Journal of Reproductive Immunology 140 (2020) 103130

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Journal of Reproductive Immunology

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Concordance between peripheral and decidual NK cell subsets and killer T

immunoglobulin-like receptors in women with recurrent spontaneous
miscarriages
Omnia El-Badawya,*, Amany S. Helmyb, Ahmed M. Abbasc, Asmaa M. Zahrand, Noha A. Afifia,
Mona H. Abdel-Rahima
a
Medical Microbiology & Immunology Department, Faculty of Medicine, Assiut University, Assiut, Egypt
b
Blood Bank, Ministry of Health, Egypt
c
Obstetrics and Gynecology Department, Faculty of Medicine, Assiut University, Assiut, Egypt
d
Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Assiut, Egypt

ARTICLE INFO ABSTRACT

Keywords: Background: The role of decidual natural killer (dNK) cells in normal and complicated pregnancy and their
RSM relation with peripheral NK (pNK) cells remains unclear. The study aim was phenotypic analysis of pNK and dNK
Miscarriage cells at time of miscarriage in recurrent spontaneous miscarriage (RSM) patients to assess whether measuring
NK levels of pNK cell populations can reflect changes in dNK cells or not.
KIR
Methods: This study included 40 middle aged pregnant women in the 1st trimester subjected to evacuation
decidua
because of a current miscarriage. They had a history of previous ≥ two unexplained miscarriages. Frequencies of
pNK and dNK cells, based on the expression of CD56, CD16, inhibitory (CD158b) and activating (CD161) Killer
immunoglobulin-like receptors (KIRs), were detected by flow cytometry.
Results: Percentages of CD56+ NK cells in peripheral blood and decidua were 17.5 % and 17.3 %, respectively.
In both blood and decidua, CD56dim NK cells were exceeding CD56bright NK cells. The CD56dim CD16− NK cells
were the predominating subset of NK cells, followed by CD56dim CD16dim. No substantial differences were de-
tected in the levels of KIRs expression by the different NK subsets between blood and decidua. Abnormal up-
regulation of both CD161 and CD158b on NK cells was observed in blood and decidua.
Conclusion: At the time of miscarriage, patients with RSM have an extremely active immune system and an
increased number of toxic NK cells both in blood and decidua. The pNK cells reflect dNK cell changes during
miscarriage and may be a useful non-invasive predicting tool in reproductive failure setting.

1. Introduction as an immunological cause of miscarriage based on the incompatibility

One of the amazing things is how the maternal immune system can
simultaneously balance between the immune tolerance against growing
semi-allogeneic fetus and the immune activation against harmful pa-
thogens (Vassiliadou and Bulmer, 1996). The immunological etiology of
recurrent spontaneous miscarriage (RSM) is growingly accepted
(Cooper et al., 2001a; Lash et al., 2010).
Natural killer (NK) cells play an essential role in the maintenance of
healthy pregnancy. During implantation and early gestation, NK cells
are the most predominant immune cells at the implantation site reg-
ulating endometrial differentiation and decidualization (Hosseini et al.,
2014). They also prevent trophoblast over-invasion (Chaouat, 2016).
Experimental and clinical researches have explored the role of NK cells
between the mother and her fetus (Campbell et al., 2001).
Two different subsets are recognized among the peripheral NK
(pNK) cells, the predominant subset (95 %) is NK cell with a dim ex-
pression of CD56, and the remaining 5 % is with bright CD56 expres-
sion (Cooper et al., 2001b). CD56dim pNK cells expressing high levels of
CD16 and Killer immunoglobulin-like receptors (KIRs) are granular and
cytotoxic. In contrast, CD56bright pNK cells are abundant cytokine
producers, with much less lytic granules, CD16, and KIRs (King et al.,
1991), thus, signifying an immunomodulatory role for the CD56bright
pNK subset and a cytotoxic role for the CD56dim pNK cells (King et al.,
1996). Meanwhile, 80 % of the decidual NK (dNK) cells are CD56bright
and CD16− (Verma et al., 1997) with low cytotoxicity resembling the
CD56bright CD16− pNK cells (Deniz et al., 1994). However, they are

⁎
Corresponding author at: Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, 71515, Egypt.
E-mail address: omniaalbadawy@aun.edu.eg (O. El-Badawy).

https://doi.org/10.1016/j.jri.2020.103130
Received 10 January 2020; Received in revised form 2 March 2020; Accepted 4 April 2020
0165-0378/ © 2020 Elsevier B.V. All rights reserved.

