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Simple enumerations of peripheral blood natural killer (CD56+ NK) cells, B cells and T cells have no predictive value in IVF treatment outcome

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Simple enumerations of peripheral blood natural killer (CD56+ NK) cells, B cells and T cells have no predictive value in IVF treatment outcome

Abstract

To evaluate the association between the absolute counts of the peripheral natural killer (NK) cells (including total CD56(+) NK cells, CD56(dim) NK cells and CD56(bright) NK cells), B cells and T cells on the implantation rate and miscarriage rate after IVF treatment. This was a prospective observation study. A total of 138 patients who underwent IVF treatment from December 2002 to July 2003 were recruited to the study. Blood samples were obtained on the day of vaginal oocyte retrieval prior to the procedure. The absolute counts of lymphocytes, NK cells, B cells and T cells were identified by flow cytometry. These absolute counts and their relationships to IVF treatment outcome and miscarriage rate were analysed. There were no significant differences with regard the mean values of absolute lymphocyte count, T cell count, B cell count and NK cell count (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells) between the pregnant and non-pregnant groups and also between the ongoing pregnancy and miscarriage groups. The cause of infertility, duration of infertility, basal FSH levels, number of previous failed IVF treatments, number of previous miscarriages and stimulation characteristics were not significantly different between the pregnant and non-pregnant groups. Previous studies have suggested that women with a history of recurrent miscarriage and those with infertility accompanied by recurrent failed IVF treatments are associated with a peripheral blood NK cell percentage >12%, therefore further analysis of peripheral CD56(+) NK cell levels <12% (group A) and >12% (group B) was performed. There was no significant difference in implantation rate (group A: 17.0%; group B: 23.2%), pregnancy rate (group A: 36.6%; group B: 47.7%) or miscarriage rate (group A: 23.3%; group B: 28.6%). There were no significant differences between simple enumerations of peripheral blood NK cells (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells), B cells and T cells with IVF treatment outcome and pregnancy outcome. Women who had a peripheral NK cell level >12% did not have higher number of previous pregnancy losses. Importantly their pregnancy rate was not reduced and their miscarriages were not increased compared to women who had a peripheral NK cells level <12%.
Simple enumerations of peripheral blood natural killer
(CD56
1
NK) cells, B cells and T cells have no predictive
value in IVF treatment outcome
M.Y.Thum
1,2,3,4
, S.Bhaskaran
2
, A.S.Bansal
2
, H.Shehata
3
, B.Ford
2
, N.Sumar
2
and H.I.Abdalla
1
1
Lister Fertility Clinic, Lister Hospital, Chelsea Bridge Road, London SW1W 8RH,
2
Immunology Department, Epsom and St Helier
University Hospital, Surrey and
3
Obstetrics and Gynaecology Department, St Helier Hospital, London, UK
4
To whom correspondence should be addressed. E-mail: mythum@doctors.net.uk
BACKGROUND: To evaluate the association between the absolute counts of the peripheral natural killer (NK)
cells (including total CD56
1
NK cells, CD56
dim
NK cells and CD56
bright
NK cells), B cells and T cells on the
implantation rate and miscarriage rate after IVF treatment. METHODS: This was a prospective observation
study. A total of 138 patients who underwent IVF treatment from December 2002 to July 2003 were recruited to
the study. Blood samples were obtained on the day of vaginal oocyte retrieval prior to the procedure. The absolute
counts of lymphocytes, NK cells, B cells and T cells were identified by flow cytometry. These absolute counts and
their relationships to IVF treatment outcome and miscarriage rate were analysed. RESULTS: There were no sig-
nificant differences with regard the mean values of absolute lymphocyte count, T cell count, B cell count and NK
cell count (including total CD56
1
NK, CD56
dim
NK and CD56
bright
NK cells) between the pregnant and non-preg-
nant groups and also between the ongoing pregnancy and miscarriage groups. The cause of infertility, duration of
infertility, basal FSH levels, number of previous failed IVF treatments, number of previous miscarriages and
stimulation characteristics were not significantly different between the pregnant and non-pregnant groups. Pre-
vious studies have suggested that women with a history of recurrent miscarriage and those with infertility
accompanied by recurrent failed IVF treatments are associated with a peripheral blood NK cell percentage >12%,
therefore further analysis of peripheral CD56
1
NK cell levels <12% (group A) and >12% (group B) was per-
formed. There was no significant difference in implantation rate (group A: 17.0%; group B: 23.2%), pregnancy
rate (group A: 36.6%; group B: 47.7%) or miscarriage rate (group A: 23.3%; group B: 28.6%). CONCLUSION:
There were no significant differences between simple enumerations of peripheral blood NK cells (including total
CD56
1
NK, CD56
dim
NK and CD56
bright
NK cells), B cells and T cells with IVF treatment outcome and pregnancy
outcome. Women who had a peripheral NK cell level >12% did not have higher number of previous pregnancy
losses. Importantly their pregnancy rate was not reduced and their miscarriages were not increased compared to
women who had a peripheral NK cells level <12%.
Key words: B cells/CD56/IVF/natural killer cells/T cells
Introduction
For the conceptus to implant and for pregnancy to be main-
tained, the conceptus has to protect itself from the maternal
immune system (Hill et al., 1992; Yokoyama et al., 1994).
This involves a balance between maternal immune defence
mechanisms and invasion by the allogenic trophoblast. Lym-
phocytes including NK cells, T cells and to a lesser extent
B cells are clearly present in the human decidua (Bulmer
and Sunderland, 1984) and the most abundant amongst these
in the endometrium is the NK cell (Moffett-King, 2002). All
lymphocytes are, however, in intimate direct contact with
trophoblast cells and have the potential to initiate an
immune response (Lachapelle et al., 1996). Yamada et al.
(1994) showed that trophoblasts can stimulate peripheral
blood mononuclear cells to proliferate and produce embryo-
toxic factors in women with recurrent miscarriage. Others
have suggested that NK cells, monocytes/macrophages, and
T cells could produce decidual toxic factors resulting in
fetal loss (Baines and Gendron, 1990; Clark, 1994). While
the factors controlling the influx, proliferation and differen-
tiation of these cells in the uterus are unknown, the pro-
cesses seem to be dependent on decidualization of the
stromal cells.
