Human Reproduction Vol.18, No.3 pp.

608615, 2003

DOI: 10.1093/humrep/deg139

Analysis of intra-uterine cytokine concentration and

matrix-metalloproteinase activity in women with recurrent
failed embryo transfer
N.Inagaki1,2,3,4, C.Stern1, J.McBain1, A.Lopata1,2, L.Kornman2 and D.Wilkinson1,5
1

Reproductive Biology Unit in the Royal Women's Hospital, 2The Department of Obstetrics and Gynaecology, University of
Melbourne, Royal Women's Hospital, Melbourne and 3The Pregnancy Research Centre in the Royal Women's Hospital, Carlton,
Victoria, Australia
4

Present address: Obstetrics and Gynecology, Saitama Municipal Hospital, 2460, Saitama City, Saitama Pref., Japan 336-8522

To whom correspondence should be addressed at: Reproductive Biology Unit, The Royal Women's Hospital, 132, Grattan Street,
Carlton, Victoria, 3053, Australia. E-mail: david.wilkinson150@mivf.com.au

BACKGROUND: In all IVF programmes, some patients fail to achieve an ongoing pregnancy, even after numerous
embryo transfer procedures. An unfavourable environment within the uterus might be a contributory factor to such
recurrent implantation failure. This question was addressed by measuring cytokine concentrations and matrix
metalloproteinase activities in uid derived from uterine irrigation of such patients. METHODS AND RESULTS:
The uterine cavities of 22 patients who had previously undergone embryo transfer of at least 10 embryos without
ongoing pregnancy were irrigated during the luteal phase. The resultant uid was assayed for the concentration of
interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, leukaemia inhibitory factor
(LIF), IL-10 and matrix metalloproteinase (MMP) -2 and -9 activity. The results were compared with those of a
control population of women known to be previously fertile (n = 16) and also with women with recurrent
spontaneous abortion (n = 13). In the recurrent implantation failure group, the MMP score and IL-1beta
concentration were signicantly higher than those in the control group, whereas concentrations of IFN-gamma and
IL-10 were signicantly lower. CONCLUSIONS: In IVF patients with recurrent implantation failure, an altered
pattern of intra-uterine cytokine concentration and MMP activity was observed.
Key words: implantation failure/interleukin-1beta/interleukin-10/matrix metalloproteinase/uterine ushing

Introduction
Despite continuing advances in assisted reproductive technology (ART), all IVF programmes include patients who fail to
become pregnant despite numerous embryo transfer procedures. Whilst it is well known that much of the failure of embryo
implantation may be explained by chromosomal abnormalities,
it has been postulated that in some patients there may be a
problem with uterine receptivity. This, hypothetically, could be
at the level of an unfavourable intra-uterine milieu or an
endometrial lining that is hostile to embryonic implantation.
In natural pregnancies, the developing embryo usually enters
the uterine cavity at the morula stage and spends 23 days
within the cavity prior to implantation at the blastocyst stage.
Successful implantation therefore depends upon appropriate
intra-uterine conditions as well as a receptive endometrium. In
the case of embryo transfer following IVF or ICSI, embryos are
commonly transferred into the uterine cavity at day 2 or 3, and
spend 4 or 3 days respectively in the uterine cavity prior to
implantation. The difculties encountered by many IVF units
608

in establishing efcient extended in-vitro blastocyst culture

systems reinforces the importance of the extra-embryonic
environment during this pre-implantation phase.
Substantial published data exist relating to possible factors
involved in successful embryo implantation, both in animal
models and in humans (Edwards, 1995; Bischof, 2000). Such
studies have implicated a variety of cytokines (Lopata, 1996;
Rice and Chard, 1998; Sharkey, 1998; Simon et al., 1998),
prostaglandin-associated enzymes (Bonventre et al., 1997; Lim
et al., 1997), matrix metalloproteinases (MMP) (Bischof et al.,
1994), adhesion molecules (e.g. integrin beta3; Lessey et al.,
1994, 1995; Lessey, 1997, 2000, 2002; Illera et al., 2000;
Damario et al., 2001), cell surface mucins (e.g. MUC-1),
altered cellular populations and pinopode formation (Martel
et al., 1991; Bentin-Ley, 2000; Nikas, 2000; Nardo et al., 2002)
in the implantation process. Despite this wealth of information,
the relative contribution of these various factors to successful
implantation remains unresolved. What can be stated is that the
process requires a number of facilitatory events which include
European Society of Human Reproduction and Embryology

