Journal of Reproductive Immunology 95 (2012) 59–66

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Journal of Reproductive Immunology

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The correlation of autoantibodies and uNK cells in women with

reproductive failure
N.G. Mariee a,∗ , E. Tuckerman b , S. Laird c , T.C. Li b
a
Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
b
Biomedical Research Unit, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
c
Biomedical Research Centre, Sheffield Hallam University, City Campus, Sheffield S1 1WB, UK

a r t i c l e i n f o a b s t r a c t

Article history: There is conflicting evidence on the role of autoimmune disorders in reproductive failure,
Received 14 December 2011 including recurrent miscarriage (RM) and recurrent implantation failure (RIF), after in vitro
Received in revised form 16 April 2012 fertilisation (IVF). Several commonly studied autoimmune markers in women with repro-
Accepted 23 April 2012
ductive failure include antiphospholipid antibodies (APAs), thyroid peroxidase antibodies
(TPA) and uterine natural killer (uNK) cells. However, there have not been any studies
that have examined the correlation of these markers in women with reproductive failure.
Keywords:
uNK cells
To determine if women who tested positive for autoantibodies (APA and thyroid perox-
Autoantibodies idase antibodies) have significantly higher uNK cell numbers than women who tested
Reproductive failure negative for these antibodies, the percentage of stromal cells that stained positive for
CD56 was identified by immunocytochemistry in endometrial biopsies from 42 women
with unexplained RM (29 women tested negative for autoantibodies and 13 women tested
positive for autoantibodies) and 40 women with unexplained RIF (30 women tested neg-
ative for autoantibodies and 10 women tested positive for autoantibodies). Biopsies were
obtained on days LH + 7 to LH + 9. There was no significant difference in uNK cell numbers
between women with unexplained RM who tested negative and those who tested posi-
tive for autoantibodies. Similarly, there was no significant difference in uNK cell numbers
between women with unexplained RIF who tested negative and those who tested posi-
tive for autoantibodies. In women with reproductive failure the presence of autoantibodies
does not appear to affect the numbers of uNK cells in the endometrium around the time of
implantation.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction recurrent first-trimester miscarriage and second-trimester

There are various causes of reproductive failure (recur-
rent miscarriage and recurrent implantation failure),
such as chromosomal aberrations, anatomic anomalies,
endocrine disorders, infections and immunological disor-
ders (Li, 1998). The role played by immunological disorders
in reproductive failure is rather controversial. The guide-
line on The Investigation and Treatment of couples with
∗ Corresponding author. Tel.: +44 07717445772.
E-mail address: najatjom@yahoo.co.uk (N.G. Mariee).
miscarriage published by The Royal College of Obste-
tricians and Gynaecologists (2011) did not recommend
immunological tests in routine clinical practice, although
it highlighted the potential for future research.
Among the various studies on the immunological
mechanisms involved with reproductive failure, the most
commonly studied immune markers are antiphospholipid
antibodies, thyroid peroxidase antibodies (TPA) and uter-
ine natural killer (uNK) cells. However, their association
with either recurrent miscarriage or recurrent implanta-
tion failure is controversial. Some studies have found a
strong association between these immune markers and

