NK and trophoblast cells interaction: cytotoxic activity on recurrent pregnancy loss

Abstract

The trial objective was to determine the peripheral blood NK cells cytotoxic activity effect on trophoblast cells at recurrent pregnancy loss (RPL). The investigation involved non-pregnant women with PRL in proliferating and secretory menstrual cycle phases (PMCPh and SMCPh, respectively); women of 6–7 weeks pregnancy with RPL in past medical history; healthy fertile non-pregnant women in PMCPh and SMCPh, women of 6–7 weeks physiological pregnancy, nulliparity healthy women with regular menstrual function in PMCPh and SMCPh. NK cells cytotoxic activity was determined using peripheral blood mononuclear cells. The target cells were JEG-3 line trophoblasts. It has been established that NK cells cytotoxic activity effect on trophoblasts is lower in SMCPh than in PMCPh in non-pregnant fertile women. The NK cells cytotoxic activity was higher in SMCPh than in PMCPh in non-pregnant women with PRL and also higher than the same value in SMCPh in non-pregnant fertile women. The increased NK cells cytotoxic activity values in SMCPh in women with RPL may be the reason for miscarriage.

Full text

Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=igye20
Gynecological Endocrinology
ISSN: 0951-3590 (Print) 1473-0766 (Online) Journal homepage: https://www.tandfonline.com/loi/igye20
NK and trophoblast cells interaction: cytotoxic
activity on recurrent pregnancy loss
Dmitriy I. Sokolov, Valentina A. Mikhailova, Alana O. Agnayeva, Dmitry
O. Bazhenov, Evgeniya V. Khokhlova, Olesya N. Bespalova, Aleksandr M.
Gzgzyan & Sergey A. Selkov
To cite this article: Dmitriy I. Sokolov, Valentina A. Mikhailova, Alana O. Agnayeva, Dmitry O.
Bazhenov, Evgeniya V. Khokhlova, Olesya N. Bespalova, Aleksandr M. Gzgzyan & Sergey A.
Selkov (2019) NK and trophoblast cells interaction: cytotoxic activity on recurrent pregnancy loss,
Gynecological Endocrinology, 35:sup1, 5-10, DOI: 10.1080/09513590.2019.1632084
To link to this article: https://doi.org/10.1080/09513590.2019.1632084
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 18 Sep 2019.
Submit your article to this journal
Article views: 54
View related articles
View Crossmark data

ORIGINAL ARTICLE
NK and trophoblast cells interaction: cytotoxic activity on recurrent
pregnancy loss
Dmitriy I. Sokolov , Valentina A. Mikhailova , Alana O. Agnayeva, Dmitry O. Bazhenov, Evgeniya V. Khokhlova,
Olesya N. Bespalova , Aleksandr M. Gzgzyan and Sergey A. Selkov
Federal State Budgetary Scientific Institution ‘Research Institute of Obstetrics, Gynecology, and Reproductology named after D.O. Ott’, Saint
Petersburg, Russia
ABSTRACT
The trial objective was to determine the peripheral blood NK cells cytotoxic activity effect on trophoblast
cells at recurrent pregnancy loss (RPL). The investigation involved non-pregnant women with PRL in pro-
liferating and secretory menstrual cycle phases (PMCPh and SMCPh, respectively); women of 6–7 weeks
pregnancy with RPL in past medical history; healthy fertile non-pregnant women in PMCPh and SMCPh,
women of 6–7 weeks physiological pregnancy, nulliparity healthy women with regular menstrual function
in PMCPh and SMCPh. NK cells cytotoxic activity was determined using peripheral blood mononuclear
cells. The target cells were JEG-3 line trophoblasts. It has been established that NK cells cytotoxic activity
effect on trophoblasts is lower in SMCPh than in PMCPh in non-pregnant fertile women. The NK cells
cytotoxic activity was higher in SMCPh than in PMCPh in non-pregnant women with PRL and also higher
than the same value in SMCPh in non-pregnant fertile women. The increased NK cells cytotoxic activity
values in SMCPh in women with RPL may be the reason for miscarriage.
KEYWORDS
NK cells; trophoblast;
cytotoxic activity;
pregnancy; recurrent
pregnancy loss; miscarriage;
peripheral blood
Introduction
The recurrent pregnancy loss (RPL) frequency is still persistently
high. The pathology is caused by genetic disorders, hormone
production imbalance, uterine structure disorders, infections, and
immune conflicts [1,2]. The immune interaction disorders in
mother–fetus system are one of the main pathogenic factors [1].