Intra-uterine cytokines and MMP after failed IVFembryo transfer

maternal immunological tolerance of the hemi-allogenic

embryo.
A number of reports have implicated altered ratios of
T-helper (Th) 1-type lymphocytes and changes in cytokine
concentration with unexplained recurrent miscarriage (Hill
et al., 1995; Makhseed et al., 1999; Raghupathy et al., 1999,
2000; Jenkins et al., 2000; Lim et al., 2000). However, these
studies have generally focused on cytokine mRNA levels
rather than the actual protein concentrations within the uterine
cavity.
In the present study the intra-uterine concentrations of a
number of cytokines thought to be important for embryo
implantation were analysed. In addition, MMP activity was
assayed, as these proteases are thought to be intimately
involved in the implantation process. The decision was made
to analyse women on the authors' IVF programme who had
failed to achieve an ongoing pregnancy despite the transfer of
at least 10 embryos. It was reasoned that in some of these
women, there might be factors other than intrinsic problems
with embryo quality that were contributing to their failure to
achieve an ongoing pregnancy. The method used to assess
cytokine and MMP concentrations within the uterine cavity
was irrigation (ushing) followed by analysis of the resultant
uid. This method has been shown previously to provide a
reproducible means of assaying both MMPs (Laird et al., 1999)
and cytokines (Licht et al., 1998). Similar analyses were
performed on two other groups of patients as controls. The rst
control group included multiparous women undergoing surgical reversal of sterilization; the reproductive history of this
group suggested that previously they had not experienced any
signicant problem with embryonic implantation. The second
control group included women with a history of recurrent
spontaneous abortion; for these patients it was reasonable to
suggest that the conditions within their uterus were facilitatory
for early implantation events, with problems arising later in
pregnancy.
The cytokines monitored in this preliminary study were
interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha,
interferon (IFN)-gamma, leukaemia inhibitory factor (LIF) and
IL-10 because of their proposed roles in embryonic implantation. The IL-1 receptor is expressed on the endometrial
surface epithelium throughout the menstrual cycle (Simon
et al., 1993), and human pre-implantation embryos have been
shown to express mRNA for IL-1beta (Krussel et al., 1998).
Embryonic secretion of IL-1beta is thought to be important to
the implantation process. However, high levels of this cytokine
have also been observed in association with intra-uterine
devices (IUD) and inammatory responses (Dechaud et al.,
1998); therefore, it is possible that high levels of this cytokine
could be detrimental to successful implantation. In conclusion,
the precise role of IL-1beta in implantation remains uncertain
as gene knockout mice not expressing the IL-1beta gene are
fertile (Abbondanzo et al., 1996).
TNF-alpha is produced by a number of cell types (including
macrophages) and is associated with inammatory responses.
IFN-gamma produced largely by T lymphocytes and natural
killer (NK) cells is a marker for activation of these cell types.
IL-10 may play a role in the suppression of immune responses

and therefore could potentially be important in maternal

immunological tolerance of the embryo (Trautman et al., 1997;
Piccinni et al., 2000; Clark and Croitoru, 2001). In addition,
LIF was assayed as gene knockout studies have demonstrated a
pivotal role for this cytokine in murine embryonic implantation
(Stewart et al., 1992).

