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Received: 9 May 2018    Accepted: 20 June 2018

DOI: 10.1111/aji.13022

REVIEW ARTICLE

Recurrent pregnancy loss and vitamin D: A review of the

literature

Daniela Reis Gonçalves1  | António Braga1 | Jorge Braga1 | António Marinho2,3

1
Department of Obstetrics and
Gynecology, Centro Hospitalar do Porto,
Porto, Portugal
2
UMIB, Instituto de Ciências Biomédicas de
Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
3
Clinical Immunology Unit, Centro
Hospitalar do Porto, Porto, Portugal
Correspondence: Daniela Reis Gonçalves,
Department of Obstetrics and Gynecology,
Centro Hospitalar do Porto, Largo da
Maternidade de Júlio Dinis, 4050-651,
Porto, Portugal (danielareisgoncalves@
hotmail.com).
Recurrent pregnancy loss (RPL) affects approximately 1%-­
2% of reproductive
women. Auto-­and cellular immune responses seem to be associated with RPL.
Vitamin D (VD) has been shown to play a role in the modulation of the immune sys-
tem. Effects of VD deficiency (VDD) in pregnancy have been associated with preec-
lampsia, gestational diabetes, fetal growth restriction, preterm labor, and sporadic
spontaneous abortion (SA). We systematically reviewed articles that studied women
with 2 or more SA and its association with VD. Eleven studies were included. Studies
reported a high prevalence of VD insufficiency (VDI) or VDD in women with RPL and
suggested that this could be associated with immunological dysregulation and conse-
quently with RPL. Immunological benefits were reported in the peripheral blood of
women with RPL after VD exposure. Thus, it is possible to speculate a beneficial role
for VD supplementation in RPL. It seems that there are not differences in the vitamin
D receptor (VDR) and CYP27B1 expression in endometrium of women with RPL but,
in villous and decidual tissues, RPL women seem to have a decreased expression of
VDR and, perhaps, a decreased expression of CYP27B1. Further randomized con-
trolled studies are required to investigate the association between VDD or VDI and
RPL.

KEYWORDS
1,25-dihydroxyvitamin D, 25-hydroxyvitamin D, habitual abortion, recurrent miscarriage,
recurrent pregnancy loss, vitamin D

1 |  I NTRO D U C TI O N
A pregnancy loss or spontaneous abortion (SA) is defined as the spon-
taneous demise of a pregnancy before the fetus reaches viability. It
includes all pregnancy losses (PL) from the time of conception until
24 weeks of gestation.1,2 Approximately 15% of pregnant women
experience sporadic loss, 2% experience 2 consecutive PL and 0.4
to 1% have 3 consecutive PL.3 Recurrent pregnancy loss (RPL) is de-
fined as the loss of 2 or more pregnancies, however there are some
discrepancy in opinions among clinicians regarding this definition,
some of them keep a definition of 3 or more consecutive losses in
their clinical practice.1,2,4 RPL affects approximately 1-­2% of repro-
ductive women but exact prevalence is very difficult to estimate and
it depends on the definition used.1 Primary RPL can be defined as
RPL without a previous viable pregnancy beyond 24 weeks’ gesta-
tion and secondary RPL as an episode of RPL after 1 or more previ-
ous pregnancies progressing beyond 24 weeks’ gestation.1
PL has an important emotional impact on women and their part-
ners. Feelings of guilt, shame, isolation, and sometimes anger are
common. These feelings can intensify with repeated losses and can
affect the marital relationship.5 Being able to listen with understand-
ing and empathy and to recognize PL as an adverse life event is es-
sential in managing these situations.6
Several pathogenic mechanisms associated with RPL have been
described, including uterine abnormalities, endocrine and meta-
bolic problems, genetic anomalies, acquired and inherited throm-
bophilia and immunological factors. Unfortunately, the cause of
RPL cannot be determined in 50% of patients.1,2,7 An association

