ORIGINAL ARTICLES: EARLY PREGNANCY

Prolactin and proinflammatory

cytokine expression at the
fetomaternal interface in first
trimester miscarriage
Emanuele Garzia, M.D.,a Roberta Clauser, M.D.,a Luca Persani, M.D., Ph.D.,b Stefano Borgato, M.D.,b
Gaetano Bulfamante, M.D.,c Laura Avagliano, M.D.,a Federica Quadrelli, M.D.,a
and Anna Maria Marconi, M.D.a
a
Unit of Obstetrics and Gynecology, Department of Health Sciences, San Paolo Hospital Medical School; b Department of
Clinical Sciences and Community Health, University of Milan, and Lab of Endocrine and Metabolic Research, IRCCS Istituto
Auxologico Italiano; and c Human Pathology, Department of Health Sciences, San Paolo Hospital Medical School, University
of Milano, Milan, Italy

Objective: To investigate the expression of prolactin (PRL), PRL-receptor (PRL-R), and the TH1 cytokines interleukin-2 (IL-2), tumor
necrosis factor-a (TNF-a), and interferon-g (IFN-g) at the maternofetal interface.
Design: Case-control study.
Setting: University hospital unit of gynecology and obstetrics and research laboratories.
Patient(s): Women undergoing suction curettage for spontaneous miscarriage (study group) and voluntary termination of pregnancy
(control group) in the first trimester.
Intervention(s): Samples of decidua and villi collected and histologically examined at the time of suction curettage.
Main Outcome Measure(s): Evaluation of all villous samples for karyotype with only euploid cases included; detection of transcripts of
PRL, PRL-R, TNF-a, IFN-g, and IL-2 by qualitative reverse-transcriptase-polymerase chain reaction (RT-PCR); investigation of pattern
and site of expression by immunohistochemistry.
Result(s): In both groups, PRL-R and IFN-g were broadly expressed. The expression of PRL was impaired or absent in the villi of the
study group compared with controls. Expression of TNF-a was reduced, although not statistically significantly, in both decidual and
villous samples of the study group. Immunohistochemical analysis showed the lack of IL-2 expression in decidual specimens of the
control group versus the full expression shown in the study group.
Conclusion(s): Our results highlight the correspondence between PRL expression and vital
pregnancy and the involvement of the TH1 cytokines with different specific roles at the implan-
tation site. Prolactin and IL-2 may reciprocally influence expression. (Fertil SterilÒ 2013;100: Use your smartphone
108–15. Ó2013 by American Society for Reproductive Medicine.) to scan this QR code
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E
mbryo implantation is currently of pregnancies, mostly before 12 weeks maternal side, but the embryonic side
considered the limiting factor of gestation, result in miscarriage; an has not been investigated to the same
for the establishment of preg- imbalance in embryo-maternal cross- extent. Cytokines are known to play
nancy. Up to 60% of all pregnancies talk may be the major reason (1, 2). A an important role in embryo-maternal
are terminated at the end of the peri- number of studies have investigated signaling. Cytokines of the embryo-
implantation period and a further 10% the physiology and pathology of the maternal complex are virtually in-
volved in the regulation of all stages
Received July 3, 2012; revised February 5, 2013; accepted February 23, 2013; published online March
29, 2013. of gestation (3) and may be directed
E.G. has nothing to disclose. R.C. has nothing to disclose. L.P. has nothing to disclose. S.B. has nothing from the embryo toward the mother
to disclose. G.B. has nothing to disclose. L.A. has nothing to disclose. F.Q. has nothing to disclose.
A.M.M. has nothing to disclose. and vice versa. The break in ‘‘cytokine
Reprint requests: Emanuele Garzia, M.D., Unit of Obstetrics and Gynecology, San Paolo Department balance’’ induced by exogenous or
of Health Sciences, University of Milano, 20142 Milan, Italy (E-mail: emgarzi@tin.it).
endogenous factors can lead to sponta-
Fertility and Sterility® Vol. 100, No. 1, July 2013 0015-0282/$36.00 neous abortion and even to pregnancy
Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. complications (4). In animal studies,
http://dx.doi.org/10.1016/j.fertnstert.2013.02.053