Materials and methods

Patients
Patients were recruited from the outpatient department. Those with
recurrent implantation failure were categorized as patients who did not
have an ongoing pregnancy after 10 or more embryos had been
transferred, without possible aetiological factors such as parental
chromosomal abnormalities, antiphospholipid antibodies and intracavitary uterine leiomyomata. The primary indication for IVF/ICSI in
these women included unexplained infertility, endometriosis and male
factor infertility. The aim was to obtain these samples during the
putative window of receptivity for implantation (i.e. mid-luteal
phase). Although 26 samples were collected for this study, four
were eliminated for inappropriate dating by endometrial biopsy, as
described below.
As controls, samples were taken from multiparous women with a
history of tubal sterilization who were undergoing surgical tubal
anastomosis. A number of these women became pregnant while the
study was in progress. In the control group, 16 samples were obtained
in the luteal phase and nine in the proliferative phase for comparative
purposes. In addition, a group of women with a history of unexplained
recurrent spontaneous abortion was studied; these included 13 patients
categorized as having three or more consecutive spontaneous
abortions without a live birth and who had been screened for thyroid
function, parental chromosomal abnormalities, antiphospholipid
antibodies, hereditary thrombophilias and infectious diseases. For
patients with recurrent miscarriage, the level of serum progesterone
was used to conrm that samples had been collected in the luteal
phase.
This study was approved by the Research and Ethics committees at
the Royal Women's Hospital. Samples were collected only from
patients who gave informed consent.
Uterine cavity irrigation
A bivalve speculum was inserted into the vagina and a exible infant
feeding tube (2.7 mm diameter) was introduced into the uterine cavity
through the cervical canal. A syringe was then used to instil 5 ml of
sterile saline into the uterine cavity. The uterine contents were
aspirated quickly and collected without contamination by vaginal
uid. Samples were centrifuged at 1000 g for 10 min, after which the
supernatants were collected and stored at 80C. Following uterine
irrigation, all patients in the recurrent failed embryo transfer group
underwent hysteroscopic examination of their uterine cavity;
endometrial biopsies were performed to conrm secretory-phase
endometrium.
Enzyme-linked immunosorbent assay (ELISA) of cytokines
A quantitative sandwich enzyme immunoassay technique was used in
accordance with the manufacturer's protocol. Quantitative ELISA kits
for detecting IL-1beta, TNF-alpha, IFN-gamma, and IL-10 were
obtained from R&D systems (Minneapolis, MN, USA). The limits of
detection for these cytokines by ELISA were <1, 4.4, 8, 8 and 3.9 pg/ml
respectively. None of the samples examined had a cytokine level
>1000 pg/ml. Unfortunately, not all cytokines were measurable in all

609

N.Inagaki et al.

samples due either to insufcient volume or to a limited number of

ELISA assays being performed.
Gelatin zymography measurement of MMP-2 and MMP-9
Gelatin zymography was used to monitor picogram levels of MMP-2
and MMP-9, according to a published method (Fridman et al., 1992),
albeit slightly modied (Kleiner and Stetler-Stevenson, 1994). Briey,
gelatin at a nal concentration of 1 mg/ml was incorporated into the
running polyacrylamide gel containing 10% acrylamide (Bio-Rad,
CA, USA), 25% Tris buffer (1.5 mol/l, pH 8.8), 0.4% sodium dodecyl
sulphate (SDS; ICN, Ohio, USA), 0.3% ammonium peroxodisulphate
(APS; ICN) and 0.1% N,N,N,N-tetramethylethylendiamine (TEMED;
ICN). The stacking gel containing 2% acrylamide, 25% Tris buffer
(Tris 0.5 mol/l, pH 6.5), 0.4% SDS, 0.4% APS and 0.1% TEMED was
placed on top of the running gel. The same volume of sample buffer
consisting of 17.5% SDS, 7% sucrose and bromophenol blue (1 mg/ml)
was added to each sample. Aliquots (10 ml) of each sample were
loaded into wells and the proteins were electrophoresed for ~1 h at
200 V. After electrophoresis, the gels were washed ve times for 5 min
in a Tris-based solution consisting of 3% Triton X-100 (ICN). Gels
were then washed three times and incubated for 48 h at 37C in a
solution containing 5.8% TrisHCl, 1.7% Tris base, 0.1% NaN3, 0.7%
CaCl22H2O and 5% 100 mmol/l ZnCl2. After incubation, the gels
were stained for 6 h with 0.1% Coomassie brilliant blue.
The presence of gelatinases was conrmed by their inhibition using
EDTA and p-phenathroline (data not shown). Other MMPs (e.g.
MMP-1, -3 and -7), which can be detected using casein zymography
including 1 mg/ml casein were not normally detected in the uterine
irrigation uid.
Quantitation of MMPs on zymograms
Although zymograms can be semi-quantitated using densitometric
analysis (Kleiner and Stetler-Stevenson, 1994; Salamonsen et al.,
1997), this is accurate only at picogram levels of MMPs when the
bands on the zymograms are faint. Analysis of stronger bands results
in underestimation of levels. Because of the very high amount of
gelatinase activity in many of the samples analysed, an alternative
semi-quantitative technique was used. The neat samples of ushing
uid were diluted 40-fold before running gels for scoring.

