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An increase in the absolute count of CD56dimCD16+CD69+ NK cells in the peripheral blood is associated with a poorer IVF treatment and pregnancy outcome

Authors:

Abstract

Our aim was to evaluate the effect of the absolute count of the activation marker (CD69), IgG Fc receptor (CD16) and inhibitor marker (CD94) expression on peripheral blood natural killer (NK) cells on implantation and miscarriage rates after IVF treatment. Prospective observational study of 138 randomly selected women who underwent IVF treatment from December 2002 to September 2003. NK cells were identified as CD56(+) (dim + bright) and CD3(-) by flow cytometry. The absolute counts of the CD69(+), CD16(+) and CD94(+)expressing NK cells were recorded and their relation to IVF treatment outcome and miscarriage rate was analysed. The mean (+/-SD) absolute count of the CD56(dim)CD16(+)CD69(+) NK cells for women who had a successful ongoing pregnancy was 0.61 x 10(6)/l (+/-0.31). For those women who failed to achieve a pregnancy, the mean value of the absolute count of CD56(dim)CD16(+)D69(+) NK cells was significantly (P=0.003) higher at 1.66 x 10(6)/l (+/-0.52). The absolute count of CD56(dim)CD16(+)CD94(+) and CD56(dim)CD16(+) NK cells did not show any statistically significant differences between those women with successful and failed IVF treatment. Receiver operating characteristic (ROC) curve analysis was performed to select a CD69 threshold for further statistical analysis. The implantation rate (IR) was significantly lower (13.1%) and miscarriage rate (MR) was significantly higher (66.7%) for women with an absolute CD56(dim)CD16(+)CD69(+) NK cell count of >1.0 x 10(6)/l compared to women with count below this value (IR 28.2% and MR 16.7%). Further analysis of the absolute count of CD56(bright)CD69(+) and CD56(bright)CD94(+) NK cells did not show any significant difference between those women with successful and failed IVF treatment. An increase in the absolute count of activated NK cells (CD56(dim)CD16(+)CD69(+)) in the peripheral blood is associated with a reduced rate of embryo implantation in IVF treatment. Furthermore, women with high CD56(dim)CD16(+)CD69(+) peripheral blood NK cell absolute count, who are able to achieve pregnancy, have a significantly higher miscarriage rate.
An increase in the absolute count of CD56
dim
CD16
1
CD69
1
NK cells in the peripheral blood is associated with a poorer
IVF treatment and pregnancy outcome
M.Y.Thum
1,2,3,4
, S.Bhaskaran
2
, H.I.Abdalla
1
, B.Ford
2
, N.Sumar
2
, H.Shehata
3
and A.S.Bansal
2
1
Lister Fertility Clinic, Lister Hospital, Chelsea Bridge Road, London SW1W 8RH,
2
Immunology Department and
3
Women Health
Department, Epsom and St Helier University Hospitals NHS Trust, Surrey, UK
4
To whom correspondence should be addressed. E-mail: mythum@doctors.net.uk
BACKGROUND: Our aim was to evaluate the effect of the absolute count of the activation marker (CD69), IgG Fc
receptor (CD16) and inhibitor marker (CD94) expression on peripheral blood natural killer (NK) cells on implan-
tation and miscarriage rates after IVF treatment. METHODS: Prospective observational study of 138 randomly
selected women who underwent IVF treatment from December 2002 to September 2003. NK cells were identified
as CD56
1
(dim 1 bright) and CD3
by flow cytometry. The absolute counts of the CD69
1
, CD16
1
and CD94
1
expressing NK cells were recorded and their relation to IVF treatment outcome and miscarriage rate was ana-
lysed. RESULTS: The mean (6 SD) absolute count of the CD56
dim
CD16
1
CD69
1
NK cells for women who had a
successful ongoing pregnancy was 0.61 3 10
6
/l (6 0.31). For those women who failed to achieve a pregnancy, the
mean value of the absolute count of CD56
dim
CD16
1
D69
1
NK cells was significantly (P 5 0.003) higher at
1.66 3 10
6
/l (6 0.52). The absolute count of CD56
dim
CD16
1
CD94
1
and CD56
dim
CD16
1
NK cells did not show any
statistically significant differences between those women with successful and failed IVF treatment. Receiver opera-
ting characteristic (ROC) curve analysis was performed to select a CD69 threshold for further statistical analysis.
