American J Rep Immunol - 2022 - Mukherjee - Immune alterations in recurrent implantation failure

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Received: 3 February 2022 Revised: 1 April 2022 Accepted: 26 April 2022
DOI: 10.1111/aji.13563
R E V I EW ART I C L E
Implantation
Immune alterations in recurrent implantation failure
NupurMukherjee Richa Sharma DeepakModi
Molecular and Cellular Biology Laboratory,
ICMR-National Institute for Research in
Reproductive and Child Health, Indian Council
ofMedical Research (ICMR), Parel, Mumbai,
Maharashtra, India
Correspondence
DeepakModi, Molecular and Cellular Biology
Laboratory, ICMR-National Institute for
Research in Reproductive and Child Health,
Indian Council ofMedical Research (ICMR), JM
Street, Parel, Mumbai 400012,Maharashtra,
India.
Email: deepaknmodi@yahoo.com
Funding information
Indian Council ofMedical Research;
Department of Biotechnology, Government of
India, Grant/Award Number:
BT/PR30401/BIC/101/1077/2018;
Department of Science and Technology,
Ministry of Science and Technology, Science
and Engineering Research Board Government
of India, Grant/Award Number: IF170222
Abstract
A failure to achieve pregnancy after three or more embryo transfer cycles with high-
quality blastocysts is referred to as recurrent implantation failure (RIF). RIF can be due
to altered uterine factors or male factors or embryo factors. Disrupted endometrial
receptivity, altered expression of genes in several pathways, immunologic disturbances
in the peripheral blood and/or the endometrium, and epigenetic alterations are associ-
ated with RIF. Amongst the immunologic disturbances, altered Th1/Th2 ratio, altered
NK cell and macrophage numbers are observed in women with RIF. However, not all
women with RIF have the same kind of immune dysfunction suggesting that RIF is a
heterogeneous condition associated with varied immune responses and one size may
not fit all. Thus, personalized therapies based on the immune status of the patient
are being tested in women with RIF. In general, women with a high Th1/Th2 ratio are
offered Tacrolimus, while intravenous IgG is recommended inwomenwith highNK cell
numbers/HLAmismatch.Womenwith hyperactivated immune status in the uterus are
offeredprogesterone support, prednisolone, vitaminE, and intralipid treatment to sup-
press inflammation and oxidative stress, while endometrial scratching and intrauterine
hCG administration are offered to women with hypo-active immune status. There is a
need for standardized tests for evaluation of immune status in patients and sufficiently
powered randomized controlled trials for personalized therapies to determine which
of thesewill be beneficial in womenwith RIF. Till then, the ART community should limit
the use of such add-on interventions in womenwith RIF.
KEYWORDS
cytokines, decidua, embryo, endometrium, inflammation, placenta, pregnancy, treatment
1 INTRODUCTION
Embryo implantation is a rate-limiting step for a successful preg-
nancy. There is an intricate cross-talk between the embryo and the
endometrium and there is a need for synchrony between embryo
development and uterine differentiation and any defects in either of
the two processes can lead to implantation failure Also, defects in
embryo implantation and early placentation are thought to be a cause
of spontaneous abortions and pregnancy-related complications like
© 2022 JohnWiley & Sons A/S. Published by JohnWiley & Sons Ltd.
preeclampsia.1–3 Assisted reproductive technology (ART) has allowed
biological parenthood in couples with involuntary childlessness. Scien-
tific and technical progress in the last three decades has significantly
improved the outcomes of ART; however, pregnancy losses in the ART
cycles cause a lot of socio-economic stress on the couples. Pregnancy
is either lost before (or around the time of implantation) or postim-
plantation until 24 weeks. The loss of two or more consecutive clinical
pregnancies until 20 or 24 weeks gestation is defined as recurrent
pregnancy loss.4 While failure to conceive (clinical pregnancy) after
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mailto:deepaknmodi@yahoo.com
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How to cite this article: Mukherjee N, Sharma R,Modi D.
Immune alterations in recurrent implantation failure. Am J
Reprod Immunol. 2023;89:e13563.
https://doi.org/10.1111/aji.13563
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iley.com
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razil, W
iley O
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erm
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onditions (https://onlinelibrary.w
iley.com
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https://onlinelibrary.wiley.com/doi/full/10.1111/aji.12217
https://pubmed.ncbi.nlm.nih.gov/26777262/
https://pubmed.ncbi.nlm.nih.gov/26777262/
https://pubmed.ncbi.nlm.nih.gov/33841443/
https://pubmed.ncbi.nlm.nih.gov/33841443/
https://doi.org/10.1111/aji.13563
	Immune alterations in recurrent implantation failure
	Abstract
	1 | INTRODUCTION
	1.1 | Established etiological factors of RIF
	1.2 | RIF is an endometrial defect
	1.3 | RIF and defects in the immune system
	1.3.1 | Changes in peripheral blood immune profiles of women with RIF
	1.3.2 | Changes in tissue-resident immune cells in women with RIF
	1.3.3 | Cytokines altered in RIF
	1.3.4 | Molecular mechanisms of altered maternal immune response in RIF
	1.4 | Endometrial epigenetic defect as a cause of local immune disturbances in RIF
	1.5 | Immunotherapies in RIF
	1.6 | Immune profiling and personalized therapies
	1.7 | Personalized therapy based on circulating Th1/Th2 ratios
	1.8 | Personalized therapy based on circulating NK cell numbers
	1.9 | Personalized treatments for RIF based on the immune status of the endometrium
	1.10 | OPtimization of thyroid function, immunity, and uterine milieu (OPTIMUM) treatment strategy for RIF
	1.11 | Limitations and pitfalls
	2 | SUMMARY AND CONCLUSION
	ACKNOWLEDGEMENTS
	DATA AVAILABILITY STATEMENT
	ORCID
	REFERENCESC
onditions (https://onlinelibrary.w
iley.com
/term
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MUKHERJEE ET AL. 3 of 16
smoking, and stress during ART are associated with RIF. Along with
these, thrombophilia has been associatedwith RIF. Studies have shown
that women with inherited thrombophilias such as methylenetetrahy-
drofolate reductase (MTHFR) deficiency, factor V Leiden, prothrombin
deficiency, and antithrombin III deficiency have a higher risk of RIF
in comparison with controls without these genetic changes.19 It has
also been shown that polymorphisms in genes such as SERPINE1which
encodes plasminogen activator inhibitor type 1 and inhibits fibrinolysis
have been linked to RIF.20 Genital infections such as Chlamydia, female
genital tuberculosis, and chronic endometritis are also associated with
RIF.21–24
Intriguingly RIF is associatedwith systemicmetabolic alterations. In
a study on women with RIF and controls, serum levels of Nitric oxide
synthase (NOS), valine, adipic acid, l-lysine, creatine, ornithine, glyc-
erol, d-glucose, and urea were lower in women with RIF.25 Glucose
metabolism in the endometrium is essential for embryo implantation
and altered GLUTs expression impairs decidualization.26 Increased
NOS activity is reported at the site of embryo implantation and mice
knockout for endothelial NOS have lowered uterine blood flow and
altered spiral artery elongation.27–29 It will be interesting to see if
these metabolites are also altered in the endometrium of women
with RIF. Beyond the factors discussed above, endometrial defects
and immunologic disbalance are thought to be major contributors
to RIF.