Am J Reprod Immunol. 2018;e13022. wileyonlinelibrary.com/journal/aji © 2018 John Wiley & Sons A/S.  |  1 of 15
https://doi.org/10.1111/aji.13022 Published by John Wiley & Sons Ltd
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between congenital uterine malformations and RPL has been well
8
documented. Acquired uterine malformations (endometrial polyps,
submucous myomas, uterine adhesions) have been found prevalent
9
in women with RPL but the clinical association is unclear. Thyroid
hormone disorders and thyroid peroxidase (TPO) antibodies are
associated with folliculogenesis, spermatogenesis, fertilization,
and embryogenesis, supporting a role for thyroid hormone disor-
ders and thyroid autoimmunity in PL.10 Polycystic ovary syndrome
seems to be associated with PL and insulin resistance is shown to
11,12
be more prevalent in women with RPL. Prolactin may play a role
in maintaining corpus luteum function and progesterone secretion
but the mechanism is still unclear and studies didn’t find consis-
tent evidence for an association between prolactin and RPL.13,14
Genetic abnormalities are a recognized cause of sporadic and RPL.
The prevalence of chromosome abnormalities in a subsequent mis-
carriage after preceding RPL is 39%, comparable to the prevalence
in a single sporadic SA.15 There isn’t a clear association between
hereditary thrombophilia and RPL, but there is an association be-
tween antiphospholipid syndrome (APS), an acquired thrombophilia,
and RPL. Antiphospholipid antibodies (APA) should be screened in
1,16
women with RPL. Approximately 20% of women with RPL have
autoimmune conditions, such as APA, but also cellular immune ab-
normalities, including increased natural-killer cell (NK) levels, NK
cytotoxicities and T helper (Th) 1/Th2 ratios.17-19 Some studies also
suggest decreased regulatory T cell (Treg) levels, which contribute
to the immune tolerance during pregnancy, and increased Th17 cell
levels, which participate in inflammatory reactions, in the peripheral
20-22
blood of RPL women. Thus, auto-­and cellular immune responses
seem to be associated with RPL.
Vitamin D (VD), or calciferol, is a fat soluble vitamin mainly syn-
thesized in the skin exposuring to the sunlight, and less obtained
23,24
from the diet. Dermal synthesis is the major natural source of
VD but the production varies with factors such as skin type, lati-
tude, season, time of day, exposed body area, exposure time, pol-
25
lution, and age. During exposure to the ultraviolet rays in sunlight
7-­dehydrocholesterol converts into VD3 (cholecalciferol). In plants
VD2 (ergocalciferol) is produced from radiation of ergosterol.
Humans can metabolize VD3 and VD2 but the last one is less potent.
VD is mainly combined with vitamin D binding protein (VDBP), but
also with serum albumin and lipoproteins, for transport to the liver.
In the liver VD undergoes its first enzymatic conversion by the mi-
tochondrial form of 25-­hydroxylase resulting in 25-­hydroxyvitamin
D or 25(OH)D (calcidiol), the major circulating form of VD. The
second enzymatic conversion occurs mainly in the kidney by 1α-­
hydroxylase (CYP27B1) and 24α-­
hydroxylase (CYP24) resulting
in 1,25-­dihydroxyvitamin D or 1,25(OH)2D (calcitriol), the active
metabolite of VD. 26 1,25(OH)2D actions are mediated through its
specificity and high affinity for vitamin D receptor (VDR), a nuclear
receptor which regulates gene transcription in target tissues. VDR
exists not only in the organs involved in the regulation of calcium
phosphate but also in other tissues and cells, such as mononuclear
cell, endometrial stroma, vascular endothelial cells, pancreatic β-­
cells, keratinocytes, and nerve tissue. 27
The active form of VD has been shown to play a classic role in the
regulation of calcium and phosphate homeostasis and bone metab-
olism. 28 Beyond this critical function VD has recently been found to
play an important role in the modulation of the immune system by
regulating hormone secretion, immune function, cell differentiation,
and cell proliferation. 29 VD participates in both innate immunity and
adaptive immunity responses, promoting the first and suppressing
the second. The innate immunity involves the activation of Toll-­like
receptors (TLR) in monocytes, macrophages and epithelial cells of
different tissues and organs, increasing the ability of macrophages
to confront with intracellular bacteria.30,31 In the adaptive immune
system VD decreases the activity oh Th1 cells, by decreasing the
production of interferon gamma (INF-­γ), interleukin (IL) 2, IL-­12 and
tumour necrosis factor alpha (TNF-­α), and increases the activity of
Th2 cells, by increasing the production of IL-­4,5,6,9,10,13.32,33 VD
has also an inhibitory effect on Th17 cells and a stimulatory effect
on Treg cells by upregulation forkhead box protein 3 (Foxp3), a factor
that supports the differentiation and expansion of these cells.32,33 In
the dendritic cells, VD inhibits their differentiation and maturation
and decreases their immune-­stimulation capacity.34 VD also down-
regulates the B cell proliferation, plasma cell differentiation, and im-
munoglobulin production.35
In recent years, there has been a growing interest on the role
of VD in the reproductive physiology. VD metabolism in pregnancy
is characterized by physiological increase in maternal 1,25(OH)2D,
maternal 25(OH)D availability for optimal neonatal 25(OH)D sta-
tus and increase in maternal VDBP levels.36,37 Maternal 25(OH)
D crosses the placenta and represents the main pool of VD in the
fetus. These changes occur in systemic circulation and placenta sug-
gesting that placenta is an important site of VD metabolism in preg-
nancy.37 During normal pregnancy, the maternal adaptive immune
system is suppressed whereas the innate immune system is rela-
tively stimulated. These changes in maternal-­fetal interface are ben-
eficial producing immune tolerance and prevent fetal rejection.38,39
Normal pregnancy is characterized by a shift in immune response
from type 1 to type 2. Regarding the immunomodulatory effects of
VD it has been suggested that VD can play a role in immune regu-
lation at the maternal-­fetal interface, promoting adequate levels of
inflammatory response for decidualization and implantation. Several
studies found VDR, VD metabolism enzymes and VDBP in human
decidua, placental tissues and endometrium.40 Furthermore, several
studies suggested that VD may have a direct impact on likelihood of
reproductive treatment success. A recent systematic review includ-
ing 11 studies investigated the association between VD status and
reproductive outcomes.41 This group demonstrated that replete VD
status is associated with greater chances of reproductive treatment
success. Thus, to diagnose and treat vitamin D deficiency in women
planning an assisted reproductive treatment may be beneficial to op-
timize pregnancy outcomes.
Several reviews have found high prevalence of VD deficiency
(VDD) worldwide. VDD seems to be a major public health problem
worldwide in all age groups and ethnicities, even in those residing in
countries with low latitude and high sun exposure.42 Also, a recent
GONÇALVES et al.
European study estimated a prevalence rate of VDD that are a mat-
43
ter of concern. Worldwide, VDD is present in more than half of
pregnant women and newborns.44 VDD is classically characterized
by osteomalacia, osteoporosis, and bone fractures but low VD levels
also has been linked to cardiovascular disease,45 multiple sclerosis,46
47 48
chronic infections, autoimmune diseases, cancers,
tes.49 Effects of VDD in pregnancy have been associated with some
adverse pregnancy outcomes, such as preeclampsia, gestational di-
abetes, fetal growth restriction, maternal infections, and preterm
labor.50-53 However, the number of high-­quality studies is limited to
assess a causal role for VDD in these pathologies. According to a
Cochrane review (2016), the evidence on whether VD supplementa-
tion should be given as a part of routine antenatal care to all women