Previous studies have suggested that an increase in circu-
lating lymphocytes might be associated with recurrent mis-
carriages. Thus Souza et al. (2002) reported that an increase
Human Reproduction Vol.20, No.5 pp. 1272–1276, 2005 doi:10.1093/humrep/deh774
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in peripheral blood absolute T cell count, but not NK cell
count, was evident in women with recurrent abortion.
Furthermore, Yamamoto et al. (1999) reported that the per-
ipheral blood NK cell percentage was not significantly differ-
ent between pregnant women and women who had a
miscarriage with a chromosomally normal or abnormal preg-
nancy, while Yamada et al. (2001) reported that NK cell
activity was only higher in women who had a miscarriage
with a chromosomally normal pregnancy. In the same publi-
cation, Yamada et al. (2001) also reported that the peripheral
NK cell percentages were not significantly different between
women who had miscarriage with a chromosomally normal
or abnormal pregnancy. However, in a later publication
(Yamada et al., 2003) the same group reported that both NK
cell activity and percentage were elevated in women who
suffered from recurrent miscarriage and had a miscarriage
with a chromasomally normal pregnancy. Coulam et al.
(1995) also suggested that the peripheral blood NK cell per-
centage could predict pregnancy outcome. Additionally, Beer
et al. (1996) showed that women with a history of recurrent
miscarriage and infertile women with recurrent failed IVF
treatments were associated with a peripheral blood NK cell
percentage . 12%. These findings have not been replicated
by any other research groups. However, numerous prac-
titioners are offering immunomodulation therapy based on
the evidence provided by Coulam et al. (1995) and Beer et al.
(1996) that the peripheral blood NK cell absolute count or
percentage can affect IVF or pregnancy outcome.
The objective of this study was to investigate, prospec-
tively, women undergoing IVF treatment to determine
whether there was any association between the peripheral
blood NK cell (including total CD56
þ
NK, CD56
dim
and
CD56
bright
NK cells), T cell and B cell percentages and
absolute counts and implantation success or miscarriage in
patients undergoing IVF treatment.
Methods and materials
Study population
From December 2002 to July 2003, 138 patients undergoing
IVF treatment cycles were recruited into the study. Indepen-
dent approval was obtained from the Local Research Ethics
Committee and informed consent was obtained from each
patient prior to the study. Ten millilitres of peripheral blood
were obtained on the day of vaginal oocyte retrieval prior to
the procedure. Exclusion criteria were: women with known
immunological disease (antiphospholipid antibodies, lupus
anticoagulant, anticardiolipin antibodies), uterine abnormal-
ity (fibroid, uterine polyp, uterine septum), fewer than two
embryos available for transfer, or endometrium thickness
, 8 mm before embryo transfer. Exclusion of candidates
was performed without the knowledge of the blood test
result. Blood samples were obtained on the day of vaginal
oocyte collection prior to the procedure. We had noted that
Giuliani et al. (1998) reported that controlled ovarian stimu-
lation in assisted reproduction did not affect the immune
system.
Stimulation protocol for IVF treatment
Stimulation protocol for IVF treatment was as previously
described (Thum et al., 2003). Briefly, pituitary down-regu-
lation 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 (1822 mm), 10 000 IU of hCG
was administered. Oocytes were aspirated using transvaginal
ultrasound guidance 34 36 h after hCG administration. All
embryos were allowed to cleave and the best two or three
embryos were selected for transfer. Embryo transfer was per-
formed on day 2 or day 3 using a soft catheter (Wallace)
with transabdominal ultrasound guidance. Progesterone sup-
plement for luteal support (Cyclogest; Shire Pharmaceuticals
Ltd, UK), 400 mg once a day per vaginum or per rectum,
was commenced 1 day before embryo transfer and continued
until a pregnancy test was performed. A pregnancy test was
performed 2 weeks after embryo transfer.
Flow cytometric NK activation and inhibition
quantification assay
Ten millilitres of peripheral blood was collected in hepari-
nized tubes and analysed within 24 h. Fifty millilitres of each
sample was incubated for 15 min at room temperature with
10 ml mouse anti-CD56 PE (BD PharMingen), anti-CD3 PE
Cy5 (Quest Biomedical) monoclonal antibodies (mAb). Iso-
typic control mAb included mouse IgG
1
PE (BD PharMin-
gen) and IgG
1
PE Cy5 (Quest Biomedical). In this lyse, no
wash procedure, 1 ml of Quicklysis lysing solution (Quest
Biomedical) was added to each tube and incubated for a
further 10 min at room temperature. A volume of 50 ml of
PerfectCount beads (Quest Biomedical) was then accurately
pipetted to each tube and samples run with BD FACSCalibur
flow cytometer.
Data analysis
All IVF data were collected prospectively in Medical System
for IVF (MedicalSys, UK) and analysed by Statistics Package
for Social Sciences (SPSS, UK). Descriptive statistical analy-
sis was performed initially to examine the normal distribution
of all continuous variables for parametric statistical tests.
Analysis of variance was then conducted to assess the dur-
ation and amount of gonadotrophin required to achieve fol-
licular maturity, number of mature follicles, number of
available embryos for transfer, number of oocytes collected,
fertilization rate, the absolute count of lymphocytes and its
subpopulation (NK cells, B cells and T cells) between the
pregnant and non-pregnant women after IVF treatment. The
x
2
cross-tabulation test was used to analyse for difference of
pregnancy rates, miscarriage rates and live birth rates
between women with NK cell percentage . 12% and , 12%
as suggested by Beer et al. (1996).