108 VOL. 100 NO. 1 / JULY 2013


TH1 immunity has been related to implantation failure and fe-
tal resorption (5, 6), whereas the TH2 cytokines produced at
the fetomaternal interface have been demonstrated to be
beneficial to the maintenance of pregnancy through the
suppression of cellular cytotoxicity (7).
In humans, the existence of a TH2 bias in the successful
pregnancy has not been clearly validated. The update of the
TH1/TH2 paradigm in a TH1/TH2/TH3 (8) or TH1/TH2/TH3/
Treg-1 (9) paradigm stems from the assumption that a TH1
proinflammatory cytokine milieu in the implantation site is
potentially hazardous for fetal development. Against the
belief that the type of immune reactivity, type 1 or type 2,
is strictly predictive of implantation outcome and pregnancy
maintenance, some investigators have suggested that
reproductive failures cannot be simply related to an inflam-
matory pathway and that some proinflammatory cytokines
are physiologically expressed in healthy pregnancies (10, 11).
Prolactin (PRL) is a peptide hormone essentially secreted
by the anterior pituitary and, to a lesser extent, by other
extrapituitary tissues (12, 13) such as the endometrium
(14–16). Endometrial PRL is synthesized by decidualized
endometrial cells in the mid-to-late secretory phase in a non-
conception cycle and throughout pregnancy (17). The effects
of PRL are mediated by a membrane-bound receptor (PRL-R),
member of the superfamily of cytokine receptors. If preg-
nancy occurs, PRL-R expression is maintained and localized
in the decidua, placental trophoblast, and amniotic epithe-
lium (18). The coordinated temporal pattern of expression of
both PRL and PRL-R in the nonpregnant and pregnant endo-
metrium suggests that PRL may have an important role in the
process of implantation, acting as a differentiation factor
rather than as mitogenic. The relationship between the proin-
flammatory TH1 cytokines, classically involved in miscar-
riage, and PRL is still poorly understood. The demonstration
of a dose-dependent inhibition of the synthesis and release
of PRL in primary decidual cell cultures by exposure to tumor
necrosis factor-a (TNF-a) (19), interleukin-2 (IL-2) (20), and
interferon-g (IFN-g) (21) suggests the presence of a network
of molecules strongly interrelated at the maternofetal inter-
face. Our study investigated the expression of PRL, PRL-R,
and the TH1 cytokines IL-2, TNF-a, and IFN-g at the implan-
tation site in decidua and villous samples collected from
women undergoing suction curettage for spontaneous abor-
tion (study group) and voluntary termination of pregnancy
(control group).
MATERIALS AND METHODS
The study was performed at the units of Obstetrics and
Gynecology and Human Pathology of the San Paolo
Department of Health Sciences in Milan in collaboration
with the Laboratory of Endocrine and Metabolic Research of
the Istituto Auxologico Italiano. The study protocol was
approved by the ethics committee of San Paolo Hospital,
and informed consent was given by all study participants.
Patient Selection
One hundred and twelve patients aged 16–41 years (median
age 31.4) undergoing suction curettage in the first trimester
VOL. 100 NO. 1 / JULY 2013
Fertility and Sterility®
of pregnancy (%13 weeks of amenorrhea) were recruited:
84 affected by spontaneous miscarriage (SM) and 28 asking
for voluntary termination of pregnancy (VTP). The inclusion
criteria were history of regular menstrual cycles, spontaneous
conception, normal uterine anatomy as demonstrated by
transvaginal ultrasonography, and gestational sac and
embryo sizes consistent with gestational age. Exclusion