In the meantime, the existing immune laboratory testes (lupus
anticoagulant test, anticardiolipin, phosphatidylserine antibody,
Factor V Leiden mutation, glucose test, C and S protein func-
tional activity, prolactin level, hyperandrogenemia, etc.) allow to
determine changes surpassing normal values only in 40% women
with RPL [3]. In 60% women with RPL, the disease cause
remains unascertained [3]. Among a variety of immune parame-
ters, the NK cells role in RPL pathogenesis, especially their cyto-
toxic function is often stressed [4].
NK cells are lymphocytes capable of virus-infected and tumor
cells contact cytolysis. Endometrial NK cells are a specific NK
population and their representation in uterus varies depending
jn menstrual cycle (MC) phase and pregnancy [5–7]. NK cells
have a regulatory function during a physiological pregnancy
which means creating optimal conditions for blastocyst invasion,
the invasion process control depending on gestation terms as
well as taking part in uterus spiral artery remodeling and ute-
rus–placenta normal blood flow establishment [7–10]. Trials in
rats showed that NK cells decrease in uterus causes disorders of
spiral artery restructuring [11].
In case of miscarriage, the NK cells activity may vary [11].
Attempts are made to work out miscarriage risk diagnostics and
assessment methods based on NK cells functional activity vari-
ation measuring [12]. There are two approaches to obtain
biological material for NK cells investigation in miscarriage cases.
The less invasive one is peripheral blood sampling with further
NK cells isolation. The other source for NK cells is endometrium
biopsy material [12]. Generally, the functional status of periph-
eral blood NK cells reflects the same of uterus NK cells [12,13].
The peripheral blood sampling variant is more preferable because
it is more standard and less invasive compared to biopsy
method. Apart from that, blood sampling is possible both within
and out of pregnancy while in case of biopsy we have a local tis-
sue sampling which is possible only out of pregnancy, offers only
sub-microgram of the material and is bound to cell loss during
the isolation procedure [14].
At the present time, chronic myeloid leukemia K562 line cells
are used as targeting cells to determine NK cells cytotoxic activ-
ity. Different ways are applied to label the targeting cells: fluores-
cent dyeing [15,16], transfection with plasmid containing green
fluorescent protein gene [17], and some others. All the men-
tioned methods simulate the cytotoxic NK cells activity leaving
out the peculiarities of NK cells and targeting cells interaction
during pregnancy and its pathologies. Invasive extravillous
trophoblast cells are the most obvious targeting cells for NK cells
in decidua. Due to that, the trial objective was to determine the
peripheral blood NK cells cytotoxic activity effect on trophoblast
cells in case of recurrent miscarriage.
Materials and methods
Materials
The trial embraced non-pregnant women with PRL in MC pro-
liferating (PPh) (n¼19) and secretory phases (SPh) (n¼23);
CONTACT Dmitriy I. Sokolov falcojugger@yandex.ru Department of Immunology and Intercellular Interactions, Federal State Budgetary Scientific Institution
‘Research Institute of Obstetrics, Gynecology, and Reproductology named after D.O. Ott’, Mendeleevskaya liniya, 3, Saint Petersburg 199034, Russia
ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
GYNECOLOGICAL ENDOCRINOLOGY
2019, VOL. 35, NO. S1, 5–10
https://doi.org/10.1080/09513590.2019.1632084

women of 6–7 weeks pregnancy with RPL in past medical history
(n¼23). The comparison groups included healthy non-pregnant
fertile women, who had normal delivery ended previous preg-
nancy (henceforward non-pregnant fertile women) in PMCPh
(n¼20) and SMCPh (n¼20), as well as women with physio-
logical 6–7 weeks pregnancy (n¼25). The women involved were
of normal karyotype, without antiphospholipid syndrome, her-
editary high risk thrombophilia, hypertensive disease, diabetes, or
adiposity. The control group included reproductive age healthy
nulliparity women with regular menstrual function and no aggra-
vated obstetric and gynecological anamnesis in PMCPh (n¼25)
and SMCPh (n¼25). Another control group consisted of healthy
men (n¼15). All the people involved in the trial were Caucasian
race coming from The North-West region of Russia. The trial
was carried out in the scientific-ambulatory and pregnancy path-
ology Departments of ‘Research Institute of Obstetrics,
Gynecology, and Reproductology named after D.O. Ott’together
with Saint-Petersburg Miscarriage prevention, diagnostics and
treatment Scientific and Practice Center. The study was prospect-
ive and cohort.
