Human Reproduction Vol.19, No.10 pp. 2395–2400, 2004
Advance Access publication August 19, 2004
An increase in the absolute count of CD56dimCD161CD691NK cells in the peripheral blood is associated with a poorerIVF treatment and pregnancy outcome
M.Y.Thum1,2,3,4, S.Bhaskaran2, H.I.Abdalla1, B.Ford2, N.Sumar2, H.Shehata3and A.S.Bansal2
1Lister Fertility Clinic, Lister Hospital, Chelsea Bridge Road, London SW1W 8RH, 2Immunology Department and 3Women HealthDepartment, Epsom and St Helier University Hospitals NHS Trust, Surrey, UK
4To whom correspondence should be addressed. E-mail: [email protected]
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 randomlyselected women who underwent IVF treatment from December 2002 to September 2003. NK cells were identifiedas CD561 (dim 1 bright) and CD3 – by flow cytometry. The absolute counts of the CD691, CD161 and CD941expressing 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 CD56dimCD161CD691 NK cells for women who had asuccessful ongoing pregnancy was 0.61 3 106/l (6 0.31). For those women who failed to achieve a pregnancy, themean value of the absolute count of CD56dimCD161D691 NK cells was significantly (P 5 0.003) higher at1.66 3 106/l (6 0.52). The absolute count of CD56dimCD161CD941 and CD56dimCD161 NK cells did not show anystatistically 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 CD56dimCD161CD691 NK cell count of > 1.0 3 106/l compared to women
with count below this value (IR 28.2% and MR 16.7%). Further analysis of the absolute count of CD56brightCD691and CD56brightCD941 NK cells did not show any significant difference between those women with successful andfailed IVF treatment. CONCLUSIONS: An increase in the absolute count of activated NK cells (CD56dimCD161CD691) in the peripheral blood is associated with a reduced rate of embryo implantation in IVF treatment. Fur-thermore, women with high CD56dimCD161CD691 peripheral blood NK cell absolute count, who are able toachieve pregnancy, have a significantly higher miscarriage rate.
Key words: activation markers/CD69/flow cytometry/IVF/natural killer cells
A previous study by Beer et al. (1996) showed that an
The natural killer (NK) cell is the most abundant immune
elevated percentage of peripheral blood NK cells was associ-
cell infiltrating the uterine implantation site (Moffett-King,
ated with recurrent failed IVF treatment cycles. Later, Fukui
2002). It is the first line cellular immune defence mechanism
et al. (1999) showed that increased peripheral blood NK cell
and has a close contact with conceptus or placenta. NK cells
cytotoxicity level was associated with an increased rate of
comprise , 15% of all lymphocytes and are defined pheno-
recurrent failed implantation after IVF treatment. More recent
typically by expression of CD56 and lack of expression of
studies have confirmed elevated CD69 expression on NK
CD3 on the cell surface (Robertson and Ritz, 1990). The
cells as being associated with recurrent miscarriage and infer-
majority (, 90%) of human NK cells have low density
tility of unknown aetiology (Ntrivalas et al., 2001). Finally, a
expression of CD56 (CD56dim) and express high levels of
recent small non-randomized study by Coulam and Roussev
Fcg receptor IIIa (FcgRIIIa; CD16þ) whereas , 10% of NK
(2003) revealed that infertile women undergoing IVF treat-
cells are CD56brightCD16dim or CD56brightCD162 (Cooper
ment also have a higher percentage of elevated CD69
et al., 2001). Uterine NK cells appear to be CD56bright and
expression on NK cells as compared to multiparous women.
increase in number during the post-ovulatory luteal phase
CD69 belongs to the C-lectin type superfamily and is a type
(King et al., 1996).
II integral membrane protein consisting of a disuphide-linked
Human Reproduction vol. 19 no. 10 q European Society of Human Reproduction and Embryology 2004; all rights reserved
M.Y.Thum et al.
homodimer with two phosphorylated chains (Ziegler et al.,
soft catheter (Wallace) with transabdominal ultrasound guidance.