to improve maternal and infant outcomes remains unclear.
rigorous randomized trials are required to confirm these effects.
Several studies were published about the relationship be-
tween VDD and sporadic SA. Zhang et al (2017) in a systematic
review and meta-­analysis, which included 5 cohort studies,
concluded that an extremely low 25(OH)D level (<20 ng/mL) was
significantly associated with an increased risk of SA in the first tri-
mester. Thus, severe maternal VDD may be a modifiable risk factor
60 61
in early embryonic development. Hou et al found that having
1 or more histories of failed clinical pregnancy in the first trimes-
ter was significantly associated with VDD, 97% of women with 1
or more SA had serum 25(OH)D concentrations below 30 ng/mL.
This study suggests that low VD levels among women with failed
clinical pregnancies history may predispose to increased risk for
SA. Therefore, the authors suggested screening this woman for
VDD. Zhou et al 62 showed that a single dose of lipopolysaccha-
ride (LPS) caused 63% mice with abortion pregnancy but the rate
of abortion dropped to 14% when pregnant mice were pretreated
with VD3. LPS is a toxic component of cell walls and the outer cell
membranes in gram-­n egative bacteria. This study suggests that
VD3 pre-­t reatment protects against LPS-­induced early pregnancy
loss. However, other studies have failed to observe an association
between VD and sporadic SA. 55,59,63
However, sporadic SA (1 SA) and RPL (≥2 SA) are considered
64
distinct disease entities. Sporadic SA is the most common compli-
cation of early pregnancy and is considered to primarily represent
failure of abnormal embryos to progress to viability. RPL is a less
common phenomenon and is thought to have multiple etiologies,
although unknown in most cases. Our knowledge about the role of
VD in RPL remains limited and there is some contradictory findings
in the literature. In this context, we aimed to present a review of the
current research literature on the role of VD in RPL.
2 | M ATE R I A L A N D M E TH O DS
This study was designed to include articles that studied women
with 2 or more SA (RPL) and its association with VD. A literature
search was performed using MEDLINE (Pubmed) and the Cochrane
Library databases. The search strategy used the following medical
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subject heading (MeSH): “abortion, habitual”, “abortion, spontane-
ous”, “vitamin D”, “calcifediol”, “25-­hydroxyvitamin D”, “calcitriol”,
“1,25-­dihydroxyvitamin D”, “receptors, calcitriol”. Articles published
between January 1978 and January 2018, in English or Portuguese,
were included. References of all included primary and review articles
and diabe- were examined to identify relevant articles not captured by elec-
tronic searches. Review articles, letters, and studies that included
women with sporadic SA (<2 SA) were excluded.
3 | R E S U LT S
The Preferred Reporting Items for Systematic Reviews and Meta-­
54
Further Analyses (PRISMA) flow diagram of the review process is presented
in Figure 1.65,66 The search strategy yields 29 articles, of which 4
were excluded as it was clear from scrutinizing the title and abstract
that they did not fulfill the selection criteria. Full manuscripts of
55-59
25 articles were obtained. A total of 14 of these publications were
excluded because 1 was a review, 1 was a letter, and 12 were not
specific to RPL. Therefore, the total number of studies included in
the review was 11. From the included studies, 7 articles were ob-
servational studies and 4 were clinical trials, all of them published
between 2011 and 2017. The studies were carried out in various
countries: 2 studies from USA, 4 from Iran, 4 from China, and 1 from
Egypt. Study characteristics of the 11 included studies are presented
in Tables 1 and 2.
3.1 | RPL and VD
The articles included in the review are discussed below divided by
local where metabolism of VD was studied: peripheral blood, endo-
metrium, maternal-­fetal interface.
3.1.1 | Peripheral blood
Ota et al and Chen et al67,68 reported a high proportion of women
with RPL and low VD levels (≤30 ng/mL). Therefore, this result raises
the possibility that VD insufficiency (VDI) or VDD in women with RPL
could be associated with this poor obstetrical outcome. Ota et al67
analyzed 133 women with RPL, these women were divided into 2
groups, normal (≥30 ng/mL) and low VD (<30 ng/mL), depending on
their serum VD level. Women with RPL and low VD levels (n = 63)
had higher prevalence of APA, total anti-­nuclear antigen antibody,
anti-­single-­stranded DNA antibodies (anti-­ssDNA) and TPO anti-
body than women with RPL and normal VD levels (n = 70). This was
consistent with previous studies that reported increased prevalence
of VDD in patients with APS and reported an inversely correlation
between VD levels and TPO antibody titer.69-72 The RPL group with
low VD levels had higher percentage of B cells, percentage of NK
cells and NK cytotoxicity. These results suggest that VD plays a role
in regulating B cell proliferation and function and regulating both
NK cell population and cytotoxicity, NK cytotoxicity by suppressing
perforin secretion. Thus, a high proportion of women with RPL have
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4 of 15       GONÇALVES et al.
low VD levels (47.4%), which is associated with increased cellular and
autoimmunity, compared to women with RPL and normal VD levels.
This result suggests that low VD serum concentrations could be an
etiological or worsening factor for immunological changes involved
in RPL. This study did not include a control group (women without
SA) to compare the immunological results with women with RPL and
normal VD levels.
68
More recently Chen et al compared 35 women with RPL and
normal VD levels with 64 women with RPL and VDI or VDD (≤30 ng/
mL). They showed that a high proportion of RPL women have VDI
or VDD (64.6%) and the number of previous SA was higher in VDI/
VDD group. Women with RPL and VDI/VDD had a tendency to in-
crease their percentage of B cells, TNF-­α producing Th cells and NK
cytotoxicity than women with RPL and normal VD levels. Therefore,
women with RPL and low VD levels have an excessive inflammatory
adaptive response, or at least more prominent than women with RPL
and normal VD serum concentrations. This response can play a role
in RPL as suggested by increased number of SA in the low VD level
group.
Since women with RPL have a high prevalence of VDI or VDD
and this insufficiency or deficiency could be associated with immu-
nological dysregulation and consequently with RPL, it is possible to
speculate a beneficial role for VD supplementation in RPL women.
67,68
Ota et al and Chen et al also evaluated the impact of VD
supplementation on the immune system, in vitro and in vivo, re-
spectively. Ota et al67 showed that in vitro VD3 reduced NK cyto-
toxicity, supressed secretion of type 1 cytokines (TNF-­α , INF-­γ) and
increased type 2 cytokines (IL-­10), promoting a shift from type 1
to type 2 immunity response which is associated with a successful
pregnancy. On the other hand, women with RPL have a tendency to
increase their type 1 immunity response suggesting that VDD can
F I G U R E   1   PRISMA flow diagram for
study selection
contribute to this unfavorable response.18 In vitro VD3 also inhibited
perforin release and interfered in the NK’s polarization, indicating a
direct effect of VD in NK cytotoxicity. This article doesn’t explain if
the VD was added to in vitro assays only in the RPL group with low
VD levels or in both groups. Chen et al68 treated women with RPL
and VDI or VDD with 1,25(OH)2D for 2 months. Supplementation
reduced significantly the percentage of B cells, TNF-­α producing Th
cells and NK cytotoxicity in this group. Thus, both studies suggest
that VD may play an important role in the modulation of the per-
centages and functions of peripheral blood lymphocyte subsets in
RPL women, inducing favorable changes for a successful pregnancy.
Ota et al73 also investigated the effects of in vitro da 1,25(OH)2D3