1.2 RIF is an endometrial defect
The process of embryo implantation is comparable to the process of
seed germination where the health of the seed (the embryo) and the
component of soil (the endometrium) are determinants of success.
While a healthy embryo is a prerequisite for successful implantation,
a healthy endometrium is also crucial for successful implantation, any
defects in either of the two compartments can cause RIF.1,30 Anatom-
ical pathologies such as polyps, myomas, adhesions, and reduced
endometrial thickness is associated with RIF.31
Since high-quality euploid embryos fail to implant in womenwith an
anatomically normal uterus, it is hypothesized that RIFmay be a defect
in the endometrium. The observation that healthy embryos failed to
implant in the endometrium of nearly healthy women fueled the idea
that RIF could be due to a failure to synchronize the embryo transfer
time with a receptive endometrium.32 Indeed, a proportion of women
withRIF have a displaced timing ofwindowof receptivity,However, the
numbers of women with RIF having a displaced window of receptivity
are not significantly different as compared to women without RIF.33
Not surprisingly, personalized embryo transfer guided by the endome-
trial receptivity test did not significantly improve outcomes in women
with RIF as compared to controls.34–35 Thus, displacement in the tim-
ing of endometrial receptivity may not be a causative factor for RIF.
However, disruption or pathology in the attainment of receptivity itself
cannot be ruled out.36 Indeed, genes induced by embryonic stimuli and
those associated with cell adhesion are disrupted in the endometrium
of womenwith RIF.37,38
To understand endometrial defects in women RIF, transcriptome
profiles of the endometrium from women with and without RIF have
been compared. The results reveal altered expression of several coding
genes,39–41 long noncoding RNA,42 andmiRNAs43 in the endometrium
of women with RIF. Altered levels of several mRNA and miRNAs
are also observed in extracellular vesicles isolated from women with
RIF.30,41,44–46 Network analysis of the transcriptome data has identi-
fied disruptions in circadian clock pathways, Hippo signaling pathway,
renin-angiotensin system pathway, cell adhesion, regulation of cell
motility, and cellular response to vascular endothelial growth factor
stimulus, and several others.39,40 Functionally these alterations can
affect endometrial receptivity, lead to defective decidualization, and
alterations in endometrial immune profiles in the endometrium of
women with RIF.7,47–49 Thus, it is becoming increasingly clear that RIF
is an endometrial defect.
1.3 RIF and defects in the immune system
Females have a normal functioning immune system that completes
with a semiallogenic conceptus during pregnancy. Thus, the acceptance
and tolerance of the semiallogenic conceptus mandates the transfor-
mation of thematernal immune system.While the precisemechanisms
by which the embryo is protected from the maternal immune assault
are poorly understood, a picture that has emerged out of the stud-
ies suggests a dramatic transformation of multiple immune cells in
the peripheral blood and endometrium. At the implantation stage,
the immune cells mainly the uterine natural killer (NK) cells, T cells,
dendritic cells, and macrophages make up half of the total number
of endometrial cells, almost 30% of the total number of endometrial
cells in early pregnancy are immune cells. The role of immune cells
NK cells, dendritic cells, macrophages, and T cell subsets in endome-
trial functions and ensuring pregnancy iswell established.50 TheCD4+
CD25+ FOXP3+ regulatory T cells (T-regs) are important in mediat-
ing maternal immune tolerance to the allogeneic fetus during embryo
implantation and early pregnancy. Although the CD8+ T cell numbers
do not change in the human endometrium through themenstrual cycle,
in the endometriumCD8+T cytotoxicity is suppressed in the secretory
phase during implantation.51
In general, pregnancy is associated with Th2 dominance, and Th1
immune response is associated with pregnancy losses.52,53 T helper 17
(Th17) cells differentiate from naive T-cells and produce the cytokine
IL-17 that has an important role in the feto-maternal interface. During
implantation, the Th17 cells are present in decidua and their numbers
increase in theperipheral blood in the first trimester. Thedecidual stro-
mal cells recruit peripheral Th17 cells into the decidua for proliferation
and invasion of human trophoblast cells.54
In the human endometrium, the CD45RA+ B cells are found all
the time during the cycle, they are considered rare lymphocyte sub-
populations in the uterus with a median of 2% in preimplantation
uterine tissue and 5% in the decidua.55 In the implantation stage
uterus, the majority are naïve or memory B cells, with few plasma
cells and as compared to the circulating B cells the endometrial B
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iley.com
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4 of 16 MUKHERJEE ET AL.
cells have an activated phenotype.55 Macrophages are also found dur-
ing all phases of the menstrual cycle their numbers increase before
implantation.50,56 Of the dendritic cells, themature dendritic cell num-
bers are significantly lower than that of immature dendritic cells,
during implantation and bothmacrophages and DCs accumulate in the
decidua.57,58 Additionally, mast cells are perpetually found during the
menstrual cycle, and mast cell activation is most pronounced immedi-
ately before menstruation59,60 However, the functions of mast cells in
the endometrium and pregnancy are yet unclear.