For each patient, gelatinolytic activity was estimated when seen as

clear bands against a blue background. The individual bands revealed
the presence of various molecular weight enzymes and bands
identied as proMMP-2, active MMP-2, proMMP-9, active MMP-9
and dimeric MMP-9, the intensities of which were individually graded
by visual inspection on a scale of 0 (no band) to 5 (very strong) and
summarized for the total score. All analyses were performed by two
independent observers who were blinded to the sample type. A total
MMP score for each sample was achieved by adding the scores for
each band in individual lanes. Inter-assay variability was assessed as
10.3%. Any gel on which the intensity of the bands in the standard
sample was not consistent was repeated.
Protein assay
Protein concentrations in the irrigation uid were monitored using the
Bio-Rad Detergent Compatible Protein Assay (Bio-Rad) according to
the product manual.
Statistical analysis
Data were analysed using the WilcoxonMannWhitney U-test,
Welch's unpaired t-test and Student's t-test where appropriate. For
the statistical analysis, P < 0.05 was considered signicant. Pearson's
correlation tests were performed using Microsoft EXCEL.

Results
When samples were obtained during the luteal phase, the aim
was to achieve this during the putative window of uterine
receptivity for implantation. The mean (6SD) for day of
sampling was day 21.1 6 2.2 (n = 16) for the control samples,
day 22.0 6 2.7 (n = 22) for the patients with implantation
failure, and day 22.2 6 3.7 (n = 12) for the patients with
recurrent miscarriage (Table I).
The control population of multiparous women undergoing
surgical reversal of sterilization showed a low MMP score, and
low levels of IL-1beta, TNF-alpha and IFN-gamma in the
luteal phase as well as in the proliferative phase (Table I). No
sample in the luteal phase contained >10 pg/ml IL-1beta. LIF

Table I. Matrix metalloproteinase (MMP) score and cytokine concentrations in irrigation uid of patients with recurrent implantation failure on unexplained
recurrent miscarriage, and in control patients. Values are mean 6 SD.
Group

Control group
Luteal phase
Proliferative phase
Recurrent implantation
failure group
Luteal phase
Unexplained recurrent
miscarriage group
Luteal phase
aP

Age (years)

Day in
menstrual
cycle

MMP
score

IL-1beta
(pg/ml)

TNF-alpha
(pg/ml)

IFN-gamma
(pg/ml)

LIF
(pg/ml)

IL-10
(pg/ml)

33.6
(n =
35.9
(n =

21.1
(n =
10.3
(n =

0.5 6 1.3
(n = 16)
1.4 6 1.6
(n = 9)

2.15
(n =
6.21
(n =

9.31
(n =
7.96
(n =

16.28 6 12.69
(n = 16)
13.73 6 8.98
(n = 9)

15.32 6 26.54
(n = 16)
4.50 6 2.65
(n = 7)

7.24
(n =
5.27
(n =

6 3.9
16)
6 5.6
9)

6 3.2
16)
6 3.6
9)

6 1.82
16)
6 10.35
9)

6 6.73
15)
6 5.82
9)

35.0 6 3.6
(n = 22)

22.0 6 2.7
(n = 22)

5.0 6 3.1a
(n = 22)

80.576 160.21b
(n = 22)

21.69 6 35.16
(n = 22)

1.65 6 2.38c
(n = 22)

5.86 6 4.25
(n = 22)

1.27 6 2.63d
(n = 15)

31.8 6 5.0
(n = 13)

22.2 6 3.7
(n = 12)

0.0 6 0.0
(n = 13)

2.66 6 2.22
(n = 7)

18.57 6 25.73
(n = 9)

15.32 6 20.56
(n = 6)

5.51 6 5.19
(n = 5)

5.75 6 2.70
(n = 8)

< 0.05 versus control group (luteal phase and proliferative phase) and the unexplained recurrent miscarriage group.
< 0.05 versus control group (luteal phase and proliferative phase) and the unexplained recurrent miscarriage group.
cP < 0.05 versus control group (luteal phase and proliferative phase).
dP < 0.05 versus control group (luteal phase and proliferative phase) and the unexplained recurrent miscarriage group.
(WilcoxonMannWhitney U-test and Welch's unpaired t-test).
IFN = interferon; IL = interleukin; LIF = leukaemia inhibitory factor; TNF = tumour necrosis factor.
bP