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O. El-Badawy, et al.
granulated and express KIRs like CD56dim pNK cells (Lachapelle et al.,
1996).
Providing there is no efficient down-regulation of the cytotoxic NK
immune activity, successful uncomplicated pregnancy is not possible.
The fine-tuning between the inhibitory and activating KIRs on dNK cells
is therefore crucial for the maintenance of pregnancy (Kurioka et al.,
2018). For a normal pregnancy, the inhibitory KIRs activity should
predominate with subsequent suppression of dNK cytotoxicity against
fetal allogenic antigens (Gonen-Gross et al., 2010). Decreased expres-
sion of inhibitory KIRs might cause excess cytotoxicity to the invading
trophoblast cells and growing fetus and thus may explain the mis-
carriage (Quenby et al., 2005). CD56bright NK cells producing IL-10
might favor the balance of the immune system by regulating cytotoxic
and inhibitory immune responses (Zhu et al., 2017).
Assessment of dNK cells during an ongoing pregnancy is inapplic-
able, as it requires an invasive procedure posing surgical and infectious
risks. Therefore, the role of dNK cells in normal and complicated
pregnancy, as well as their relation with pNK cells, remains unclear
(Park et al., 2010; Moffett and Colucci, 2014). Further characterization
of phenotypic and functional differences between dNK and pNK cells is
needed.
The study aim was phenotypic analysis of peripheral and decidual
NK cells and their expression of inhibitory (CD158b) and activating
(CD161) KIRs at the time of miscarriage in RSM patients, trying to
answer the question whether we can rely on measuring the level of pNK
cell populations to reflect changes in dNK cells or not.
2. Patients and methods
This was a cross-sectional study including 40 middle-aged pregnant
women in the first trimester presenting with vaginal bleeding to a
tertiary University hospital. They had a history of previous two or more
unexplained miscarriages (Anon, 2015) (more than 8 wks. and less than
20 wks. of gestation). They were subjected to evacuation and curettage
of uterine content of conception because of a currently missed mis-
carriage (diagnosed by ultrasound). All included patients had regular
marital life with the same partner, regular menstruation before current
pregnancy, and spontaneous conception. Patients with any detected
cause for recurrent miscarriage (anatomical, infections, autoantibodies,
endocrine abnormalities, or chromosomal abnormalities) were ex-
cluded.
The Institutional Ethical Committee reviewed and accepted the
study (IRB No. 17,100,791). Informed consent was taken from all
participants.
2.1. Sample preparation
Decidua specimens (about 1−7 grams) were collected by uterine
curettage, washed in phosphate buffered saline (PBS), and then pre-
served in Rosewell Park Memorial Institute (RPMI) 1640 medium
supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin and
5 % fetal bovine serum (GIBCO BRL, Thermo Fisher Scientific, USA)
until transfer to the laboratory. At the flow cytometry laboratory, the
tissue samples were minced finely and enzymatically digested by
adding 3 mL collagenase enzyme type Ia and 2 mL PBS for 40 min at 37
°C in shaking water path at 100 r.p.m. Cell suspensions obtained were
filtered, then the pellet after centrifugation was washed with PBS.
Afterward, a lysing solution was added (Neller et al., 2014). Peripheral
blood samples were also treated with red blood cells lysing solution.
Samples were rewashed with PBS.