The implantation rate (IR) was significantly lower (13.1%) and miscarriage rate (MR) was significantly higher
(66.7%) for women with an absolute CD56
dim
CD16
1
CD69
1
NK cell count of > 1.0 3 10
6
/l compared to women
with count below this value (IR 28.2% and MR 16.7%). Further analysis of the absolute count of CD56
bright
CD69
1
and CD56
bright
CD94
1
NK cells did not show any significant difference between those women with successful and
failed IVF treatment. CONCLUSIONS: An increase in the absolute count of activated NK cells (CD56
dim
CD16
1
CD69
1
) in the peripheral blood is associated with a reduced rate of embryo implantation in IVF treatment. Fur-
thermore, women with high CD56
dim
CD16
1
CD69
1
peripheral blood NK cell absolute count, who are able to
achieve pregnancy, have a significantly higher miscarriage rate.
Key words: activation markers/CD69/flow cytometry/IVF/natural killer cells
Introduction
The natural killer (NK) cell is the most abundant immune
cell infiltrating the uterine implantation site (Moffett-King,
2002). It is the first line cellular immune defence mechanism
and has a close contact with conceptus or placenta. NK cells
comprise , 15% of all lymphocytes and are defined pheno-
typically by expression of CD56 and lack of expression of
CD3 on the cell surface (Robertson and Ritz, 1990). The
majority (, 90%) of human NK cells have low density
expression of CD56 (CD56
dim
) and express high levels of
Fcg receptor IIIa (FcgRIIIa; CD16
þ
) whereas , 10% of NK
cells are CD56
bright
CD16
dim
or CD56
bright
CD16
2
(Cooper
et al., 2001). Uterine NK cells appear to be CD56
bright
and
increase in number during the post-ovulatory luteal phase
(King et al., 1996).
A previous study by Beer et al. (1996) showed that an
elevated percentage of peripheral blood NK cells was associ-
ated with recurrent failed IVF treatment cycles. Later, Fukui
et al. (1999) showed that increased peripheral blood NK cell
cytotoxicity level was associated with an increased rate of
recurrent failed implantation after IVF treatment. More recent
studies have confirmed elevated CD69 expression on NK
cells as being associated with recurrent miscarriage and infer-
tility of unknown aetiology (Ntrivalas et al., 2001). Finally, a
recent small non-randomized study by Coulam and Roussev
(2003) revealed that infertile women undergoing IVF treat-
ment also have a higher percentage of elevated CD69
expression on NK cells as compared to multiparous women.
CD69 belongs to the C-lectin type superfamily and is a type
II integral membrane protein consisting of a disuphide-linked
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homodimer with two phosphorylated chains (Ziegler et al.,
1993). It is a functional triggering molecule on activated NK
cells and is one of the earliest cell surface activation markers
expressed (Yokoyama, 1999). It is capable of inducing cyto-
toxicity and stimulating cytokine production (Zingoni et al.,
2000). Besides mediating NK cell cytotoxicity, it also medi-
ates other NK cell functions such as proliferation, tumour
necrosis factor (TNF-a) production and expression of other
activation antigens (Borrego et al., 1999; Pisegna et al.,
2002).
CD94 is an inhibitory marker of NK cell function. It is
part of the killing inhibitory receptor (KIR) family which is a
sub-group of the C type lectin superfamily (Lopez-Botet
et al., 1997). Borrego et al. (1999) demonstrated that NK cell
cytotoxicity could be blocked by the CD94 inhibitory recep-
tor. Previous studies have shown that imbalances in CD69
and CD94 expression on NK cell could result in infertility of
unknown aetiology or recurrent miscarriage (Ntrivalas et al.,
2001; Coulam et al., 2003).
CD16 (also classified as FcgRIIIa) is one of the low affi-
nity receptors for the Fc region of IgG. FcgRIIIa is an inte-
gral membrane protein expressed on NK cells, on a subset of
T lymphocytes, and on a subpopulation of monocytes and
macrophages (Ravetch and Perussia, 1989). Previously Fukui
et al. (1999) showed that an increased percentage of peri-
pheral blood CD16
þ
NK cells was associated with failed
implantation after IVF treatment.
