Journal of Reproductive Immunology 138 (2020) 103080

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Journal of Reproductive Immunology

journal homepage: www.elsevier.com/locate/jri

The uterine immunological changes may be responsible for repeated T

implantation failure
Fatemehsadat Amjadia,1, Zahra Zandieha,1, Mehdi Mehdizadeha,*, Samaneh Aghajanpoura,
Ehsan Raoufib, Azin Aghamajidic, Reza Aflatooniand,**
a
Cellular and Molecular Research Center, Department of Anatomical Sciences, Iran University of Medical Sciences, Tehran, Iran
b
Department of Medical Biotechnology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
c
Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
d
Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: A significant part of couples in IVF-ICSI cycles experience Repeated Implantation Failure (RIF). Screening of the
Endometrium embryos with new methods like Next Generation Sequencing and arrays showed that even euploid embryos fail
Implantation window to implant. Immunology is a potent window maybe resolve the RIF problem. In this investigation we employed
RIF innate and adaptive immune system PCR array to compare the transcriptome profiles of endometrium in un-
QPCR array
explained RIF and healthy fertile women.
A total of 21 women were enrolled in the present study, 11women with unexplained RIF and 10 healthy
fertile women. After RNA extraction and cDNA synthesis PCR array was performed using RT2 profiler PCR array
human innate and adaptive immune responses kit (Qiagen, Cat.No: PAHS-052A). PCR Array data analysis
identified significantly greater expression of IL6, IFNG, IL17A, IL23A, IFNA1, IFNB1, CD40 L, CCR4, CCR5,
CCR6, CXR3, CCL2, IL2, TLR4, IRF3, STAT3, RAG1, IFNAR1 in unexplained RIF women than in controls
(P < 0.05). However, expression of IL1B, IL8, NFKB, HLA-A, HLA-E, CD80, CD40 was significantly lower in
unexplained RIF group than in controls (P < 0.05). Our results showed that modulation of immune system in RIF
patient is shifted to inflammatory responses as pNK cells, Th17 signaling pathway and TLR signaling pathway
are activated. So, by stimulation of immune system and initiation of humoral immune responses the panel of
immunity and immunotolerance is completely changed in RIF patients comparing normal. It seems that attention
to these alterations individually help physician to manage RIF patients better.

1. Introduction
Despite advances in the field of reproductive medicine such as im-
provements in embryo culture, embryo screening prior to transfer and
stimulation treatments, implantation rate remains suboptimal in sti-
mulated cycles (Liang et al., 2015; Pathare et al., 2017). A significant
proportion of couples undergoing IVF experience repeated implantation
failure (RIF) (Pathare et al., 2017; Lédée et al., 2016).
RIF usually is defined as the absence of clinical pregnancy after
transferring 6 top‑quality embryos in at least three consecutive IVF
cycles (Simon and Laufer, 2012; Comins-Boo et al., 2016). The mole-
cular mechanism underlying RIF are still unclear (Simon and Laufer,
2012; Comins-Boo et al., 2016). Several etiologies have been suggested
but until recently the primary focus has been on the embryo, and
especially embryonic aneuploidy (Hashimoto et al., 2017; Franasiak
and Scott, 2017). However, even the transfer of euploid embryos, al-
ways does not yield successful implantation (Garcia-Velasco, 2017). It
might suggest that inadequate endometrial receptivity is a major lim-
iting factor for achieving pregnancy (Shi et al., 2017).
“Endometrial receptivity”, is referred to the state of the

Abbreviation: AMH, anti-Mullerian hormone; dNK, decidual; FSH, follicle stimulating hormone; LH, luteinizing hormone; pNK, peripheral NK; RIF, repeated
implantation failure; uNK, Uterine NK cells, NK; WOI, window of implantation
⁎
Corresponding author at: Cellular and Molecular Research Center, Department of Anatomy, School of medicine, Iran University of medical science, Tehran, PO
Box: 15875-1454, Iran.
⁎⁎
Corresponding author at: Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive
Biomedicine, ACECR, Tehran, PO Box: 16635-148, Iran.
E-mail addresses: mehdizadeh.m@iums.ac.ir (M. Mehdizadeh), R.Aflatoonian@gmail.com (R. Aflatoonian).
1
Fatemehsadat Amjadi and Zahra Zandieh have equal contribution.

https://doi.org/10.1016/j.jri.2020.103080
Received 23 July 2019; Received in revised form 29 December 2019; Accepted 9 January 2020
0165-0378/ © 2020 Elsevier B.V. All rights reserved.