Investigating the disturbances in the local uterine immune environ-
ment is the key to understanding the molecular mechanisms that are
associated with pregnancy and the underpinnings of RIF. Underactive
immune cell activity mayfail to create the necessary implantation
reaction while hyperactive immune cells may lead to rejection of the
embryo. Several studies have associated altered endometrial/decidual
immune profiles in women experiencing first-trimester pregnancy
losses. There is a growing body of evidence reporting the defects in
the immune signaling in the endometrium and peripheral blood of
women with RIF.61,62 Some of the commonly known altered immune
factors associated with RIF include changes in T helper 1: T helper
2 (Th1:Th2) response, altered Natural Killer (NK) cell levels,5 and
altered macrophage cell numbers.63 Also, the levels of cytokines,
chemokines, and their receptors such as IL6, IFNG, IL17A, IL23A,
IFNA1, IFNB1, CD40 L, CCR4, CCR5, CCR6, CXR3, CCL2, IL2, TLR4,
IRF3, STAT3, RAG1, IFNAR1,64 are different in RIFwomen than in con-
trols. Whether these are causative of RIF or just a causal association is
yet to be established.
Programmed cell death protein 1 (PD-1, CD279) and its ligand PD-
L1 (CD274) are immune checkpoint-pathway that play a significant
role in maintaining the immune-suppressed microenvironment. Stud-
ies have shown that there are dynamic alterations of PD-1/PD-L1 on
peripheral blood anddecidual T cells during pregnancy suggesting their
involvement in regulatingmaternal immune tolerance.65
Human leukocyte antigen (HLA)-G is a key for the immunomodula-
tory system, especially during pregnancy. HLA-G is mainly expressed
by the trophoblast cells and regulates its interactions with different
immune cells. Several studies have shown that levels of soluble serum
levels of HLA-G and some polymorphisms of the HLA-G gene are
associated with RIF.66,67 Besides, a high rate of human leukocyte
antigen (HLA) gene loci sharing (HLA-A, B, C, DR, DQ) in couples has
been associated with RIF.67–69 It is believed that sharing of more than
one HLA loci may lead to immune responses that may act to inhibit
the development of maternal immune-tolerant response toward the
semiallogeneic embryo leading to implantation failure.
1.3.1 Changes in peripheral blood immune profiles
of women with RIF
For ease of accessibility, immunophenotyping of the peripheral blood
leukocytes is the most used tool for the evaluation of the immune
status of women susceptible to RIF (Table 1, Figure 2). Differ-
ences in the levels of immune cell types like NKG2D+ γδ-T cells,
NKp30/TIGIT-expressing cytotoxic NK cells Myeloid-Derived Sup-
pressor Cells (MDSC), regulatory T (Treg) cells, NK cells subtypes, and
subpopulations of CD8+ T cells (IFN-γ+ and TNF-α+ CD8+ T cells)
have been observed in RIF vs. fertile controls.70–75 Lower frequency
of ILT4+ DCs was observed in peripheral blood lymphocytes in RIF
patients compared to the numbers in the fertile control group.76 The
study suggested that higher numbers of ILT4 expressing DC cells were
required formounting immune tolerant responses in themother during
pregnancy via induction of Foxp3+ Treg cells, a process that was found
to be impaired in womenwith RIF.
As the PD-1/PD-L1 pathway plays an essential role in immune
tolerance,65 the roles in recurrent pregnancy losses have been well
reported,77 but their roles in mediating RIF is however not investi-
gated. A single study has reported low levels of PD-L1 transcripts
in B cells from women with RIF as compared to healthy controls,78
indicating a possible involvement of the PD-1/PD-L1 pathway in RIF
pathogenesis. Further studies are needed to clarify the role of PD-1
pathway and implantation failure.
A considerable number of studies have indicated quantitative
and/or functional abnormalities of NK cells to be associated with
recurrent implantation failures.73,75,79,80 In one such study, a lower
percentage of CD56+CD16 CD56+CD16+ NK cell populations mea-
sured in the early follicular phase was reported to be an indicator of
reduced pregnancy and implantation success rates in RIF patients.75
A higher percentage of NK cells were observed in peripheral blood
lymphocytes of women with idiopathic RIF in the midluteal phase
compared to fertile controls,79 thereby suggesting that testing for
blood NK cells might be a useful strategy for defining women with
idiopathic RIF. Different subpopulations of NK cells are known to dif-
fer in their cytotoxic functions based on the relative expression of
CD16/CD56 markers. For instance, the cytotoxic activity of CD56dim
NK cells is significantly higher than that of CD56bright NK cells while
CD57expression onNKcells correlateswith high cytolytic potential.81
So different investigators have tried to evaluate whether testing dif-
ferent subpopulations of NK cells in peripheral blood can help in
better diagnosis of RIF patients. In a study by Jiang et al. 2017,82
the percentage of CD45+CD56bright NK cells in CD45+ lympho-
cytes was found to be significantly lower in women with RIF than
fertile controls. The authors further observed that the ratio of cells
expressing CD57+/CD56+ was higher in RIF patients compared to
fertile controls indicative of activation of high cytolytic functions of
NK cells in women with RIF. In another study by Fukui et al. 200874
that aimed to profile the cytokine expression by different peripheral
blood NK cell subsets in RIF patients, a significantly higher percent-
age of CD56bright/interferon-gamma(+)/TNF-alpha (+) expressing NK
cells was observed in women with recurrent implantation failures as
compared to fertile controls. Also, the TNF-alpha/GM-CSF express-
ing CD56 bright cell ratio was significantly higher in women with RIF
than in controls. The authors suggested that profiling Tumor necrosis
factor-alpha/GM-CSF expressing CD56bright NK cell population to be a
useful strategy for the diagnosis of RIF patients. Although recent stud-
ies (Table 1) indicate different immune cell parameters to be associated
with different types of RIF patients,41,69–74 evaluating the immune cell
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iley.com
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MUKHERJEE ET AL. 5 of 16
T
A
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gn
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tl
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cr
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se
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in
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IF
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it