criteria were an extrauterine location of the gestational sac,
the presence of known causes of miscarriage, and the
presence of vaginal bleeding at the time of hospitalization.
In the SM group, miscarriage was defined as a fetal pole
lacking fetal heart rate activity in women without clinical
signs of expulsion; in the VTP, group the fetal heart rate
activity was present before surgery.
Sample Collection
Within 3 hours of surgery, all women were given 1 mg of
intravaginal gemeprost (Cervidil; Serono). At the time of
suction curettage, the gestational tissues were removed in
sterile conditions and macroscopically assessed through
several washes in sterile phosphate-buffered saline solution
to separate chorionic villi from decidua and embryo. For
each patient, a sample of decidua and villi were collected
and histologically examined by an experienced gynecologic
pathologist; only tissues in agreement with the macroscopic
assessment were selected for the study. Tissues collected for
the RNA extraction were stored in RNAasi-free test tubes,
snap-frozen in dry ice, and stored in liquid nitrogen. The
samples collected for immunohistochemical (IHC) analysis
were initially stored in formalin. In all cases, the analysis of
fetal karyotype was performed using direct preparation of
chorionic villi, and only samples with a normal karyotype
were used for further analysis.
Immunohistochemistry
Immunohistochemical studies were carried out on 4 mm thick
tissue sections using a Novolynk Polymer Detection System
(Novocastra Laboratories Ltd.) with rabbit anti-human
polyclonal prolactin antibody (DakoCytomation), mouse
anti-human tumor necrosis factor-a monoclonal antibody
(clone 195; Chemicon International), mouse anti-human
monoclonal IL-2 antibody (clone 297C16F11, BioSource
Europe S.A.) and rabbit anti-human IFN-g polyclonal
antibody (sc-8308; Santa Cruz Biotechnology). Sections
were deparaffinized in Bio-Clear (Bio Optica) for 20 minutes
then washed twice in ethanol.
For the TNF-a analysis, the slides were placed in a water
bath containing 9 mM sodium citrate at pH 6.0 for 30 minutes
at 95
C. Endogenous peroxidase activity was quenched with
3% hydrogen peroxide in distilled water for 10 minutes.
Staining was performed with 3,30 diaminobenzidine (DAB)
as a chromogen.
For the PRL staining, the primary antibody was applied at
a concentration of 1:500 in 0.5% bovine serum albumin (BSA)
and sodium azide and incubated 30 minutes at room
temperature. For IL-2 staining, the primary antibody was
applied at a concentration of 1:50 in 0.5% BSA and sodium
azide and incubated 30 minutes at room temperature. For
109
ORIGINAL ARTICLE: EARLY PREGNANCY
TNF-a staining, the primary antibody was applied at
a concentration of 1:200 in 0.5% BSA and sodium azide
and incubated 30 minutes at room temperature. For IFN-g
staining, the primary antibody was applied at a concentration
of 1:300 in 0.5% BSA and sodium azide and incubated 30
minutes at room temperature.
For each batch, slides with an absence of the primary
antibody were included as negative controls. Immunohisto-
chemical analysis detected the site and pattern of expression
of PRL, TNF-a, IL-2, and IFN-g.
Ribonucleic Acid Extraction and RT-PCR
Decidua and villous tissue samples stored at 70
C were
homogenized in phenol-guanidine isothiocyanate (TRIzol;
GIBCO BRL). The total RNA was extracted in chloroform and
precipitated by centrifugation in isopropanol (12,000  g for
10 minutes, at 4
C). The RNA pellet was washed with 75%
ethanol, resuspended in diethyl pyrocarbonate-treated water,
and quantified by absorbance at 260/280 nm. For detection of
PRL, PRL-R, TNF-a, and IL-2 messenger RNA (mRNA), 3 mg of