Methods
To determine the peripheral blood NK cells cytotoxic activity
effect on trophoblast cells, mononuclear cells were applied which
were isolated from patients’peripheral blood by standard density
gradient centrifugation Histopaque
V
R
-1077 (Sigma Aldrich, St.
Louis, MO) [18]. After isolation, the peripheral blood mono-
nuclear cells containing NK cells were incubated in a complete
culture medium containing DMEM, 10% fetal calf serum (FCS).
100 U/ml of penicillin and 100 mkg/ml of streptomycin, 2 mM of
L-glutamine and 10 mM of sodium pyruvate (Sigma, St. Louis,
MO). Then, the peripheral blood mononuclear cells were culti-
vated for four days both in presence or absence of recombinant
IL-2 (‘Roncoleukine’NPK ‘BIOTECH’, Saint Petersburg, Russia).
The applied trophoblast cells were of JEG-3 line (ATCC,
Manassas, VA), simulating morphological, phenotypic and func-
tional characteristics of first trimester pregnancy trophoblasts
[19]. Twenty-four hours before the experiment (on the third
incubation day), the trophoblast cells were prepared, the mono-
layer disintegrated through exposition in trypsin–Versene solu-
tion (1:1), then half of the obtained cell suspension was placed in
a new flask for adhesive culture in a complete culture medium,
containing 1% of HEPES solution and 10% FCS (Sigma, St.
Louis, MO). On the next day, the trophoblast cells were disinte-
grated, treated by carboxyfluorescein succinimidyl ester (CFSE)
5-6 solution then incubated for 4 h with peripheral blood mono-
nuclear cells in effector–target 10:1 rate. A part of trophoblast
cells was incubated in a similar cultural medium without mono-
nuclear cell addition to determine the base trophoblast cell death
and then the number of viable and inviable trophoblast cells was
measured by propidium iodide solution staining in a final
0.01 mg/ml concentration (Sigma Aldrich, St. Louis, MO). Part
of trophoblast cells were not treated by CFSE and propidium
iodide solutions but were used as negative control for cell auto-
fluorescence estimation. For each patient, NK cells cytotoxic
activity was measured both in presence or absence of IL-2.
During each NK cells cytotoxic activity estimation, the base
trophoblast cell death was also measured in absence of mono-
nuclear cells (Figure 1).
Statistics
The data obtained were statistically processed by Statistica 10
program. For data comparison, the Mann–Whitney U-criteria
along with Wilcoxon’s criteria were applied. Differences at
p<.05, p<.01, p<.001 were estimated statistically relevant.
Results
It was established that the NK cells cytotoxic effect on tropho-
blast cells was higher in presence of IL-2 compared to the same
indicator after incubation without IL-2 in all tested patient
groups (Figure 2).
The groups of healthy non-pregnant women without pregnan-
cies in past history in PMCPh and SMCPh did not have differen-
ces from the healthy men group in NK cells cytotoxic effect on
trophoblast cells both in IL-2 presence and absence.
The NK cells cytotoxic activity without IL-2 in healthy non-
pregnant women in PMCPh with no pregnancies in past history
was higher than in those with 6–7 weeks physiological pregnancy
(Figure 3). In the meantime, the NK cells cytotoxic activity in
healthy non-pregnant women in SMCPh was lower compared to
the same group in PMCPh and did not differ from the same
value in women with physiological pregnancy (Figure 3). The
comparison of healthy non-pregnant fertile women group in
both phases to the group of physiologically pregnant women
showed similar tendencies. The peripheral blood NK cells cyto-
toxic activity in case of incubation without IL-2 was higher in
healthy non-pregnant fertile women in PMCPh compared to the
values in physiologically pregnant women group (Figure 3). The
NK cells cytotoxic activity in case of incubation without IL-2 was
lower in fertile non-pregnant women in SMCPh compared to the
same in PMCPh (Figure 3). No difference was indicated between
the group of non-pregnant fertile women in SMCPh and of those
physiologically pregnant (Figure 3).
In presence of IL-2, the NK cells cytotoxic activity in non-
pregnant women without previous pregnancies in PMCPh
(51.7% (44.2%; 58.4%)) and SMCPh (48.8% (42.7%; 59.8%)) was
higher than in those with 6–7 weeks term physiological preg-
nancy (43.7% (34.8%; 49.0%); p˂.001).
