Medical Hypotheses (2004) 63, 250–253

http://intl.elsevierhealth.com/journals/mehy

1a,25-dihydroxy-vitamin-D3 as
new immunotherapy in treatment of
recurrent spontaneous abortion
I. Bubanovic*

Department of Gynecology and Obstetrics – “MEDICA CENTER” – Novosadska 1/c, 18000 Nis, Serbia and
Montenegro

Received 26 September 2003; accepted 23 November 2003

Summary Recurrent spontaneous abortion (RSA) is serious health problem affecting 2–5% of reproducing couples
worldwide. It has long been suspected that nearly 80% of the unexplained RSAs are due to immunologic causes.
Although the major tissue confronting the mother’s immune system is the placental villous trophoblast, the
immunological risk to the developing embryo is not great until the time of implantation. In addition, trophoblast is not
sensible to lysis by NK cells, TNF-a or macrophages, but may be killed by lymphokine activated NK cells (LAK) and may
undergo apoptosis in response to TNF-a and/or IFN-c in vitro. The two most commonly used treatments for RSA are
intravenous immunoglobulin (IVIg) and alloimmunization with partner’s leukocytes (LIT). We promote vitamin D3 as
new immunomodulatory agent in treatment of RSA. Different mechanisms have been proposed to account for the
immunosuppressive effect of 1a, 25-dihydroxy-vitamin-D3 (VD3). Portion of the VD3 activity involves the downreg-
ulation of IL-2, IFN-c and TNF-a genes transcription. Because immunomodulatory effects of VD3 are very similar to IL-
10 effects, acting of VD3 in immunotherapy of RSA syndrome, preeclamptic and eclamptic pregnancy, as well as PIH
syndrome, is very reasonable. We propose using of VD3 as immunotherapy or adjuvant therapy in combination with
classic immunotherapies of endangered pregnancies.
c 2004 Elsevier Ltd. All rights reserved.

Mechanisms of recurrent spontaneous cental cells play important immunoregulatory role,

abortion as they are much less sensitive to NK cell mediated
lysis. In addition, HLA-G may confuse, trap or de-
fuse the T cell receptor (TCR) complex. As none of
In most mammals, cells of the syncytotrophoblast
the forms of trophoblast carries class II MHC mol-
are totally MHC I and II negative, but the underlying
ecules they cannot stimulate T helper cells directly
cytotrophoblasts are weakly MHC I positive [1].
to begin immunological reactions. Consequently,
Extravillous cytotrophoblasts express a kind of MHC
without any Class I or II MHC molecules, the villous
class I molecules, so-called HLA-G and its expres-
trophoblast cells cannot function as the targets for
sion is limited to the placenta and the epithelial
MHC directed cytotoxic T cells [2,3].
cells of the thymus [2]. The HLA-G positive pla-
Cytokines are seen to have a complex role in
*
Tel: +38-1184-9178; fax: +38-1642-951-004. post-implantation pregnancy. In normal preg-
E-mail address: ibubanovic@yahoo.com. nancy, trophoblastic cells are resistant to lysis by


0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mehy.2003.11.037
1a,25-dihydroxy-vitamin-D3 as new immunotherapy in treatment of recurrent spontaneous abortion 251