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 F. Amjadi, et al.
endometrium during the window of implantation (WOI), a short period
of the menstrual cycle in the mid- luteal phase in which the en-
dometrium acquires new adhesive properties that supports embryo
implantation in a synchronic way (Pathare et al., 2017; Dekel et al.,
2014). Some studies have reported that impaired endometrial re-
ceptivity is responsible for around two‑thirds of implantation failures
(Pathare et al., 2017; Shi et al., 2017).
The cross-talk between the embryo and endometrium is mediated by
many substances, including cytokines, adhesion molecules, growth
factors and hormones (Dekel et al., 2014). A large proportion of these
molecules are involved in immunological process which plays key role
in acceptance of semi-allogenic embryo by maternal endometrium and
establishment of a suitable environment during implantation (Pathare
et al., 2017; Piccinni et al., 2015).
Among all different elements that affect implantation failure of high
grade quality embryos, the immune system has been both the most
reasonable and the most debated (Liang et al., 2015; Franasiak and
Scott, 2017). In most of the previous studies, only a single or a few
markers was examined, which has a restricted value especially in the
case of a complex process such as implantation (Huang et al., 2017;
Gong et al., 2017). Limited studies have been reported the differential
gene expression profiles of endometrium in RIF patients compared with
healthy fertile women (Pathare et al., 2017; Huang et al., 2017). In
recent years, it has been proposed that the main cause of RIF is immune
imbalance and reproductive immunology takes a leading role after re-
peated implantation failure, however till date the mechanisms of this
alterations, remains ill-defined (Franasiak and Scott, 2017; Garcia-
Velasco, 2017). Thus, endometrial immune profile may help un-
explained RIF women in IVF cycles (Lédée et al., 2016). In this in-
vestigation we employed PCR array focusing on distinct immune actors
to compare the transcriptome profiles of endometrium in unexplained
RIF and healthy fertile women. It was worthwhile to identify the dys-
regulation of genes and pathways leading to failure of implantation in
these patients undergoing stimulated IVF cycle.
2. Materials and methods
2.1. Patient selection and sample collection
A total of 21 women were enrolled in the present study, 11women
with unexplained RIF and 10 healthy fertile women (Table 1). Un-
explained RIF was defined as failure to achieve a pregnancy after
transfer of at least four morphologically high‑grade embryos in a
minimum of three cycles. In this group, there were no other obvious
cause, such as polycystic ovary syndrome, ovarian tumors, autoimmune
disorders, chromosomal anomaly, sexually transmitted diseases, polyps,
fibroids, endometriosis, hydrosalpinx, adenomyosis, and uterine mal-
formation. Healthy oocyte donors with proven fertility undergoing COS
were recruited as the “control” group. Controls had one or more live
birth following natural conception.
Inclusion criteria for all cases and controls were age < 40 years
regular menstrual cycles; normal uterine cavity, normal endometrial
thickness of 7 mm and above in ultrasound, normoresponders to the
stimulation protocol and normal sperm from the partners, without in-
trauterine adhesions or inflammation. They had normal levels of serum
follicle stimulating hormone (FSH), luteinizing hormone (LH),
Table 1
characteristics of the RIF and control groups.
PValue control group RIF group Column1
10 11 patients
0.711 33.1 32.6 Age
0.31 22.70 23.77 BMI (kg/m2)
– 6.8 Length of infertility
2
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Journal of Reproductive Immunology 138 (2020) 103080
estradiol, progesterone and anti-Mullerian hormone (AMH) and pro-
lactin.
For ovarian stimulation, Gonadotropins (Gonal-F; Merck, Geneva,
Switzerland) were administrated by starting dose of 150 IU from day 3
of the following cycle then GnRH antagonist (Cetrotide Merk Serono,
Germany) was administered with a daily dose of 0.25 mg beginning
when the largest follicles had reached a diameter of 13–14 mm. Oocytes
were retrieved 36 h after HCG administration under vaginal ultrasound
guidance. Endometrial biopsies were performed during window of im-
plantation, 6 or 7 days after HCG administration by Pipelle catheters
under sterile conditions as described previously (Amjadi et al., 2018).
In case group, all embryos were frozen to transfer then in sub-
sequent FET cycles.
Each sample was divided into two pieces; one portion was placed
immediately in RNAlater (Ambion, Austin, TX), frozen in liquid ni-
trogen and stored at −80 °C for RNA extraction, another portion was