h
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it
am
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D
in
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ff
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ie
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cy
vs
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o
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o
l(
ii)
C
D
6
8
+
m
ac
ro
p
h
ag
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n
u
m
b
er
s
si
gn
if
ic
an
tl
y
h
ig
h
er
in
R
IF
p
at
ie
n
ts
w
it
h
vi
ta
m
in
D
in
su
ff
ic
ie
n
cy
vs
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o
n
tr
o
l
M
ar
ro
n
et
al
8
0
E
n
d
o
m
et
ri
al
b
io
p
si
es
N
K
ce
ll,
T
ce
lls
u
N
K
(C
D
1
6
-,
C
D
5
6
b
ri
gh
t)
;p
N
K
ce
lls
(C
D
3
−
,C
D
1
6
+
,C
D
5
6
d
im
),
N
K
-T
(C
D
3
+
,C
D
1
6
−
,C
D
5
6
d
im
),
C
D
8
+
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ce
lls
1
7
8
3
5
Si
gn
if
ic
an
t
in
cr
ea
se
(P
1
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n
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%
o
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IF
p
at
ie
n
t
h
av
in
g
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ig
h
im
m
u
n
e
ac
ti
va
ti
o
n
si
gn
at
u
re
 16000897, 2023, 2, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1111/aji.13563 by U
niv of Sao Paulo - B
razil, W
iley O
nline L
ibrary on [26/02/2023]. See the T
erm
s and C
onditions (https://onlinelibrary.w
iley.com
/term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the applicable C
reative C
om
m
ons L
icense
MUKHERJEE ET AL. 7 of 16
F IGURE 2 Potential candidates of altered immune responses in RIF patients. Red arrow: increased number/expression; Green arrow:
decrease in cell number/expression of cytokines; RIF, recurrent implantation failure; Treg, Regulatory T cells; uNK, uterine NK cells, pNK,
peripheral NK cells. Figure prepared in BioRender.com
types seems to be a useful tool for screening specific subgroups of RIF
patients.
1.3.2 Changes in tissue-resident immune cells in
women with RIF
The utility of evaluating immune cell distribution in peripheral blood
as a reliable parameter to identify patients susceptible to RIF has
been contradicted by many investigators as the immune parameters
measured in blood could be reflective of other additional conditions
and may not necessarily be relevant for RIF.83 Further, the uterine
immune cells have gene signatures distinct from their peripheral blood
counterparts. In contrast to the majority of peripheral blood NK cells,
uterine NK cells are CD56brightCD16−, while NK cells found in periph-
eral blood are mainly CD56dimCD16+83,84 In the T cell subsets, the
expressionofHLADRandCD69 is higher in endometrial CD3+, CD4+,
CD8+T cells as compared to the T cells in the peripheral blood.84 Thus,
relying solely on changes in the peripheral blood immune profiles may
not be a reliable way to address the problems in RIF.
Several studies (Table 1) have associated altered immune cell type
numbers in uterine tissueswith the occurrence of RIF (Figure 2). These
mainly include alterations in uterine natural killer (uNK) cells, T cells,
Dendritic cells, and Foxp3+ Treg cells.79,81–76 Some of these stud-
ies have tried to compare the utility of testing immune cell profiles
in both blood and uterine tissue biopsies from the same RIF patients
to understand their effectiveness as a tool to define and diagnose
RIF patients76,79 In one such study by Liu et al. 2018,76 a significant
decrease in ILT4-expressing DCs was observed in the endometrial tis-
sue of patients with RIF compared to fertile controls. Interestingly, in
the same study, a significant correlation was reported between the
number of ILT4+ cells (monocytes/macrophages/DCs) and immuno-
suppressive Foxp3+Tregs in endometrial tissues from healthy fertile
controls, but not in patients with RIF indicating that disbalance in
ILT4+ DCs could lead to disruption of immunosuppressive responses
in endometrium leading to implantation failure. Also, a similar trend
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8 of 16 MUKHERJEE ET AL.
of decreased ILT4-expressing DCs was observed in peripheral blood
lymphocytes from the same RIF patients compared to fertile controls,
although slightly more expansion of ILT4+ DCs was observed in the
endometrium compared to numbers in peripheral blood lymphocytes
of the RIF patients. These observations suggest that testing for the
ILT4+DCs in whole blood could serve as a noninvasive technique to
evaluate the ILT4+DCs mediated immune signature in endometrial
tissue of women susceptible to RIF. In another study by Ding et al
2017,87 the authors observed a significant increase in the percentage
of CTLA4+ cells in the endometrium of RIF patients compared to that
of fertile controls indicating that this marker could play a negative role
in influencing the immune responses.
Several studies have correlated alteredNK cell numbers in endome-
trial tissues of RIF patients compared to fertile controls (Table 1).
A significantly higher frequency of endometrial CD56+ cells was
reported in the midluteal phase of women with idiopathic RIF79 than
in controls; there is a positive correlation between blood and endome-
trial CD56 cell numbers in women with idiopathic RIF.79 Thus, testing
for CD56+ NK cells could be a useful strategy for screening cases
of women with idiopathic RIF by evaluating blood/endometrial tissue
samples collected in the midluteal phase of the reproductive cycle.
More such studies are needed to validate this screening strategy for
idiopathic RIF patients. In another comprehensive study on NK and
T cell profiling of endometrial tissue samples from women with a his-
tory of RIF and fertile controls, changes in immune cell milieu specific
to RIF patients were identified that included a significant increase
in the number of CD56+ cells, uterine NK cells (CD16-, CD56bright),
and peripheral NK cells (pNK: CD3−, CD16+, CD56dim), with signifi-
cantly lowered CD8+ T cells and NK-T (CD3+, CD16−, CD56dim) cell
numbers in endometrial tissues of RIF patients compared to fertile
women.83,85 In contrast to the findings in this study, another CD56
evaluating CD56+ NK cells in endometrial tissues on days 21–24 of
the patients’ menstrual cycle showed a significant decrease in CD56+
NK cells in endometria of women with RIF as compared to that in the
fertile controls.88 Although this study did not analyze the CD56bright
(uNK) and CD56dim expressing (peripheral NK) subpopulations of NK
cells. These studies indicate that the precise roles of NK cells toward
contributions to RIF are unclear.