total RNA, 2 mL of dNTPs, and 1.5 mL of random hexamer
primers (Promega) were denatured at 65
C for 10 minutes,
and the reverse-transcriptase (RT) reaction was performed at
37
C for 60 minutes in the presence of M-MLV Reverse-
transcriptase (Moloney murine leukemia virus reverse
transcriptase; Promega) and recombinant ribonuclease
inhibitor (Promega). Complementary DNA fragments were
amplified by using intron spanning primers designed to
prevent the amplification of contaminating genomic DNA.
The primer sequences are available upon request. The amplifi-
cation of the ubiquitously expressed HGPRT gene was used as
the reverse-transcriptase polymerase chain reaction (RT-PCR)
control, as previously described elsewhere (22). The qualitative
PCR protocol comprised 30 amplification cycles in all cases
(annealing temperature: 56
C for PRL and 62
C for PRL-R).
The PCR products were electrophoresed on a 3% polyacryl-
amide gel stained with ethidium bromide and visualized under
ultraviolet light. The detection of bands corresponding to the
amplification products was confirmed by means of software
analysis (Quantity One; Bio-Rad). The RT-PCR analysis
detected transcripts of PRL, PRL-R, TNF-a, and IL-2. The
laboratory workers for the immunohistochemical and
RT-PCR assays were blinded to the origin of the samples.
FIGURE 1
84 SM
14 no mitosis
24 SM in study group
Study population: analysis of fetal karyotype.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.
110
Statistical Analysis
Data are presented as mean  standard deviation (SD).
Differences between the mean were analyzed with Student's
t test for unpaired samples; differences between decidua
and villous expression of cytokines in SM and VTP were
analyzed by a Fisher two-tailed exact test corrected
chi-square using Statistica for Windows. P< .05 was
considered statistically significant.
RESULTS
In 14 (16.7%) of 84 villous samples of the study group and 6
(21.4%) of 28 of the control group karyotyping failed whereas
46 (65.7%) of 70 cases in the study group and 1 (4.5%) of 22 in
the control group had abnormal karyotypes (P< .001) (Fig. 1).
Thus, further analyses were performed in 24 samples from the
SM and 21 from the VTP groups. No statistically significant
differences were found in women's age (SM 30  5.2 years,
range 21–39 years; VTP 30.1  6.5 years, range 17–41 years)
or in gestational age (SM 9.2  1.5 weeks, range: 6.6–13
weeks; VTP 9.5  1.4 weeks, range: 6.5–12.5 weeks) between
the two groups. The male/female ratio among the euploid
samples was 1.02. The absence of a female excess in the
products of conception supports the lack contamination of
maternal cells.
In six cases of the study group and one of the control
group, the tissue samples were inadequate for RT-PCR; thus,
the analysis was performed in 18 of 24 cases in the SM group
and in 20 of 21 cases of the VTP group. However, the
immunohistochemical analysis was performed in all cases
of both groups. The RT-PCR and immunohistochemical
studies demonstrated that PRL-R, PRL, TNF-a, IL-2, and
IFN-g were all expressed at the implantation site and were
detected in the proteins of various tissues from the
maternofetal interface.
Prolactin and PRL-R
Expression of PRL-R mRNA was detected in all villous
samples of both groups. In the decidua, it was present in
16 (88.9%) of 18 specimens of the SM group and in 20 of
20 controls (Table 1).
Transcripts of PRL were identified in 17 (94.4%) of 18
decidua samples of the study group and 19 (95%) of 20 of the
112 women
28 VTP
46 abnormal karyotype 6 no mitosis 1 abnormal karyotype
21 VTP in control group
VOL. 100 NO. 1 / JULY 2013

