Keywords
pregnancy - recurrent spontaneous miscarriage - progesterone - dydrogesterone - immunomodulation
Introduction
Our immune system with its exquisite specificity and versatile capabilities is what
stands between us and the bewildering range of pathogens. The immune system manages
to prevent a whole host of foreign invaders such as viruses and bacteria from infecting
us using its arsenal of innate and adaptive immunity, molecules like antibodies, complement,
and cytokines, and cells such as macrophages, lymphocytes, and granulocytes. However,
much as the immune system serves as a protective defense barrier, it is also responsible
for adverse reactions such as autoimmune diseases and hypersensitivities. One such
problem, based on the interactions between the immune system and the reproductive
system, occurs when the immune system interferes with fertilization and causes autoimmune
infertility.[1]
[2] Another problem is the sometimes adversarial effect that the maternal immune system
has on pregnancy. Several potential complications may arise between the long period
of pregnancy from fertilization to delivery; these include threatened miscarriage,
spontaneous miscarriage, pre-eclampsia, preterm rupture of fetal membranes, and preterm
delivery. A large number of studies have examined the roles of the maternal immune
system in contributing to the pathogenesis of many of these conditions but in this
review, we will focus on the immunology of spontaneous miscarriage.
Methodology of the Review
Methodology of the Review
A comprehensive search strategy was used to identify articles relevant to this review.
Literature searches were done by using PRISMA-IPD (Preferred Reporting Items for a
Systematic Review and Meta-analysis of Individual Participant Data)[3] in the following databases: PubMed, Google Scholar, and MEDLINE via OVID. These
searches were conducted by the first author (S.R.) and then examined by both authors
(S.R. and R.R.). References for this review were identified by searches from January
1, 1990 until March 1, 2020 by the use of the search terms “recurrent spontaneous
miscarriage,” “cytokines,” “progesterone,” “progestogen,” “dydrogesterone,” and “immunomodulation.”
Only articles in English were included. The articles included original research papers
and reviews.
Recurrent Spontaneous Miscarriage
Recurrent Spontaneous Miscarriage
Spontaneous miscarriage, defined as a pregnancy loss in the first 20 weeks of gestation,
occurs in one out of every four pregnant women. Recurrent spontaneous miscarriage
(RSM) is the occurrence of two or more pregnancy losses prior to the 20th week of
gestation and is one of the most challenging disorders of pregnancy.[4] It may come as a surprise that despite decades of research only 40 to 50% of the
cases of RSM are due to “known” causes such as chromosomal abnormalities, endocrinologic
aberrations, infections, and anatomical problems.[5] As much as 60% of the cases of RSM are due to “unknown” or “unexplained” etiologies
([Fig. 1]).[4] Thus the cause of RSM has remained unexplained in a majority of the cases and this
has stimulated research into possible immunologic etiologies of RSM. Immunologists
have played valuable roles in research in obstetrics and gynecology, by exploring
immunologic factors that may be responsible for RSM unexplained in terms of genetic,
infectious, and endocrinologic factors.
Fig. 1 Etiologies of recurrent spontaneous miscarriage.
Immunologic Etiologies of Recurrent Spontaneous Miscarriage
Immunologic Etiologies of Recurrent Spontaneous Miscarriage
Both humoral immunity and cell-mediated immunity have been investigated as possibly
contributing to RSM. Interestingly, the fetoplacental unit is resilient to attack
by humoral immune factors except for anti-phospholipid antibodies which are clearly
implicated in a proportion of RSM.[6]
Much attention has been focused on cell-mediated immune effectors as possible etiologic
factors; these include maternal T lymphocytes, macrophages, and natural killer (NK)
cells in maternal peripheral blood and in uteroplacental tissues. A significant boost
to research in this area came from the revolutionary discovery of the different subsets
of T helper (Th) lymphocytes and the cytokines produced by them. As important effectors
of cell-mediated immunity, cytokines have received a great deal of attention in the
field of maternal–fetal immunology.
What are Cytokines?
What are cytokines and what is the relevance of cytokines to pregnancy and to pregnancy
loss? Cytokines are the most crucial mediatory molecules of the immune system; they
play extremely vital roles as communication signals primarily between cells of the
immune system, and also between other cells in the body. Cytokines are pluripotent
mediators of an impressive range of immune responses including the initiation and
sustenance of normal immune responses to infections, rejection of allografts, autoimmune
diseases, and hypersensitivity.[7]
[8]
Cytokines synthesized and secreted by immune cells such as Th lymphocytes, macrophages,
and NK cells have been studied extensively in the context of the maternal–fetal relationship.