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Journal of Reproductive Immunology 140 (2020) 103130
2.2. Flow cytometric detection of CD56, CD16, CD158b, and CD161 on
peripheral and decidual NK cells
One hundred μl of the treated whole blood or decidua samples and
10 μL of fluoroisothiocyanate (FITC)-conjugated Anti-CD3,
Allophycocyanin (APC) conjugated Anti-CD56, and Peridinin-chlor-
ophyll Protein Complex (PerCP) conjugated Anti-CD16 were incubated
with Phycoerythrin (PE) conjugated Anti-CD161 in one tube and with
PE-conjugated Anti-CD158b in another tube (All monoclonal antibodies
were purchased from R&D Systems, Minneapolis, MN, USA). After in-
cubation for 20 min, washing with PBS and analysis by FACS Calibur
flow cytometry with CellQuest software (BD Biosciences, USA) were
done. Appropriate isotype negative control was processed similarly and
used with each sample. About 50,000 cells were acquired. Forward/
side scatter dot plot was used to define the lymphocyte population. CD3
expression was assessed on lymphocytes and then CD3− lymphocytes
were gated for further expression of CD56 to identify the CD56+ NK
cells. Further analysis was done to assess the NK cell subsets based on
the differential expression of CD56 and CD16. The percentages of NK
subsets (CD56dimCD16bright, CD56dimCD16dim, CD56dimCD16−,
CD56brightCD16bright, CD56brightCD16dim and CD56brightCD16−) were
assessed within the CD56+ cells. Then the levels of the expression of the
activating (CD161) and inhibitory (CD158b) receptors were measured
after gating on each NK cell subset as shown in Fig. 1.
2.3. Statistical analysis of the data
Statistical package for social sciences (SPSS), version 24, was used
for data analysis. Data were expressed as mean ± standard error (SE) or
standard deviation (SD). The paired t-test was used for calculation of
differences in mean values between blood and decidua. One-way
ANOVA was employed for comparison between NK subsets. P-value was
considered significant when less than 0.05. Pearson correlation was
applied to assess the relations between different variables.
3. Results
3.1. The demographic data of patients
The current study included 40 patients. Their demographic data are
shown in (Table 1). The mean age of the study participants was
28 ± 3.1 years and the mean body weight was 72 ± 7.2 kg. The ge-
stational age recorded in their current miscarriage ranged from 7 to 12
weeks with a mean age of 8 ± 2 weeks. History of early miscarriage was
found in 26 (65 %) patients, and previous late miscarriage was also
detected in 26 (65 %) patients. Also, four (10 %) patients had previous
preterm labor.
3.2. Analysis of the proportions of NK subsets in peripheral blood and
decidua
The total percentages of NK (CD56+) cells from the total lympho-
cytes population in both peripheral blood and decidua of women with
RSM at the time of miscarriage were 17.5 % and 17.3 %, respectively.
In (Table 2), comparison of the proportions of different NK cell subsets
(from CD56+ NK cells) between blood and decidua has shown that
CD56dim NK cells were significantly higher in the peripheral blood than
in the decidua (91.6 % ± 0.9 vs. 85.7 % ± 2, p= 0.003), whereas
CD56bright NK cells were significantly higher in the decidua (9.7 % ± 2
vs. 5.1 % ± 1, p= 0.01), especially those not expressing CD16 (5.8
% ± 1 vs. 1.9 % ± 0.3, p= 0.01). Still, no significant differences were
O. El-Badawy, et al. Journal of Reproductive Immunology 140 (2020) 103130