The aim of this study was to document any association
between the absolute count of activation marker (CD69), IgG
Fc receptor (CD16) and inhibitor marker (CD94) expressing
NK cells on the implantation and miscarriage rate after IVF
treatment.
Materials and methods
Study population
From December 2002 to July 2003, 138 patients undergoing IVF
treatment cycles were recruited into the study. Independent ethical
approval was obtained from the Local Research Ethics Committee.
Exclusion criteria: women with known immunological disease (anti-
phospholipid antibodies, lupus anticoagulant, anticardiolipin anti-
bodies), uterine abnormality (fibroid, uterine polyp, uterine septum),
fewer than two embryos available for transfer or endometrium thick-
ness , 8 mm before embryo transfer. All transvaginal ultrasound
scans were performed by a sonographer and exclusion of candidates
was performed without the knowledge of the NK cell blood test
result. Blood samples were obtained on the day of vaginal oocyte
collection prior to the procedure. Informed consent was provided by
all subjects at recruitment.
Stimulation protocol
Pituitary down-regulation was achieved with either nafarelin or
buserelin at mid-luteal phase. Ovarian stimulation was carried out
with either recombinant FSH, hMG or urinary FSH. When follicles
reached pre-ovulatory size (18 22 mm), 10 000 IU of hCG was
administrated. Oocytes were aspirated using transvaginal ultrasound
guidance 3436 h after hCG administration. All embryos were
allowed to cleave and the best two or three embryos were selected
for transfer. Embryo transfer was performed on day 2 or 3 using a
soft catheter (Wallace) with transabdominal ultrasound guidance.
Progesterone supplement for luteal support (Cyclogest; Shire Phar-
maceuticals Ltd, UK), 400 mg once a day per vaginum or per rec-
tum, was commenced 1 day before embryo transfer and continued
until a pregnancy test was performed 2 weeks after embryo transfer.
Flow cytometric NK activation and inhibition quantification assay
Peripheral blood was collected in heparinized tubes and analysed
within 24 h. Fifty millilitres of blood was placed in flow cytometric
tubes (Becton Dickinson) and each incubated for 15 min at room
temperature with mouse anti-human CD16 fluorescein isothio-
cyanate (FITC), anti-CD56 phycoerythrin (PE) (BD PharMingen),
anti-CD3 PE Cy5 (Quest Biomedical), together with CD69 or CD94
APC (BD PharMingen) monoclonal antibodies (mAb). Isotypic con-
trol mAb included mouse IgG
1
FITC, IgG
1
APC, IgG
1
PE (BD
PharMingen) and IgG
1
PE Cy5 (Quest Biomedical). In this lyse, no
wash procedure, 1 ml of Quicklysis lysing solution (Quest Biomedi-
cal) was added to each tube and incubated for a further 10 min at
room temperature. Fifty millilitres of PerfectCount beads (Quest
Biomedical) were then accurately pipetted to each tube and samples
run with BD FACSCalibur flow cytometer. Cells negatively staining
for CD3, but positively for CD56, were selected and their CD69 and
CD94 expression analysed using a Cell Quest software (BD) using a
four-colour protocol.
Data analysis
All IVF data were collected in Medical System for IVF (Medical-
Sys, UK) and analysed by Statistics Package for Social Sciences
(SPSS, UK). Descriptive statistical analysis was performed initially
to examine the normal distribution of all continuous variances for
parametric statistical tests. The t-test was then used to compare the
mean value in two groups: pregnant and not pregnant. Receiver
operating characteristic (ROC) curve and area under curve (AUC)
analysis were performed. The ROC curve represents the probability
of true positive results (sensitivity) as a function of the probability
of false positive results (1 - specificity). The AUC is a measure of
the accuracy of a test. In order to perform the ROC curve calcu-
lation, set threshold values for the state variable (CD69 absolute
count) were selected. These are arbitrary values selected to analyse
the association of treatment outcome. For each CD69 threshold, sep-
arate curves were produced for treatment outcome and pregnancy
outcome, making a total of 14 curves. x
2
cross-tabulation test was
used to analyse the significance of differences in pregnancy rates,
miscarriage rates and live birth rates between groups. Analysis of
variance was then conducted to assess the duration and amount of
gonadotrophin required to achieve follicular maturity, estradiol
levels on hCG day, number of mature follicles, number of available
embryos for transfer, number of oocytes collected and fertilization
rate between groups.
