LIF on endothelial cells are described. Leukemia inhibi-
only detected VEGF-A 165. The ELISA we used detected tory factor, either or not in the presence of an angiogenic
both the 121 and 165 isoform. Furthermore, Krüssel et al. factor, was found to inhibit (34 –36) or stimulate (31, 37)
used a larger volume of conditioned medium per embryo (50 endothelial cell proliferation and tube formation. Also on the
␮L vs. 10 –30 ␮L in our experiments). influence of LIF on the proteolytic potential of endothelial
cells, an important phase in angiogenesis, opposite results
The human embryos in our study were the only source of
were found (29, 31, 34). These discrepancies can be attrib-
VEGF-A production, because little or no VEGF-A protein
uted to the biologic versatility of LIF which depends on cell
could be detected in the control IVF medium in which no
species and origin.
embryos were cultured. After 24 (pools A to C) or 72 (pool
D) h of culture the medium was changed. This eliminates Although the embryo only produces small amounts of
other potential sources of cytokine production (by granulosa angiogenic factors, locally high concentrations are reached
cells, cumulus cells, and sperm cells). Krüssel et al. (22, 24) owing to close contact between the embryo and maternal
almost ruled out the possibility of paternal contamination by blood vessels. Together with the increased sensitivity of the
using embryos, which resulted from intracytoplasmic sperm endometrial endothelium to angiogenic factors at the time of
injection. They found that unfertilized oocytes did not ex- implantation, the embryo might very well cause an increased
press VEGF-A mRNA, which proved that the VEGF-A angiogenic response at the implantation site.
mRNA was truly embryonic (23).
In conclusion, VEGF-A has been recognized as an important
The amount of VEGF-A detected by ELISA varied in the mediator in the process of endometrial angiogenesis during the
pools. This could not be explained only by the difference in menstrual cycle (10, 38, 39). The results of this study support a
numbers of embryos from which the pools were derived. crucial role for embryonic VEGF-A in the process of angio-
Differences in the culture media used in pools A to C on the genesis during the peri-implantation phase. By the expression
one hand and pool D on the other, differences in embryonic of VEGF-A, the embryo enables itself to induce angiogenesis
stage and viability, or reuptake of VEGF-A by the blastocyst directly at its implantation site, and as such creates an environ-
itself (25) may contribute to and explain the phenomenon. ment necessary for its survival and growth.
It should be noted that the proliferation and outgrowth of In contrast to tumor angiogenesis, endometrial angiogen-
tubular structures was reduced at high concentrations of esis at the time of implantation and placentation seems to be
embryo-conditioned medium. It is plausible that in addition strictly orchestrated. Further studies should focus on the
to the stimulatory VEGF-A, angiogenesis-inhibiting com- exact nature of the interactions between the human embryo
pounds also are present, which only become effective at and the endometrium regarding angiogenesis in the peri-
relatively high concentrations and point to a delicate balance implantation phase and during the formation of placenta and

1238 Kapiteijn et al. Embryo-conditioned medium and angiogenesis Vol. 85, Suppl 1, April 2006
on the role of defective angiogenesis in implantation failure, molecule secreted by human blastocysts modulates regulation of
which affects the outcome of gestation. HOXA10 expression in an epithelial endometrial cell line. Fertil Steril
2003;80:1169 –74.
19. Geva E, Ginzinger DG, Zaloudek CJ, Moore DH, Byrne A, Jaffe RB.
Acknowledgments: We wish to thank Ellen Rijnders, Ilse de Croo, and their
Human placental vascular development: vasculogenic and angiogenic
colleagues from the IVF Department of the Reinier de Graaf Group (Diacon-
(branching and nonbranching) transformation is regulated by vascular
essenhuis, Voorburg, The Netherlands) and the Infertility Centre from the Gent
endothelial growth factor-A, angiopoietin-1, and angiopoietin-2. J Clin
University Hospital (Gent, Belgium) for collecting the IVF culture media.
Endocrinol Metab 2002;87:4213–24.
Furthermore we would like to thank Dr. R. A. Verwey and colleagues of the
20. Abberton KM, Rogers PA. Production of an endothelial cell migratory
Bronovo Hospital (Den Haag, The Netherlands), who provided us with endo-
signal in rat endometrium during early pregnancy. Cell Tissue Res
metrial tissue, and Erna Peters for her technical assistance.
1995;279:215–20.
21. Goodger AM, Rogers PA. Uterine endothelial cell proliferation before
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