Fig. 1. (A) A representative forward scatter (FSC)/side scatter (SSC) dot plot showing R1 used to select lymphocytes. (B) A representative dot plot showing CD3\SSC
dot plot in which the region R2 was drawn to select CD3− lymphocytes (C) A representative dot plot showing CD56\SSC dot plot where CD56+ NK cells were
selected by drawing the region R3 (D) A representative dot plot showing NK cell subsets based on the differential expression of CD56 and CD16. Each subset was
further analyzed for activating (CD161) and inhibitory (CD158) KIRs expression levels using the following regions: R4 represents CD56dim CD16bright, R5 represents
CD56dim CD16dim, R6 represents CD56dim CD16-, R7 represents CD56bright CD16bright, R8 represents CD56bright CD16dim, and R9 represents CD56bright CD16-. (E) A
representative dot plot showing the expression of CD161 by one of the previous NK subsets and (F) represents the expression of CD158 by the same NK subset.

observed among the three CD56dim NK cell subsets and the two
CD56brightCD16+ subsets between blood and decidua.
In both blood and decidua, the CD56dim NK cells were significantly
exceeding those with CD56bright expression (p < 0.0001). The
CD56dimCD16− NK cells were the predominating subset of NK cells
(blood: 64.2 % ± 3 and decidua: 61.4 % ± 2) followed by
CD56dimCD16dim (blood: 27.4 % ± 3 and decidua: 25.8 % ± 2) and
afterwards, was the CD 56brightCD16- NK subset in decidua (5.8 % ± 1)
and CD56dimCD16bright NK cells in blood (2.4 % ± 0.4).

Table 1
3.3. Differential expression of killer immunoglobulin-like receptors (KIR)
Demographic data of patients.
CD161 and CD158b among the different NK subsets in blood and decidua
Parameter Patients
Analysis of the differential expression of CD161 (activating) KIR and
Age (years)* 28 ± 3.1
Body weight (kg)* 72 ± 7.2 CD158b (inhibitory) KIR by the different NK subsets is shown in
BMI (Kg/m2)* 27 ± 3.2
Underweight (< 18.5) 0
Normal weight (18.5- < 25) 9 (22.5 %) Table 2
Overweight (25- < 30) 22 (55 %) Comparisons of the proportions of NK subsets between blood and decidua.
Obese (≥30) 9 (22.5 %)
Gestational age (weeks)* 8±2 NK subsets (%) Blood Decidua p-value
Previous early miscarriage 26 (65 %) (n = 40) (n = 40)
Previous late miscarriage 26 (65 %)
Preterm labor 4 (10 %) CD56+ NK cells 17.5 ± 2 17.3 ± 2 0.6
CD56dim 91.6 ± 0.9 85.7 ± 2 0.003
BMI body mass index, definitions were in accordance with the CDC CD56dimCD16− 64.2 ± 3 61.4 ± 2 0.1
CD56dimCD16dim 27.4 ± 3 25.8 ± 2 0.5
(Centers for Disease Control and Prevention, 2017).
CD 56dimCD16bright 2.4 ± 0.4 1.7 ± 0.4 0.2
Early miscarriage: a nonviable, intrauterine pregnancy with either an
CD56 bright 5.1 ± 1 9.7 ± 2 0.01
empty gestational sac or a gestational sac containing an embryo or CD 56brightCD16− 1.9 ± 0.3 5.8 ± 1 0.01
fetus without fetal heart activity within the first 12 6/7 weeks of ge- CD 56brightCD16dim 2 ± 0.3 2 ± 0.2 0.99
station (National Institute for Health and Clinical Excellence, 2012). CD56brightCD16 bright 1.1 ± 0.2 0.6 ± 0.2 0.06
Late miscarriage: a miscarriage that happens when a baby dies be-
tween 13 and 26 weeks of gestation (as calculated from the last (%) The percentage of CD56+ NK cells were calculated from the total lym-
menstrual period) (American College of Obstetricians and phocytes and the percentages of CD56+ NK subsets were calculated from the
Gynecologists, 2013). CD56+ NK cells.
Preterm labor: birth between 20 0/7 weeks of gestation and 36 6/7 Results expressed as mean ± SE.
weeks of gestation (Simhan, 2016). Paired t-test, significant p-value < 0.05.
Results expressed as number (percentage), * expressed as mean ± SD.