1993). It is a functional triggering molecule on activated NK
Progesterone supplement for luteal support (Cyclogest; Shire Phar-
cells and is one of the earliest cell surface activation markers
maceuticals Ltd, UK), 400 mg once a day per vaginum or per rec-
expressed (Yokoyama, 1999). It is capable of inducing cyto-
tum, was commenced 1 day before embryo transfer and continued
toxicity and stimulating cytokine production (Zingoni et al.,
until a pregnancy test was performed 2 weeks after embryo transfer.
2000). Besides mediating NK cell cytotoxicity, it also medi-ates other NK cell functions such as proliferation, tumour
Flow cytometric NK activation and inhibition quantification assay
necrosis factor (TNF-a) production and expression of other
Peripheral blood was collected in heparinized tubes and analysed
activation antigens (Borrego et al., 1999; Pisegna et al.,
within 24 h. Fifty millilitres of blood was placed in flow cytometric
tubes (Becton Dickinson) and each incubated for 15 min at roomtemperature with mouse anti-human CD16 – fluorescein isothio-
CD94 is an inhibitory marker of NK cell function. It is
cyanate (FITC), anti-CD56 phycoerythrin (PE) (BD PharMingen),
part of the killing inhibitory receptor (KIR) family which is a
anti-CD3 PE Cy5 (Quest Biomedical), together with CD69 or CD94
sub-group of the C type lectin superfamily (Lopez-Botet
APC (BD PharMingen) monoclonal antibodies (mAb). Isotypic con-
et al., 1997). Borrego et al. (1999) demonstrated that NK cell
trol mAb included mouse IgG1 FITC, IgG1 APC, IgG1 PE (BD
cytotoxicity could be blocked by the CD94 inhibitory recep-
PharMingen) and IgG1 PE– Cy5 (Quest Biomedical). In this lyse, no
tor. Previous studies have shown that imbalances in CD69
wash procedure, 1 ml of Quicklysis lysing solution (Quest Biomedi-
and CD94 expression on NK cell could result in infertility of
cal) was added to each tube and incubated for a further 10 min at
unknown aetiology or recurrent miscarriage (Ntrivalas et al.,
room temperature. Fifty millilitres of PerfectCount beads (Quest
2001; Coulam et al., 2003).
Biomedical) were then accurately pipetted to each tube and samples
CD16 (also classified as FcgRIIIa) is one of the low affi-
run with BD FACSCalibur flow cytometer. Cells negatively staining
nity receptors for the Fc region of IgG. FcgRIIIa is an inte-
for CD3, but positively for CD56, were selected and their CD69 andCD94 expression analysed using a Cell Quest software (BD) using a
gral membrane protein expressed on NK cells, on a subset of
T lymphocytes, and on a subpopulation of monocytes andmacrophages (Ravetch and Perussia, 1989). Previously Fukui
et al. (1999) showed that an increased percentage of peri-
pheral blood CD16þ NK cells was associated with failed
All IVF data were collected in Medical System for IVF (Medical-
implantation after IVF treatment.
Sys, UK) and analysed by Statistics Package for Social Sciences(SPSS, UK). Descriptive statistical analysis was performed initially
The aim of this study was to document any association
to examine the normal distribution of all continuous variances for
between the absolute count of activation marker (CD69), IgG
parametric statistical tests. The t-test was then used to compare the
Fc receptor (CD16) and inhibitor marker (CD94) expressing
mean value in two groups: pregnant and not pregnant. Receiver
NK cells on the implantation and miscarriage rate after IVF
operating characteristic (ROC) curve and area under curve (AUC)
analysis were performed. The ROC curve represents the probabilityof true positive results (sensitivity) as a function of the probabilityof false positive results (1 - specificity). The AUC is a measure of
Materials and methods
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
From December 2002 to July 2003, 138 patients undergoing IVF
the association of treatment outcome. For each CD69 threshold, sep-
treatment cycles were recruited into the study. Independent ethical
arate curves were produced for treatment outcome and pregnancy
approval was obtained from the Local Research Ethics Committee.