Results
During the study period, from December 2002 to July 2003,
138 women who underwent IVF were recruited. Twelve
women were excluded from statistical analysis; four had
NK cell numbers and IVF outcome
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failed fertilization, four had only one embryo available for
transfer, one had OHSS therefore did not have embryo trans-
fers, two had an endometrial thickness , 7.5 mm and one had
poor quality embryos. The overall pregnancy rate was 38.6%,
the implantation rate 19.1% and the miscarriage rate 24.5%
for all the patients recruited. Out of 128 cycles, 82 (64.1%)
were conventional IVF and 46 (35.9%) were IVF in conjunc-
tion with ICSI. There were no differences regarding ovarian
stimulation characteristics and cycle outcomes between con-
ventional IVF and IVF with ICSI.
There were no significant differences between the two
groups with regard to patients’ characteristics (Table I).
Table II confirms the absence of any significant differences
in absolute lymphocyte count, T Cell, B cell, CD56
þ
NK
cell, CD56
dim
and CD56
bright
NK cells in the pregnant and
non-pregnant groups. This was also the case for those women
with ongoing pregnancy and miscarriage (Table III).
Table IV examines the relationship between outcome of
ovarian stimulation, IVF treatment outcome and pregnancy
outcome with CD56
þ
NK cell percentage equal to or , 12%
and . 12%. There were no significant differences with regard
to the outcome of ovarian stimulation, IVF treatment out-
come and pregnancy outcome between the two groups. The
pregnancy rate and miscarriage rate were higher in CD56
þ
NK cell . 12% group but not significantly so.
Discussion
Maternal immunological mechanisms have been postulated
to explain recurrent miscarriage, unexplained infertility and
recurrent failed IVF cycles (Norwitz et al., 2001). A success-
ful pregnancy depends on protection or down-regulation of
potentially harmful maternal immune responses at the utero-
placental interface to enable a functional placenta to develop
(King et al., 1997). Although implantation and development
and maintenance of the placenta are typically localized pro-
cesses, it is interesting to evaluate whether any systemic
immunological changes can have a detrimental effect on
implantation, placentation or on the conceptus. However, it is
difficult to examine the local environment at the implantation
site. Clearly a biopsy of the endometrium cannot be per-
formed near to the time of implantation or before embryo
Table I. Patients’ demographic and stimulation characteristics
Not pregnant Pregnant
No. of patients 75 51
Age (years) 35.45 ^ 3.8 34.16 ^ 4.0
Tubal factor (%) 23.7 23.5
Male factor (%) 25.0 27.6
Other
a
(%) 16.3 15.6
Unexplained (%) 35.0 33.3
Duration of infertility in years (mean ^ SD) 4.60 ^ 2.7 3.57 ^ 2.3
Basal FSH level (IU/l) (mean ^ SD) 7.82 ^ 2.9 7.71 ^ 3.8
Mean no. of previous failed IVF attempts 1.88 1.37
No. of previous miscarriages (^ SD) 0.25 0.33
Gonadotrophin
b
(IU) 3067.0 2531.6
Estradiol (IU) on hCG day 8599.87 7015.88
No. of oocytes collected (mean ^ SD) 12.3 ^ 5.5 12.9 ^ 6.6
Fertilization rate (%) 66.7 66.5
Embryos available for transfer (mean ^ SD) 7.96 ^ 4.4 8.76 ^ 5.7
Mean no. of embryos transferred 2.18 2.04
a
Anovulatory and endometriosis.
b
Mean amount of gonadotrophin used for stimulation (recombinant FSH,
hMG or urinary FSH).
No differences between groups were statistically significant (P . 0.05).
Table II. The absolute counts of lymphocyte, T-cell, B-cell, NK cell and its
sub-groups between pregnant and not pregnant groups
Non-pregnant Pregnant
No. of patients 75 51
Lymphocyte absolute
count ( £ 10
9
)
a
2.23 ^ 0.76 2.07 ^ 0.61
T lymphocyte absolute
count ( £ 10
9
)
a
(%)
1.62 ^ 0.53 (73.1) 1.54 ^ 0.50 (74.2)
B lymphocyte absolute
count ( £ 10
9
)
a
(%)
0.38 ^ 0.02 (16.2) 0.29 ^ 0.04 (13.7)
CD56
þ
NK cell absolute
count ( £ 10
9
)
a
(%)
0.232 ^ 0.02 (10.5) 0.256 ^ 0.01 (11.8)
CD56
dim
NK cell absolute
count ( £ 10
9
)
a
(%)
0.211 ^ 0.07 (9.7) 0.238 ^ 0.03 (10.9)
CD56
bright
NK cell absolute
count ( £ 10
9
)
a
(%)
0.017 ^ 0.008 (0.82) 0.018 ^ 0.007 (0.95)
a
Mean ^ SD.
No differences between groups were statistically significant (P . 0.05).
Table III. The absolute counts of lymphocyte, T-cell, B-cell, NK cell and
its sub-groups between on going pregnancy and miscarriage groups
Ongoing pregnancy Miscarriage
No. of patients 38 13
Lymphocyte absolute
count ( £ 10
9
)
a
2.05 ^ 0.6 2.13 ^ 0.6
T lymphocyte absolute
count ( £ 10
9
)
a
(%)
1.54 ^ 0.5 (74.7) 1.53 ^ 0.4 (72.4)
B lymphocyte absolute
count ( £ 10
9
)
a
(%)
0.267 ^ 0.01 (12.8) 0.34 ^ 0.02 (16.1)
CD56
þ
NK cell absolute
count ( £ 10
9
)
a
(%)
0.243 ^ 0.02 (11.9) 0.249 ^ 0.02 (11.4)
CD56
dim
NK cell absolute
count ( £ 10
9
)
a
(%)
0.224 ^ 0.01 (10.9) 0.231 ^ 0.02 (10.6)
CD56
bright
NK cell absolute
count ( £ 10
9
)
a
(%)
0.018 ^ 0.008 (1.0) 0.017 ^ 0.007 (0.8)
a
Mean ^ SD.
No differences between groups were statistically significant (P . 0.05).