TABLE 1
RT-PCR expression of PRL, PRL-R, TNF-a, and IL-2 and immunohistochemical expression of PRL, TNF-a, IL-2, and IFN g in decidual and
villous samples of spontaneous miscarriage and voluntary pregnancy termination.
Spontaneous miscarriage (study group)
Decidua (%) Villi (%)
RT-PCR expression
PRL 94
PRL-R 89 100
TNF-a 72
IL-2 56
Immunohistochemistry expression
PRL 96
TNF-a 79
IL-2 100*
IFN-g 100 100
Note: IFN-g ¼ interferon g; IL-2 ¼ interleukin 2; PRL ¼ prolactin; PRL-R ¼ prolactin receptor; RT-PCR ¼ reverse-transcriptase polymerase chain reaction; TNF-a ¼ tumor necrosis factor a.
* P< .001.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.
control group. In contrast, in the villous samples even though
a broad expression was present in the control group (16 of
20, 80%), the expression of PRL-mRNA was present only in 4
(22.2%) of 18 SM cases (Table 1; P< .001) (Supplemental
Fig. 1). Immunohistochemistry confirmed the different villous
expression pattern: immunostaining for PRL was detected in
all the specimens from the VTP group (21 of 21) and none
from the SM group (Table 1; P< .001) (Supplemental Fig. 2).
In the controls, PRL was clearly detected in cytotrophoblasts,
macrophages, and stromal cells (Fig. 2). In the decidua, PRL im-
munoexpression was present in all the specimens of the control
group and in 23 (95.8%) of 24 of the study group. The compar-
ison of the different slides revealed stronger staining in the
superficial stromal layer than in the deeper stroma (Fig. 3).
TNF-a
Messenger RNA expression of TNF-a was detected in 20 of 20
villous samples of the VTP group and 14 (77.8%) of 18 of the SM
group (Supplemental Fig. 1). Similarly, all decidua samples of
the control group showed expression of TNF-a when compared
with 13 (72.2%) of 18 of the study group. Immunostaining re-
lated to TNF-a protein was demonstrated in the villous samples
of 15 (71.4%) of 21 controls and in 14 (58.3%) of 24 study group
cases (see Table 1). When it was detected, staining for TNF-a
was shown in syncytiotrophoblasts, cytotrophoblasts, macro-
phages, endovascular trophoblast, and stromal cells. The
expression pattern was similar for both groups (see Fig. 2).
In the decidua, the expression of TNF-a was detected in 20
(95.2%) of 21 samples of the VTP group and in 19 (79.2%) of 24
samples of the SM group (Table 1). In contrast with the expres-
sion of PRL, in both groups TNF-a immunostaining was stron-
ger in the deep decidua than in the surface decidua (see Fig. 3).
Even though the results were not statistically significant,
immunohistochemical analysis confirmed the deficient ex-
pression of TNF-a in the study group compared with controls.
IL-2
We detected IL-2 mRNA in 9 (45%) of 20 decidual samples
from the VTP group and in 10 (55.6%) of 18 from the SM
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Fertility and Sterility®
Voluntary pregnancy termination (control group)
Decidua (%) Villi (%)
22* 95 80
100 100
78 100 100
17 45 0
0* 100 100
58 95 71
17 57 0
100 100
group. In the villous tissues, expression of transcripts of
IL-2 was demonstrated in 3 (16.6%) of 18 specimens of
the study group and in none of the controls (see Table 1).
Immunostaining of IL-2 was detected in 12 (57.1%) of 21
decidual samples from the control group and in all samples
from the study group (Table 1; P< .001) (Supplemental Fig.
2). No differences were shown in the villous samples expres-
sion [4 of 24 study group; 0 of 21 controls]. Thus, the IL-2 re-
sults were diametrically opposed to those for PRL and TNF-a:
a more substantial expression of IL-2 in samples from SM
compared with VTP. The immunohistochemical IL-2 expres-
sion is shown in Figures 2 and 3. In both groups, when pres-
ent, the expression pattern was similar.
IFN-g
The immunohistochemical signal related to IFN-g was
detected in all decidual and villous samples of the study and
control groups (Table 1). In villous specimens, immunostain-
ing of IFN-g was clearly present in syncytiotrophoblasts, cy-
totrophoblasts, macrophages, and stromal cells (see Fig. 2).
The signal was strongly evident also in the decidual samples,
both in the stromal cells and the glandular epithelium
(see Fig. 3).
DISCUSSION
In our study, an undetectable level of PRL expression in the
villi and a reduced level of TNF-a expression in both the
decidua and the villi were a frequent finding in samples
from SM when compared with VTP. In contrast, we found
a reduced expression of IL-2 in vital pregnancies compared
with spontaneous miscarriages. The expression of PRL-R in
villous and decidual samples as well as of PRL in the decidua
has been previously reported (17, 18, 23); we found no
statistically significant differences between the study and
control groups.
In tissues from SM, one would expect that the presence of
hypoxia, necrosis, apoptosis, and inflammation would alter
the protein expression in the villi and that the mere absence
of vaginal bleeding at the time of hospitalization might be
111
ORIGINAL ARTICLE: EARLY PREGNANCY