Results
Of the 138 women who underwent IVF, 12 were excluded
from statistical analysis. Of these 12, four had failed fertiliza-
tion, four had only one embryo available for transfer, one
had ovarian hyperstimulation syndrome and therefore did not
have embryo transfer, two had an endometrial thickness
, 7.5 mm and one woman had poor quality embryos. None
of the women who participated in the study were excluded
due to abnormal uterine anatomy or known previous abnor-
mal immunological tests.
M.Y.Thum et al.
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Table I shows patients’ treatment outcome, mean age,
causes of infertility, duration of infertility, basal FSH levels,
mean number of previous IVF attempts, number of previous
miscarriages and outcome of ovarian stimulation in the preg-
nant and non-pregnant groups. There were no significant
differences between the two groups with regard to any of the
parameters.
Table II examines the relationship between the absolute
count of CD69, CD94 and CD16 expressing CD56
dim
cells
and the absolute count of CD69 and CD94 expressing
CD56
bright
cells with IVF treatment outcome; pregn-
ant versus non-pregnant. For women who had a success-
ful ongoing pregnancy, the mean (^ SD) value of
the absolute CD56
dim
CD16
þ
D69
þ
NK cells was
0.61 £ 10
6
/l ^ 0.31 £ 10
6
. For those women who failed to
achieve pregnancy, the mean value of the absolute count of
CD69-expressing NK cells was significantly higher at
1.66 £ 10
6
/l ^ 0.52 £ 10
6
(P ¼ 0.003). The absolute count
of CD94 and CD16 expressing CD56
dim
NK cells and
CD56
bright
NK cells showed no significant difference between
those women with successful and failed IVF treatment.
For the ROC analysis, 14 curves were produced, one for
treatment outcome (pregnancy rate) and one for pregnancy
outcome (live birth rate) for each of the seven chosen CD69
absolute count threshold. Table III illustrates the test charac-
teristics and AUC for each selected CD69 absolute count
threshold. The AUC values were maximum at a CD69 absol-
ute count of 1.0 £ 10
6
/l for both treatment outcome (preg-
nancy rate) and pregnancy outcome (live birth rate). This
indicated that 1.0 £ 10
6
/l is a better level for further statisti-
cal analysis as compared to the other selected levels. Increas-
ing the threshold level improved specificity and positive
predictive value at the expense of sensitivity. Therefore
the threshold value of 1.0 106/1 was selected for further
statistical analysis without selecting a threshold level for the
test.
The study population was then divided into two groups
based on the absolute count of CD69-expressing NK cells.
Group A women (n ¼ 87) had a count , 1.0 £ 10
þ
6/l while
group B women (n ¼ 39) had a count . 1.0 £ 10
6
/l.
Table IV shows number of women in each group, their mean
age, causes of infertility, duration of infertility, basal FSH
levels, mean number of previous IVF attempts and previous
miscarriages, stimulation characteristics and treatment out-
come in both groups. There were no significant differences
between group A and group B with regard to age, causes and
duration of infertility and basal FSH levels. The mean num-
ber of previous failed IVF attempts and the mean number of
previous miscarriages were significantly higher in group B as
compared to group A. There was no significant difference
between the two groups with regard to amount of gonado-
trophin used for stimulation, estradiol levels on hCG day,
number of oocytes collected, fertilization rate, number of
available embryos for transfer or number of embryos trans-
ferred. In group B, the implantation rate, pregnancy rate and
live birth rate were significantly lower, and the miscarriage
rate was significantly higher as compared to group A.
Discussion
CD69 is one of the earliest specific activation markers
expressed during large granulated lymphocyte activation,
which includes the NK cell (Craston et al., 1997; Marzio
et al., 1999; Llera et al., 2001). Activated CD69
þ
NK cells
will release cytokines which will further activate other NK
cells and the cellular immune system (Marzio et al., 1999).