610

6 3.95
13)
6 2.36
6)

Intra-uterine cytokines and MMP after failed IVFembryo transfer

Figure 1. Matrix-metalloproteinase (MMP) scores in patients with recurrent implantation failure following embryo transfer. Lane 1: Standard
from the supernatant of BHK9 breast carcinoma cell culture. Lanes 2, 3, 4, 5, 6, 7 and 8: Samples that were scored l, 12, 0, 3, 7, 4 and 0
respectively. Lane 2: proMMP-2: 1, activeMMP-2: 0, proMMP-9: 0, activeMMP-9: 0, dimericMMP-9: 0. Lane 3: proMMP-2: 1, activeMMP2: 0, proMMP-9: 5, activeMMP-9: 2, dimericMMP-9: 4. Lane 4: proMMP-2: 0, activeMMP-2: 0, proMMP-9: 0, activeMMP-9: 0,
dimericMMP-9: 0. Lane 5: proMMP-2: 1, activeMMP-2: 0, proMMP-9: 2, activeMMP-9: 0, dimericMMP-9: 0. Lane 6: proMMP-2: 1,
activeMMP-2: 0, proMMP-9: 3, activeMMP-9: 1, dimericMMP-9: 2. Lane 7: proMMP-2: 1, activeMMP-2: 0, proMMP-9: 2, activeMMP-9:
0, dimericMMP-9: 1. Lane 8: proMMP-2: 0, activeMMP-2: 0, proMMP-9: 0, activeMMP-9: 0, dimericMMP-9: 0. The details of scoring are
described in the Materials and methods.

Table II. Analysis of the individual matrix metalloproteinase (MMP) bands in patients with recurrent implantation failure. Values are mean 6 SD.

All patients with

recurrent implantation
failure (n = 22)
Patients with a total
MMP score >4 (n = 12)

Pro MMP-2

Active
MMP-2

Total
MMP-2

Pro
MMP-9

Active
MMP-9

Dimeric
MMP-9

Total
MMP-9

Total
MMP score

1.4 6 1.2

0.1 6 0.3

1.5 6 1.3

1.9 6 1.4

0.4 6 0.7

1.2 6 1.1

3.5 6 2.9a

5.0 6 3.1

1.8 6 1.3

0.1 6 0.3

1.9 6 1.5

2.7 6 0.8

0.8 6 0.8b

1.9 6 1.0

5.3 6 2.6c

7.3 6 2.0

Total MMP-2 = pro MMP-2 + active MMP-2.

Total MMP-9 = pro MMP-9 + active MMP-9 + dimeric MMP-9.
aP < 0.05 versus total MMP-2.
bP < 0.05 versus active MMP-2.
cP < 0.05 versus total MMP-2.
(WilcoxonMannWhitney U-test, Welch's unpaired t-test and Student's t-test)

and IL-10 concentrations in the control group were 15.32 6

26.54 and 7.24 6 3.95 pg/ml in the luteal phase. The mean age
of the control patients was 33.6 6 3.9 years in the luteal phase,
and 35.9 6 5.6 years in the proliferative phase.
The patients in the unexplained recurrent miscarriage group
also showed low levels of MMP score, IL-1beta, TNF-alpha
and IFN-gamma (Table I).
Patients in the recurrent implantation failure group showed
signicantly higher levels of MMPs and IL-1beta than those in
the control group (Table I; Figures 1 and 2). When the
irrigation uid was diluted 40-fold for scoring, some 54.5% of
women (12/22) showed MMP scores >5 in the recurrent
implantation failure group, compared with 6% (1/16) in the
control luteal group. A detailed analysis of each MMP band
showed that the total score of MMP-9 was signicantly higher
than the total score of MMP-2 (Table II). For samples with a

total MMP score >5, the individual scores of active MMP-9

and total MMP-9 were signicantly higher than those of active
MMP-2 and total MMP-2 (Table II). The score of proMMP-9
also tended to be higher than that of proMMP-2, although the
difference was not signicant.
In the recurrent implantation failure group, 14 of 22 samples
showed an IL-1beta level >10 pg/ml, and nine samples showed
a level >30 pg/ml, whereas in the control group (luteal phase)
no sample showed a level >10 pg/ml. Some of the patients in the
recurrent implantation failure group showed very high level of
IL-1beta (Figure 2b). The MMP score was signicantly
correlated (r = 0.595; P = 0.0033) with IL-1beta in the
recurrent implantation failure group (Figure 3a), but no
correlations were detected between MMP score and protein
concentration, nor between IL-1beta and protein concentration
(Figure 3b and c). This implies that, in the recurrent implant611

N.Inagaki et al.