Table IV. Stimulation characteristics and cycle outcomes
Group A CD56
þ
NK cell # 12%
Group B CD56
þ
NK cell . 12%
No. of patients 82 44
Gonadotrophin (IU)
a
2902.2 2590.3
No. of oocytes
collected (mean ^ SD)
12.32 ^ 5.9 12.27 ^ 6.0
Fertilization rate (%) 64.15 ^ 23.5 67.8 ^ 22.9
Embryos available for
transfer (mean ^ SD)
7.75 ^ 4.8 8.39 ^ 5.2
Mean no. of embryos
transferred
2.07 2.16
Implantation rate (%) 17.0 23.2
Pregnancy rate per cycle
started (%)
36.6 (30/82) 47.7 (21/44)
Live birth rate per cycle
started (%)
28.1 (23/82) 34.1 (15/44)
Miscarriage rate (%) 23.3 (7/30) 28.6 (6/21)
a
Mean amount of gonadotrophin used for stimulation.
No differences between groups were statistically significant (P . 0.05).
M.Y.Thum et al.
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transfer during IVF treatment. Moreover, a biopsy prior to
the treatment cycle/pregnancy may not reflect the condition
at the time of implantation. In consequence it is difficult to
evaluate the local immunological milieu in this situation. The
objective of the study was to evaluate whether variations in
the absolute numbers of different lymphocyte subsets had a
detrimental effect on implantation, placentation or on the
conceptus after IVF treatment.
The results of this study showed no significant differences
in the peripheral T cell count between women with a positive
or negative IVF treatment outcome or with pregnancy out-
come, i.e. live birth and miscarriage. This finding is in accord
with previous studies. Thus Quenby et al. (1999) showed no
significant difference in the endometrial T cell count between
women with recurrent miscarriage and multiparous women
with no history of recurrent miscarriage. Additionally,
Vassiliadou and Bulmer (1998) showed similar number of
T cells in normal first trimester deciduas compared to decid-
uas obtained after spontaneous miscarriage. However,
Yamada et al. (1994) showed that peripheral blood T cells
from women with recurrent miscarriage were sensitive to tro-
phoblasts when cultured in vitro and were able to proliferate
and produce embryotoxic factors. However, this sensitivity
may be the result of previous miscarriages rather than the
cause, as even non-viable pregnancy tissue can initiate a
maternal immune response. Regarding peripheral blood B
cells, these were also not significantly different between
pregnant and non-pregnant women after IVF treatment or
between women with ongoing pregnancy and miscarriage.
This finding is in keeping with Quenby et al. (1999), who
revealed that endometrial B cells were not significantly
different between women with a history of recurrent miscar-
riage and multiparous women with no history of recurrent
miscarriage. These findings suggest that variation in periph-
eral blood T cell and B cell counts have no significant influ-
ence on IVF treatment outcome or pregnancy outcome.
In this study, we explored the relationship between the
IVF treatment outcome, pregnancy outcome and peripheral
blood CD56
þ
NK cell values. Our results revealed the
absence of any significant relationship between peripheral
blood CD56
þ
NK cell values (absolute count and percen-
tage) and IVF treatment outcome or pregnancy outcome.
Therefore, an increase in the peripheral blood CD56
þ
NK
cell percentage or absolute count may not be associated
with increased failed implantation or an increased rate of
miscarriage. This finding is in accord with previous studies
(Yamamoto et al., 1999; Michimata et al., 2002) in show-
ing that the peripheral blood NK cell percentage has no
association with miscarriages and no predictive value for
pregnancy outcome. Our finding is in contrast with that of
Coulam et al. (1995), where the authors suggested that the
peripheral blood NK cell percentage could predict preg-
nancy outcome. However, the flow cytometric method used
to identify NK cells in the Coulam study may not have
been accurate due to the lack of CD3 antigen assessment to
exclude CD56 expressing T cells. Consequently the lym-
phocytes examined were a mixture of T cells and NK cells;
therefore one could argue that their conclusion that the
peripheral blood NK cell percentage could predict preg-
nancy outcome may not be accurate. Moreover, the study
group used by Coulam et al. (1995) was not homogeneous
and included women receiving donor oocytes or intravenous
immunoglobulin G (IV IgG) treatment. In our study we
analysed both the absolute and percentage lymphocyte sub-
set counts with a particular focus on the absolute count. It
is well known that the latter is more accurate than the per-
centage, as the percentage is heavily influenced by the pre-
sence of the other lymphocyte subsets in a sample. Our
finding is also in contrast with that of Yamada et al.
(2003), where the authors reported that NK cell percentage
was elevated in women who suffered from recurrent mis-
carriage and had a miscarriage with a chromosomally nor-
mal pregnancy. However, the statistical analysis in
Yamada’s study included women with known endocrine or
autoimmune disorders.
We further evaluated the IVF outcome and pregnancy out-
come for patients with peripheral blood CD56
þ
NK cell per-
centages , 12% and $ 12%. This subdivision of the study
group with a 12% threshold for analysis was based on the
work by Beer et al. (1996). They reported that women with
peripheral blood CD56
þ
NK cell percentages . 12% had a
reduced pregnancy rate after IVF treatment and increased
miscarriage risk. This work has encouraged many prac-
titioners to offer immunomodulation therapy to patients,
including paternal lymphocyte immunization, i.v. IgG and
anti-tumour necrosis factor (TNF) therapy, based on the per-
centage of their peripheral blood NK cells. The results of our
study revealed no significant difference with regard to preg-
nancy or miscarriage rate in patients with peripheral blood
CD56
þ
NK cell percentages , 12% or $ 12%. Therefore,
our data suggest that the 12% threshold has no predictive
value in IVF treatment outcome and risk miscarriage. It is
important to consider some gaps in the work reported by
Beer et al. (1996). Thus it was unclear at what stage the
blood samples were obtained from the study subjects. This is
clearly important as the results of NK analysis can vary
according to the point in a treatment cycle at which the
sample was obtained. Furthermore, part of their analysis
compared the NK cell percentage in pregnant women with
non-pregnant women having a history of recurrent miscar-
riage. This comparison may also not be valid as lymphocyte
composition can vary during a pregnancy. There was also no
explanation of how the arbitrary threshold level of 12% was
selected. Our finding suggests that the peripheral blood NK
cell count and percentage have no bearing on IVF treatment
outcome, pregnancy outcome or risk of miscarriage. More
importantly the 12% arbitrary threshold has no predictive
value in determining IVF treatment outcome and risk of mis-
carriage. Finally, it is worth noting that the NK cell percen-
tage range in a healthy individual can be $ 20% (Cooper
et al., 2001). Therefore based on the findings of our study it
may be inappropriate to offer immunomodulation therapy for
this group of patients. Beer et al. (1996), however, reported
that no women with CD56
þ
NK cells . 18% had delivered a
live-born child. This is in contrast to the results of our study
in which 15 women with CD56
þ
NK cells . 18% completed
NK cell numbers and IVF outcome
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an IVF treatment cycle and six (40%) became pregnant.