From these studies, it appears that the immune profile of endome-
trial tissue differs in different RIF patients in terms of differences in
NK cell, T cell, and macrophage numbers. Thus, diagnosis and treat-
ment based on the immune activation status of endometrial tissue
from individual RIF patients could be a better strategy for improv-
ing implantation rates and successful outcomes of pregnancy. More
studies are warranted to understand the effectiveness of screening
different immune cell types as an etiological factor for RIF and for
selecting appropriate therapy for RIF patients.
1.3.3 Cytokines altered in RIF
Cytokines are key modulators of immune functions. Produced by a
plethora of cells, the immune cells are considered the key source
of many cytokines. Several studies (Table 2) have discussed the
relevance of cytokines released by different cell types during preg-
nancy, their role in embryo development, and the continuation of
pregnancy.89–92 Dysregulation of cytokine profiles is associated with
different pregnancy-associated disorders including RIF.93,94 In one
such study,93 compared to fertile women, the ratio of IFN-γ:IL-4 and
TNF-α:IL-4 were higher in serum of women with RIF. This data sug-
gested that a Th1 bias exists in RIF patients that could lead to an
implantation failure in these patients. In another study,95 high levels
of IL-1β cytokine were observed in the uterine fluid of RIF patients
compared to fertile controls. Increased expression of proinflamma-
tory cytokines like resistin, leptin, sTNF-R, MPO, and IL-22 have been
observed in serum samples of RIF patients compared to fertile women.
Downregulation in the levels TGF-β, IL19, IL4, CXCL13, CCL5, and the
cytokine receptors IL4R is reported in the endometriumofRIF patients
compared to fertile control.86 However, in most studies the sample
size is limited (Table 2). The expression of these cytokines should be
tested inmoreRIFpatients tounderstand their importanceaspotential
biomarkers for diagnosing/ treating RIF patients.
1.3.4 Molecular mechanisms of altered maternal
immune response in RIF
Different studies have tried to elucidate the underlying molecular
mechanisms by which altered immunological status of endometrial tis-
sue causesRIF.Disbalance in different subsets of immune cell types are
identified in RIF patients (Figure 2) It is known that ILT4 expression on
dendritic cells and HLA-G/ILT4 pathway inductionmakes the dendritic
cells tolerogenic and suppresses cytotoxicity of NK and T cells.96 Inter-
estingly, in peripheral blood samples, the percentage of ILT4+dendritic
cells was significantly higher in healthy fertile controls, compared with
RIF. Even in the endometrial tissues, the percentage of ILT4+ dendritic
cells is higher in fertile controls as compared to women with RIF.76
Thus, it appears that theexpansionof ILT4+dendritic cellsmayoccur in
healthy fertile controls to facilitatematernal immune tolerance toward
developing embryos that are impaired in patients with RIF.
Besides, several biomolecules have been identified to be necessary
for endometrial receptivity, embryo implantation, and maintenance
of tolerogenic immune response necessary for positive pregnancy
outcomes.97,98 Interleukins, such as interleukin-6 (IL-6), leukemia
inhibitory factor (LIF), IL-1α, IL-1β IL-Ira, and IL-1 cytokines, secretary
factors like Glycodelin-A, angiogenic factors like Vascular endothe-
lial growth factor (VEGF), and placental growth factor (PlGF) have
been suggested to be crucial for embryo implantation and develop-
ment and immune-modulation at the feto-maternal interface.90,97 Any
perturbations in the expression of these cytokines and growth fac-
tors could lead to altered signaling responses leading to alterations in
downstream signaling pathways leading to RIF. For instance, in a uter-
ine transcriptome study of patients with RIF, LIF-(JAK)-STAT signaling
pathway was found to be one of the most significantly downregulated
pathways.90,99 Indeed, the mRNA levels of inflammatory cytokines
IL6, IL2, IFNG, IL17A, IL23A, IFNA1, IFNB1, and their modulators like
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MUKHERJEE ET AL. 9 of 16
TABLE 2 Altered cytokine expression in RIF patient’s vs. fertile women
Author Cytokine
RIF
(N)
Controls
(N) Tissue Method Expression in patient vs control
Guo et al,98 TGFB1 22 18 Endometrial
biopsies
qRTPCR,Western
blot
TGFBmRNA and protein Low in RIF
vs. ctrl
Choi et al,99 CXCL13 15 7 Endometrial
biopsies
mRNA-microarray Expression of CXCL13 significantly
downregulated in endometrial
tissue in 75% of RIF patients
compared to fertile controls
Jiang et al,72 CCL5, TGF-β 26 30 Serum ELISA Reduced expression of CCL5 and
TGF-β in RIF vs. ctrl
Galgani et al,86 resistin, leptin,
sTNF-R,MPO
and IL-22
15 13 Serum ELISA Reduced expression of resistin,
leptin, sTNF-R,MPO, and IL-22 in
serum of RIF vs. ctrl
CD40 L, CCR4, CCR5, CCR6, CXR3, CCL2, TLR4, IRF3, STAT3, RAG1,
IFNAR1 are reported to besignificantly higher in the endometrium
of women with RIF as compared to controls,64 suggesting that there
is an inflammatory response where the NK cells, Th17 signaling path-
way, and TLR signaling pathway are activated. With such an activated
immune response, the immune tolerance in the uterinemilieuwould be
altered resulting in RIF.