outcome, making a total of 14 curves. x2 cross-tabulation test was
Exclusion criteria: women with known immunological disease (anti-
used to analyse the significance of differences in pregnancy rates,
phospholipid antibodies, lupus anticoagulant, anticardiolipin anti-
miscarriage rates and live birth rates between groups. Analysis of
bodies), uterine abnormality (fibroid, uterine polyp, uterine septum),
variance was then conducted to assess the duration and amount of
fewer than two embryos available for transfer or endometrium thick-
gonadotrophin required to achieve follicular maturity, estradiol
ness , 8 mm before embryo transfer. All transvaginal ultrasound
levels on hCG day, number of mature follicles, number of available
scans were performed by a sonographer and exclusion of candidates
embryos for transfer, number of oocytes collected and fertilization
was performed without the knowledge of the NK cell blood test
rate between groups.
result. Blood samples were obtained on the day of vaginal oocytecollection prior to the procedure. Informed consent was provided byall subjects at recruitment.
Of the 138 women who underwent IVF, 12 were excluded
from statistical analysis. Of these 12, four had failed fertiliza-
Pituitary down-regulation was achieved with either nafarelin or
tion, four had only one embryo available for transfer, one
buserelin at mid-luteal phase. Ovarian stimulation was carried out
had ovarian hyperstimulation syndrome and therefore did not
with either recombinant FSH, hMG or urinary FSH. When follicles
have embryo transfer, two had an endometrial thickness
reached pre-ovulatory size (18 – 22 mm), 10 000 IU of hCG wasadministrated. Oocytes were aspirated using transvaginal ultrasound
, 7.5 mm and one woman had poor quality embryos. None
guidance 34 – 36 h after hCG administration. All embryos were
of the women who participated in the study were excluded
allowed to cleave and the best two or three embryos were selected
due to abnormal uterine anatomy or known previous abnor-
for transfer. Embryo transfer was performed on day 2 or 3 using a
mal immunological tests.
CD691 peripheral NK cells and IVF outcome
CD69-expressing NK cells was significantly higher at
Table I. Patients' demographics and stimulation characteristic between
1.66 £ 106/l 0.52 £ 106 (P ¼ 0.003). The absolute count
pregnant and non-pregnant women
of CD94 and CD16 expressing CD56dim NK cells and
CD56bright NK cells showed no significant difference between
those women with successful and failed IVF treatment.
Age (years) (mean SD)
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-
Duration of infertility (years)
teristics and AUC for each selected CD69 absolute count
(mean SD)Basal FSH levels (IU/l)
threshold. The AUC values were maximum at a CD69 absol-
ute count of 1.0 £ 106/l for both treatment outcome (preg-
Mean no. of previous
nancy rate) and pregnancy outcome (live birth rate). This
failed IVF attemptsMean No. of previous
indicated that 1.0 £ 106/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
Estradiol (IU) on hCG
predictive value at the expense of sensitivity. Therefore
No. of oocytes collected
the threshold value of 1.0 106/1 was selected for further
statistical analysis without selecting a threshold level for the
Fertilization rate (%)
No. of available embryos
for transfer (mean SD)
The study population was then divided into two groups
Mean no. of embryos
based on the absolute count of CD69-expressing NK cells.
Group A women (n ¼ 87) had a count , 1.0 £ 10þ6/l while
a Anovulation and endometriosis.
group B women (n ¼ 39) had a count . 1.0 £ 106/l.
bMean amount of gonadotrophin used for stimulation in IU (recombinant
Table IV shows number of women in each group, their mean
FSH, hMG or urinary FSH).