on NK-­cell immunity in women with RPL (n = 18) and compared it to a
control group (n = 16), independently of VD serum levels. NK cells of
both groups were treated with 1,25(OH)2D3 and vehicle only (with-
out VD). When exposed to the vehicle, women with RPL had signifi-
cantly higher NK cytotoxicity and decreased CD107a expression on
NK cells as compared with that of control group, which is consistent
with previous studies.17 1,25(OH)2D3 inhibited NK-­cell cytotoxic-
ity and decreased CD107a expression in women with RPL. These
changes were not seen in controls. CD107a is a surface marker of
NK-­cell functional activity and its induction is in concert with cyto-
kine secretion and target cell lysis. In addition, CD107a may identify
activated NK cells that may degranulate in the absence of cytokine
secretion. Thus, 1,25(OH)2D3 may suppress NK cell cytotoxicity via
interfering with the NK cell degranulation process regardless of NK
cell cytokine secretion. In the RPL group, VD also suppressed CD69
expression, a receptor which induces the cytotoxic activity and co-
stimulates cytokine production; upregulated CD158a and CD158b
expression, inhibitory NK-­cell receptors; downregulated NKp30 and
NKp44 expression, natural cytotoxicity receptors; downregulated
GONÇALVES et al.
TNF-­α and INF-­γ secretion; downregulated TLR4 expression, sup-
pressing inflammatory immune responses that are normally acti-
vated by these receptors as seen in previous studies.74 In this study,
VD reduced perforin-­mediated cytotoxicity of NK cells, via prohib-
iting perforin polarization, although the capacity to form conjugates
with their targets was not affected by 1,25(OH)2D3. These results
suggest that women with RPL have various NK-­cell dysfunction and
1,25(OH)2D3 can regulate NK cell functions via various mechanisms.
This raise the possibility that VD could be used in women with RPL
and dysregulated NK cell immunity.
Rafiee et al75 investigated the effect of VD3 on the balance of
Th17 and Treg cells. Two groups were compared: 22 women with
RPL treated with lymphocyte immune therapy (LIT) and VD3 (study
group) and 22 women with RPL treated with LIT alone (control
group). The mean percentage of Th17 and Treg cells and ratio Th17/
Treg significantly decreased post-­LIT in the study and control groups.
However the decline in Th17 cell frequency and in Th17/Treg ratio
was significantly more in the study group than in control group when
compared to baseline values. Thus, once again, VD showed bene-
ficial therapeutic effects in women with RPL. Therefore, adequate
VD levels may be important to maintain proper innate and acquired
immunity for successful pregnancy. In view of all of these findings,
VD might be a new therapeutic option for RPL women with abnor-
mal immunity.
This review includes 2 randomized controlled trials, published
in journals not indexed in MEDLINE and identified through refer-
ences.76,77 Ibrahim et al76 evaluated the immunomodulatory role
of VD3 in prevention of SA in cases of RPL. Forty pregnant women
(≤6 weeks) with history of RPL were included. They were randomly
assigned to 2 groups: study group which received oral VD3 analog
supplementation (0.25 mcg twice daily), progesterone rectal sup-
positories (400 mg daily), low dose aspirin (81 mg daily) and folic acid
(5 mg daily), and control group which received the same therapy ex-
cept VD3. IFN-­γ levels were evaluated until 14 weeks. After 10 weeks
of gestation, the control group was found to have significantly higher
levels of IFN-­ƴ compared to the study group. In the study group,
there was a significant decrease in IFN-­γ levels compared to pre-­
treatment value. IFN-­γ cut-­off value at or below 57.3 pg/mL was
considered diagnostic of successful pregnancy. The present study
shows that IFN-­γ when it is downregulated significantly after treat-
ment by VD3 is associated significantly with successful pregnancy.
VD oral supplementation has resulted in reducing risk of PL up to
15% among women with RPL but the result was statistically insig-
nificant, so the authors suggest that large-­scale studies with larger
sample size need to be carried out.
77
Samimi et al examined the effect of VD supplementation on
the occurrence of RPL and the serum level of IL-­23 in Th17 cells.
Seventy-­seven pregnant women with history of RPL were included.
They were randomly assigned to 2 groups: study group which re-
ceived oral VD3 (400 IU/d) and vaginal progesterone (400 mg daily),
and control group which received tablets that were similar to VD3
with no active ingredient and vaginal progesterone (400 mg daily).
Before the start of the study the serum level of VD3 (on average
|
      5 of 15
12 ng/mL) and IL-­23 were not statistically different between both
groups. At the end of the study serum level of VD3 increased in both
groups, but more in the study group, and serum level of IL-­23 de-
creased in the study group and increased in the control group. VD3
and IL-­23 showed an inverse relationship and there was a significant
relationship between the serum level of IL-­23 and the incidence of
SA. There were more SA in the control group but the effect of VD3
and the incidence of SA is not statistically significant when applying
the confounding factors. These results suggest that VD can reduce
the level of IL-­23 and subsequently the incidence of SA in women
with RPL.
Both trials studied VD supplementation starting after pregnancy
confirmation. Samimi et al77 don’t explain when supplementation
was started (after biochemical and/or ultrasound confirmation). Each
study only evaluated 1 immunological factor in the peripheral blood.
Therefore, in these 6 studies immunological benefits were re-
ported in the peripheral blood after VD exposure, in vitro e in vivo.
Ota et al, 67,73 Ibrahim et al76 and Samimi et al77 reported benefits in
women with RPL, independently of VD serum levels, Chen et al68 and
Rafiee et al75 reported benefits in women with RPL and VDI or VDD.
It is still unclear if women with RPL and normal VD levels have im-
munological benefits with VD supplementation. Further studies are
required to investigate if these laboratory changes that occur after
VD exposure are associated with lower probability of SA. Ibrahim
et al76 and Samimi et al77 failed to show a significant correlation.
All this studies were conducted in the peripheral blood but the
maternal-­
fetal interface is where direct contact occurs between
mother and fetus and this microenvironment is crucial for normal
pregnancy. Therefore, it is important to investigate whether low VD
levels and laboratory changes after VD exposure that were observed
in the peripheral blood of women with RPL also occur in endome-
trium and fetal-­maternal interface.
3.1.2 | Endometrium
Tavakoli et al studied the VD metabolism in the endometrium of
women with RPL.78,79 In 2011, they hypothesized that endome-
trial cells of women with RPL have impaired capacity to produce or
respond to this hormone.78 To elucidate this possibility they com-
pared endometrial samples, obtained through biopsy curette, of 8
women with RPL with 8 age matched women with at least 1 healthy
live birth, without SA or infertility. Endometrial stromal cells (ESC)
expressed high levels of VDR in their nucleus and CYP27B1 was
expressed predominantly in the cytoplasm, showing that ESC are
target cells for VD3 action. Whole endometrial cells (WEC) from RPL
group produced significant amounts of IFN-­γ prior to VD3 expo-
sure, contrary to those in control group. The WEC from both groups
had the same trend of changes in cytokine production after treat-
ment with 1,25(OH)2D3, except IL-­8. After VD3 exposure, WEC from
women with RPL significantly lowered IFN-­γ and IFN-­γ:IL-­10 ratio.
Both groups, RPL and control, converted 25(OH)D3 to 1,25(OH)2D3
at comparable levels, suggesting that both have comparable capacity
to produce the active form of VD3. Thus, endometrial cells of women
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6 of 15       GONÇALVES et al.