fixed in formalin for histological dating.
All participants donating tissue for this study provided written in-
formed consent prior to sample collection and the study was approved
by the Ethics Committee of Iran University of Medical Sciences.
2.2. RNA extraction
Tissue samples in each case and control group were pooled as three
biological repeats. The pooled samples were subjected to RNA extrac-
tion. RNA extraction and purification was performed using RNeasy mini
kit Qiagen, Cat.No: 73,304 according to the manufacturer’s instruc-
tions. RNA quality and concentration was measured using Nanodrop
2000 spectrophotometer Thermoscientific. Then cDNA synthesis was
performed with the RT2 first strand kit Qiagen, Cat.No: 330,404.
2.3. PCR-Array
PCR array was performed using RT2 profiler PCR array human in-
nate and adaptive immune responses kit (Qiagen, Cat.No: PAHS-052A)
and RT2 SYBR green ROX qPCR mastermix Qiagen, Cat.No: 330,502 on
step one plus real time PCR system ABI. The 84 gene involved in the
human innate and adaptive immune responses relative to the level of
GAPDH as the endogenous control was calculated using the 2–ΔΔCt
algorithm.
2.4. Statistical analysis
The statistical significance of difference in expression levels be-
tween groups was analyzed using Student’s t-test. P-value < 0.05 was
considered statistically significant. PCA (Principal Component analysis)
was carried out using the Minitab 16 statistical software. Hierarchical
clustering was implemented by correlation coefficient and complete
linkage in Minitab 16 statistical software.
3. Results
Profiler PCR-array containing primer sets targeting human innate
and adaptive immune responses related genes used to evaluate the
changes in their expression between endometrial tissues from un-
explained RIF and healthy fertile women. The genes that their expres-
sion were significantly (P < 0.05) altered in unexplained RIF compared
to control group are shown in Figs. 1–3.
PCR Array data analysis identified significantly greater expression
of innate pro-inflammatory and adaptive immune cytokines including
IL6, IFNG, IL17A, IL23A, IFNA1, IFNB1, CCL2, IL2 in unexplained RIF
women than in controls (P < 0.05). However, expression of IL1B, IL8
was significantly lower in unexplained RIF group than in controls
(P < 0.05) (Fig. 1).
The relative expression of cytokine and chemokine receptors in-
cluding CD40 L, CCR4, CCR5, CCR6, CXR3, TLR4, IFNAR1 in
 F. Amjadi, et al.
Fig. 1. The relative mRNA level of innate pro-inflammatory and adaptive im-
mune cytokines in RIF and normal groups. Bar represent by mean ± SD and the
p value ≤ 0.05 was considered as significant.
Fig. 2. The relative expression of cytokine and chemokine receptors at the
mRNA level in RIF group versus normal control. Bar represent by mean ± SD
and the p value ≤ 0.05 was considered as significant.
Fig. 3. The relative expression of the immune transcription factors for the in-
nate and adaptive immune activation. Bar represent by mean ± SD and the p
value ≤ 0.05 was considered as significant.
unexplained RIF women than in controls (P < 0.05). However, ex-
pression of HLA-A, HLA-E, CD80, CD40 was significantly lower in un-
explained RIF group than in controls (P < 0.05) (Fig. 2).
The relative expression of immune transcription factors for the in-
nate and adaptive immune activation including IRF3, STAT3, RAG1 in
unexplained RIF women than in controls (P < 0.05). However,
3
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Journal of Reproductive Immunology 138 (2020) 103080
expression of NFKB1 was significantly lower in unexplained RIF group
than in controls (P < 0.05) (Fig. 3).
4. Discussion
Implantation, placentation and the first and early second trimester
of pregnancy are pro-inflammatory phases that need strong in-
flammatory responses. Normal pregnancy is accompanied by a shift in
the ratio of Th2 to Th1 cytokines. So pregnancy is a Th2- dominant state
(Franasiak and Scott, 2017) that induced by progesterone and resulted
in decreasing the amount of inflammatory cytokines and finally im-
munotolerance.
The RIF patients have criteria include failure to achieve a pregnancy
following two to six IVF/ICSI–ET cycles, in which more than 10 high-
grade embryos were transferred. By the entrance of the embryo to the
uterus the innate immune system as the first line of defences is acti-
vated and responded to the embryo by various type of modulation in
adaptive and innate immunity. The natural killer cells − macrophage,
dendritic cell and Treg cell − migrate and increase in the endometrium
during the implantation window. Deletion of these cells has deleterious
effects on implantation and placental (Liang et al., 2015).