0165-0378/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jri.2012.04.003
60 N.G. Mariee et al. / Journal of Reproductive Immunology 95 (2012) 59–66
reproductive failure (Coulam et al., 1997; Geva et al., 1994;
Ledee-Bataille et al., 2005; Nielsen and Christiansen, 2005;
Stern et al., 1998; Tuckerman et al., 2007; Wilson et al.,
1999). However, others have failed to confirm the finding
(Lachapelle et al., 1996; Shimada et al., 2004; Thum et al.,
2005; Denis et al., 1997; Kowalik et al., 1997). One of the
important reasons for the inconsistent results is the great
variability in how the tests are performed and the results
interpreted (Kallen and Arici, 2003).
Those who are supportive of an immune basis for repro-
ductive failure, are also divided on how best to investigate
these women with reproductive failure. Some investigators
focus on immunological derangements associated with B-
cell function, whereas others prefer to examine changes
associated with T-cell function. Commonly used tests in
relation to B-cell function include autoantibody testing in
peripheral blood, whereas the marker most often exam-
ined in relation to T-cell function is uterine natural killer
cells. Nevertheless, there have not been any studies that
have simultaneously examined both the B-cell and T-cell
markers in women with reproductive failure. The aim of
this study was to investigate whether or not women who
tested positive for autoantibodies (APAs and thyroid per-
oxidase antibodies [TPA]) had significantly different uNK
cell numbers than women who tested negative for these
antibodies.
2. Materials and methods
2.1. Subjects
2.1.1. Recurrent miscarriage group
The recurrent miscarriage (RM) group consisted of 42
women with a history of three or more consecutive mis-
carriages in the first trimester. All women had completed
investigations according to our established protocol (Li,
1998). All of the following investigations were normal:
paternal karyotypes, FSH, LH, prolactin, progesterone, free
androgen index, pelvic ultrasound and hysterosalpingog-
raphy.
In this group of women, 13 women tested positive
for autoantibodies. Five out of 13 women tested positive
for thyroid peroxidase antibodies (levels >50 IU/ml), while
eight women tested positive for lupus anticoagulant or
anticardiolipin (IgM and IgG) antibodies on two occasions,
six weeks apart. The latter group of subjects therefore ful-
fil the diagnostic criteria for anti-phospholipid syndrome.
The remaining 29 women tested negative for the autoanti-
bodies.
2.1.2. Recurrent implantation failure group
The recurrent implantation failure (RIF) group consisted
of 40 women who had repeated IVF failure cycles despite
replacement of good quality embryos. The definition of
recurrent implantation failure in this study includes:
1. The age of the female partner is less than 40 years.
2. Failure to achieve a clinical pregnancy after three
or more IVF treatment cycles in which good quality
embryos had been replaced.
3. At least four or more good quality embryos had been
replaced during the IVF treatment cycles.
They also underwent investigations similar to the RM
subjects and all parameters in this group of women were
also normal (other than autoantibodies described below).
In this group of women with RIF, ten subjects tested
positive for autoantibodies, but all the other investigations
were normal. Two women had high thyroid peroxidase
antibodies (>50 IU/ml), while eight women tested positive
for lupus anticoagulant or anticardiolipin (IgM and IgG)
antibodies on two occasions, six weeks apart. The remain-
ing 30 subjects tested negative for the autoantibodies.
2.2. Endometrial biopsy
All women participating in the study underwent daily
urine LH measurement to identify the LH surge. Women
were advised not to have unprotected sexual intercourse
during the biopsy cycle to avoid undergoing a biopsy in
the conception cycle. The endometrial biopsies were col-
lected using a Pipelle sampler (Prodimed, France) in the
luteal phase of the menstrual cycle between day 7 and day 9
after the LH surge. Unlike the use of a curette, which obtains
specimens from both the superficial and the deep layers of a
localised area of the endometrial cavity, the Pipelle sampler
is designed to obtain specimens, via a suction mechanism,
from the superficial layers of a much wider (almost entire)
area of the upper uterine cavity. Samples were then imme-
diately fixed in formalin overnight and automatically wax
embedded for immunocytochemistry study. All the biopsy
specimens examined showed evidence of secretory trans-
formation.
2.3. Immunocytochemistry
Sections measuring 5 ␮m were de-waxed in xylene,
rehydrated through the alcohols to Tris-buffered saline
pH 7.6 (TBS), and quenched in 0.3% hydrogen peroxide
in methanol for 20 min. After washing, unmasking was
performed in an 800W microwave oven in 10 mmol/l cit-
rate buffer pH 6.0. Buffer was heated in the microwave
oven until boiling. Slides were added to the buffer, and
left covered at a high heat for 3 min. Slides were fur-
ther incubated for 12 min at a medium heat and allowed
to cool for 20 min. An ABC kit (Vector Laboratories, UK)
was used according to the manufacturer’s instructions
with some adaptations. Slides were washed in TBS and
blocked in blocking buffer containing 250 ␮l avidin/ml
(Vector Laboratories) for 1 h at room temperature, and
incubated overnight at +4 ◦ C with a mouse monoclonal
primary anti-CD56 antibody (NCL-CD56-504; Novacas-
tra Laboratories Ltd, UK) diluted 1:50 in antibody buffer
containing 250 ␮l/ml biotin. Slides were washed in TBS
throughout, and after application of secondary antibody
and Vectorstain, binding was visualised by incubation with
peroxidase substrate DAB (3,3 -diaminobenzidene terahy-
drochloride; Vector Laboratories). Slides were washed in
distilled water and counterstained with 20% haematoxylin
for 30 min, differentiated, dehydrated through the alcohols,
 N.G. Mariee et al. / Journal of Reproductive Immunology 95 (2012) 59–66 61