TA B L E   1   Characteristics of studies in the peripheral blood

Author (year)
Location and
period Aim of the study Study design Study population Timing of VD assessment

Ota et al. (2014) To investigate if Retrospective cross-­ 133 women with 3 or more consecutive Early follicular phase
USA women with RPL sectional study (in vitro) SA of unknown cause:
01/2011-­ and VDI or VDD 70 women with RPL and VDN levels
12/2012 have increased 63 women with RPL and low VD levels
prevalence of
auto-­and cellular
immune abnormali-
ties when
compared with
women with RPL
and VDN levels
To investigate if VD
in vitro has any
effect on cellular
immunity

Chen et al. To investigate the Clinical trial (in vivo) 99 women with 2 or more SA of Previous VD treatment,
(2016) modulatory effects unknown cause: in the middle luteal phase
China of VD on peripheral 35 women with RPL and VDN levels After VD treatment, in
01/2014-­ blood cellular 64 women with RPL and VDI or VDD the middle luteal phase
03/2016 immune response in
women with RPL

Otaet al. (2015) To investigate the Case-­control study (in 18 women with 3 or more consecutive x
USA effects of VD on vitro) SA of unknown cause RPL (study
(NA) NK-­cell immunity group)
16 age matched women without a
history of RPL or infertility (control
group)

Rafiee et al. To investigate the Double blind placebo-­ 44 women with 3 or more consecutive Previous treatment, in
(2015) effect of VD3 on controlled clinical trial (in SA of unknown cause and VDD: the follicular phase
Iran the balance of Th17 vivo) 22 women treated with LIT and VD3 3 months after treatment,
10/2013-­ and Treg cells (study group) in the follicular phase
09/2014 22 women treated with LIT alone
(control group)
GONÇALVES et al.
Method of VD VD cut-­offs
assessment utilized Summary results
Liquid chromatogra- Low 47.4% of women with RPL had low VD levels
phy/tandem mass <30 ng/mL Women with RPL and low VD levels had
spectrometry (LC/ Normal higher prevalence of APA, total anti-­nuclear
MS/MS) ≥30 ng/mL antigen antibody, anti-­ssDNA antibody, and
TPO antibody than women with RPL and
VDN levels
Women with RPL and low VD levels had
higher % of B cells, % of NK cells and NK
cytotoxicity than women with RPL and VDN
levels (no difference in the % of T cells)
In vitro VD3 reduced NK cytotoxicity,
supressed secretion of type 1 cytokines
(TNF-­α , INF-­γ) and increased type 2
cytokines (IL-­10)
In vitro VD3 inhibited perforin release and
interfered in the NK’s polarization
Chemiluminescent Deficiency <20 ng/ 64.6% of women with RPL had VDI or VDD
immunoassay mL Number of miscarriages was higher in VDI
Insufficiency and VDD groups
20-­3 0  ng/mL Women with RPL and VDI or VDD had a
Replete tendency to increase their % of B cells,
>30 ng/mL TNF-­α producing Th cells and NK cytotoxic-
ity (no difference in the % of T cells, NK cells
and INF-­γ) than women with RPL and VDN
levels
Supplementation with 1,25(OH)2D reduced
significantly % of B cells, TNF-­α producing
Th cells and NK cytotoxicity in women with
RPL and VDI or VDD
x x • Women with RPL had significantly higher
NK cytotoxicity and decreased CD107a
expression on NK cells as compared with
that of control group
• In vitro 1,25(OH)2D3, in women with RPL:
o Inhibited NK cell cytotoxicity, in
dose-dependent manner
o Decreased CD107a expression
o Suppressed CD69 expression
o Upregulated CD158a and CD158b
expression (inhibitory NK-cell receptors)
o Downregulated NKp30 and NKp44
expression
o Reduced perforin-mediated cytotoxicity
of NK cells, via prohibiting perforin
polarization
o Downregulated the expression of TLR4
and TNF-α and INF-γ secretion in NK
cells
Enzyme-­linked Deficiency <20 ng/ The mean % of Th17, mean % of Treg and ratio
immunosorbent assay mL Th17/Treg significantly decreased post-­LIT in
(ELISA) the study and control groups
The decline in Th17 cell frequency and in
Th17/Treg ratio was significantly more in the
study group than in control group when
compared to baseline values (no difference
in % of Treg)
|
      7 of 15
Conclusions
High proportion of RPL women have low VD
levels, which is associated with increased
cellular and autoimmunity
An adequate level of VD or supplementation
might be important to maintain proper
innate and acquired immunity for successful
pregnancy
VD might be a new therapeutic option for
reproductive failure
High proportion of RPL women have VDI or
VDD
Women with RPL might have excessive
inflammatory adaptive immunity
VD might have a potential beneficial role as
adjuvant therapy in women with RPL
1,25(OH)2D3 migh:
supress NK-­cell cytotoxicity via interfering
with the NK-­cell degranulation process
regardless of NK-­cell cytokine secretion
interfere in the early stage of NK-­cell
activation
regulate inhibitory NK-­cell receptors and in
turn NK-­cell cytotoxicity
decrease NK cytotoxicity and NK-­cell
degranulation via reduction of NKp30 and
NKp44 expression
change the expression pattern of NK-­cell
receptors (downregulation of killer
activating receptors and upregulation of
killer inhibitory receptor) and therefore
reduce perforin-­mediated NK cytotoxicity
Combinatorial therapy with VD3 and LIT
might play a more important role than LIT
alone in Th17/Treg ratio reduction
Th17 decrease subsequent to VD3 consump-
tion is of more importance in Th17/Treg
ratio alteration, while Treg cell frequency
showed no significant change following VD3
(Continues)
 |
8 of 15       GONÇALVES et al.