Th1 and Th2 cells are the major subsets of Th cells, with different patterns of cytokine
production and thus different roles in immune responses.[9]
[10] Th1 cells secrete the cytokines interferon (IFN) γ, tumor necrosis factor (TNF)-β,
TNF-α, and interleukin (IL)-2; these so called Th1 cytokines stimulate and maintain
strong cell-mediated and inflammatory reactions such as cytotoxicity and delayed-type
hypersensitivity. These inflammatory cytokines mediate graft rejection, autoimmune
disease pathology, and inflammatory tissue damage. On the other hand, Th2 cells secrete
the cytokines IL-4, IL-5, IL-6, IL-10, and IL-13 which stimulate robust antibody production.
Th1 and Th2 cells are mutually antagonistic to each other; some Th1 cytokines suppress
the activation and/or the function of Th2 cells and vice versa.[9]
[10]
Cytokines and Pregnancy Loss
Cytokines and Pregnancy Loss
Healthy pregnancy is associated with a natural pregnancy-induced immunomodulation
in which there is a downregulation of Th1 responses and upregulation of Th2 responses,
manifested as a suppression of cell-mediated immunity and an enhancement of humoral
immunity.[11]
[12]
[13] Indeed, cellular immunity mediated by effector cells and/or cytokines released by
them have been demonstrated to harm the conceptus. The administrations of single low
doses of the inflammatory Th1 cytokines TNFα, IFNγ, and IL-2 into pregnant mice cause
abortions while the injection of anti-TNFα antibodies reduces abortion rates in a
murine model of natural, immunologically-mediated abortion.[14] TNFα and IFNγ block the outgrowth of human trophoblast cells in vitro[15] and synergistically induce the apoptosis of human primary villous trophoblast cells.[16]
Considering that pro-inflammatory or Th1 cytokines have so many cytotoxic and tissue-damaging
effects, as well as anti-pregnancy capabilities, it is not surprising that unexplained
RSM is associated with a greater bias toward Th1or pro-inflammatory cytokines.[17] Peripheral blood cells from women with a history of RSM when stimulated with human
trophoblast antigens were shown to secrete much higher levels of Th1 cytokines with
embryotoxic activity.[18] We have demonstrated that mitogen-stimulated peripheral lymphocytes from women with
a history of healthy pregnancy produce significantly higher levels of the anti-inflammatory
Th2 cytokines IL-4, IL-5, and IL-10; on the other hand, women with unexplained RSM
produce significantly elevated levels of the pro-inflammatory cytokines IL-2, IFNγ,
and TNF-α ([Figs. 2]
[3]).[19]
[20] We went on to confirm this by studying maternal immune reactivity to placental antigens
stimulated either by co-culturing maternal lymphocytes with autologous placental cells
and by exposing maternal lymphocytes to a trophoblast antigen preparation.[21] The ratios of inflammatory cytokines to anti-inflammatory cytokines were higher
in women undergoing RSM compared with women undergoing healthy pregnancy; this supports
the contention of Th1 or pro-inflammatory cytokine dominance in RSM as opposed to
a stronger Th2-bias in healthy pregnancy.[22] Marzi and colleagues studied cytokine production 1 to 2 weeks before any upcoming
pathology could be detected; they found decreased production of the anti-inflammatory
cytokines IL-4 and IL-10 and increased production of the pro-inflammatory cytokines
IFNγ and IL-2 by antigen-stimulated lymphocytes from women with RSM as opposed to
those from normal pregnancy.[23]
Fig. 2 Effects of progestogens on Th1 cytokine production. Effects of progesterone (PHA+P),
dydrogesterone (PHA+D), and dihydrodydrogesterone (PHA+DHD) on the production of IFN-α
(A) and TNF-α (B) by peripheral blood lymphocytes stimulated with a mitogen (PHA).
The levels of these two inflammatory Th1 cytokines are higher in recurrent miscarriage
than in normal pregnancy and these are suppressed by progesterone (P), dydrogesterone
(D), and dihydrodydrogesterone (DHD). IFN, interferon; TNF, tumor necrosis factor.
Fig. 3 Effects of progestogens on Th2 cytokine production. Effects of progesterone (PHA+P),
dydrogesterone (PHA+D), and dihydrodydrogesterone (PHA+DHD) on the production of IL-4
(A), IL-10 (B), and IL-13 (C). The levels of these three anti-inflammatory Th2 cytokines
are lower in RSM than in normal pregnancy, and progesterone, dydrogesterone, and dihydrodydrogesterone
increase the levels of IL-4 and IL-13. DHD, dihydrodydrogesterone; IL-4, interleukin-4;
RSM, recurrent spontaneous miscarriage.