The NK cells cytotoxic activity effect on trophoblast in
women with RPL was higher in SMCPh than in PMCPh in case
of incubation without IL-2 (Figure 4). Apart from that, the cyto-
toxic activity of NK cells obtained from non-pregnant women
with PRL was higher in SMCPh compared to the same value in
healthy fertile women also in SMCPh (Figure 4). The NK cells
cytotoxic activity effect on trophoblasts in pregnant women with
RPL in case of incubation without IL-2 was lower compared to
that in non-pregnant women with RPL in SMCPh (Figure 4).
Along with that, the NK cells cytotoxic activity effect on tropho-
blasts in case of incubation in presence of IL-2 was also lower in
pregnant women with RPL (44.4% (37.0%; 50.8%)) compared to
non-pregnant women with RPL in SMCPh (48.6% (39.7%;
53.8%); p˂.05).
Discussion
Chronic myeloid leukemia K562 line cells are used as targeting
cells to determine the NK cells cytotoxic activity in the most
widely applied methods [15–17,20,21]. Some attempts were pre-
viously made to estimate the NK cells cytotoxic activity effect on
trophoblasts by using BeWo linear cells together with placenta
isolated trophoblast cells. Those methods showed similar activity
6 D. I. SOKOLOV ET AL.

of NK cells compared to cytotoxiceffect on K562 cells [21].
However, the placenta isolated trophoblast cells contained
macrophage and B-lymphocyte admixtures [21] which could
affect the trial results. Along with that placenta isolated tropho-
blast cells usage is not reasonable because the surface receptor
expression of population difference by trophoblast cells may also
affect the obtained results [22]. Immune histochemical studies
showed BeWo line cells inclination to syncytium fanning which
is not observed in case of JEG-3 line cells [23]. It is likely to get
distorted results due to BeWo line cells damage in process of
preparing them as target cells for NK cells cytotoxicity testing.
For this reason, we preferred JEG-3 line trophoblast cells as tar-
get cells for the present trial.
We have established that the NK cells cytotoxic activity effect
on trophoblast cells was higher in presence of IL-2 in all tested
patient groups. Thus, the peripheral blood NK cells ability to
increase the cytotoxic activity in presence of an activator (IL-2)
within our simulating system affirms the possibility of applying
the suggested experimental scheme to estimate cells func-
tional status.
We have also established that NK cells cytotoxic activity was
lower in non-pregnant women, both without previous pregnan-
cies and the fertile ones in SMCPh, and did not differ from NK
cells cytotoxic activity in women with physiological pregnancy.
Taking into consideration the uterine NK cells cytopoiesis possi-
bility on the base of migration from peripheral blood, the per-
formed difference may be the effect of endometrium preparation
for blastocyte implanting, which normally takes place in
6–7 days after ovulation and ovum fertilization [10]. During this
period, NK cells number increase in uterus is observed [24,25]
along with variance both of NK cells and macrophage secretion
and adhesive molecule expression by endometrium cells [24]. It
Figure 1. JEG-3 line trophoblast cells gating strategy after incubation with peripheral blood NK cells. (a) Trophoblast cells in FSC-SSC and FSC-FITC coordinates (nega-
tive control); (b) CFSE and propidium iodide solution treated trophoblast cells after incubation in complete culture medium in FSC-SSC and FSC-FITC coordinates (base
death); (c) CFSE and propidium iodide solution treated trophoblast cells after incubation in presence of peripheral blood mononuclear cells containing NK cells in FSC-
SSC and FSC-FITC coordinates (NK cells cytotoxic activity caused death).
GYNECOLOGICAL ENDOCRINOLOGY 7

Figure 2. Cytotoxic activity effect on trophoblasts of NK cells incubated both in presence and in absence of IL-2. Data are presented in box diagrams, where the mid-
line corresponds to the median and the upper and lower box borders –to 75% and 25% quartiles, respectively. The group differences within incubation condi-
tions: p<.001.
Figure 3. Cytotoxic activity effect on trophoblasts of NK cells incubated without IL-2 in non-pregnant and pregnant women. Data presented in box diagrams, where
the midline corresponds to the median and the upper and lower box borders –to 75% and 25% quartiles, respectively. Group differences: p<.05,
p<.01, p<.001.
8 D. I. SOKOLOV ET AL.

is likely that hormonal context variance taking place during MC
affects not only cell population representation in uterus [10,25],
but also peripheral blood NK cells functional characteristics.