TA B L E 1   (Continued)

Author (year)
Location and
period Aim of the study Study design Study population Timing of VD assessment

Ibrahim et al. To investigate the Randomized Controlled 40 pregnant women with ≤6 wk with x
(2013) role of VD3 as Trial (in vivo) history of 2 or more consecutive SA (of
Egypt immunomodulator unknown cause):
09/2010-­ in prevention of 20 pregnant women treated with folic
03/2012 pregnancy loss in acid, low dose aspirin, progesterone
cases of recurrent and VD3 (study group)
missed miscarriage 20 pregnant women treated with folic
acid, low dose aspirin and progester-
one (control group)

Samimi et al. To investigate the Double-­Blind Randomized 77 pregnant women with 2 consecutive Start of the study
(2017) effect of VD3 Controlled Trial (in vivo) or at least 3 non-­consecutive SA (of (previous treatment)
Iran supplementation on unknown cause): End of the study (time
11/2013-­ the occurrence of 39 pregnant women treated with of abortion in women
03/2015 RPL and the serum progesterone and VD3 (study group) with SA, twentieth
level of IL-­23 in 38 pregnant women treated with week in other women)
Th17 cells progesterone and placebo (control
group)

CLIA: Chemiluminescent immunoassay; VD: Vitamin D; RPL: Recurrent pregnancy loss; VDI: Vitamin D insufficiency; VDD: Vitamin
D deficiency; VDN: Vitamin D normal; SA: Spontaneous abortion; APA: Antiphospholipid antibody; ssDNA: Single-­stranded DNA; TPO:
Thyroperoxidase; NK: Natural Killer; TNF-­α: Tumor necrosis factor-­alpha; INF-­γ: Interferon-­gamma; IL: Interleukin; Th: T helper; NA:
Information not available; 1,25(OH)2D: 1,25-­Dihydroxyvitamin D; TLR: Toll-­like receptor; Treg: T regulatory; LIT: Lymphocyte Immune
Therapy.

with RPL do not seem to have impaired capacity to produce or re-
spond to VD. This study also showed that VD might have positive
immunologic effects in controlling inflammatory responses. In 2015,
using the same study groups, Tavakoli et al79 published an article
about the differences in endometrial expression of VDR, CYP27B1
and CYP24A1 between women with RPL and a control group. No
difference was seen between the groups. In view of the fact that
CYP27B1 expression was similar in both groups, this result are con-
sistent with the previous study (2011) showing that endometrial cells
from RPL and control groups converted 25(OH)D3 to 1,25 (OH)2D3
at comparable levels in vitro. Comparable VDR expression in this
study and comparable trend of changes in cytokine production after
1,25(OH)2D3 exposure in the previous study (2011) could, therefore,
be interpreted as RPL and normal women having the same quantity
and function of this receptor.
3.1.3 | Fetal-­maternal interface
Other authors investigated the expression of VDR and CYP27B1
in the chorionic villi and decidua.80-82 Yan et al80 hypothesized
that VDR expression is decreased in the fetal-­maternal interface in
women with RPL. They compared the chorionic villi and decidua, ob-
tained during surgical evacuation of uterus of 40 women with RPL
and 40 gestational age matched women who underwent voluntary
GONÇALVES et al. |
      9 of 15

Method of VD VD cut-­offs
assessment utilized Summary results Conclusions

x x There was no statistically significant IFN-­γ is down regulated significantly after

difference between both groups at 6th and treatment with oral VD3 supplementation
8th wk of gestation while at 10th, 12th, and IFN-­γ down regulation is associated
14th wk the control group was found to have significantly with successful pregnancy
significantly higher levels of IFN-­γ while miscarriage was associated with
There was a significant decrease failure of IFN-­γ down regulation
in IFN-­γ level at 10th, 12th, and 14th wk in The effect of VD3 on outcome of current
the study group compared to pre-­treatment pregnancy was no statistically different
value at 6th wk (unlike the control group) between both groups but large-­scale studies
After treatment with VD3 IFN-­γ cut-­off value with larger sample size may result in more
≤57.3 are considered diagnostic of successful significant results
pregnancy
(88.2% sensitivity, 93.1% specificity)
Although statistically insignificant, VD oral
supplementation has resulted in reducing
risk of SA to 15% among women with RPL
Comparing symptoms suggesting
side effects of VD3 showed no significant
difference between both groups
NA x Before the start of the study, the serum level The serum level of IL-­23 decrease when the
of VD3 and Il-­23 were not statistically serum level of VD3 increase
different between both groups VD in women with RPL can reduce the level
At the end of the study, serum level of VD3 of IL-­23 and subsequently the incidence of
increased in both groups but more in the SA
study group
At the end of the study, serum level of IL-­23
decreased in the study group and increased
in the control group
VD3 and IL-­23 showed an inverse relationship
There was a significant relationship between
the serum level of IL-­23 and the incidence of
SA
There were more SA in the control group but
the effect of VD3 and the incidence of SA is
not statistically significant when applying the
confounding factors

pregnancy termination and had at least 1 successful pregnancy (with-
out SA). Chorionic villous and decidual tissues co-­expressed VDR
mRNA and protein confirming that VD metabolism and localized au-
tocrine or paracrine regulatory signaling occurs in fetal-­maternal in-
terface in early human pregnancy. However, the expression levels of
VDR were reduced in the chorionic villi and decidua in the RPL group
compared with the controls. Thus, there are evidence for disrupted
VDR metabolic homeostasis in early pregnancy loss but the con-
sequences are not known. Serum VDR levels were also decreased
in women with RPL compared with the control group suggesting
that serum VDR levels may predict the level of VDR expression at
the fetal-­maternal interface. These results raise the possibility that
reduced VDR expression at fetal-­maternal interface in women with
RPL may contribute to this poor outcome.
Wang et al,81 the same study group, investigated the expres-
sion and localization of CYP27B1 at the fetal-­maternal interface in
the first trimester pregnancy. They hypothesized that RPL may be
associated with aberrant expression of the CYP27B1 at the fetal-­
maternal interface. Three groups were compared: 20 women with
RPL, 20 women with 1 SA of unknown cause, and 20 age matched
women who underwent voluntary pregnancy termination and had
at least 1 successful pregnancy. CYP27B1 was expressed in both
villous and decidual tissues in the first trimester pregnancy, suggest-
ing that these tissues are an extrarenal site of VD synthesis and VD
 |
10 of 15       GONÇALVES et al.