Previous studies have also shown that elevated CD69
expression on NK cells is associated with an increase in cyto-
toxicity of NK cells towards target cells, which induces target
cell lysis (Lanier et al., 1988; De Maria et al., 1994). Chao
et al. (1999) have suggested that maternal NK cell CD69
expression is involved in recognition of HLA-G and HLA-C
on the allogeneic embryonic and trophoblast cell surface. In
theory, recognition of HLA-G and HLA-C expression is
Table II. Natural killer cell sub-population and CD69 expression between
pregnant and non-pregnant women
Non-pregnant Pregnant P-value
No. of patients 75 51 NA
CD56
dim
CD16
þ
CD69
þ
1.66 ^ 0.52 0.61 ^ 0.31 0.003
CD56
dim
CD16
þ
CD94
þ
142.1 ^ 87 149.9 ^ 117 NS
CD56
dim
CD16
þ
212.2 ^ 149 233.0 ^ 168 NS
CD56
bright
CD69
þ
0.31 ^ 0.34 0.28 ^ 0.31 NS
CD56
bright
CD94
þ
15.9 ^ 8.9 16.9 ^ 7.5 NS
Values are mean ^ SD absolute count ( £ 10
6
/l) unless otherwise stated.
NA ¼ not applicable; NS ¼ difference not statistically significant
(P . 0.05).
Table I. Patients’ demographics and stimulation characteristic between
pregnant and non-pregnant women
Non-pregnant Pregnant P-value
No. of patients 75 51 NA
Age (years) (mean ^ SD) 35.45 ^ 3.8 34.16 ^ 4.0 NS
Tubal factor (%) 23.7 23.5 NS
Male factor (%) 25.0 27.6 NS
Other
a
(%) 16.3 15.6 NS
Unexplained (%) 35.0 33.3 NS
Duration of infertility (years)
(mean ^ SD)
4.60 ^ 2.7 3.57 ^ 2.3 NS
Basal FSH levels (IU/l)
(mean ^ SD)
7.82 ^ 2.9 7.71 ^ 3.8 NS
Mean no. of previous
failed IVF attempts
1.88 1.37 NS
Mean No. of previous
miscarriages
0.25 0.33 NS
Gonadotrophin
b
(IU) 3067.0 2531.6 NS
Estradiol (IU) on hCG
day
8599.87 7015.88 NS
No. of oocytes collected
(mean ^ SD)
12.3 ^ 5.5 12.9 ^ 6.6 NS
Fertilization rate (%) 66.7 66.5 NS
No. of available embryos
for transfer (mean ^ SD)
7.96 ^ 4.4 8.76 ^ 5.7 NS
Mean no. of embryos
transferred
2.18 2.04 NS
a
Anovulation and endometriosis.
b
Mean amount of gonadotrophin used for stimulation in IU (recombinant
FSH, hMG or urinary FSH).
NA ¼ not applicable; NS ¼ difference not statistically significant
(P . 0.05).
CD69
1
peripheral NK cells and IVF outcome
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thought to protect the embryo from destruction by NK cells
(Ellis et al., 1989; Kovats et al., 1990; King et al., 1997). Ho
et al. (1996) showed that NK cell cytotoxicity is decreased in
a normal healthy pregnancy compared with an anembryonic
pregnancy. He suggested that activated NK cells, with CD69
expression on their cell surface, play an important role in the
control of trophoblast growth and placental development. In
support of this, in vitro models show that activated CD69
positive NK cells are capable of lysing trophoblasts (Helige
et al., 2001; Avril et al., 2003).