Producing IL-10 and TGF-β by regulatory T cells play an im-
munosuppressive role in normal pregnancy. Increased IL-1,IL-6, IL-21
or IL-23 and decreased TGF-β production might cause increased Th17
cells and decreased Treg cells in the uterus (Saini et al., 2020). The
important factor for producing Treg cells is TGF β that our results
showed reducing TGFβ in RIF patients comparing with normal ones.
(Fig. 4).
Toll Like Receptors are key parts of innate immunity, results showed
high level of transcriptions in TLR4 (p < 0.002), IRF3 (p = 0.022) and
IRF7 (p = 0.08) in RIF patients in comparison with normal ones. As a
results of the expression of TLR4, IRF3 and IRF7 signaling pathway, the
level of inflammatory cytokine as well as interferon type 1 also in-
creased (O’Neill et al., 2020). So, it seems that innate immunity is al-
tered in RIF patients.
One of the important cell feature in normal pregnancy is human
decidual NK (dNK) cells that in contrast to peripheral NK (pNK) cells
express high level of CD56 and lack the expression of CD16 (Santoni
et al., 2007). They are an important subset of NK cells that play im-
munomodulatory role in implantation (Koopman et al., 2003). dNK
cells display about 15% cytotoxic activity of the pNK. But some factors
like IL-2 can promote their cytotoxic activity. So, during pregnancy, the
cytotoxicity of dNK cells toward trophoblast should be firmly regulated
(Sun et al., 2020). (Fig. 4)
Conversion of pNK phenotype to dNK cell is induced by TGFβ. dNK
cells also produce TGFβ, IL6, IL8, TNFα and IL1β (Sun et al., 2020) that
all of them are decreased in RIF patients.
Another important cells in implantation and early stage of preg-
nancy is Uterine NK cells(uNK) that have key roles in angiogenesis and
spiral artery remodeling (Sun et al., 2020).
uNK cells in early stage of pregnancy have high potential for se-
creting angiogenic factors like VEGF, VEGF-C, IL8 and etc. results in
this study demonstrated significant decrease in IL8 (p < 0.0001) or
CXCL8 level.
Increasing in the level of inflammatory cytokines like IL2 (p =
0.0068), TNFα and IFNγ (p < 0.0001) by macrophages and activated
NK-cells was demonstrated in RIF than control. These cytokines sti-
mulate APC cells that presents antigens to adaptive immune system like
T cell and convert them to Th1 cells. Th1 cells activation resulted in
chemokine secretion like CCL2 (MCP1) (p < 0.0001) in RIF than con-
trol. In addition to, CCL2 caused increasing in mono nuclear phagocy-
tosis and more inflammatory situation (Fig. 4).
High expression of CD86 in APC cells also was shown in RIF patients
in our results that help activating T cells that resulted in more in-
flammatory cytokine production and more cytotoxic activity of NK-cells
(Robertson et al., 2020).
 F. Amjadi, et al.
Additionally, comparing RIF patients to control, it seems that Th17
cells are increased in response to IL-6, IL-12 and IL-23 (p < 0.001) and
chemokine receptor CCR4 (P = 0.005) and CCR6 (P = 0.019) ex-
pression. STAT3 also has high level in RIF than control (p < 0.0001).
activation of STAT3 is associated with IL-23 and IL-6 increasing that
study showed may also be involved in pathogenesis of repeated spon-
taneous abortion (Mesquita et al., 2009). IL-17, has high level in RIF
patients (p < 0.0001) is a potent inducer of inflammation that raise the
cellular infiltration and produce numerous pro-inflammatory cytokines
and chemokines (Cai et al., 2016). High level of expression in CCR6,
CCR4, IL-6, IL-17, IL-21 and IL-23 in results showed that Th17 and its
inflammatory signaling pathway are active in RIF and play a role in
implantation failure (Fig. 4).
It can be noted that significant increase in RIG-I (DDX58) in RIF
patients showed ssRNA and short double strand RNA infection that can
be another cause of invading immune system to embryo.
In conclusion, modulation of immune system in RIF patient is
shifted to inflammatory responses as pNK cells, Th17 signaling pathway
and TLR signaling pathway are activated. So, by stimulation of immune
system and initiation of humoral immune responses the panel of im-
munity and immunotolerance is completely changed in RIF patients
comparing normal. It seems that attention to these alterations in-
dividually help physician to manage RIF patients better. Also, further
study will be designed to explore the mechanism and cutoff for these
factors precisely.
Declaration of Competing Interest
Authors declare no conflict of interests
Acknowledgement
This research was supported by Iran University of Medical Sciences
(grant no. 25717).
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Journal of Reproductive Immunology 138 (2020) 103080
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