TA B L E   2   Characteristics of studies in the endometrium, chorionic villi, and decidua

Author (year)
Location and Study Type of Timing and technique
period Aim of the study design Study population sample of sampling

Tavakoli et al. To investigate immu- Case-­ 8 women with 2 or more consecu- Endometrium Biopsy curette after
(2011) nomodulatory effect of control tive SA of unknown cause (study hysteroscopy
Iran 1,25(OH)2D3 on study group)
NA cytokine production by 8 age matched women with at least
endometrial cells of one healthy live birth, without SA
women with RPL or infertility (control group)

Tavakoli et al. To investigate the Case-­ 8 women with 2 or more consecu- Endometrium Biopsy curette after
(2015) endometrial expression control tive SA of unknown cause (study hysteroscopy
Iran of VDR, CYP27B1 and study group)
NA CYP24A1 in women 8 age matched women with at least
with RPL 1 live birth, without SA (control
group)
Yan et al. Test the hypothesis that Case-­ 40 women with 2 or more Chorionic villi Chorionic villi and decidua:
(2016) VDR expression is control consecutive SA of unknown cause and decidua Surgical evacuation of uterus
China decreased in the study (study group) Blood In the study group within the
10/2013-­ fetal-­maternal 40 gestational age matched first 24 h after diagnosis
10/2014 interface in women women who underwent voluntary -­Blood:
with RPL pregnancy termination and had at same day of surgical
least one successful pregnancy, evacuation of uterus
without SA (control group)

Wang et al. To investigate the Case-­ 20 women with 2 or more Chorionic villi Surgical evacuation of uterus
(2016) expression and control consecutive SA of unknown cause and decidua In the study group on
China localization of study (study group) the day of or the second
10/2013-­ CYP27B1 at the 20 women with 1 SA of unknown day of SA diagnosis
10/2014 fetal-­maternal cause (control group 1)
interface in the first 20 age matched women who
trimester pregnancy underwent voluntary pregnancy
termination and had at least one
successful pregnancy (control
group)

Li et al (2017) To investigate 25(OH)D Case-­ 30 women with 2 or more Decidua Surgical evacuation of uterus
China concentration, VDR control consecutive SA of unknown cause
01/2016-­ and CYP27B1 study (study group)
12/2016 expression in the 30 women who underwent
decidual tissues of voluntary pregnancy termination
women with RPL and had at least one successful
pregnancy, without SA (control
group)