cytotoxic T lymphocytes, NK cells and antibody The cytokines TNF-a and IFN-c play an important
dependent cytotoxicity [3]. The lymphokines, role in abortions, as their administration increases
which can activate NK cells into LAK cells, include the abortion rate and specific antagonists decrease
TNF-a, IL-2, IFN-, IL-12 and IL-18. Another cyto- the abortion rate. It has been proposed that
kines have been shown to prevent LAK cell activa- macrophage derived TNF-a stimulates NK cells to
tion and abortion. These include IL-3, GM-CSF, CSF- produce IFN-c, which further activates the macro-
1, IL-10 and TGF-b produced by CD8+ cells ex- phages, as occurs in the early defense response to
pressing progesterone receptors [3,4]. TGF-b has infectious agents. There are other potential sources
been shown to be a competitive antagonist of IL-2, of TNF-a and IFN-c, and systemic Th1 type re-
where IL-2 induces the activation of NK cells and sponses may cause abortions via augmenting levels
the secretion of TNF from these cells [4,5]. of such cytokines; IL-2 may also cause abortions by
T helper type 1 (Th1) cells produce abortogenic contributing to NK macrophage activation at the
cytokines such as IL-2 and INF-c which are seen to feto-maternal interface. TNF-a is thought to be
cause abortion in mice; T helper type 2 (Th2) cells part of the mechanism, which brings about preg-
however produce IL-3, IL-4 and IL-10 which pro- nancy loss through its effects on the placenta. In
mote antibody formation, put off inflammation and women experiencing RSA, circulating IFN-c pro-
NK cell activation [6–8]. The NK derived INF-c may mote mechanisms of MHC class I and II molecules
activate the macrophages of the feto-maternal expression. Consequently, MHC expressing cells
interface or other TNF-a secreting cells whereas become apparently target cells. In addition, IFN-c
Th2 cells would suppress this activation [4,5,8]. stimulated macrophages produce TNF-a. This in
Some authors represent opinion that the major effect would cause the recognition of the induced
inhibitor of the abortogenic reaction of the NK cells MHC expression and thus rejection of the fetal al-
is seen to be the trophoblast dependent natural lograft by the maternal immune system would re-
suppressor (NS) cell. During pregnancy, there are sult [4–6]. Maternal recognition of the conceptus as
an increased number of NK cells in the decidua and foreign is seen by some as the primary or possibly
these cells account for about 40% of the decidua the only step in preventing its rejection. In animal
lymphocytes. These cells are uniquely CD56+CD16) models, TNF-a and IFN-c coadministration aborted
and are seen to produce suppressor factors and Th2 >80% of the embryos, whether or not NK cells or
cytokines. It is possible that these NK cells are NS macrophages had been depleted or estradiol and
cells, which are responsible for maintenance of the progesterone was injected to correct potential re-
fetal allograft and initiating the appropriate im- duction in ovarian function by cytokines. Some au-
mune response [6,7,9]. thors have shown that the embryos die from
The CD8+ T cells with the ab-TCR appear to be ischemia due to activation of vascular endothe-
the more protective T cell subset. Production of IL- lial cell procoagulant, which causes thrombosis
4 and IL-7 by the trophoblast would be expected to and inflammation [6,8,9]. This appears similar to
deviate T cell differentiation along the Th2 path- the mechanism whereby TNF-a causes ischemic
way whatever the TCR phenotype and along with necrosis of nonantigenic tumors [12].
the trophoblast cell derived factors, such as GM-
CSF, may boost NS cell activity. Independently,
IL-10 and IL-4 may inhibit NK cell activation into
LAKs [8,10]. In these processes, maybe the most Current models of immunotherapy in
important mechanism is extrathymic lymphocyte patients with RSA past
maturation pathway. In pregnancy, the site of ex-
trathymic lymphocyte maturation is decidua Various forms of immunotherapy have been intro-
[5,6,10]. duced to treat couples suffering from recurrent
The basis for pregnancy failure is thus centered unexplained abortions. IVIg is seen to suppress anti-
on the activation of the NK cell into a LAK cell. phospholipid antibodies and is the therapy used
Some of the data obtained from studies of RSA when conventional anti-coagulant or immunosup-
seem to suggest that abortion occurs due to failure pressive treatment is ineffective. It has been noted
of the activation of NS cells. These cells carry the that the IVIg infusion contains anti-idiotypic anti-
cd-TCR rather than the ab-TCR that requires HLA- bodies, which inhibit the binding of anti-phospho-
A, B and C for efficient recognition and binding lipid antibodies to corresponding antigens and
[11]. As well as showing a deficiency in inactivate idiotype bearing B cells. Alteration of T
CD56+CD16) cells, women experiencing RSA show cell subsets, modulation of cell mediated responses,
an increased level of the CD56+CD16+ NK activated and blockade of the immunoglobulin FcgR on mono-
cells in the decidua and blood [2,3]. cytes, as well as reduction of the NK cytotoxicity
252 Bubanovic