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Table 3
Differential expression of activating (CD161) and inhibitory (CD158b) KIR by different NK cell subsets in blood and decidua.
KIR Sample CD56dim CD56dim CD16dim CD56dim CD56brightCD16− CD56brightCD16dim CD56brightCD16bright p- value1
CD16− CD16bright

CD161+ Blood 62.9 ± 2.8 89.9 ± 1.2 99.2 ± 0.1 78.6 ± 5.1 96.6 ± 1.4 98.2 ± 0.2 < 0.0001
Decidua 63.1 ± 3.3 87.4 ± 1.3 99 ± 0.3 68.3 ± 8.4 97.4 ± 1 97.7 ± 0.5 < 0.0001
p- value2 0.9 0.1 0.6 0.3 0.2 0.3 –
CD158b+ Blood 58.9 ± 3.2 87.8 ± 0.9 98.7 ± 0.2 79.2 ± 5.1 95.9 ± 1.5 97.2 ± 0.4 < 0.0001
Decidua 56.3 ± 3.2 84.6 ± 1.5 97.2 ± 1.5 65.2 ± 8.7 96.5 ± 1.2 95.9 ± 0.8 < 0.0001
p- value3 0.5 0.07 0.3 0.2 0.2 0.1 –

Significant p-value < 0.05.

p- value1 comparing the levels of expression of CD161 or CD158b among different natural killer cells (NK) subsets in blood and decidua (ANOVA).
p- value2 comparing the cells expressing CD161 in each NK subset between blood and decidua (Paired t-test).
p- value3 comparing the cells expressing CD158b in each NK subset between blood and decidua (Paired t-test).

(Table 3). No substantial differences were detected in the levels of KIR
expression by the different NK subsets between blood and decidua
samples. In both blood and decidua samples, the CD16− NK cells were
the least subsets expressing CD158b or CD161 (p < 0.0001) followed by
CD56brightCD16- and CD56dimCD16dim NK cells.
3.4. Correlations among different NK cell subsets and KIR expression and
other parameters
3.4.1. Correlations between different NK cell subsets in blood and decidua
Significant correlations observed between different NK subsets in
the peripheral blood and decidua were illustrated in Fig. 2. The

Fig. 2. Correlations among NK cell subsets in blood and decidua: (A) Between CD56dim NK cells in blood and CD56bright NK cells in decidua (B) Between CD56dim NK
cells in blood and CD56dim NK cells in decidua (C) Between CD56dimCD16dim NK cells and CD56dim CD16− in blood. (D) Between CD56dimCD16dim NK cells and
CD56dimCD16- NK cells in decidua (E) Between CD56dimCD16bright and CD56dimCD16- NK cells in blood (F) Between CD56dimCD16bright and CD56dimCD16- NK cells
in decidua (G) Between CD56dimCD16dim NK cells in blood and decidua.