Of these six women, five delivered and one woman
had a miscarriage. This gives a live birth rate of 30% and
a miscarriage rate of 20% for this group of women.
In conclusion, our data suggest that there is no significant
association between simple enumerations of peripheral blood
NK cells (including total CD56
þ
NK, CD56
dim
NK and
CD56
bright
NK cells), B cells and T cells with IVF treatment
outcome and pregnancy outcome. Women who have a per-
ipheral NK cell level . 12% do not have either a reduced
rate of achieving pregnancy or higher rate of pregnancy loss
after IVF treatment. Even with a markedly elevated periph-
eral blood CD56
þ
NK cell of . 18%, there was no associ-
ation with poorer IVF treatment outcome or pregnancy
outcome.
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... In contrast, the elevated NK counts were suggested as indicators of increased risk of reproductive failure in women. Undoubtedly, the predictability of prognosis using NK frequency as well as the role of elevated NK% in reproductive outcome are still up for debate [48,49]. Many investigators agree that measurement of the functional activity of NK cells is more important for clinical prognosis [1]. ...
... In contrast, the characterization of NK phenotype markers is much easier and possesses comparable clinical utility. It was shown, for example, that misbalance between KIR receptor activation and ligand subsets is associated with reproduction failures [48,50]. ...
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Natural killer (NK) frequency and NK cytotoxicity (NKc) are key determining factors of a clinical outcome. In our previous study, we showed the prognostic clinical significance of immune parameters when they are beyond the optimal range (accentuated). In this study, we attempted to explain the disparity of accentuated but physiologically and immunologically normal NK parameters that might serve as negative clinical prognostics indications of failed pregnancies. We have analyzed NK%, NKc levels, and their reciprocal correlation in 2,804 patients with reproductive failures. In the entire clinical population, NK% correlates with NKc. Interestingly, we found this relationship to be strongly dependent on NK level's status. NK%-NKc correlation was the strongest (r = 0.2021, p < 0.0001) in a patient group with high NK% (> 17.5%). Patients with NK% between 15-17.5% manifested lower but still significant correlation NK%-NKc (r = 0.1213, p = 0.0155). Additionally, significant correlation (r = 0.2689, p < < 0.0001) between NK% and NKc was observed in a group of patients with NK levels < 7% (1.7-7%). While patients' groups with NK% (7-15%) did not reveal NK%-NKc association. This led us to hypothesize that the qualitative-quantitative status of NK population is responsible for their cytotoxic activity. Consistent with our hypothesis, the "balanced zone" NK% is tightly controlled, and thus does not correlate directly with NKc. In contrast, the "accentuated zones" of NK% escape this control and directly affecting NKc. Demonstrated phenomena supports our idea about the clinical significance of immune accentuation and explains its novel physiological role.
... Research into local endometrial immunological variations as causative factors for 58 reproductive failure is widely considered to be controversial (Thum et al., 2005). 59 Natural killer cell assessment has been widely used by some physicians over the last 60 20 years despite a lack of conclusive evidence, and many treatments dispensed 61 based on these results (Sacks, However, quite dramatic increases in cell percentages were observed relative to 171 control tissue. ...
Article
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Purpose Can a comprehensive flow cytometry panel be used to assess immunophenotype profiles in menstrual blood of patients experiencing reproductive failure and age matched controls of proven fertility? Methods 58 recurrent pregnancy loss and repeated implantation failure patients, along with 15 age matched controls of proven fertility, had menstrual blood samples obtained within the first 24 hours of the onset of menstruation to non-invasively assess the local immunophenotype. Using a comprehensive multi-parameter flow panel the lymphocyte sub-populations were described and compared. Results Relative to well established peripheral blood immunophenotyping values, distinct lymphocyte population differences were noted between the subgroups. The ratios of CD4+ and CD8+ T-cells were inverted relative to peripheral blood and uterine NK cells represented by CD56bright were distinctly visualised, emphasising the distinction of menstrual and peripheral blood. Relative to controls there were marked increases in CD3+ve T-cells (p=0.009), CD4:CD8 ratio (p=0.004), CD19 B-cells (p=0.026) and CD56dim NK’s (p=0.002) in the reproductive failure cases. Conclusions Flow cytometric evaluation can provide a rapid and objective analysis of lymphocyte subpopulations in many forms of tissue and fluid. The findings show significant variations in cellular composition of immune cells indicating a distinct compartment, with differences between cases and controls. Immunological assessment of the menstrual blood immunophenotype, in clinically appropriate patients, may provide insight into the aetiology of adverse reproductive outcome, without the risks and inconveniences associated with a more invasive endometrial biopsy.
... Flow cytometry enumeration of NK lymphocytes is much simpler, and is used in many investigations. Unfortunately, the determination of the number of NK lymphocytes is not enough to evaluate their functional status [16,18] and seems to have limited clinical significance [32]. It is hoped that the identifying of NK phenotypes and NK subset structures may be more likely to assess NK function, but right now, the NK cytotoxicity test remains the golden standard [33]. ...