FIGURE 2

Immunohistochemical cytokine expression in villous samples of spontaneous miscarriage (SM) and voluntary termination of pregnancy (VTP).
(A) Prolactin (PRL) immunoreaction was negative in SM cases [A1] while, in VTP cases a strong PRL immunostaining was detected in
cytotrophoblast, macrophages and stromal cells [A2]. (B) No differences in the immunoreaction of tumor necrosis factor a (TNF-a) were present
between cases [B1] and controls [B2]: syncytiotrophoblast, cytotrophoblast, macrophage, and stromal cells were TNF-a positive. (C) Interleukin-
2 (IL-2) immunoreaction was negative in VTP [C2] samples while positive IL-2 immunostaining has been shown in a few SM samples in
cytotrophoblast, macrophages and stromal cells [C1]. (D) No differences in the immunoreaction of interferon gamma (IFN-g) were present
between cases [D1] and controls [D2]: syncytiotrophoblast, cytotrophoblast, macrophage, and stromal cells were IFN-g positive. Original
magnification: 20.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.

insufficient in preventing the effects of these potential
confounding inflammatory changes. Our data, however,
show different results in SM villous samples compared with
controls for any cytokine expression: from a greatly impaired
expression, as seen for PRL, to a null variation as
demonstrated in PRL-R and IFN-g expression. Therefore, it
seems unlikely that the differences between study and control
group are related only to the inflammatory changes
associated to necrosis.
Prolactin protein is expressed in all vertebrates, and more
than 300 different actions have been reported, many of which
are directly or indirectly associated with the process of
reproduction (24). Prolactin acts on its receptors in the
glandular compartment to up-regulate the expression of
target genes such as IRF-1 (25). In animal models, IRF-1
regulates the expression of a wide array of genes associated
with cellular proliferation, growth inhibition, and differentia-
tion (26). The target genes of PRL via IRF-1 in the human

112 VOL. 100 NO. 1 / JULY 2013

 Fertility and Sterility®

FIGURE 3

Immunohistochemical cytokine expression in decidua basalis and parietalis. The decidual stromal cells (arrow) and glandular epithelium (arrowhead)
expressed all cytokines investigated in our study, albeit with different intensity of staining. *Trophoblastic cell columns of anchoring villi. $Spiral
arteries. Original magnification: A1, B1, C1, D1 ¼ 20; A2, B2, C2, D2 ¼ 40.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.

endometrium remain to be elucidated. In patients affected by
unexplained infertility and repeated miscarriages, impaired
PRL expression during the mid-to-late secretory phase has
been clearly demonstrated (27). In agreement with our results,
recently Salker et al. (28) showed higher prokinecitin-1 mRNA
levels (PROK-1, a recently identified key regulator of
endometrial receptivity) and approximately 100-fold lower
PRL levels in endometrial biopsy samples timed in the
implantation window of patients affected by recurrent
pregnancy loss. The favorable role that has been proposed
for PRL in embryo implantation is explained through the
modification of the immune environment of the maternofetal
interface or the regulation of factors that may control
trophoblast proliferation and invasion of the endometrium
(18, 27, 29).
In addition, our current data suggest the prominence of
the trophoblast in maternofetal signaling. To our knowledge,
ours is the first study to investigate PRL expression in human
villous samples: the demonstration of PRL villous expression
is clearly related to vital pregnancies.
The broad expression of TNF-a and IFN-g shown in the
samples from the control group is strongly in contrast
with the theory that a TH1 type cytokine bias is related to
reproduction failure. The Wegmann paradigm ‘‘pregnancy is