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O. El-Badawy, et al.
Table 4
Correlations among different NK cell subsets expressing activating (CD161) and inhibitory (CD158b) KIR in blood and decidua.
NK subsets
dim − + dim − +
CD56 CD16 CD161 CD56 CD16 CD158b
CD56dimCD16dimCD161+ CD56dimCD16dimCD158b+
CD56dimCD16brightCD161+ CD 56dimCD16brightCD158b+
CD56brightCD16−CD161+ CD 56brightCD16−CD158b+
CD56brightCD16dimCD161+ CD 56brightCD16dimCD158b+
CD56brightCD16brightCD161+ CD56brightCD16brightCD158b+
r correlation coefficient, Pearson correlation, Significant p-value < 0.05.
CD56dim NK cells in blood showed a positive correlation with the
CD56dim NK cells in the decidua (r = 0.5, p < 0.0001) and negative
correlation with CD56bright NK cells in the decidua (r=-0.5,
p < 0.0001). Also, a direct relation was detected between the levels of
CD56dimCD16dim NK cells in blood and the same subset in the decidua
(r = 0.6, p < 0.0001).
The increase in the levels of CD56dimCD16− NK cells was inversely
associated with the levels of CD56dim16dim NK cells and CD56dim16bright
NK cells in blood (r=-0.8, p < 0.0001 and r=-0.7, p < 0.0001, re-
spectively) and in decidua (r=-0.5, p < 0.0001 and r=-0.4, p = 0.008,
respectively).
3.4.2. Correlations among different NK cell subsets expressing activating
(CD161) and inhibitory (CD158b) KIR in blood and decidua
In both blood and decidua (Table 4), very strong direct correlations
were found between the NK cells expressing activating (CD161) and
cells expressing inhibitory (CD158b) KIR in the same subset.
CD56brightCD16bright was the only subset showing a moderate correla-
tion between NK cells expressing CD161 and cells expressing CD158b.
3.4.3. Relations between the blood and decidua levels of the different NK
subsets and the number of previous early and late miscarriages and preterm
deliveries
Among the different NK subsets, only CD56dimCD16− NK cells in
both blood and decidua have shown positive correlations with the
number of previous early miscarriages (r = 0.4, p=0.01 and r = 0.5,
p=0.001, respectively) and negative correlations with number of pre-
vious late miscarriages (r= -0.4, p=0.003 and r= -0.3, p=0.04, re-
spectively). On the contrary, CD56dimCD16dim NK cells in both blood
and decidua have shown positive correlations with the number of
previous late miscarriages (r = 0.4, p=0.02 and r = 0.6, p < 0.0001,
respectively) and negative correlations with number of previous early
miscarriages (r= -0.3, p=0.04 and r= -0.5, p < 0.0001, respectively).
Furthermore, a positive correlation was observed between the decidua
levels of CD56dimCD16bright NK cells and the number of previous pre-
term deliveries (r = 0.5, p=0.001).
3.4.4. Relations between the blood and decidua levels of the different NK
subsets and the body weight and the body mass index (BMI)
The decidua levels of CD56dimCD16dim NK cells have shown positive
correlations with both the body weight and BMI (r = 0.3, p=0.04 and r
= 0.3, p=0.02, respectively). Meanwhile, the decidua levels of
CD56brightCD16− NK cells had a negative correlation with the BMI (r=
-0.3, p=0.04).
4. Discussion
In this study, we aimed to appraise whether the levels of pNK cells
and their subtypes are related to levels of dNK cells at implantation site
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Journal of Reproductive Immunology 140 (2020) 103130
Blood Decidua
r = 0.9 r = 0.9
p < 0.0001 p < 0.0001
r = 0.8 r = 0.95
p < 0.0001 p < 0.0001
r = 0.9 r = 0.8
p < 0.0001 p < 0.0001
r = 0.9 r = 0.995
p < 0.0001 p < 0.0001
r = 0.99 r = 0.9
p < 0.0001 p < 0.0001
r = 0.5 r = 0.6
p = 0.005 p = 0.001
or not. Parallel studies of pNK and dNK cells have been difficult because
of the complexities in accessing dNK cells during an ongoing human
pregnancy. In agreement with previous studies (Lachapelle et al., 1996;
De Maria et al., 2011; Wang et al., 2014; Varla-Leftherioti and
Keramitsoglou, 2016), our results showed that the percentage of
CD56dim NK cells, which are considered toxic to pregnancy, was sig-
nificantly exceeding CD56bright NK cells in both blood and decidua.
Moreover, among different CD56dim subsets, CD56dimCD16−NK cells
were the predominating subset of NK cells followed by
CD56dimCD16dim, both in blood and decidua samples of RSM patients.
Besides, negative correlations were found between the CD56dimCD16−
subset in blood and decidua with the other CD56dim subsets in blood