1.4 Endometrial epigenetic defect as a cause
of local immune disturbances in RIF
A study37 compared the methylome of the endometrium from women
with RIF and controls undergoing controlled ovarian stimulation
and identified 448 differentially methylated loci of which 242 were
hypomethylated whereas 206 were hypermethylated. Interestingly,
the most significant biological processes associated with the differen-
tially methylated genes were associated with antigen processing and
presentation including endogenous and exogenous peptide presenta-
tion withMHC class I andMHC class II. The pathways enriched belong
to those involved in phagosomes, allograft rejection, graft vs host
disease, antigen presentation, and inflammatory bowel disease. More-
over, the differentially methylated loci at the transcription initiation
sites were predominantly enriched for genes involved in immunologi-
cal functions like antigenprocessing andpresentation, immunoglobulin
production, IL-10 and IL-4 regulation, T-helper cell immune response,
and inflammatory response comprising of HLA-DRB1 and HLA-DRB5
genes.37 When overlayed with the gene expression profile most of the
downregulated genes had roles in innate immune and cellular defense
response, regulation of T-cell proliferation, chemokine-mediated sig-
nalingpathway, regulationof complement activation, acute andchronic
inflammatory response, and cell chemotaxis.37 The antigen processing
and presentation pathways, Natural killer cell-mediated cytotoxicity
pathways, and complement and coagulation cascades were the top
three pathways enriched among the downregulated genes. These
results imply that an epigenetic defect in the endometrium could be
the cause of local immune disturbances in the endometrium leading
to RIF.
Theabove studies demarcate thatwomenwithRIFhaveadisruption
in immune profiles, in the peripheral blood and the tissues. However,
there is no unifying immune signature that can discern RIF from the
controls. It is yet not clear if RIF is a sequel of a hyperactivated or
underactive immune response. This is because only limited aspects (or
arms) of the immune system are addressed in each of the studies and
comprehensive profiling of all the immune cells and their subtypes is
lacking.
1.5 Immunotherapies in RIF
The fact that RIF is a defect associated with immunomodulation in the
endometrium prompted researchers to try different immunomodula-
tors for improving the success rates of ART in women with RIF. These
include prednisolone, Intravenous Immunoglobulin Therapy (IVIg),
Tacrolimus, intentional endometrial injury, subcutaneous or intralumi-
nal administration of Granulocyte-colony stimulating factor (G-CSF),
intrauterine autologous platelet-rich plasma (PRP) infusion, intra-
venous intralipid, intrauterine human chorionic gonadotropin (hCG)
injection, low-molecular-weight heparin (LMWH), aspirin and several
others.
Lymphocyte immunotherapy (LIT) with paternal or third-party
lymphocytes has been employed by several investigators since long as
a possible immunotherapy treatment for breaking the uterine immune
response toward allogenic fetus for improving clinical outcomes of
pregnancy in women with recurrent implantation failures or recurrent
miscarriages. Mechanisms such as reduction in the Th1/Th2 ratios
and their cytokines production, altered antipaternal cytotoxic anti-
bodies, enhanced expression of progesterone-induced blocking factor,
anti-idiotypic antibodies, and mixed lymphocyte reaction blocking
antibodies are proposed to be involved in improving outcomes of LIT in
RIF and recurrent pregnancy losses.100 However, controversies exist
on the beneficial effect of LIT treatment in patients with RIF and recur-
rent pregnancy loss.101–102,103–104 Thus, the roles of LIT in improving
clinical outcomes in RIF needmore systematic investigations.
The outcomes in most therapeutic interventions are from obser-
vation studies with very few from randomized controlled trials and
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10 of 16 MUKHERJEE ET AL.
there is a strong element of bias. A detailed meta-analysis of most of
these interventions was reported recently105,106 and hence will not
be detailed here. As per the results, there is a beneficial effect of
intrauterine infusion of peripheral blood mononuclear cells, subcuta-
neous G-CSF, intrauterine platelet-rich plasma on clinical pregnancy
and/or live birth rates in women with RIF. Observational studies
have also demonstrated a positive effect of IVIg and intrauterine
hCG infusion on clinical pregnancy and/or live birth rates in women
with RIF. However, in most studies including meta-analysis, there
is only a marginal improvement in the outcomes and most women
do not seem to be having a beneficial effect. These results imply
that all women with RIF may not have the same abnormality in the
immune milieu in their endometrium and RIF may be a heteroge-
neous disorder with only a subset of women having an immunological
disturbance.
1.6 Immune profiling and personalized therapies
Considering that RIF is a heterogeneous condition, and one size may
not fit all has prompted investigators to undertake personalized thera-
pies based on the immune status of the patient. To date, several clinical
trials are reporting personalized immunotherapy in women with RIF.
These are based on the immune dysregulation in the peripheral blood
or the endometrium. (Figure 2).
1.7 Personalized therapy based on circulating
Th1/Th2 ratios
As T cells are prime mediators of the adaptive immune response and
elevated Th1 response is observed in women with RIF, it was hypoth-
esized that women with RIF due to elevated Th1/Th2 ratio could be
benefit from immunosuppressive therapy (Figure 3). Tacrolimus is an
immune-suppressive drug that reduces the alloreactivity of a recipi-
ent’s immune system and subdues the immunological rejection of an
allograft by inhibiting lymphocyte proliferation, cytotoxic T cell genera-
tion, and the production of T cell-derived cytokines like IL-2 and IFN-γ.
Thus, Tacrolimus is considered to be a possible treatment for women
with elevated Th1/Th2 ratio. Three studies by the same group107–109
administered 1-3 mg of Tacrolimus daily starting from 2 days before
embryo transfer and continued until the day of the pregnancy test
completing a 16-day treatment regimen. They reported that 40% of
womenwith RIF had elevated Th1/Th2 cell ratios (based on the ratio of
peripheral IFN-γ(+)/IL-4(+) CD4+ cells), While tacrolimus treatment
per se did not improve the pregnancy rates, the authors observed that
post-therapy a proportion of women responded to the treatment and
Th1 levels reduced. In the responders, the authors observed a higher
clinical pregnancy rate (48.8%) over the nonresponders (33.3%). How-
ever, the differences were not statistically significant suggesting that
the Th1/Th2 imbalance in the peripheral blood may not be a cause of
RIF or at least correcting it does not improve pregnancy outcomes in
ART. Also, caution must be exerted while administering Tacrolimus as
it is known to cross the placental barrier and has teratogenic effects in
animals.
A reason for aberrant T helper cell functions and/or numbers ratio in
somewomenwith RIF isthought to be due to chronic endometritis.110
Thus, suppressing the T cell function using tacrolimus might not help
in improving pregnancy rates until the primary defect is corrected, and
antibiotics are proposed to be the first line of treatment.111 However,
correctionofT cell responsespostantibiotic therapy inwomenwithRIF
has not been reported.