The situation in the maternal periphery is mirrored by developments in the local environment,
i.e., at the maternal–fetal interface; lower levels of anti-inflammatory cytokine-producing
T cell clones were found in the decidua of women with unexplained RSM than in the
decidua of women with normal pregnancy.[24] Endometrial expression of pro-inflammatory cytokines are upregulated, and anti-inflammatory
cytokines downregulated, in women with idiopathic recurrent miscarriage as compared
with controls.[25] Most reports thus support the notion that women with recurrent miscarriage have
increased levels of Th1 cytokines, while women with healthy pregnancy have decreased
levels of Th1 cytokines and increased levels Th2 cytokines; there is thus an increased
pro-inflammatory cytokine bias in unexplained recurrent miscarriage.[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Mechanisms of Cytokine Action in Recurrent Miscarriage
Mechanisms of Cytokine Action in Recurrent Miscarriage
Pro-inflammatory Th1 cytokines have been reported to mediate damage to the fetoplacental
unit in different ways. NK cells in the blood and the uterus show elevated levels
and activity in RSM; NK cells, like activated Th1 cells, produce cytokines that are
known to be injurious to the trophoblast.[26] Th1 cytokines also have the ability to convert NK cells into lymphokine-activated
killer (LAK) cells that have been shown to lyse trophoblast cells; systemic levels
of LAK-like cells correlate with high miscarriage rates.[27] Hill explains that a Th1-dominated response could be detrimental to early placental
differentiation and growth and thus harm embryonic development.[28] Decidual NK cells are not cytotoxic, but secrete high levels of the Th1 cytokine
IFNγ[29] which activates decidual macrophages which are known to kill trophoblasts.[30]
TNF-α and IFNγ both Th1 inflammatory cytokines kill trophoblast cells by apoptosis,
and may thus directly damage the conceptus[18]
[31] and also inhibit the secretion of the growth-stimulating cytokine granulocyte-macrophage
colony-stimulating factor which contributes to the growth of placental cells. A potent
mechanism of damage to the placenta was described by Knackstedt and colleagues[32] who termed it “cytokine-triggered vascular autoamputation”; they showed that Th1
cytokines activate coagulation mechanisms that can lead to vasculitis which limits
blood supply to the implanted embryo.
Manipulation of Cytokine Profiles
Manipulation of Cytokine Profiles
If pro-inflammatory cytokines are associated with recurrent miscarriage, can we downregulate
these cytokines, and upregulate the beneficial anti-inflammatory cytokines? This would
help create a milieu that is conducive to the success of pregnancy. This rationale
is already being used successfully for diseases such as rheumatoid arthritis[33] and inflammatory bowel disease[34] using antibodies to cytokines and/or cytokine receptors. In the field of maternal–fetal
immunology we can consider the use of the pregnancy-related hormone progesterone,
which fascinatingly enough has been shown as early as in 1983, to have anti-inflammatory
and immunosuppressive properties.[35] Progesterone is of course indispensable for the establishment of the receptive endometrium
and this has been known for a long time; what is less widely known is that progesterone
contributes immunologically to the success of pregnancy by interacting with the maternal immune system. In fact
progesterone was referred to as “Nature’s immunosuppressant” 43 years ago, based on
studies that demonstrated immunosuppressive properties of progesterone.[36] Progesterone suppresses the activation and proliferation of lymphocytes,[37] suppresses the oxidative burst of activated monocytes, and has been shown to prolong
the survival of allografts when administered locally.[38]
Modulation of Cytokine Profiles by Progestogens
Modulation of Cytokine Profiles by Progestogens
Considering that pro-inflammatory cytokines are associated with recurrent pregnancy
loss on the one hand[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] and that progesterone has interesting immunomodulatory capabilities,[35]
[36]
[37]
[38] it stands to reason that progestogens can be considered for downregulating Th1/pro-inflammatory
cytokine reactivity.