NA: Information not available; 1,25(OH)2D: 1,25-­Dihydroxyvitamin D; RPL: Recurrent pregnancy loss; SA: Spontaneous abortion; VDR:
Vitamin D receptor; ESC: Endometrial stromal cells; WEC: Whole endometrial cells; VD: Vitamin D; INF-­γ: Interferon-­gamma; IL: Interleukin;
25(OH)D: 25-­Hydroxyvitamin D; mRNA: messenger ribonucleic acid; TNF-­α: Tumor necrosis factor-­alpha; TGF-­β: Transforming growth
factor beta; Treg: T regulatory; Th: T helper.
GONÇALVES et al.
Parameters Summary results
Cytokines ESC expressed high levels of VDR in their nucleus with some
CYP27B1 scattered pattern of reactivity in their cytoplasm
(1α-­hydroxylase) CYP27B1 was expressed predominantly in the cytoplasm of
-­VDR ESC
WEC from RPL group produced significant amounts of IFN-­γ
prior to VD3 exposure (contrary to those in control group)
WEC from both groups had the same trend of changes in
cytokine production after treatment with 1,25(OH)2D3,
except IL-­8 which was increased in RPL group and sup-
pressed in control group
Treatment with in vitro VD3 of WEC from women with RPL
significantly lowered IFN-­γ and IFN-­γ:IL-­10 ratio
Both RPL and control groups converted 25(OH)D3 to 1,25
(OH)2D3 at comparable levels
VDR No difference in the endometrial expression of VDR,
CYP27B1 CYP27B1, and CYP24A1 was seen between RPL group and
(1α-­hydroxylase) controls
CYP24A1 No significant difference in VDR and CYP27B1 expression
(24-­hydroxylase) pattern was found between RPL and control group
VDR Chorionic villous and decidual tissues co-­expressed VDR
mRNA and protein
The expression levels of VDR were reduced in the chorionic
villi and decidua in the RPL group compared with the
controls
VDR expression was mostly decreased in cytotrophoblasts,
decidual glandular epithelial cells, and villous and decidual
stromal cells
Serum VDR levels were decreased in women with RPL
compared with the control group
CYP27B1 CYP27B1 was expressed in both villous and decidual tissues
(1α-­hydroxylase) in the first trimester pregnancy
mRNA and protein for CYP27B1 were significantly decreased
in villous and decidual tissues from RPL group compared to
those from the control group
CYP27B1 was significantly decreased in villous trophoblasts
and decidual glandular epithelial cells in RPL group
IL-­10 markedly decreased and TNF-­α , Il-­2, and INF-­γ markedly
increased in villous and decidual tissues from RPL group
compared to those from the control group
No significant differences in the localization of CYP27B1,
Il-­10, INF-­γ, TNF-­α e IL-­2 expression were identified between
the groups
VDR 25(OH)D and TGF-­β concentrations and VDR expression were
CYP27B1 significantly decreased in women with RPL compared to
(1α-­hydroxylase) control group
25(OH)D IL-­17 and IL-­23 concentrations were significantly increased in
TGF-­β women with RPL compared to control group
IL-­17 CYP27B1 expression showed no significant difference
IL-­23 between the study and control groups
RPL was significantly negatively correlated with 25(OH)D
25(OH)D showed a significant negative correlation with IL-­23
IL-­23 showed a significant positive correlation with IL-­17
|
      11 of 15
Conclusions
ESC are the target of VD3 action
VD might have positive immunologic effects in
controlling inflammatory responses
VD might have potential therapeutic effect on
controlling immune-­mediated pregnancy losses
Endometrial expression of VDR and the enzymes
involved in VD metabolism was comparable in RPL and
control group
VD metabolism and localized autocrine or paracrine
regulatory signaling occurs in fetal-­maternal interface
in early human pregnancy
There are evidence for disrupted VDR metabolic
homeostasis in early pregnancy loss but the conse-
quences are not known
Decreased VDR expression during the first trimester
of pregnancy might be associated with
RPL
Villous and decidual tissues were an extrarenal site of
VD synthesis and VD functioning target
CYP27B1 is a local regulator of levels of 1,25(OH)2D3
Reduced CYP27B1 might be associated with RPL
It was not clear whether increase of TNF-­α , Il-­2, and
INF-­γ and decrease in IL-­10 expression in RPL were
due to decreased expression of VDR
Low 25(OH)D concentrations and reduced VDR
expression in the fetal-­maternal interface might be
associated with a higher risk of RPL
VD might play a vital role in influencing the IL-­23/IL-­17
axis
Decreased VD concentrations might be one of the
causes of the Treg/Th17 imbalance leading to abortion
 |
12 of 15       GONÇALVES et al.
functioning target. In this study, mRNA and protein for CYP27B1
were significantly decreased in villous and decidual tissues from RPL
group, raising the possibility that this enzyme played a local role in
embryo implantation. In RPL group, IL-­10 was markedly decrease
and TNF-­α , IL-­2, and INF-­γ were markedly increase in villous and
decidual tissues compared to those from the control group. Thus,
CYP27B1 seems to play an important role in controlling expression
of 1,25(OH)2D3 at fetal-­maternal interface, suggesting that reduced
CYP27B1 expression may be associated with RPL.
82
Li et al also evaluated 25(OH)D concentration, VDR and
CYP27B1 expression in decidual tissues of women with RPL. Thirty
women with RPL were compared to 30 women who underwent vol-
untary pregnancy termination and had at least 1 successful pregnancy
(without SA). 25(OH)D and TGF-­β concentrations and VDR expres-
sion were significantly decreased and IL-­17 and IL-­23 concentrations
were significantly increased in women with RPL compared to con-
trol group. 25(OH)D showed a significant negative correlation with
IL-­23 and IL-­23 showed a significant positive correlation with IL-­17. It
is known that IL-­23 contributes to the proliferation and differentia-
tion of Th17 cells and promotes the secretion of IL-­17. The increased
expression of IL-­23 and IL-­17 in women with RPL was reported in
83
previous studies. In this study, RPL was significantly negatively
correlated with 25(OH)D, suggesting that low VD levels in decidual
tissues were associated with RPL and VD might play a vital role in
influencing the IL-­23/IL-­17 axis. Low VD levels might be one of the
causes of Treg/Th17 imbalance leading to abortion. VDR expression
were significantly decreased in RPL group, as found by Yan et al,80 but
CYP27B1 expression showed no significant difference between the 2
groups, contrary to what was reported by Wang et al.81
Therefore, it seems that there are not differences in the VDR
and CYP27B1 expression in endometrium of women with RPL but,
in villous and decidual tissues, RPL women seem to have a decreased
expression of VDR (2 concordant studies) and, perhaps, a decreased
expression of CYP27B1 (1 concordant study and 1 discordant study).
4 |  CO N C LU S I O N
Our review identified 7 observational studies and 4 clinical trials in-
vestigating the association between RPL and VD. However, only 2
clinical trials were randomized controlled studies.
Worldwide, VD deficiency during pregnancy is prevalent and
recent studies reported an association between low VD levels and
RPL.44,67,68 The role of VD in reproductive health is corroborated
by the presence of VDR and enzymes which participate in VD me-
tabolism in endometrium and decidua.40,80-82 The studies suggest
that VD might play an immunoregulatory role, promoting implan-
tation and a successful pregnancy. Furthermore, women with RPL
seem to have more auto and cellular immune abnormalities espe-
cially if they have low VD levels.17,19,67,68,73,84 VD has been show
to positively regulate this immune abnormalities, in vitro and in
67,68,73,75
vivo. However, it remains unclear if there is a benefit of
VD supplementation only for women with RPL and low VD levels
or immunological abnormalities or for all women with RPL, this
information is important to clinical practice. Indeed, it is still un-
clear whether laboratory changes induced by VD in women with
RPL are associated with fewer SA, since the reported randomized
controlled trials failed to show a significant correlation between
VD supplementation and incidence of SA. In practice, it remains
the doubt if VD should be recommended to women with RPL who
are pregnant or desire to be pregnant.
We also think that further study is needed to investigate whether
VD supplementation has positive immunological or clinical effects
when given in preconception, since in all studies included VD was
given after pregnancy confirmation. Supplementation during this
critical period might lead to different results.
Furthermore, studies about VD levels, expression and function
of VDR and other molecules which participate in VD metabolism in
fetal-­maternal interface are limited, some of them had different re-
sults and used small samples.78-81
In addition, the 11 studies included in this review were con-
ducted in USA,67,73 Iran,75,77-79 China,68,80-82 and Egypt,76 coun-
tries where food intake patterns, UV exposure, skin color, latitude,
and other factors that affect VD levels are probably different from
Europe, Mediterranean, or Portugal.
Of note, VD is not the only immunomodulatory agent reported
in RPL. Intravenous immunoglobulin (IVIG) seems to act through a
multitude of mechanisms that include reducing the activity of NK
cells, increasing the activity of suppressor T cells, elimination of im-
mune complexes, down-­regulation of stimulatory receptors, and up-­
regulation of inhibitory receptors on the surface of different immune
cells.85,86 Such as for VD, several studies have indicated that treatment
with IVIG in RPL patients may improve pregnancy outcomes through
an immunological effect.87-89 However, this studies are underpow-
ered for definitive conclusions and ESHRE Guidelines considered that
IVIG cannot be recommended for clinical use at this moment.1 Thus,
although therapies with immunomodulatory properties may seem
beneficial in RPL, an agent with proven effectiveness is lacking.
Therefore, further randomized controlled studies are required
to investigate immunomodulatory therapies in RPL, the association
between VDD, VDI, and incidence of RPL, the benefit of VD supple-
mentation in women with RPL and how this supplementation should
be implemented.
C O N FL I C T S O F I N T E R E S T
The authors declare no conflict of interest.
ORCID
Daniela Reis Gonçalves  http://orcid.org/0000-0002-9925-4670
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How to cite this article: Gonçalves DR, Braga A, Braga J,
Marinho A. Recurrent pregnancy loss and vitamin D: A
review of the literature. Am J Reprod Immunol. 2018;e13022.
https://doi.org/10.1111/aji.13022