1.8 Personalized therapy based on circulating
NK cell numbers
Studies have reported that the endometrium of women with RIF
has too few or too many NK cells resulting in a disbalance in
cytokine production causing RIF. In a retrospective, observational
study of 428 women (217 with recurrent pregnancy loss and 211
with RIF), the authors analyzed NK cells (CD3CD56brightCD16-,
CD3CD56+CD16+) and NKT-like cells (CD3+CD56+) in peripheral
blood by flow cytometry.112 By this method, 18% of their patients
(combined RIF and RPL) had dysregulated immune profiles. The
patients with the expansion of blood NK Cells (> 12% of total lympho-
cytes) or expansion ofNKT like cells (> 10%of total lymphocytes) were
recommended Intravenous Immunoglobulin Therapy (IVIg). In the RIF
group with NK cell expansion and IVIg therapy, the clinical pregnancy
rate was 93.8% while only 26.2% of women in the control group (with
NK cell expansion without IVIg). The live birth rate was 80.0% in the
personalized treatment arm and 17.9%, in the untreated arm. Similar
success rates were reported in other studies where women with
NK cell expansion (CD56+ cells) in peripheral blood were given IVIg
therapy.113,114 A meta-analysis was conducted that demonstrated
that personalized IVIg therapy based on immunological disturbances
increases pregnancy rate in the cohort and cross-sectional studies, it
also results in a high live birth rate in both cohort and cross-sectional
studies. However, we must await prospective, randomized controlled
trials of sufficient size to ensure if this form of personalized medicine
is useful in womenwith RIF.115
1.9 Personalized treatments for RIF based on the
immune status of the endometrium
Presently, there is no unifying immune signature that can discern RIF
from the controls; it is yet not clear if RIF is a sequel of a hyperac-
tivated or underactive immune response. At present, it appears that
some women with RIF may have a hypo-immune response while oth-
ers may have a hyperimmune response. These observations prompted
researchers to investigate if immunological profiling of the uterus may
help to discriminate these two conditions and treat the women based
on the hypo and hyperactive immune responses. Thus, in women with
altered NK cell numbers IVIg therapy could prove to be beneficial
(Figure 3).
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MUKHERJEE ET AL. 11 of 16
F IGURE 3 Personalized therapy for RIF patients based on endometrial biopsy. RIF, recurrent implantation failure; ART, assisted reproductive
technology; OPTIMUM,OPtimization of Thyroid function, IMmunity, and UterineMilieu100
A study by Lédée et al., 2016,116 analyzed the endometrial immune
profile of 394 women with a history of RIF. They measured the
expression levels of IL-15/Fn-14 mRNA as a marker for uNK cell
activation/maturation, IL-18/TWEAK mRNA levels as a measure of
Th1/Th2 balance, and numbers of CD56+ NK cells. Based on these
markers the women could be classified into three categories. Accord-
ing to their cutoffs, the endometrium in 56% of women was in an
overactivation state, 25% was in an underactivated state, and in 18%
there was no dysregulation. The authors then administered a therapy
based on endometrial profiles. The women in the hyperactivated arm
were given progesterone support and treated with prednisolone, vita-
min E, and intralipid (to suppress inflammation and oxidative stress),
and exposure to seminal plasma was curtailed by avoiding sexual
intercourse. Prednisolone and Intralipids are unknown to improve
pregnancy rates in a subset of RIF patients.117–119 In the group that
had under activated endometrium profile, the endometrial scratching
was performed in the midluteal phase of the cycle preceding the
embryo transfer cycle, there was supplementation of the luteal phase
with human chorionic gonadotrophin (hCG), and sexual intercourse
was suggested for exposure to seminal plasma (to induce inflammation)
Thewomenwithout any dysregulation of the immune profileswere not
treated and followed conventional protocols for assisted reproduction.
The study showed that these therapies normalized the endometrial
immune profiles in half the patients. The personalized therapy led to
live birth (after ART) in 37% of patients in the hyperactivated arm,
46.5% in the underactivated arm while those left untreated and with-
out any dysregulated endometrium the live birth rates were only 19%.
The miscarriage rates in the three groups were comparable (∼8-9%).
The authors concluded that the personalized therapy led to live birth
in 40% of women diagnosedwith a form of immune dysregulation.
Encouraged by these results, the authors recently published their
data on additional 1738 infertile women.120 Using the same diag-
nostic and personalized therapy strategy, the authors reported that
17% of infertile patients had no immune dysregulation, 28% had an
immune underactivation, 45% had an immune overactivation. They
also uncovered a group of 10% of women who had amixed profile. The
results revealed that the pregnancy rates were two-fold higher if an
endometrial dysregulation was diagnosed and the personalized plan
applied, compared to the patients with an apparent balanced immune
profile. The group recently published a third study121 in 104 patients
with recurrent pregnancy loss (two ormore consecutivemiscarriages).
Independent of age and AMH level, personalized therapy for dysregu-
lated immune profiles increased the live birth rates by almost five-fold
in the women recurrent pregnancy loss.
The results from these studies are very encouraging and suggest
that one solution will not fit all in women experiencing RIF. Not sur-
prisingly endometrial scratching, the administration of corticosteroids,
or any type of immunotherapy failed to find any beneficial effects in
women with RIF. In a prospective randomized control trial on women
with RIF, unguided endometrial scratching also does not have any
beneficial effects on clinical pregnancy rates, implantation rate, abor-
tion rate, ongoing pregnancy rate, and cumulative pregnancy rates.122
These results are however not surprising as based on the available data
only 20–30% of the women with RIF would benefit from these ther-
apies. While we await the results of their randomized controlled trial
on endometrial immune profiling-based personalized therapy (as per
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12 of 16 MUKHERJEE ET AL.
the authors, the trial should have been completed by the end of 2021).
It must be borne in mind that these studies are not blinded and come
from a single group and a single center. Independent confirmations are
necessary till we apply these personalized therapy protocols in ART.