NA ¼ not applicable; NS ¼ difference not statistically significant
age, causes of infertility, duration of infertility, basal FSH
levels, mean number of previous IVF attempts and previousmiscarriages, stimulation characteristics and treatment out-
Table I shows patients' treatment outcome, mean age,
come in both groups. There were no significant differences
causes of infertility, duration of infertility, basal FSH levels,
between group A and group B with regard to age, causes and
mean number of previous IVF attempts, number of previous
duration of infertility and basal FSH levels. The mean num-
miscarriages and outcome of ovarian stimulation in the preg-
ber of previous failed IVF attempts and the mean number of
nant and non-pregnant groups. There were no significant
previous miscarriages were significantly higher in group B as
differences between the two groups with regard to any of the
compared to group A. There was no significant difference
between the two groups with regard to amount of gonado-
Table II examines the relationship between the absolute
trophin used for stimulation, estradiol levels on hCG day,
count of CD69, CD94 and CD16 expressing CD56dim cells
number of oocytes collected, fertilization rate, number of
and the absolute count of CD69 and CD94 expressing
available embryos for transfer or number of embryos trans-
ferred. In group B, the implantation rate, pregnancy rate and
ant versus non-pregnant. For women who had a success-
live birth rate were significantly lower, and the miscarriage
rate was significantly higher as compared to group A.
0.61 £ 106/l 0.31 £ 106. For those women who failed to
achieve pregnancy, the mean value of the absolute count of
CD69 is one of the earliest specific activation markersexpressed during large granulated lymphocyte activation,which includes the NK cell (Craston et al., 1997; Marzio
Table II. Natural killer cell sub-population and CD69 expression between
et al., 1999; Llera et al., 2001). Activated CD69þ NK cells
pregnant and non-pregnant women
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 CD69expression is involved in recognition of HLA-G and HLA-C
Values are mean SD absolute count ( £ 106/l) unless otherwise stated.
on the allogeneic embryonic and trophoblast cell surface. In
NA ¼ not applicable; NS ¼ difference not statistically significant(P . 0.05).
theory, recognition of HLA-G and HLA-C expression is
M.Y.Thum et al.
Table III. The test characteristics and area under the curve (AUC) for each selected CD69 absolute count threshold
per treatment cycle
AUC ¼ area under the ROC (receiver operating characteristic) curve; PPV ¼ positive predictive value; NPV ¼ negativepredictive value
thought to protect the embryo from destruction by NK cells
significantly higher level of activated CD56dimCD16þCD69þ
(Ellis et al., 1989; Kovats et al., 1990; King et al., 1997). Ho
NK cells in the peripheral blood. This was evident despite
et al. (1996) showed that NK cell cytotoxicity is decreased in
the fact that there were no significant differences in patients'
a normal healthy pregnancy compared with an anembryonic
demographic details, number of previous failed IVF attempts
pregnancy. He suggested that activated NK cells, with CD69
or miscarriage, ovarian stimulation outcome, embryo quality
expression on their cell surface, play an important role in the
or number of embryos transferred. This appears to be the first
control of trophoblast growth and placental development. In
study to reveal elevated CD56dimCD16þCD69þ peripheral
support of this, in vitro models show that activated CD69
blood NK cells in women who experience failed IVF treat-
positive NK cells are capable of lysing trophoblasts (Helige
ment. It has however been reported that women with inferti-
et al., 2001; Avril et al., 2003).
lity needing IVF treatment and women who experience
In this study, we explored the relationship between the
recurrent spontaneous miscarriage have significantly higher
IVF treatment outcome with the quantification of activation
receptor (CD69) and inhibitory receptor (CD94) on peri-
(Ntrivalas et al., 2001; Coulam et al., 2003). The mechanism
pheral blood NK cells. Our results revealed that women who
of implantation and the precise role of NK cells in implan-
failed to achieve a pregnancy after IVF treatment have a
tation are still not fully understood, but it can be speculated
Table IV. Patients' demographics, stimulation characteristic, treatment and pregnancy outcome in group A and B
CD69 # 1.0 £ 106/l
CD69 . 1.0 £ 106/l
Age (years) (mean SD)
Duration of infertility (years) (mean SD)
Basal FSH levels (mean SD)
No. of previous failed IVF attempts (mean SD)
No. of previous miscarriages (mean SD)
Gonadotrophinb (IU) (mean SD)
Estradiol (IU) on hCG day
No. of oocytes collected (mean SD)
Fertilization rate (%) (mean SD)
No. of available embryos for transfer (mean SD)
No. of embryos transferred (mean SD)
Implantation rate, % (n)
Pregnancy rate per cycle started, % (n)
Live birth rate per cycle started, % (n)
Miscarriage rate, % (n)
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).