It has been also established that NK cells cytotoxic activity
effect on trophoblast cells in non-pregnant women with recur-
rent miscarriage is higher in SMCPh compared to PMCPh and
also compared to the same value in non-pregnant fertile women
in SMCPh. Thus, contrary to the case of non-pregnant fertile
women where NK cells cytotoxic activity decrease in SMCPh is
observed, we see cytotoxic activity increase in SMCPh in women
with RPL.
Pregnant women with RPL in past history show decreased
NK cells cytotoxic effect on trophoblast compared to the same
value in non-pregnant women with RPL in SMCPh. This differ-
ence may be seen as pregnancy consequence together with
macrophage and regulatory T-cell decidua affect outcome
[26,27], stimulating implantation and placentation, and also that
of yellow body and chorion secreted progesterone [4,10].
Following the results obtained we may suppose that NK cells
cytotoxic activity effect on trophoblast varies during the MC.
It is higher in the proliferative phase and decreases in the secre-
tion phase. This variation may be determinative for blastocyst
implantation and pregnancy success. NK cells cytotoxic activity
increased values in women with RPL in SMCPh may stipulate
the miscarriage. It should be noted that according to the research
literature the NK cells content as well as NK cells cytotoxic activ-
ity investigation has not been seen yet as top priority for RPL
patients’treatment [12,28,29]. Basing on data obtained, we would
recommend to introduce immune disorders diagnostics including
NK cells cytotoxic activity effect on trophoblast cells monitoring
into the treatment practice of patients with RPL.
Acknowledgments
The authors express thanks to L.P. Viazmina for technical assistance.
Disclosure statement
The authors declare no conflict of interest.
Funding
This work was supported by the Grant Board of President of
Russian Federation under Grant NSh-2873.2018.7 (NK cells cytotox-
icity analysis); Russian Federation State Program No. aaaa-a19-
119021290116-1 (cell culturing).
Figure 4. Cytotoxic activity effect on trophoblasts of NK cells incubated without IL-2 in non-pregnant and pregnant women with RPL. Data are presented in box dia-
grams, where the midline corresponds to the median and the upper and lower box borders –to 75% and 25% quartiles, respectively. Group differences:
p<.05, p<.001.
GYNECOLOGICAL ENDOCRINOLOGY 9

ORCID
Dmitriy I. Sokolov http://orcid.org/0000-0002-5749-2531
Valentina A. Mikhailova http://orcid.org/0000-0003-1328-8157
Olesya N. Bespalova http://orcid.org/0000-0002-6542-5953
Aleksandr M. Gzgzyan http://orcid.org/0000-0003-3917-9493
Sergey A. Selkov http://orcid.org/0000-0003-1560-7529
References
[1] Kwak-Kim J, Bao S, Lee SK, et al. Immunological modes of preg-
nancy loss: inflammation, immune effectors, and stress. Am J Reprod
Immunol. 2014;72:129–140.
[2] El Hachem H, Crepaux V, May-Panloup P, et al. Recurrent preg-
nancy loss: current perspectives. IJWH. 2017;9:331–345.
[3] Jaslow CR, Carney JL, Kutteh WH. Diagnostic factors identified in
1020 women with two versus three or more recurrent pregnancy
losses. Fertil Steril. 2010;93:1234–1243.
[4] Pandey MK, Rani R, Agrawal S. An update in recurrent spontaneous
abortion. Arch Gynecol Obstet. 2005;272:95–108.
[5] Tabiasco J, Rabot M, Aguerre-Girr M, et al. Human decidual NK
cells: unique phenotype and functional properties –a review.
Placenta. 2006;27:34–39.
[6] Keskin DB, Allan DS, Rybalov B, et al. TGFbeta promotes conversion
of CD16þperipheral blood NK cells into CD16–NK cells with simi-
larities to decidual NK cells. Proc Natl Acad Sci USA. 2007;104:
3378–3383.
[7] Zhang J, Dunk C, Croy AB, et al. To serve and to protect: the role of
decidual innate immune cells on human pregnancy. Cell Tissue Res.
2016;363:249–265.
[8] Wallace AE, Fraser R, Cartwright JE. Extravillous trophoblast and
decidual natural killer cells: a remodelling partnership. Hum Reprod
Update. 2012;18:458–471.