and decidua, respectively. The previous findings may denote the over-
activation of the highly cytotoxic CD56dimCD16bright NK with sub-
sequent loss of CD16 expression. This was supported by earlier studies
(Romee et al., 2013; Amand et al., 2017) which have demonstrated that
upon activation, several CD56dimCD16bright NK cells lose the expression
of CD16 through metalloprotease-mediated shedding and become
CD56dimCD16dim/−.
Both inhibitory and activating KIRs on dNK cells are essential for the
maintenance of pregnancy (Keramitsoglou et al., 2011). Compared to a
prior study on peripheral blood of healthy individuals (Konjevic et al.,
2009), increased levels of CD161+ cells and CD158b+ cells were de-
tected among all NK cell subsets in both blood and decidua of our RSM
patients. Additionally, in both samples, the CD16− NK cells were the
least subsets expressing CD158b or CD161. Expression of CD161 as-
sociates with the cytotoxic function of CD16+ NK cells (Konjevic et al.,
2009) and identifies NK cells with retained capability of responding to
pro-inflammatory cytokines; IL-12 and IL-18, during differentiation
particularly within CD56dim NK cells (Kurioka et al., 2018).
A previous study reported that under certain stimulatory conditions,
CD161 expression decreases with NK proliferation (Kurioka et al.,
2018). This may support the previously mentioned hypothesis that the
CD56dimCD16− cells that were found in high percentages in our pa-
tients and showed the lowest levels of CD161; represent the over-acti-
vation of the highly cytotoxic CD56dimCD16bright NK with subsequent
loss of CD16 expression.
Previous studies analyzing the expression levels of the CD161-acti-
vating and CD158-inhibitory KIR have shown contradictory findings.
While some studies didn't find significant associations between RSM
and NK expressing CD161 (Soderstrom et al., 1997; Ntrivalas et al.,
2005) and CD158b (Soderstrom et al., 1997) in women with RSM
compared with healthy controls, other studies reported that CD56+ NK
cells expressing CD161 receptor significantly decreased in women with
RSM (Baltadzheiva et al., 2010).
On the contrary, some studies in agreement with our results re-
ported increased levels of CD161 (Ghafourian et al., 2014; Kurioka
et al., 2018) and CD158 (Zhu et al., 2018) expression by NK cells in
patients with recurrent miscarriages. These findings support the
O. El-Badawy, et al.
hypothesis of Zhu et al (Zhu et al., 2017, 2018) that in RSM patients,
the immune system is unbalanced with an abnormal up-regulation of
activating and inhibitory KIRs that is not observed in healthy control
females. The highly activated cytotoxic NK cells are probably inducing
a potentially counter-regulatory immune response consisting of in-
hibitory NK cells detected in the blood of RSM patients. Thereby, down-
regulation of the stimulated immune system might be an option for
restoring the balance of the immune system in those patients (Zhu et al.,
2019). This was endorsed by the almost perfect correlations observed in
our patients between cells expressing CD161 and cells expressing
CD158b in each NK subset.
The three toxic CD56dim subsets in our patients' decidua were as-
sociated with an increasing number of previous pregnancy losses. Yet, it
is striking that CD56dimCD16−, CD56dimCD16dim and
CD56dimCD16bright NK cells were directly related to the number of
preceding early miscarriages, late miscarriages, and preterm deliveries,
respectively. We speculate that in the women with repeated early
miscarriages, the CD56dimCD16bright NK has the highest toxic activity
and hence the complete loss of CD16 (CD16−). Whereas, in women
experiencing repeated late miscarriages the CD56dimCD16bright NK cy-
totoxic activity is lower with less loss of CD16 (CD16dim), and thereby
the pregnancy survived longer. The least toxic activity was in women
with repeated preterm labor and hence the least loss of CD16
(CD16bright) and the longest pregnancy duration. This explanation was
supported by finding a negative correlation between CD56dimCD16- and
the number of previous late miscarriages, and between
CD56dimCD16dim and the number of preceding early miscarriages both
in blood and decidua. Our findings agree with that of previous studies
(Zhu et al., 2017, 2019), in that RSM patients still show an aberrantly