Figure 2. Dot plotting of MMP scores and cytokine levels of the control and study patients. (a) MMP score; (b) interleukin (IL)-1beta; (c)
interferon (IFN)-gamma; (d) leukaemia inhibitory factor (LIF); (e) IL-10. 1: control patients in luteal phase; 2: control patients in
proliferative phase; 3: patients with recurrent implantation failure in luteal phase; 4: patients with unexplained recurrent spontaneous abortion.
The individual number of patients in each group is the same as that in Table I.

ation failure group, the high levels of MMP score and IL-1beta
were independent of protein concentration in the irrigation
uid.
There was a tendency for the concentration of TNF-alpha in
the recurrent implantation failure group to be higher than that
in the control group, but this was not statistically signicant
(Table I). The concentration of IFN-gamma in the recurrent
implantation failure group was signicantly lower than that in
the control group (Table I; Figure 2c).
There was no statistically signicant difference in the
concentration of LIF between the implantation failure and
control groups (Table I; Figure 2d).
612

The concentration of IL-10 in the recurrent implantation

failure group was signicantly lower than that in control group
(Table I; Figure 2e); however, the absolute values were very
low and approaching the lower level of sensitivity of the assay.
Discussion
The relatively low rate of successful implantation following
embryo transfer after IVF and ICSI can be partly explained by
intrinsic problems within the embryos transferred. This has
been estimated to be as high as 6080% (Schelesseman, 1979;
Rudak et al., 1985; Papadopoulos et al., 1989; Edirisinghe

Intra-uterine cytokines and MMP after failed IVFembryo transfer

Figure 3. Correlations between: (a) MMP score and IL-1beta; (b) between MMP score and protein concentration; and (c) between IL-1beta
and protein concentration in the recurrent implantation failure group. Correlation coefcients (r) were: between MMP score and IL-1beta (r =
0.595); between MMP score and protein concentration (r = 0.267); and between IL-1beta and protein concentration (r = 0.134).

et al., 1992; Jamieson et al., 1994). However, even when the

embryos are screened for chromosomal abnormalities using
pre-implantation genetic diagnosis (PGD) prior to transfer, the
resulting implantation rate is generally <50% (Gianaroli et al.,
1997; Kahraman et al., 2001). This observation raises the
possibility that additional factors might contribute towards
failed implantation, particularly for younger women whose
embryos would be expected to have a lower incidence of
karyotypic abnormality.
A number of studies have investigated the role of
endometrial factors in implantation. For example, a correlation
has been noted between decreased expression of the vitronectin
receptor avb3 (Lessey et al., 2000) and decreased uterine
receptivity (Lessey et al., 1994, 1995; Lessey, 1997, 1998;
Illera et al., 2000; Damario et al., 2001). MUC-1 expression
has also been implicated in uterine receptivity (Braga and
Gendler, 1993; Hey et al., 1994; Croy et al., 1997; Aplin, 1999;
Horne et al., 2001). Similarly, pinopode formation is thought to
be important for embryonic implantation (Martel et al., 1991;
Nikas and Psychoyos, 1997; Nikas, 1999, 2000; StavreusEvers et al., 2001; Nardo et al., 2002). In contrast, there is a
relative paucity of information regarding the in-vivo conditions
within the uterine cavity around the time of implantation. For
this reason, cytokine concentrations and MMP activities were
analysed in women who had undergone multiple unsuccessful
embryo transfers. These women (and partners) had been
screened for other possible causes of implantation failure such
as karyotypic abnormalities, antiphospholipid antibodies, thyroid function and inherited thrombophilias.
In the present study, levels of cytokines and MMPs were
measured in the uterine ushings of women with recurrent
failed embryo transfer, and also in two control groups.
Following uterine irrigation, a hysteroscopy was performed
to exclude any intra-uterine abnormality and an endometrial
biopsy was taken to conrm secretory-phase endometrium. In
the recurrent implantation failure group, statistically signicant
elevated levels of IL-1beta and reduced levels of IFN-gamma
and IL-10 were found. Although not statistically signicant,