A landmark study showed that progesterone inhibits the production of Th1 cytokines
by trophoblast antigen-activated peripheral blood cells from women with unexplained
RSM.[39] In addition to progesterone we have also investigated the immunomodulatory capabilities
of dydrogesterone (6-dehydro-9β, 10α-progesterone) (Duphaston®, Abbott Laboratories,
United States), an orally-administered progestogen, which is similar to endogenous
progesterone in its molecular structure and pharmacological effects, but more potent
than natural progesterone, with a high affinity for the progesterone receptor.[40]
We studied the production of cytokines by peripheral blood mononuclear cells (PBMCs)
stimulated with a mitogen; we observed that in the presence of dydrogesterone or progesterone,
PBMCs from women with a history of unexplained RSM produce significantly lower levels
of the Th1 (pro-inflammatory) cytokines IFNγ and TNFα as compared with women with
a history of healthy pregnancy ([Fig. 2]). On the other hand, levels of the Th2 cytokines IL-4 and IL-6 were significantly
elevated in the presence of dydrogesterone or progesterone ([Fig. 3]).[41] We found a significant reduction in Th1/Th2 cytokine ratios upon exposure to dydrogesterone,
indicating a decrease in dominance of Th1 or pro-inflammatory cytokines. We also showed
that the progesterone-receptor antagonist RU486 mitigates the effects of dydrogesterone
and progesterone indicating that the effect of dydrogesterone and progesterone is
mediated via the progesterone receptor.[41]
Several studies have focused on the potent pro-inflammatory cytokine IL-17 secreted
by a subset of T lymphocytes called Th17. IL-17 is a powerful chemoattractant and
activator of monocytes and neutrophils. The administration of IL-17 into pregnant
mice leads to embryonic loss indicating that this inflammatory cytokine is antagonistic
to pregnancy.[42] Increased levels of IL-17 have been observed in the peripheral blood and decidua
of recurrent aborters.[43] The incidence of uRSM is associated with an increase in the level of serum IL-17
and the number of Th17 cells in peripheral blood.[44] We have recently demonstrated that dydrogesterone also suppresses the production
of this inflammatory cytokine.[45] Thus, dydrogesterone suppresses not just Th1 cytokines but also the Th17 cytokine,
IL-17.
Thus, dydrogesterone has potent immunomodulatory properties; however, we were concerned
about the fact that soon after ingestion, dydrogesterone is converted into 20[α]-dihydrodydrogesterone
(DHD); in circulation dydrogesterone is actually present as DHD. Interestingly, we
found that this metabolite also inhibits the production of the pro-inflammatory cytokines
IFNγ and TNFα and upregulates the production of the anti-inflammatory cytokine IL-4
showing that major metabolite of dydrogesterone retains the immunomodulatory effects
of the parent molecule dydrogesterone.[46] which is an important feature if we are to consider dydrogesterone as a potential
therapeutic immunomodulator. Progesterone has also been shown to preferentially support
the development of human T cells producing Th2 cytokines in vitro leading to the inference
that progesterone promotes fetal survival by inducing the production of Th2 cytokines.[24] Subjects with a history of recurrent miscarriage having higher levels of serum progesterone
were shown to have lower Th1/Th2 cytokine ratios suggesting that progesterone levels
modulate cytokine production patterns in vivo.[47]
Progesterone modulates cytokine production profiles in a direct manner as described
above; however, it is also capable of indirectly modulating cytokine profiles via
a protein called progesterone-induced blocking factor (PIBF) discovered by Szekeres-Bartho
et al 35 years ago.[48] Fetal wastage induced by the transfer of NK cells into mice is reversed by the administration
of PIBF.[49] The blocking of endogenous PIBF with anti-PIBF antibodies, or the inhibition of
PIBF synthesis by blocking progesterone receptors results in Th1-dominant cytokine
production, significantly increased NK activity, and fetal loss which is corrected
by the treatment with anti-NK antibodies.[50]
PIBF is present in human serum and urine during normal gestation but urinary PIBF
levels fail to increase in pathological pregnancies as they do in normal gestation.[51] Failure to detect PIBF at 3 to 5 weeks of gestation was shown to be associated with
a higher rate of miscarriage.[52] Treatment of women undergoing threatened miscarriage with dydrogesterone proved
to be beneficial; this was associated with increased levels of PIBF and it appears
to be mediated through cytokines.[53] We have observed that PIBF induces a Th2-dominant cytokine response, by facilitating
the production of IL-4 and IL-10, thus altering the Th1/Th2 balance in favor of pregnancy.[54]
The cytokines IFNγ and TNFα that are downregulated by dydrogesterone and progesterone
are the very mediators that are detrimental to the development of the embryo, implantation,
and trophoblast proliferation[15] and that are toxic to trophoblast cells.[16] These two inflammatory cytokines cause fetal death when injected into pregnant mice.[14] Thus, the ability of progestogens to inhibit the production of these inflammatory
cytokines can be expected to be conducive to the success of pregnancy ([Fig. 4]).
Fig. 4 The impact of cytokines, progesterone, PIBF, and other factors and cells on the success
of pregnancy. Other immunological and nonimmunological factors, of both maternal and
placental origins, have been shown to influence pregnancy, but these are not discussed
in this review. PIBF, progesterone-induced blocking factor.