Table 1
Demographic data for the study populations. RM = recurrent miscarriage; RIF = recurrent implantation failure; AAB = autoantibodies; LH = luteinising hor-
mone; FSH = follicle-stimulating hormone.

Parameter RM, AAB +ve n = 13 RM, AAB −ve n = 29 RIF, AAB +ve n = 10 RIF, AAB −ve n = 30

Age (years) 34 (27–40) 34 (23–40) 37 (32–40) 34 (24–39)

Median (range)
Body mass index 26.2 (23.6–40.1) 26.3 (21.5–30.4) 24.6 (20.7–34.4) 22.5 (20–26.6)
(kg/m2 )
Median (range)
Parity 1 (0–1) 0 (0–2) 0 (0–1) 0 (0–1)
Median (range)
Gestational age at 8 (4–23) 9 (6–12) – –
miscarriage (weeks)
Median (range)
Number of previous 3 (3–6) 3 (3–4) – –
miscarriages
Median (range)
Number of failed IVF – – 4 (3–11) 4 (3–8)
cycles
Median (range)
D5 LH (IU/L) 3.8 (2–7.4) 4.7 (1.7–7.6) 4.4 (2.3–7.4) 5.3 (2.1–8.3)
Median (range)
D5 FSH (IU/L) 4.5 (3.2–9.9) 5.6 (1.0–9.9) 4.5 (3.5–8.4) 6.5 (3.7–9.8)
Median (range)
Oestradiol (pmol/L) 129 (73–262) 151 (73–307) 240 (174–278) 196 (73–285)
Median (range)
D21 Progesterone 48.3 (22.7–67.1) 43.5 (20–119) 51.1 (30.9–60) 49 (15–104)
(nmol/L)
Median (range)

cleared in xylene and mounted in Vectormount (Vector
Laboratories).
2.4. Analysis
The number of uNK cells (positively stained stromal
cells) and the number of negatively stained stromal cells
were counted in ten random non-overlapping microscopic
fields at a magnification of 400×. The percentage of positive
cells per total stromal cells for each biopsy was calculated.
2.5. Statistical methods
Data were analysed using the Statistical Package for
Social Science (SPSS) version 14. The non-parametric
Mann–Whitney U test was used to compare numbers of
CD56+ cells between groups. The Fisher’s exact test was
used to examine the relationship between the results of
autoantibody testing and uNK cell count in a two by two
contingency table. Differences were considered to be sta-
tistically significant when the p value was <0.05.
2.6. Ethics approval
Local ethics approval was obtained for this study and
written informed consent was obtained from all women
participating in the study.
3. Results
3.1. Demographic data
The age and other demographic details in the differ-
ent groups of women are shown in Table 1. There was no
significant difference in the mean age, body mass index
(BMI), parity, gestational age at miscarriage, number of pre-
vious miscarriages, number of failed IVF treatment cycles,
FSH, LH, oestradiol and progesterone levels between those
who tested positive and those who tested negative for
autoantibodies.
3.2. Women with RM
There was no significant difference (p = 0.268) in the
median (range) numbers of CD56+ uNK cells between
women with unexplained RM who tested negative (n = 29,
10.6% [1.7–38.6%]) or positive for autoantibodies (n = 13,
7.1% [1.5–14.9%]).
3.3. Women with RIF
Similarly, there was no significant difference (p = 0.286)
in the median (range) number of CD56+ uNK cells between
women with unexplained RIF who tested negative (n = 30,
14.6% [2.2–71.4]) and those who tested positive for autoan-
tibodies (n = 10, 11.9% [4.4–24.5]).
3.4. Women with RIF and RM combined
When the two groups of subjects with reproductive fail-
ure (RM & RIF) are combined together, there was also no
significant difference (p = 0.108) in the number of CD56+
uNK cells between women who tested negative (n = 59;
median [range], 11.7% [1.7–71.4]) and those who tested
positive for autoantibodies (n = 23; median [range], 10.6%
[1.5–24.5]).
When only women who tested positive for antiphos-
pholipid syndrome were considered (n = 16), the median
62 N.G. Mariee et al. / Journal of Reproductive Immunology 95 (2012) 59–66