high cytotoxic NK cell response after miscarriage. This high cytotoxic
response is mostly a long-term immune response directed against a
persistent fetal antigen in the RSM patients. Thus, to restore the missing
balance in the immune system and enable successful pregnancy, those
patients would mostly benefit from immunosuppressive therapy.
Obesity was found to be related to higher frequencies of poor
pregnancy outcomes including recurrent miscarriages (Lashen et al.,
2004). Perdu and colleagues, recorded lower levels of CD56brightuterine
NK cell in the uterus of obese women, and that uterine NK cell survival
was impaired with obesity. They suggested that obesity establishes a
potentially hostile environment to uterine NK cells, limiting their sur-
vival (Perdu et al., 2016). Supporting the previous findings, our results
revealed a negative correlation between the decidua levels of
CD56brightCD16− NK cells and BMI and positive correlations between
CD56dimCD16dim NK cells and both the body weight and BMI. These
findings reflect the potential role played by NK cells in RSM in obese
pregnant women.
Comparing NK cell subsets and KIR expression between blood and
decidua has shown many important outcomes. No significant differ-
ences were observed among almost all NK subsets between blood and
decidua. Even though CD56brightCD16− levels were slightly higher in
decidua than in blood, marked reduction was observed in its level in
decidua that was more or less comparable with its level in blood. In the
meantime, CD56dim NK cells in blood showed direct relation with the
CD56dim NK in the decidua and inverse relation with the CD56bright NK
cells in the decidua. Also, the CD56dimCD16dim NK cells subset in blood
showed significant positive correlation with the CD56dimCD16dim NK
subset in the decidua. Furthermore, no significant differences were
detected in the levels of activating and inhibiting KIR expression by the
different NK subsets between blood and decidua. These findings support
that pNK cell level reflects dNK cell levels in women with RSM.
Therefore, pNK cell level could be a useful clinical marker to select
women with RSM of immune etiologies.
Taken all into consideration, there is strong evidence that at the
time of abortion, patients with RSM have increased number of toxic NK
cells with an extremely active immune system that can barely be sti-
mulated further, both in blood and decidua. The immune system in
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Journal of Reproductive Immunology 140 (2020) 103130
those patients appears to be unable to form normal balance of cytotoxic
and inhibitory cell subsets even with marked parallel up-regulation of
the inhibitory KIR. This hostile cytotoxic NK response may persist even
after pregnancy loss resulting in recurrent miscarriages.
5. Strengths and limitations
Despite the presence of prior researches concerned with NK subsets
in patients with RSM, detailed data about subset distribution in the
periphery compared with decidua is still controversial and lacking.
Additionally, as far as we know it is the first study to address the link
between CD161 (activating) and CD158 (inhibitory) KIR in the per-
iphery compared with decidua in the context of RSM. Nevertheless, the
study has some potential limitations; we didn’t measure the cytotoxicity
and degranulation activity of each natural killer cell subset. This would
have given a better insight of the disturbances in NK subsets functions
in patients with RSM. Additionally, a larger scale study will strengthen
our results.
6. Conclusion
The NK cells in blood reflect dNK cell changes during miscarriage
and may be a useful non-invasive predicting tool in the reproductive
failure setting. This may pave the way for new immunotherapeutic
approaches to down-modulate the high toxicity of NK cells to prevent
RSM, if handled properly.
Funding
This work was supported by the Faculty of Medicine, Grant Office,
Assiut University [grant number 2016/08/03-001].
Acknowledgments
Many thanks are due to all technicians in Flow Cytometry
Laboratories, Medical Research Center, and South Egypt Cancer
Institute, Assiut University, for their valuable assistance. We also would
like to thank Dr. Mohamed A. El-Mokhtar, Medical Microbiology and
Immunology Department, Faculty of Medicine, Assiut University, for
his technical help.
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