[9] Sharkey AM, Xiong S, Kennedy PR, et al. Tissue-specific education of
decidual NK cells. J Immunol. 2015;195:3026–3032.
[10] Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of
early pregnancy. N Engl J Med. 2001;345:1400–1408.
[11] Chakraborty D, Rumi MA, Konno T, et al. Natural killer cells direct
hemochorial placentation by regulating hypoxia-inducible factor
dependent trophoblast lineage decisions. Proc Natl Acad Sci USA.
2011;108:16295–16300.
[12] Templer S, Sacks G. A blessing and a curse: is high NK cell activity
good for health and bad for reproduction? Hum Fertil. 2016;19:
166–172.
[13] Park DW, Lee HJ, Park CW, et al. Peripheral blood NK cells reflect
changes in decidual NK cells in women with recurrent miscarriages.
Am J Reprod Immunol. 2010;63:173–180.
[14] Tang AW, Alfirevic Z, Quenby S. Natural killer cells and pregnancy
outcomes in women with recurrent miscarriage and infertility: a sys-
tematic review. Hum Reprod. 2011;26:1971–1980.
[15] Thum MY, Bhaskaran S, Abdalla HI, et al. Prednisolone suppresses
NK cell cytotoxicity in vitro in women with a history of infertility
and elevated NK cell cytotoxicity. Am J Reprod Immunol. 2008;59:
259–265.
[16] Roussev RG, Dons’koi BV, Stamatkin C, et al. Preimplantation factor
inhibits circulating natural killer cell cytotoxicity and reduces CD69
expression: implications for recurrent pregnancy loss therapy. Reprod
Biomed Online. 2013;26:79–87.
[17] Kantakamalakul W, Jaroenpool J, Pattanapanyasat K. A novel
enhanced green fluorescent protein (EGFP)-K562 flow cytometric
method for measuring natural killer (NK) cell cytotoxic activity.
J Immunol Methods. 2003;272:189–197.
[18] Boyum A. Separation of leukocytes from blood and bone marrow.
Introduction. Scand J Clin Lab Investig Suppl. 1968;97:7.
[19] Kohler PO, Bridson WE. Isolation of hormone-producing clonal lines
of human choriocarcinoma. J Clin Endocrinol Metabol. 1971;32:
683–687.
[20] Karami N, Boroujerdnia MG, Nikbakht R, et al. Enhancement of per-
ipheral blood CD56(dim) cell and NK cell cytotoxicity in women
with recurrent spontaneous abortion or in vitro fertilization failure.
J Reprod Immunol. 2012;95:87–92.
[21] Ferry BL, Sargent IL, Starkey PM, et al. Cytotoxic activity against
trophoblast and choriocarcinoma cells of large granular lymphocytes
from human early pregnancy decidua. Cell Immunol. 1991;132:
140–149.
[22] Al-Nasiry S, Spitz B, Hanssens M, et al. Differential effects of
inducers of syncytialization and apoptosis on BeWo and JEG-3
choriocarcinoma cells. Hum Reprod. 2006;21:193–201.
[23] Zidi I, Rizzo R, Bouaziz A, et al. sHLA-G1 and HLA-G5 levels
are decreased in Tunisian women with multiple abortion. Hum
Immunol. 2016;77:342–345.
[24] van Mourik MS, Macklon NS, Heijnen CJ. Embryonic implantation:
cytokines, adhesion molecules, and immune cells in establishing an
implantation environment. J Leuk Biol. 2008;85:4–19.
[25] Lee SK, Kim CJ, Kim DJ, et al. Immune cells in the female repro-
ductive tract. Immune Netw. 2015;15:16–26.
[26] Sokolov DI, Selkov SA. Decidual macrophages: the role in immuno-
logic dialogue of mother and the fetus. Immunologia (Russia)/
Immunol. 2014;35:113–117.
[27] Sokolov DI, Stepanova OI, Selkov SA. The role of the different sub-
populations of CD4þT-lymphocytes during pregnancy. Med
Immunol. 2016;18:521–536.
[28] Kwak-Kim J, Han AR, Gilman-Sachs A, et al. Current trends of
reproductive immunology practices in in vitro fertilization (IVF) –a
first world survey using IVF-Worldwide.com. Am J Reprod
Immunol. 2013;69:12–20.
[29] Moffett A, Shreeve N. First do no harm: uterine natural killer (NK)
cells in assisted reproduction. Hum Reprod. 2015;30:1519–1525.
10 D. I. SOKOLOV ET AL.