there was also a trend towards TNF-alpha being higher and LIF
lower in this group. The MMP score was signicantly higher in
the recurrent implantation failure group, and also correlated
with the level of IL-1beta. Differentially increased MMP-9
activity made the major contribution to the increased total
MMP score in these patients. These observations could not be
explained by differences in the protein concentration of the
uterine ushings from the different patients.
The pattern of cytokine expression in the recurrent implantation failure group was intriguing. Raised IL-1beta levels may
occur as a result of chronic inammation, for example chronic
endometritis, but there was no histopathological evidence of
endometritis in any of the endometrial biopsy specimens. There
also appears to be an association between IL-1beta levels and
MMP score. This observation is biologically plausible as
MMP-2 and MMP-9 production by endometrial stromal cells
can be induced by IL-1beta (Huang et al., 1998; Zhang et al.,
1998). It was also conrmed that IL-1beta induced the
expression of proMMP-2, active MMP-2, proMMP-9 and
active MMP9 bands by cultured endometrial stromal cells (data
not shown).
The low level of IL-10 in the implantation failure group is an
intriguing observation, as it might be hypothesized that
underexpression of this immunosuppressive cytokine could
be detrimental to the immunological tolerance of the embryo.
However, the low measurable concentrations of IL-10 in all the
groups suggests that caution be taken against overinterpretation.
In the present study, cytokine concentrations and MMP
activities were measured during `natural' menstrual cycles. It is
possible that the high estrogen and progesterone levels found in
the setting of an IVF-stimulated cycle might inuence the
results. However, it should also be noted that in the present
group of patients with recurrent implantation failure a signicant number of thawed embryos had been replaced during
`natural' cycles without success. It is believed therefore that the
natural cycle results obtained are relevant to the implantation
failure in this group.
613

N.Inagaki et al.

A recent report has shown that increased intra-uterine

concentrations of LIF and TNF-alpha was a negative
prognostic factor for subsequent pregnancy (Ledee-Bataille
et al., 2002). These results are only partially consistent with
those of the present study, but it should be noted that the former
study included patients undergoing intra-uterine insemination
as well as IVFembryo transfer. Moreover, another report
described a signicantly lower expression of LIF in patients
with unexplained infertility by uterine ushing (Lass et al.,
2001).
In any biological study, a distinction must be drawn between
correlation and cause. In the present preliminary study, the
initial aim was to determine if there was any detectable
difference in the intra-uterine expression of MMPs and
cytokines in patients with recurrent implantation failure
compared with normally fertile women. With this in mind, a
number of cytokines were selected, the known roles of which in
implantation and immunology raised the possibility that their
differential expression could have an impact upon embryonic
implantation. Having observed some differences in the pattern
of cytokine expression, the next challenge would be to
elucidate the biological basis for these observations. With the
expansion of PGD, it would be interesting to see if these
ndings are borne out for women who continue to have failed
implantation following embryo transfer despite the demonstration of chromosomally normal embryos. Additionally, it
would be interesting to determine, in a prospective trial,
whether there is any correlation between intra-uterine cytokine/MMP expression and reproductive outcome. Ultimately,
of greatest interest would be to identify whether modulation of
the pattern of cytokine expression could improve implantation
rates in women with recurrent implantation failure. These
studies will be the focus of ongoing research.
Acknowledgements
The authors gratefully acknowledge the support and assistance of
doctors and nurses in the Reproductive Biology Unit, particularly
Professor H.W.G.Baker and Sister Helen Norris. The authors also
wish to thank Linh Ung for the assistance in the research laboratory,
Dr Lois Salamonsen of Prince Henry's Institute of Medical Research
for providing BHK9, breast carcinoma cell culture supernatant, and
Professor S.Brennecke and Dr S.Higgins for providing research
samples.

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Submitted on June 12, 2002; resubmitted on October 2, 2002; accepted on
December 3, 2002

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