VOL. 100 NO. 1 / JULY 2013 113

ORIGINAL ARTICLE: EARLY PREGNANCY
a TH2 phenomenon’’ seemed, for a long time, to be
a convincing model of the maternal immune system approach
to a semiallogenic graft (5). In fact, our detection of TNF-a
and IFN-g in the fetomaternal interface of vital pregnancies
is in agreement with previous findings (30–32), and
the demonstration of strong immunostaining for TNF-a in
the endovascular trophoblast supports a direct role of this
cytokine in the decidual invasion process.
Several experimental animal studies suggest the presence
of a dual-dose effect for the TH1 cytokines, with a high dose
being abortive and a total absence inversely affecting local
vasculogenesis and hindering proper spiral arteries
generation (33–36). Fluhr et al. (37) have shown that human
endometrial stromal cells, primarily resistant to Fas-
mediated apoptosis, can be sensitized just by TNF-a and
IFN-g, becoming apoptotic in response to the Fas-ligand
secreted by the first trimester trophoblast cells. The
coexistence of decidual inflammation and evolutive
pregnancy is otherwise shown by studies that, to maximize
the receptivity of the uterine cavity to embryo implantation,
caused an injury-induced endometrial inflammation through
several biopsies (38, 39). These studies have shown a positive
correlation between the increased expression of TNF-a and
MIP-1B (macrophage inflammatory protein 1B) after
endometrial biopsies and the pregnancy outcome.
The lack of IL-2 expression in villous samples, both
transcripts and protein, is consistent with previous evidence
(30, 31). Immunostaining of IL-2 was broadly expressed in
the decidual samples of the study group and significantly
defected in the controls. This result is important in view of
the fact that IL-2 is a potent inducer of decidual natural killer
(NK) cell cytotoxicity and proliferation (40, 41). The
discrepancies between our immunohistochemistry and RT-
PCR data need an explanation; it seems likely that the immu-
nohistochemical expression reveals the early production of
the protein and its storage into the stromal cells of the decidua
of the SM group while the transcript has been degraded. Nev-
ertheless, the IL-2 decidual expression is strongly related to
implantation failure. Kanda et al. (20) demonstrated the inhi-
bition of PRL synthesis and release by adding IL-2 in primary
decidual cell cultures media. Our data show in vivo a similar
inverse relationship between PRL (villi) and IL-2 (decidua) ex-
pression: in the control group, PRL was strongly expressed
and IL-2 was lacking; in the SM group, PRL expression was
reduced/absent and IL-2 was present. This particular
expression pattern suggests a role of the trophoblast in
determining the physiologic evolution or failure of embryo
implantation due to the reciprocal influence of PRL and
IL-2 at the interface. In the decidua of PRL-knockout mice,
Bao et al. (42) demonstrated the expression of IL-6,
a proinflammatory cytokine highly detrimental for
pregnancy: as a consequence, these mice were infertile.
In vitro PRL inhibits IL-6 expression; similarly, in vivo it
represses the cytokine expression, restoring physiologic
fertility.
There is mounting evidence that, in the current view of
the fetomaternal ‘‘seed and soil’’ interplay, the seed has
heavier weight. The positive correlation between villous PRL
expression and successful implantation strongly suggests
114
a pivotal role for fetal PRL in determining endometrial
differentiation and receptivity. Prolactin may inhibit peptides
detrimental to implantation (e.g., IL-2) and/or stimulate
cytokines (e.g., TNF-a) that may recruit immune cells,
sensitize the endometrial stromal cells to Fas-mediated
apoptosis, and trigger the production of molecules that
interact with the blastocyst facilitating its apposition and
attachment to the uterine wall.
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ORIGINAL ARTICLE: EARLY PREGNANCY

SUPPLEMENTAL FIGURE 1

RT-PCR of PRL-R, PRL, TNF-a, and IL-2 in the villi (A) and decidua (B) in
samples from spontaneous miscarriage (white bars) and voluntary
termination of pregnancy (grey bars). *P<.04; **P<.02; ***P<.001.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.

115.e1 VOL. 100 NO. 1 / JULY 2013

 Fertility and Sterility®

SUPPLEMENTAL FIGURE 2

Immunostaining of PRL, TNF-a, IL-2, and IFN-g in the villi (A) and
decidua (B) samples from spontaneous miscarriage (white bars) and
voluntary termination of pregnancy (grey bars). ***P<.001.
Garzia. Prolactin expression and miscarriage. Fertil Steril 2013.

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