Supplementation with Progesterone and Dydrogesterone: Clinical Applications
Supplementation with Progesterone and Dydrogesterone: Clinical Applications
Supplementation with oral progesterone, compared with no treatment or treatment with
a placebo, has been shown in Cochrane reviews to result in a reduction in miscarriage
rate.[55]
[56] A systematic review of progesterone supplementation in RSM has shown a significant
beneficial effect.[57] Moreover, a randomized double-blind placebo-controlled trial showed that progesterone
supplementation results in significantly lower miscarriage rate, enhanced live birth
rate, and increased rate of pregnancy continuation beyond 20 weeks. Administration
of progesterone in the luteal phase of the cycle before confirmation of pregnancy
in women with history of unexplained RM reduces the risk of miscarriage.[58] Progesterone is currently being used in assisted reproductive technology to reduce
the occurrence of miscarriage chiefly for threatened and/or recurrent miscarriage.[59] Choi et al have proposed that progesterone may benefit women in whom the etiology
of RSM is related to maternal Th1 cytokine predominance.[39]
However, while supplementation with progesterone is beneficial, orally-administered
progesterone is absorbed poorly, is subject to first-pass mechanism, has a short biologic
half-life,[60] loses much of its bioactivity,[61] and is cleared quickly.[62] On the other hand, the orally-active progestogen dydrogesterone is highly selective
for the progesterone receptor is thus a more attractive alternative as it circumvents
these drawbacks.[40] As mentioned earlier, dydrogesterone retains its immunomodulatory activity even
after it is converted to its major metabolite after oral administration.[46] Another important feature of dydrogesterone is that its anti-androgenic properties
avoid the masculinization of female fetuses and reduces the chances of insufficient
masculinization of male fetuses.[63]
Several researchers have reported that supplementation with dydrogesterone is beneficial
in recurrent miscarriage. El-Zibdeh observed that dydrogesterone-treated women with
unexplained RSM had fewer miscarriages compared with placebo controls.[64] An important randomized, double-blind, placebo-controlled study on dydrogesterone
supplementation by Kumar et al recently demonstrated a significant decrease in the
number of miscarriages as well as an increase in the mean gestational age at delivery.[65] On the basis of a systematic review of randomized trials on dydrogesterone, Carp[66] concluded that supplementation with dydrogesterone results in a 13% absolute reduction
in the miscarriage rate, with minimal side-effects. The results of this systematic
review demonstrate a significant reduction of 29% in the odds for miscarriage as compared
with conventional care, thus confirming a real treatment effect.
Summary and Future Perspectives
Summary and Future Perspectives
The data presented in this review indicate that dydrogesterone, a progestogen that
is widely used for progesterone-related endocrinologic disorders of pregnancy, may
find use from an immunologic perspective as well. Dydrogesterone shifts the balance
from a Th1 or pro-inflammatory cytokine dominance toward a Th2 or anti-inflammatory
bias; this is a cytokine pattern that is favorable to the success of pregnancy ([Fig. 5]). Thus, dydrogesterone may be considered for effective, safe, and orally-administered
therapeutic intervention in unexplained RSM.
Fig. 5 Summary of direct and indirect mechanisms of the immunological actions of progestogens
on pregnancy.
Recent evidence suggests that dydrogesterone may be well worth considering for therapeutic
or preventive intervention in other pregnancy complications such as preterm delivery
and pre-eclampsia both of which have been shown to be linked to increased Th1 reactivity.[13]
[67]
[68]
[69]
[70] We have demonstrated that dydrogesterone suppresses the production of Th1 cytokines
and upregulates the production of Th2 cytokines by lymphocytes from women undergoing
preterm delivery.[71] A recent study showed that the administration of dydrogesterone in women at risk
for preterm labor resulted in increased production of Th2 cytokines and PIBF, as well
as decreased production of Th1 cytokines, suggesting that it could be considered for
prevention or treatment of preterm labor and delivery.[72] Similarly, supplementation with dydrogesterone from 6 to 20 weeks of pregnancy significantly
decreased the incidence of pre-eclampsia in women with high-risk pregnancy.[73] Thus, while this review focuses primarily on recurrent miscarriage, recent evidence
suggests that dydrogesterone can be considered for the prevention and/or treatment
of two other major complications of pregnancy: preterm delivery and pre-eclampsia.
The fact that dydrogesterone has a well-established safety profile in addition to
its attractive feature of being orally administered makes dydrogesterone a promising
candidate for immunomodulation of pregnancy complications.