80.00

60.00
CD56/Total stromal cells

40.00

20.00

0.00

Autoantibody -ve APS +ve

Diagnosis

Fig. 1. Box plot showing the percentage of CD56+ uNK cells in women with reproductive failure who tested +ve and −ve for lupus anticoagulant or
anti-cardiolipin antibodies.

(range) number of CD56+ uNK cells (11.1% [1.5–24.5])
was not significantly different (p = 0.310) from that of
women who tested negative for antibodies (n = 59; 11.7%
[1.7–71.4]; Fig. 1).
When only women who tested positive for lupus anti-
coagulant were considered (n = 8), the median (range)
number of CD56+ uNK cells (7.1% [1.5–24.5]) was not sig-
nificantly different (p = 0.629) from that of women tested
negative for antibodies (n = 59; 11.7% [1.7–71.4]).
When only women who tested positive for ACA were
considered (n = 8), the median (range) number of CD56+
uNK cells (13.4% [7.1–14.6]) was not significantly differ-
ent (p = 0.159) from that of women who tested negative for
antibodies (n = 59; 11.7% [1.7–71.4]).
Using a two by two contingency table, there was no sta-
tistically significant association between APS and uNK cell
Table 2
Two by two contingency table analysis showing the relationship between:
(a) APS test results and uNK cell count results (normal or high), p > 0.05;
and (b) the anti-peroxidase antibody (TPA) test results and uNK cell count
results (normal or high), p > 0.05.
APS −ve
(a)
Normal uNK cell numbers 38
High uNK cell numbers 21
TPA −ve
(b)
Normal uNK cell numbers 38
High uNK cell numbers 21
count (two-tailed p value using Fisher’s exact test = 0.242;
Table 2a).
Similarly, when only women who tested positive for
anti-thyroid peroxidase antibodies (n = 7) were consid-
ered, the median (range) number of CD56+ uNK cells (5.9
[2.9–14.9]) was not significantly different (p = 0.109) from
that of women who tested negative for antibodies (n = 59;
11.7% [1.7–71.4]; Fig. 2).
There was also no significant association between TPA
and uNK cell count (two-tailed p value using Fisher’s exact
test = 1.0; Table 2b).
4. Discussion
Successful implantation and pregnancy requires a
healthy immune system to prevent rejection of the alloim-
mune fetus. Immunological mechanisms are considered to
be one of the causes of reproductive failure. The relation-
ship between autoimmunity and reproductive failure has
been well established (Geva et al., 1995). Some immune
factors, such as the presence of ACAs, thyroid peroxi-
dise antibody and increased number of uNK cells have
APS +ve been associated with reproductive failure. In this study
we investigated whether APAs and TPA correlate with
13 uNK cell numbers in women with reproductive failure.
3 We found no significant correlation between the presence
of autoantibodies and uNK cell numbers in these women.
TPA +ve Nevertheless, we acknowledge that we only selected a few
commonly tested autoantibodies for this study; some other
5 autoantibodies, such as ANA and dsDNA, have not been
2
included.
 N.G. Mariee et al. / Journal of Reproductive Immunology 95 (2012) 59–66
80.00
CD56/Total stromal cells 60.00
40.00
20.00
0.00
Autoantibody -ve
Diagnosis
Fig. 2. Box plot showing the percentage of CD56+ uNK cells in women with reproductive failure who tested +ve and −ve for anti-peroxidase antibody.
4.1. APA and reproductive failure
Antiphospholipid syndrome (APS) is the most common
autoimmune disease associated with recurrent pregnancy
loss (RPL) (Cervera and Balasch, 2008). It is an acquired
syndrome characterised by the formation of autoantibod-
ies to a variety of phospholipids and phospholipid-binding
proteins (Baker and Bick, 2008). The prevalence of anti-
cardiolipin antibodies (ACAs) and lupus anticoagulant (LA)
in a healthy population is 1–5% (Baker and Bick, 2008).