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Problem Many studies have demonstrated the negative impact of high rates of NK cytotoxicity (NKc) on reproductive processes, but there is no agreement as to the appropriateness and feasibility of using the NKc for routine diagnostic in IVF patients. This study conducted a retrospective analysis of embryo transfer (ET) success rates and live birth rates (LBR) in patients with different NKc values. Method of study 1854 cycles of ET were selected and divided into three groups according to NKc levels, and randomized by anamnesis and age: normal (nNKc, n = 871), elevated (eNKc, n = 759), and reduced NKc (rNKc, n = 123). ET with donors’ embryos (n = 101) were analyzed separately. NKc-to-K562 was measured in PBMC (peripheral blood mononuclear cells) by flow cytometry before ET. The patients did not obtain any additional treatments. Results Patients with eNKc, in addition to having reduced clinical pregnancy rates (OR1.59, p < 0.0001), had increased levels of subsequent pregnancy failures (OR2.545, p < 0.0001) when compared to nNKc patients. As a result, patients with eNKc had almost half the LBR than patients with nNKc (OR2.2, p < 0.0001). In patients with rNKc, LBR was also lowered. eNKc was equally unfavorable for implantation and delivery in cryo- or fresh cycles. Markedly, eNKc was much more unfavorable for reproduction than slightly elevated NKc. The donor’s embryos were implanted irrespective of the recipient’s NKc levels, but the later stages of pregnancy were worse in patients with eNKc. Conclusions Our findings highlighted the negative impact of high levels of NK cytotoxicity on pregnancy outcomes.
... Peripheral blood NK cells, on the other hand, are cytotoxic, and an increase in number and activity has been associated with miscarriages and implantation failure [81,82]. Previous studies have shown that pNK cells of pregnant women in the first trimester are reduced compared to non-pregnant women [83,84]. This was also observed by Kuhnert et al. (1998) and may be viewed as a logical adaption of the immune system to a healthy pregnancy [84,85]. ...
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Purpose of Review This review highlights the impact of TB mono-infection and TB-HIV co-infection on the pathogenesis of adverse maternal outcomes such as hypertensive disorders of pregnancy (HDP) and adverse fetal outcomes such as recurrent spontaneous abortion (RSA), fetal growth restriction (FGR), and low birth weight. Recent Findings Research has shown that HDP, such as severe pre-eclampsia (PE) and eclampsia, as well as adverse fetal outcomes such as recurrent spontaneous abortion, fetal growth restriction, and low birth weight, are higher in women diagnosed with TB mono-infection and even higher in TB-HIV co-infection compared to those without TB. This is speculated to occur due to exaggerated activation of both angiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO), angiotensin 2, (Ang 2), intracellular adhesion molecules (ICAMs), and inflammatory cytokines such as interleukin 2 (IL-2), (IL-17), and interferon-gamma (INF-γ). Summary There is a lack of information with regard to the pathogenesis of adverse maternal and fetal outcomes upon TB mono-infection and TB-HIV co-infection; therefore, further investigations on the impact of TB mono-infection and TB-HIV co-infection on adverse maternal and fetal outcomes are urgently needed. This will assist in improving diagnostic procedures in pregnant women affected with TB as wells as TB-HIV co-infection.
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Objective: To report the prevalence of pNK≥12% and its relationship with RPL in a population of Mexican women. Methods: Retrospective, cross sectional and observational study which included 98 women with history of RPL, who were subjected to a blood test sample to measure pNK cell and prevent complications in further pregnancies. Two groups were formed: 1) CONTROL and 2) RPL: Women with history of 2 or more miscarriages. Results: Women in the RPL group were older and had higher BMI compared to the Control group. Prevalence of patient whit pNK≥12% increase in the RPL vs Control. RPL group showed a significant increase of pNK≥1% compared to the Control (11.3±0.5 vs 9.5±0.6%, p=0.002). Finally, the median level of pNK≥12% in the PRL group was higher than Control (15.1±0.5 vs 13.5±0.8%). Conclusion: RPL may be the result of increased pNK concentrations and as observed in this study, slightly more than 50% of the Mexican population could be susceptible to abortions.
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Fetal implantation requires carefully orchestrated involvement of the maternal immune system. Aberrant function within implantation has been suggested as a cause of implantation failure. The emergence of immunological theories of miscarriage has led to immunological testing as an adjuvant treatment in assisted reproductive technology; however, it remains controversial, with mixed evidence both for immunological cause and the benefits of immunological testing. Literature on common methods of immunological testing within assisted reproductive technology is reviewed including those of peripheral and uterine natural killer cells, chronic endometritis, and T-helper cells cytokine ratio. There is little consensus in the evidence on immunological testing in the context of recurrent implantation failure. The field is limited by a lack of uniformity in approach to testing and heterogeneity of the pathophysiological cause. Nevertheless, the maternal immune system is heavily involved in implantation and the new era of personalized medicine ensures that a more defined approach to immunological testing will be achieved.
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Problem Unexplained recurrent miscarriage (uRM) is defined as two or more spontaneous abortions prior to 20 weeks of gestation with unknown etiology. Peripheral blood natural killer (pNK) cells contact with the villus and exert important role in normal pregnancy. However, it is still controversial about the association between pNK cytotoxicity and uRM, and the underlying mechanism remains unknown so far. Method of Study In this study, we aim to compare the percentage, immunophenotype and function of pNK cells between patients with uRM and fertile controls. The peripheral blood was collected from 49 patients with uRM and 11 fertile women in their middle luteal phase of the menstrual cycle. pNK cells were co‐cultured with K562 cells at different cell ratios to measure the cytotoxicity. The percentage of CD3‐CD56⁺, CD3‐CD56bright, CD3‐CD56dim pNK was analyzed by flow cytometry and quantified to evaluate the expression of cytotoxic granules (granzyme B, granulysin, and perforin), and the cell surface receptors related to pNK cell cytotoxicity (NKG2D, NKp30, NKp46, CD158a, CD158b) were also detected. Results The general linear model analysis showed that pNK cell cytotoxicity in patients with uRM was significantly lower than that in fertile controls. In addition, the ratios of NKG2D/CD158a, NKp30/CD158a, and NKp46/CD158a in CD3‐CD56bright pNK subsets were significantly lower in uRM group than that in fertile control. The logistical regression analysis showed that the reduced NKp30/CD158a, NKp46/CD158a ratios in CD3‐CD56bright pNK subsets were significantly associated with uRM. Conclusion Our results suggested that a low pNK cytotoxicity, which is mediated by inhibitory signals, might be associated with uRM.