CD691 peripheral NK cells and IVF outcome
that an excess of activated CD69þ NK cells might play a
women with spontaneous recurrent miscarriages. However,
negative role in successful implantation.
we cannot exclude the possibility that previous miscarriage
We further evaluated CD56brightCD69þ cells, a sub-
and failed IVF treatment may be the cause of the elevated
population of NK cells, which are phenotypically similar to
CD69þ NK cell count, nor that the selected threshold value
uterine NK cells. The level of peripheral CD56brightCD69þ
chosen may, by chance, divide the patients into two groups
NK cells was not significantly different between the pregnant
with very different clinical prognostic factors that determine
and non-pregnant groups. In contrast, Kodama et al. (1998)
the poorer outcome in the group with high CD69þ NK cell
reported that increased numbers of CD56brightCD69þ NK
count. This needs to be examined further in a new prospec-
cells were found in the decidua in women with spontaneous
tive study using logistic regression analysis adjusting for the
miscarriage compared with normal pregnancies. This finding
impact of these clinical variables.
may suggest that although phenotypically similar, uterine
In conclusion, our data suggest that an elevated level of
CD56bright NK cells may not be related to peripheral blood
CD56dimCD16þCD69þ peripheral blood NK cells is associ-
CD56bright NK cells. Our study demonstrates that there is no
ated with a reduced implantation rate of embryos in IVF
association between the absolute count of CD56dimCD16þ
treatment. Those women with an elevated peripheral blood
CD94þ NK cells and CD56brightCD94þ NK cells with the
CD56dimCD16þCD69þ NK cell count who achieve a preg-
outcome of IVF treatment. However, further functional cyto-
nancy after IVF manifest a significantly higher miscarriage
rate. A high level of peripheral blood CD69þ NK cells may
expression and cytotoxicity of NK cells. In contrast to a pre-
play a negative role in IVF treatment outcome but this has to
vious study by Fukui et al. (1999), the present study did not
be explored in a new prospective study.
demonstrate any statistical difference in the absolute count ofCD56dimCD16þ NK cells and CD56brightCD16þ NK cellsbetween successful and failed IVF treatment. However, for
statistical analysis these authors used percentage of CD16þ
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Submitted on December 4, 2003; accepted on May 24, 2004
Beck-Engeser et al. Retrovirology 2011, 8:91http://www.retrovirology.com/content/8/1/91 An autoimmune disease prevented by anti-retroviral drugs Gabriele B Beck-Engeser1, Dan Eilat2 and Matthias Wabl1* Background: Both Aicardi-Goutières syndrome, a Mendelian mimic of congenital infection, and the autoimmunedisease systemic lupus erythematosus can result from mutations in the gene encoding the enzyme Trex1. In mice,the absence of Trex1 causes severe myocarditis. The enzyme is thought to degrade endogenous retroelements,thus linking them to autoimmune disease. However, inhibition of reverse transcription by the inhibitor zidovudine(AZT) did not ameliorate the disease, weakening the link to retroelements.
Pré-ASTERIDAE CORNALES ERICALESASTERIDAE I GARRYALES Blackstonia, Swertia, - 1 - LAMIALES SOLANALESASTERIDAE II APIALES AQUIFOLIALES ASTERALES DIPSACALES - 2 - Ordre des GENTIANALES Pas mal de ligneux tropicaux. Leur unité est basée sur le fait que ces plantes réalisent la synthèse d'alcaloïdes qui résultent de la condensation du tryptophane et d'un reste isoprénique provenant d'un iridoïde, le loganoside. Les fleurs sont généralement régulières, en préfloraison tordues ou valvaires. Souvent de type 4 par réduction évolutive. L'ovaire supère chez les Apocynacées (qui incluent les Asclépiadacées) devient infère chez les Rubiacées.