Previous studies suggested that APS is associated with pre-
eclampsia, recurrent miscarriage (Farquharson et al., 2002;
Donohoe et al., 2002), intrauterine growth retardation
(Gleicher et al., 1993), placental abruption and thrombo-
sis of the placenta, causing impairment of the fetal blood
supply, leading to fetal death (Subrt et al., 2008).
Whilst it is generally accepted nowadays that there is an
association between APS and RM (Chogle and Bichile, 1999;
Clifford et al., 1994; Hill and Choi, 2000; Pattison et al.,
1993; Cohn et al., 2010; Quenby et al., 2005), the association
between ACAs and infertility or RIF is rather more contro-
versial. Some researchers reported an increased prevalence
of ACAs antibodies in patients with infertility (Birkenfeld
et al., 1994; Gleicher et al., 1994), while others failed
to show such a relationship (Carp and Shoenfeld, 2007;
Cervera and Balasch, 2008). There is also conflicting evi-
dence regarding the association of ACAs and IVF outcome
(Bellver et al., 2008; Cervera and Balasch, 2008; Hornstein
et al., 2000; Mardesic et al., 2000; Birkenfeld et al., 1994;
Coulam et al., 1997; Geva et al., 1994). A meta-analysis
63
TPA +ve
of ACAs and IVF outcome found no significant association
between the presence of ACAs and the clinical pregnancy
rate or live birth rate in IVF patients (Hornstein et al., 2000).
The association between ACAs and peripheral blood NK
cells has been investigated by Sher et al. (2000) who found
a correlation between the presence of ACAs and increased
peripheral blood NK cells activity in non-male factor infer-
tility (Sher et al., 2000). They found that 88% of women who
tested positive for ACAs had increased peripheral blood NK
cell activity whereas only 12% of those who tested negative
for ACAs had increased peripheral blood NK cells activ-
ity. In a more recent study, Perricone et al. (2007) showed
that women who had APS and RM had significantly higher
peripheral NK cell numbers than women who had APS,
but without RM, RM due to anatomical factors, RM asso-
ciated with hypothyroidism, or healthy control subjects
(Perricone et al., 2007). However, peripheral NK cells are
different phenotypically and functionally from uNK cells
(Nagler et al., 1989, Nishikawa et al., 1991). Phenotypically,
uNK cells express CD56, CD38 and CD69, but do not express
CD3, CD4, CD8, CD16 and CD57 (Arcuri et al., 2006; Bulmer
et al., 1991; Moffett-King, 2002). There is no correlation
between peripheral and uterine NK cell counts (Moffett
et al., 2004). Whilst uNK cells are considered to be directly
involved in implantation, the relevance of peripheral blood
NK cells in the implantation process and hence reproduc-
tive failure is questionable (Moffett et al., 2004). In contrast
to previous studies correlating peripheral blood NK cells
and autoantibody status, our study examined the relation-
ship between uNK cell counts and autoantibodies status.
64 N.G. Mariee et al. / Journal of Reproductive Immunology 95 (2012) 59–66
We were unable to show a correlation between these two
measurements.
4.2. TPA and reproductive failure
Thyroid antibodies are antibodies to the thyroid perox-
idase enzyme, which is responsible for the iodination of
tyrosine residues to form thyroid hormones (Ghazeeri and
Kutteh, 2001). TPAs were found in women with autoim-
mune diseases, such as systemic lupus erythematosus (SLE)
and autoimmune-induced premature ovarian failure.
Several reports have linked thyroid autoimmunity with
RM (Bussen and Steck, 1995; Iijima et al., 1997; Dendrinos
et al., 2000; Matalon et al., 2001), but others have failed
to show this association (Esplin et al., 1998; Bellver et al.,