1.10 OPtimization of thyroid function, immunity,
and uterine milieu (OPTIMUM) treatment strategy
for RIF
While studies have indicated that dysfunctions in the endometriumlead to RIF in most instances, the underlying cause in these patients
is multifactorial (Figure 1). Altered thyroid hormone and Vitamin
D status, altered immunological milieu (systemic as well as tissue
level), and uterine issues are associated with RIF.111 Considering the
multifactorial nature of RIF an OPTIMUM management modality
is proposed. This strategy includes screening the patient by hys-
teroscopy, endometrial CD138 immunostaining, intrauterine bacterial
culture; serum 25-hydroxyvitamin D3, TSH, and thyroid peroxidase
antibody (TPOAb) levels, numbers of interferon-γ-producing Th cell
(Th1 cell), IL-4-producing Th cell (Th2 cell), and thrombophilia test-
ing. Based on these, a correction treatment plan is to be executed
where the primary problem is treated, e.g., antibiotic therapy for
endometritis, vitamin D supplementation, immunosuppressants for
Th1 high patients so on and so forth. In this study, 59 patients were in
the control arm and 88 patients underwent personalized OPTIMUM.
While the clinical pregnancy rate was higher but not significantly
different between the OPTIMUM group and the control group, the
ongoing pregnancy rate was significantly higher and miscarriage rates
were significantly lower in the OPTIMUM group as compared to the
control group; a higher proportion of patients in the OPTIMUM group
also reported spontaneous pregnancies.103 However, these are very
early studies and need independent validations.
1.11 Limitations and pitfalls
From the above discussions, it is evident that immunological distur-
bances are a cause of RIF in a subset ofwomen and immunomodulators
are promising candidates for therapy. While these results are tanta-
lizing, there are several pitfalls and limitations to the work described
herein. In most studies, the definition of RIF is highly variable. Some
authors have defined RIF as more than two failed transfers, others
have considered > 3 failed embryo transfers. Further, some authors
have considered a failure to achieve clinical pregnancy as RIF while
others have considered a failure to achieve biochemical pregnancy as
an inclusion criterion. Second, in most studies, it is not clear if women
with other causes of RIF are screened and excluded. Most therapeutic
intervention studies arenotblinded randomizedcontrolled trials, there
is an element of bias that cannot be overlooked. The control group
across the studies is highly variable. While some studies have consid-
ered fertile women as controls, others have compared the outcomes
in RIF women with and without successful pregnancy outcomes thus
making a comparison across studies difficult. Unfortunately, in most
clinical studies, the rates of live births are not clearly reported, and
this impairs the convincingness of the analysis. Thus, considering these
limitations, and the absence of good randomized multicentric blinded
trials, the results at best are considered preliminary and the therapies
experimental in nature. Indeed, scientific bodies like American College
ofObstetricians andGynecologists (ACOG), Royal College ofObstetri-
cians and Gynaecologists (RCOG), American Society for Reproductive
Medicine (ASRM), American Society for Reproductive Immunology, or
Indian Societry of Assisted Reproduction (ISAR) have not yet endorsed
the use of such "add-on" immunotherapy interventions in assisted
reproduction.
2 SUMMARY AND CONCLUSION
RIF is a complex problem with a wide variety of etiologies (Figure 1).
Among these, changes in peripheral blood and endometrial immune
cell numbers and phenotypes are an underlying cause in a subset of
women. Based on immune profiling, the women could have a hyper-
activated immune state, a hypoactivated immune state, or even a
mixed response (Figure 2). These alterations could cause a disbal-
ance in the immune response required for implantation resulting in
implantation failure. Since immunedisbalance are treatable conditions,
these women are excellent candidates for immunotherapies. However,
there will not be a singular treatment option, but this will depend
on the etiology of the problem (Figure 3). Perhaps the best but most
complex solution will be personalized medicine, where each patient
depending on her immune profile will receive therapy. However, we
need to evolve standardized tests for evaluation which unfortunately
are not yet available. Learning lessons from cancer therapies, future
research in this area must focus on identifying the most appropri-
ate markers/immune panels that will be able to accurately classify
the RIF women with an immune disturbance and also predict the
type of immune response. This can then be implemented in well-
designed studies and establish new personalized therapy protocols.
Until then, the ART community should be careful regarding the use of
many adjunct or add-on immunotherapy interventions in women with
RIF.
ACKNOWLEDGEMENTS
DM and NM are funded by grants from the Indian Council of Medi-
cal Research (ICMR), Government of India and Department of Science
and Technology, Ministry of Science and Technology, Science and Engi-
neering Research Board Government of India (CRG/2018/002314).
NM is a recipient of a grant from the Department of Biotechnology,
Government of India (BT/PR30401/BIC/101/1077/2018). RS is thank-
ful to the DST-Inspire fellowship (# IF170222), Government of India.
Themanuscript bears the NIRRCH ID: IR/1181/12-2021.
DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were
created or analyzed in this study.
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ORCID
NupurMukherjee https://orcid.org/0000-0002-8303-8375
Richa Sharma https://orcid.org/0000-0001-8490-9761
DeepakModi https://orcid.org/0000-0002-4230-4219
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https://orcid.org/0000-0002-8303-8375
https://orcid.org/0000-0002-8303-8375
https://orcid.org/0000-0001-8490-9761
https://orcid.org/0000-0001-8490-9761
https://orcid.org/0000-0002-4230-4219
https://orcid.org/0000-0002-4230-4219
https://www.brjnmims.org/article.asp?issn=2349-3666;year=2015;volume=2;issue=1;spage=83;epage=104;aulast=Modi;type=0
https://www.brjnmims.org/article.asp?issn=2349-3666;year=2015;volume=2;issue=1;spage=83;epage=104;aulast=Modi;type=0
https://www.brjnmims.org/article.asp?issn=2349-3666;year=2015;volume=2;issue=1;spage=83;epage=104;aulast=Modi;type=0
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https://link.springer.com/article/10.1007/s43032-021-00784-z
https://academic.oup.com/humrep/article/15/suppl_3/96/652078
https://academic.oup.com/humrep/article/15/suppl_3/96/652078
http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA
http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA
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