Article
Problem To evaluate pregnancy‐compatible phenotypic and functional changes in peripheral blood natural killer (pNK) cells during frozen embryo transfer (FET) cycles. Method of Study Peripheral blood was collected from patients undergoing frozen embryo transfer cycles at three separate time points in the cycle. pNK cell phenotype was analyzed by flow cytometry. Impact of pregnancy status on pNK cell cytotoxicity was characterized by two methods: 1) a three‐dimensional endovascular tube formation approach and 2) a NK cell‐specific K562 cell kill assay. Results A total of 35 patients were enrolled, 15 with clinical pregnancies and 20 with negative serum β‐hCG levels. Overall percentage of CD45⁺CD3‐CD56⁺ pNK cell did not change during the FET cycle. Pregnancy resulted in an increase in CD45⁺CD3‐CD56⁺ pNK cell population on the day of serum β‐hCG. pNK cells from non‐pregnant patients caused significant tube disruption when compared to pregnant patients. Addition of serum from pregnant women reduced the tube disruption by pNK cells from non‐pregnant patients. pNK cells from pregnant patients showed significantly lower cytotoxicity toward K562 cells in serum‐free conditions. The addition of pregnancy serum decreased non‐pregnant pNK cells cytotoxicity. Pregnancy status had no impact on VEGF‐A and VEGF‐C serum levels. Recombinant hCG added to non‐pregnant serum resulted in a significant reduction in non‐pregnant pNK cell‐mediated K562 cell kill. Conclusion There was no difference in pNK cell populations based on timing of the FET cycle. However, pregnancy increased the percentage of CD45⁺CD3‐CD56⁺ pNK cells. Additionally, pNK cells from pregnant women have reduced cytotoxicity and this is possibly mediated by hCG.
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Natural killer (NK) cell assessment has been attempted since the 1990s and, apart from antibody testing, is probably the commonest immune test available to clinicians. It is clear that simple enumeration of uterine NK cells by immunohistochemistry is inadequate, although better methodology such as flow cytometry may prove to be more beneficial in the future. Blood testing is an appealing noninvasive test that may be a marker for immune dysfunction, rather than a guide to uterine numbers per se. It is currently performed in women with repeated reproductive failure and should be done using tests of activation. Patients value investigation and clinicians should prefer it to blind empirical immune therapy in repeated reproductive failure cases. But, in addition to blood NK testing, a fundamental new NK genetic test (the KIR/HLA‐C interaction) is likely to provide an even more effective diagnostic tool. Insights from KIR/HLA‐C analysis imply that new immune therapy trials will need to take KIR/HLA‐C results into account.
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BACKGROUND: To evaluate the effect of egg‐sharing and the outcome of assisted reproductive treatment (ART) in standard IVF/ICSI patients, egg‐sharing donors and egg‐sharing recipients. METHODS: Descriptive cohort study to evaluate 276 egg‐sharing cycles involving 192 egg‐sharers, 274 recipient cycles receiving eggs from egg‐sharers and 1098 non‐egg‐sharing standard IVF/ICSI cycles from January 1998 to December 2002. Patients were divided into three groups: group A, egg‐sharers; group B, non‐egg‐sharers, age <36 years, FSH <10 IU/l, BMI <30 kg/m2; and group C, egg‐sharing recipients, all ages. Duration and amount of gonadotrophin required to achieve follicular maturity, number of eggs collected and donated, fertilization rate, pregnancy rates and live birth rates were compared between donor, recipient and standard IVF/ICSI patients. The χ2 test was used to test for statistical significance (P < 0.05). RESULTS: There was no significant difference in pregnancy rate and live birth rate of egg‐sharers, group A (42.0%, 33.0%), non‐egg‐sharers, group B (40.0%, 30.9%) and recipients, group C (41.4%, 28.6%). The number of oocytes collected, number of mature follicles and amount of gonadotrophin used was not significantly different between the two groups (A and B). The average number of embryos transferred and the mean number of eggs allocated between egg‐sharers and recipients was not statistically different. CONCLUSION: Egg‐sharing does not compromise the chance of achieving a pregnancy or live birth for the egg‐sharer or the recipient as compared to standard IVF/ICSI patients. The egg‐sharers were not at a higher potential risk of ovarian stimulation syndrome and there was no imbalance of egg allocation.
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T lymphocyte subpopulations were compared in normal first trimester human decidua and in decidua associated with spontaneous abortion. Cryostat sections were labelled using a panel of monoclonal antibodies specific for CD3, CD8, CD4 and for the alphabeta and gammadelta heterodimers of the T cell receptor using an avidin-biotin complex peroxidase method. All the endometrial T cell subsets which have been demonstrated in normal early pregnancy were detected in similar numbers and proportions in spontaneous abortion. The findings suggest that adverse pregnancy outcome is not influenced by altered proportions of T cell subpopulations; nevertheless, the possibility remains that these cells may have an altered antigenic phenotype in spontaneous abortion which could contribute to pregnancy success or failure.
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The purpose of this study was to determine whether reproductive antigens stimulate lymphocytes and macrophages from women with recurrent abortion to secrete factors that are toxic to preimplantation embryos or trophoblast cells in vitro. Mononuclear cells were isolated from 30 fertile controls and 300 nonpregnant women being evaluated for recurrent abortion. Supernatants generated from these cells after separate culture with sperm and trophoblast antigen extracts were added to two-cell mouse embryo cultures and trophoblast proliferation assays. Toxicity was assumed when the median percentage of embryos developing to blastocysts or trophoblast proliferation was less than or equal to 50% of control values. Both parametric and nonparametric statistical methods were used where appropriate. Mouse embryo development and/or trophoblast proliferation were significantly inhibited by supernatants from trophoblast and/or sperm antigen-activated peripheral blood leukocyte cultures from a majority of 300 women with recurrent abortion but not from 30 women with normal reproductive histories. The mouse blastocyst development assay was more sensitive than the trophoblast proliferation assay in determining toxic factor production. Embryo-toxic factors were produced by activated leukocyte cultures from 90% of 180 women with a history of recurrent abortion of unexplained etiology, whereas trophoblast-inhibitory factors were detected in 50% of women from the same group. The embryo-toxic factor(s) was heat labile, had a molecular weight(s) between 10 and 30 kd, and was absorbed out by passage through affinity columns containing anti-interferon gamma beads. We conclude that recurrent abortion in some women is associated with embryo- and/or trophoblast-toxic factor production in response to stimulation by sperm or trophoblast antigens and that the principal factor may involve the 18 kd, heat-labile, T-lymphocyte cytokine interferon gamma. This study suggests a new cause of recurrent abortion.