2008; Muller et al., 1999). Similarly, some authors reported
an increase in the prevalence of TPA in women with infer-
tility and RIF (Bellver et al., 2008; Bussen et al., 2000; Geva
et al., 1995; Kim et al., 1998), while others did not find any
difference in the likelihood of biochemical pregnancies or
clinical pregnancy losses between women undergoing ART
who tested positive and those who tested negative for TPA
(Kutteh et al., 1999; Negro et al., 2007).
Our study found that women who tested positive for
TPA did not have different uNK cell counts from women
who tested negative for TPA.
4.3. uNK cells and reproductive failure
Natural killer cells are part of the innate immune sys-
tem. Ninety percent of NK cells in peripheral blood are
CD56 + CD16 + (CD56dim ) and these cells play an impor-
tant role in the innate immune system (Eriksson et al.,
2006) by producing immunoregulatory cytokines against
several types of pathogens (Sentman et al., 2004). Their
main function is cytolysis via specific cell surface molecules
that detect target cells deficient in class 1 major histocom-
patibility complex (MHC) molecules (Moretta et al., 2000,
2003).
Several studies have confirmed that women with recur-
rent miscarriage had an increased number of uNK cells
in endometrial biopsies obtained in the peri-implantation
period (Clifford et al., 1999; Kodama et al., 1998; Quenby
et al., 1999). Furthermore, we have recently reported that
women with recurrent implantation failure after IVF also
had an increased number of uNK cells in endometrial biop-
sies obtained in the peri-implantation period (Tuckerman
et al., 2010). There does seem to be good evidence from
various independent centres that the uNK cell count is ele-
vated in women with reproductive failure. However, there
is conflicting evidence on the prognostic value of uNK cell
measurement on the outcome of a subsequent pregnancy.
In a relatively small study, Quenby et al. (1999), found that
the number of pre-pregnancy uNK cells was significantly
higher in women with RM who miscarried again in a sub-
sequent pregnancy than in women who had a live birth in
a subsequent pregnancy. However, in a more recent and
larger study, Tuckerman et al. (2007) found no difference
in the number of uNK cells in women with RM who had a
live birth and those who miscarried again in a subsequent
pregnancy.
In a recent study, we examined the relationship
between uNK cells count and expression of IL15 and
LIF. We found altered expression of LIF and IL-15 in
the endometrium of women with RIF, and a correlation
between uNK cell numbers and stromal cell IL-15 expres-
sion (Mariee et al., 2012).
In this study, we did not directly address the contro-
versy of whether or not there is an immunological basis
for recurrent reproductive failure; rather, we have simply
examined an area that has not been previously explored,
i.e. whether there is an association between the results of
several commonly used autoantibodies and uterine NK cell
count in the endometrium.
In conclusion, we found that there is no significant cor-
relation between the presence or absence of autoantibodies
(ACAs, TPAs) and the number of uNK cells in women with
reproductive failure. Whilst the presence of ACAs and an
increase in the number of uNK cells are both associated
with reproductive failure, the lack of correlation between
these two measurements suggest that they affect implan-
tation via separate and different mechanisms.
Acknowledgements
We would like to thank the staff in the recurrent mis-
carriage clinic, particularly Staff Nurses Barbara Anstie and
Katherine Wood, for help in collecting endometrial biopsy
specimens.
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