In this study, we explored the relationship between the
IVF treatment outcome with the quantification of activation
receptor (CD69) and inhibitory receptor (CD94) on peri-
pheral blood NK cells. Our results revealed that women who
failed to achieve a pregnancy after IVF treatment have a
significantly higher level of activated CD56
dim
CD16
þ
CD69
þ
NK cells in the peripheral blood. This was evident despite
the fact that there were no significant differences in patients’
demographic details, number of previous failed IVF attempts
or miscarriage, ovarian stimulation outcome, embryo quality
or number of embryos transferred. This appears to be the first
study to reveal elevated CD56
dim
CD16
þ
CD69
þ
peripheral
blood NK cells in women who experience failed IVF treat-
ment. It has however been reported that women with inferti-
lity needing IVF treatment and women who experience
recurrent spontaneous miscarriage have significantly higher
levels of peripheral CD56
dim
CD16
þ
CD69
þ
NK cells
(Ntrivalas et al., 2001; Coulam et al., 2003). The mechanism
of implantation and the precise role of NK cells in implan-
tation are still not fully understood, but it can be speculated
Table III. The test characteristics and area under the curve (AUC) for each selected CD69 absolute count threshold
CD69 threshold
( £ 10
6
/l)
Outcome parameter
per treatment cycle
AUC Sensitivity (%) Specificity (%) PPV (%) NPV (%)
0.7 Pregnancy rate 0.593 51.3 67.3 70.4 47.8
Live birth rate 0.614 50.6 72.2 81.1 38.2
0.8 Pregnancy rate 0.614 47.3 75.5 74.5 48.7
Live birth rate 0.652 47.1 83.3 87.0 40.0
0.9 Pregnancy rate 0.604 43.2 77.6 74.4 47.5
Live birth rate 0.628 42.3 83.3 85.7 38.0
1.0 Pregnancy rate 0.635 40.0 82.4 77.8 48.3
Live birth rate 0.667 40.9 92.1 92.3 40.2
1.1 Pregnancy rate 0.625 39.2 81.3 76.6 48.9
Live birth rate 0.660 37.6 91.8 91.7 39.1
1.2 Pregnancy rate 0.618 29.4 89.8 83.3 47.3
Live birth rate 0.651 32.9 97.2 96.5 38.0
1.3 Pregnancy rate 0.625 31.1 83.3 88.5 47.4
Live birth rate 0.633 29.4 97.2 96.1 36.8
AUC ¼ area under the ROC (receiver operating characteristic) curve; PPV ¼ positive predictive value; NPV ¼ negative
predictive value
Table IV. Patients’ demographics, stimulation characteristic, treatment and pregnancy outcome in group A and B
Group A
CD69 # 1.0 £ 10
6
/l
Group B
CD69 . 1.0 £ 10
6
/l
P-value
No. of patients 87 39 NA
Age (years) (mean ^ SD) 35.54 ^ 3.7 35.85 ^ 4.6 NS
Tubal factor (%) 21.3 20.0 NS
Male factor (%) 22.3 30.0 NS
Other
a
(%) 17.0 12.5 NS
Unexplained (%) 39.4 37.5 NS
Duration of infertility (years) (mean ^ SD) 4.11 ^ 2.6 4.20 ^ 2.5 NS
Basal FSH levels (mean ^ SD) 8.12 ^ 3.8 7.82 ^ 3.3 NS
No. of previous failed IVF attempts (mean ^ SD) 1.29 ^ 1.5 2.60 ^ 2.77 0.001
No. of previous miscarriages (mean ^ SD) 0.20 ^ 0.48 0.45 ^ 0.75 0.023
Gonadotrophin
b
(IU) (mean ^ SD) 2579.8 ^ 979.1 3489.5 ^ 576.7 NS
Estradiol (IU) on hCG day 8430.60 7727.84 NS
No. of oocytes collected (mean ^ SD) 12.59 ^ 6.0 11.67 ^ 5.9 NS
Fertilization rate (%) (mean ^ SD) 63.75 ^ 22.7 70.49 ^ 21.2 NS
No. of available embryos for transfer (mean ^ SD) 8.16 ^ 5.3 7.82 ^ 4.0 NS
No. of embryos transferred (mean ^ SD) 2.08 ^ 0.37 2.15 ^ 0.43 NS
Implantation rate, % (n) 28.2 (53/188) 13.1 (11/84) 0.004
Pregnancy rate per cycle started, % (n) 48.3 (42/87) 23.1 (9/39) 0.006
Live birth rate per cycle started, % (n) 40.2 (35/87) 7.7 (3/39) , 0.0001
Miscarriage rate, % (n) 16.7 (7/42) 66.7 (6/9) 0.005
a
Anovulation and endometriosis.
b
Mean amount of gonadotrophin used for stimulation (recombinant FSH, hMG or urinary FSH).
NA ¼ not applicable; NS ¼ difference not statistically significant (P . 0.05).