have been reported [13]. Down-regulation of CD56+ duced by IL-4 [17,18]. The T cell response to allo-
and CD56+ CD16+ NK cells have been seen in women antigen is dependent on T cell receptor activation
treated with IVIg infusion. The infusion is effective in and costimulation via engagement of CD28 and
enhancing the percentage of live births among wo- CD40. A short treatment with fusion proteins and
men experiencing RSA. Recent data suggest that IVIg antibodies disrupting these co-stimulatory path-
therapy is useful in maintaining pregnancies among ways has been shown to prevent indefinitely acute
women with a history of RSA who lose karyotypically and chronic allograft rejection in rodents and pri-
normal embryos and who demonstrate elevated mates, stimulating the search for low molecular
levels of circulating NK cells [11,13]. weight compounds able to achieve tolerance in-
The application of LIT involves immunizing the duction by co-stimulation blockade. The unique
mother with leukocytes from either paternal or capacity of dendritic cells (DC) to activate naive T
third party origin. The LIT has been implicated cells correlates with elevated expression of MHC
in an attempt to produce a maternal immunoglob- antigens and costimulatory molecules, rendering
ulin effectors believed necessary for pregnancy them attractive targets for co-stimulation block-
maintenance [5,14]. The foundations for LIT is ade. VD3 inhibits the ability of antigen presenting
composed of three suppositions; (a) there is a ma- cells (APCs) to induce T cell activation and down-
ternal immune response to the conceptus that de- regulate APCs costimulatory molecules expression
velops in all pregnancies that must be blocked, (b) [15,18]. Treatment of human DC during their
blocking factors develop in all successful pregnan- differentiation from monocytes in the presence of
cies and (c) in the absence of blocking antibodies, GM-CSF and IL-4 with VD3 inhibited markedly the
rejection of the fetus occurs [5,14]. expression of CD80, CD86 and CD40, and partially of
The rationale for using seminal plasma in treat- class II MHC molecules, leading to an immature DC
ment of RSA is provided in the concept that the phenotype characterized by high mannose receptor
mammalian female responds to antigens present and low CD83 expression. The inhibitory effect of
not only on the trophoblast but also in seminal VD3 on DC maturation was comparable to that in-
plasma. The antiphospholipid syndrome, in women duced by IL-10, a cytokine which inhibits APC at
who suffer from RSA, has been successfully treated different levels, including secretion of IL-12
using aspirin, heparin, and prednisone or combi- [16,18]. The reduced expression of class II MHC and
nation of the three [5–7]. co-stimulatory molecules decrease the capacity of
DC to activate alloreactive T cells, as determined by
the decreased proliferation and abrogation of IFN-a
secretion in MLR. These results suggest that the
Immunomodulatory effects of VD3 ability of VD3 to decrease expression of co-stimu-
latory molecules on human DC might contribute to
Over the past decade, clinical evidence has been its inhibitory effect on APCs dependent T cell acti-
accumulating that VD3 and its analogs are effective vation and its immunosuppressive properties in al-
in the treatment of Th1 immunity mediated dis- lograft and trophoblast rejection. In the absence of
ease. Our opinion is that RSA is also Th1 immunity ConA stimulation, peripheral blood mononuclear
disease. The mechanism of VD3 activity, however, cells (PBMC) did not secrete detectable levels of
is not yet fully understood since this vitamin is IFN-c. When ConA was employed in the stimulation
pleiotropic. VD3 is thought to exhibit anti-inflam- of the cells, these cells synthesized detectable
matory properties, and has been shown to inhibit T levels of IFN-c [18]. If VD3 was presented in the
cell proliferation and the production of cytokines, medium, the vitamin significantly suppressed the
such as interleukin IL-2 and interferon IFN-c, and ConA stimulated IFN-c production by PBMC. In
TNF-a. Some authors have already reported that the absence of ConA, PBMC secreted small amounts
VD3 downregulate the production of inflammatory of TNF-a. VD3 significantly downregulate the ConA
cytokines, such as IL-1, IL-6 and IL-8, stimulated stimulated TNF-a by PBMC. Pichler et al. [18] used
with TNF-a and IFN-c [15,16]. RT-PCR to investigate the effects of VD3 on the
Human naive Th and cytotoxic (Tc) T cells, which transcription of cytokines [18]. Although IL-6 and IL-
only produce IL-2, may differentiate into Th1/Tc1 8 mRNA was detected in the freshly isolated PBMC,
or Th2/Tc2 like lymphocytes, characterized by their in the PBMC cultured for 24 and 48 hrs in the absence
cytokine production profile. VD3 has been reported of ConA stimulation, VD3 decrease the expression of
to inhibit Th1/Tc1 related, but increase Th2/Tc2 IFN-c, IL-8, TNF-a, IL-2 and IL-6 mRNA to below
associated cytokines in T cells from adults. VD3 also detectable levels [17,18].
inhibits not only IL-12 generated IFN-c production, Because effects of VD3 are very similar with
but also suppresses IL-4 and IL-13 expression in- immunomodulatory effects of IL-10, we have tested
1a,25-dihydroxy-vitamin-D3 as new immunotherapy in treatment of recurrent spontaneous abortion 253

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