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The distribution of leukocytes in first trimester human decidual tissue has been studied by using a panel of monoclonal antibodies in an indirect immunoperoxidase technique on acetone-fixed cryostat sections. The results indicate that bone marrow-derived cells are abundant in the placental bed and a proportion of these are HLA-DR positive. However, a major leukocyte population in the decidua of early pregnancy is of cells which carry the E-rosette receptor but which do not express peripheral pan-T cell antigens nor HLA-DR. The distribution of these cells suggests that they are endometrial granulocytes. A similar large number of cells express OKT 10, a marker of immature or activated cells. The presence of this unusual T lineage cell raises the possibility that a form of lymphocyte processing is occurring in the decidua in early pregnancy, perhaps in response to foetal antigens presented on trophoblast.
Article
To evaluate differences in circulating CD56+ cells between successful and unsuccessful pregnancies, 114 pregnant women were studied prospectively. Seventy women had a history of infertility (INF) and 44 had two or more previous spontaneous abortions (RSA). Among the infertile women, 12 were donor egg recipients (DER) and 15 underwent intracytoplasmic sperm injection (ICSI) for treatment of male factor infertility. Nineteen women were carrying multiple gestations (MG) and 55 had singleton gestations (SG). Thirteen additional women were receiving intravenous immunoglobulin (IVIg). The percentage of CD56+ cells was determined in 310 blood samples from 114 pregnant women by flow cytometry. The prevalence of women with persistent elevation of percent of 56+ cells (> 12%) was 58% among DER, 73% among ICSI, 37% among MG, 22% among SG, 18% among RSA, and 39% among INF. Thirteen women with SG received IVIG, 10 had CD56+ cells greater than 12% and all 13 experienced live births. Women with percentage CD56+ cells persistently greater than 12% who were not DER, not ICSI, not receiving IVIg, and not carrying MG had a live birth rate of 11%. Women with greater than 12% CD56+ cells had normal karyotype in 78% of concepti studied in contrast to women less than 12% CD56+ cells who had 68% abnormal karyotypes (P = 0.04). Elevated CD56+ cells in pregnant women who are not DER, not ICSI, not receiving IVIg, and not carrying MG predicts loss of a karyotypically normal conceptus with a specificity of 87% and positive predictive value of 78%. While the specificity value of this test is high in both infertile and RSA populations, the sensitivity is 86% in RSA and only 54% in INF suggesting this test does not identify all losses among INF. It may identify a subset of pregnancies at risk for loss of a karyotypically normal embryo that may respond to treatment with IVIg.
Article
From the clinical point of view, it has been proposed that an immunological imbalance between the mother and the fetus might exist in one of the mechanisms for human habitual abortion. However, in the definition of habitual abortion, we have no distinct immunological criteria for this clinical entity at the moment. We employed aborted placental cells as the target cells in an in vitro terminal labeling (IVTL) assay system, in which the cytotoxic activity of maternal peripheral blood mononuclear cells (PBMCs) against the placental cell was examined. Our results showed that the cytotoxic activity of maternal PBMCs was significantly higher in the habitual aborters (the mean target cell destruction: %TCD = 34.9%, N = 14) than that in the women with a normal first trimester (the mean % TCD = 8.9%, N = 14, P < .01). The results from the IVTL assay did not correlate with other assays using paternal lymphocytes as the target cells. The surface marker analyses revealed that CD16+ cells, CD14+ cells, and CD5+ cells were involved in the cytotoxic response against the placental cells in various degrees among the cases. The above evidence suggests that a variety of cytotoxic cells participate in the phenomenon of human habitual abortion.
Article
The purpose of this study was to determine whether trophoblast antigens activate cell-mediated immune responses in women with recurrent abortion of unknown cause by inducing lymphocyte proliferation and to correlate this with embryotoxic factor production. Mononuclear cells were isolated from 57 nonpregnant women with recurrent abortion of unknown cause and from 10 fertile controls with normal reproductive histories. Lymphocyte proliferation assays were performed with trophoblast antigen extracts from the human choriocarcinoma cell lines Jeg-3 and JAR, fractionated trophoblast antigens, and for control, red blood cell membrane antigens. A stimulation index > 3 was considered significant stimulation. Trophoblast-stimulated culture supernatants were also tested for embryotoxic activity. Thirty (52.6%) women with recurrent abortion of unknown cause responded to trophoblast antigen stimulation with a stimulation index > 3, whereas none of the control group responded. Neither patients with recurrent abortion nor women with normal reproductive histories responded to red blood cell membrane antigen. The mean stimulation index in the trophoblast antigen lymphocyte proliferation assay was significantly higher in women with recurrent abortion than in women with normal reproductive histories (3.99 +/- 2.82 vs 1.64 +/- 0.34, p < 0.01). Antigen extracts from the cytosol and membrane fractions of Jeg-3 provided the strongest stimulation. Lymphocyte proliferation correlated with embryotoxic factor production; 90.0% of women with a stimulation index > 3 and none of the controls produced embryotoxic factors after stimulation with trophoblast antigens. In this study 52.6% of women with recurrent abortion of unknown cause compared with none in controls exhibited in vitro evidence of T-cell immunity to trophoblast antigen stimulation. Further work is needed to determine whether T-cell immunity to trophoblast is involved in the pathogenesis of recurrent spontaneous abortion.