M.Y.Thum et al.
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that an excess of activated CD69
þ
NK cells might play a
negative role in successful implantation.
We further evaluated CD56
bright
CD69
þ
cells, a sub-
population of NK cells, which are phenotypically similar to
uterine NK cells. The level of peripheral CD56
bright
CD69
þ
NK cells was not significantly different between the pregnant
and non-pregnant groups. In contrast, Kodama et al. (1998)
reported that increased numbers of CD56
bright
CD69
þ
NK
cells were found in the decidua in women with spontaneous
miscarriage compared with normal pregnancies. This finding
may suggest that although phenotypically similar, uterine
CD56
bright
NK cells may not be related to peripheral blood
CD56
bright
NK cells. Our study demonstrates that there is no
association between the absolute count of CD56
dim
CD16
þ
CD94
þ
NK cells and CD56
bright
CD94
þ
NK cells with the
outcome of IVF treatment. However, further functional cyto-
toxicity studies would be needed to evaluate CD94
expression and cytotoxicity of NK cells. In contrast to a pre-
vious study by Fukui et al. (1999), the present study did not
demonstrate any statistical difference in the absolute count of
CD56
dim
CD16
þ
NK cells and CD56
bright
CD16
þ
NK cells
between successful and failed IVF treatment. However, for
statistical analysis these authors used percentage of CD16
þ
NK cells instead of the absolute count, which may not be
accurate, since the percentage can vary depending on the
composition of other lymphocytes in a sample.
Further statistical analysis was carried out based on the
absolute count of CD56
dim
CD16
þ
CD69
þ
NK cells above and
below 1.0 £ 10
6
/l in order to examine other variables. The
value of 1.0 £ 10
6
/l was selected for analysis after ROC test-
ing. This study is not intended to establish a threshold level
for the CD69 NK cell testing procedure. Estimating a
threshold level and testing it in the same sample will create
bias. The selected threshold value should be tested and its
value confirmed or rejected in a separate study with a new
set of samples. The result of the ROC analysis revealed that
the sensitivity and negative predictive value were rather low,
perhaps because there are numerous factors affecting IVF
outcome. For example, a woman with a low CD69 count
may not achieve a pregnancy because her embryos are
genetically abnormal.
The results show that for those women with
CD56
dim
CD16
þ
D69
þ
NK cells . 1.0 £ 10
6
/l, the implan-
tation rate and the pregnancy rate were significantly lower
and the miscarriage rate was significantly higher as compared
to women with CD56
dim
CD16
þ
D69
þ
NK cells below this
level. The combination of lower implantation rate and higher
miscarriage rate result in a significant lower live birth rate.
With this finding, we speculated that elevated peripheral acti-
vated NK cells may play a detrimental role in IVF treatment
outcome. The data also reveal that women with elevated peri-
pheral CD56
dim
CD16
þ
D69
þ
NK cells had a significantly
higher number of previous miscarriages and previous failed
IVF treatments despite the fact that there were no statistically
significant demographic differences between pregnant and
non-pregnant groups (Table I). This finding is in accord with
previous studies (Emmer et al., 2000; Ntrivalas et al., 2001)
showing that levels of activated NK cells were raised in
women with spontaneous recurrent miscarriages. However,
we cannot exclude the possibility that previous miscarriage
and failed IVF treatment may be the cause of the elevated
CD69
þ
NK cell count, nor that the selected threshold value
chosen may, by chance, divide the patients into two groups
with very different clinical prognostic factors that determine
the poorer outcome in the group with high CD69
þ
NK cell
count. This needs to be examined further in a new prospec-
tive study using logistic regression analysis adjusting for the
impact of these clinical variables.
In conclusion, our data suggest that an elevated level of
CD56
dim
CD16
þ
CD69
þ
peripheral blood NK cells is associ-
ated with a reduced implantation rate of embryos in IVF
treatment. Those women with an elevated peripheral blood
CD56
dim
CD16
þ
CD69
þ
NK cell count who achieve a preg-
nancy after IVF manifest a significantly higher miscarriage
rate. A high level of peripheral blood CD69
þ
NK cells may
play a negative role in IVF treatment outcome but this has to
be explored in a new prospective study.
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Chapter
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