Luciano G. Nardo1 and Spyridon Chouliaras2
(1)
Reproductive Health Group, Centre for Reproductive Health, Daresbury Park, Daresbury, WA4 4GE, Cheshire, UK
(2)
Centre for Reproductive Medicine, Kenton & Lucas Wing, St Bartholomew’s Hospital, London, EC1A 7BE, UK
Luciano G. Nardo
Email: lnardo@reproductivehealthgroup.co.uk
Keywords
ProgesteroneRecurrent miscarriageHabitual abortionImplantation failureLuteal phase defectImmunomodulationProgesterone receptorsProgesterone induced blocking factorCytokines
1 Introduction
Progesterone has been implicated as being essential for successful embryo implantation, and for the prevention of miscarriage. In fact, progesterone was one of the first pharmacological agents to be prescribed for managing recurrent miscarriage (RM) as early as 1950 [1]. The role of progesterone for the treatment of threatened miscarriage is covered elsewhere in this book and will not be discussed here. In the current chapter we will attempt to discuss the available evidence and review the rationale for the use of progestational agents in cases of recurrent miscarriage alone.
Recurrent miscarriage (RM) affects approximately 1 % of couples trying to conceive [2]. RM has been defined as three or more consecutive spontaneous pregnancy losses with the same biological father. However, in some parts of the world including the US, RM is defined as two or more consecutive losses [3]. The fact that RM can be defined as two or more losses, complicates the interpretation of epidemiological studies and makes the available research on this subject heterogeneous. Miscarriage is defined in North America as pregnancy loss prior to 20 weeks. However, in Europe, the term miscarriage includes all pregnancy losses from the time of implantation until 24 weeks of gestation, although advances in neonatal care have resulted in babies surviving before this gestation. Primary RM refers to women with no prior successful pregnancy, while secondary RM refers to recurrent losses following a live birth.
The difference between RM and sporadic miscarriage is that statistically as the demonstrated effect is being repeatedly observed the chances are that the causality is due to a systemic and recurring factor, most likely maternal in nature. Women with recurrent miscarriages tend to lose genetically normal pregnancies compared to women with sporadic miscarriage [4]. The observed incidence of recurrent miscarriage is higher than would be expected by chance alone (0.34 %) [5]. This in itself indicates that RM is a separate clinical entity to sporadic miscarriage. However, the 0.34 % chance was calculated assuming a sporadic miscarriage rate of 15 %, which is probably an underestimation.
The etiology of RM has been extensively researched. The underlying causes could be either maternal or fetal in origin. Maternal causes may include uterine factors such as endometrial pathology or congenital uterine anomalies, hormonal imbalance and insufficiency, infections, defective immunoregulation, hereditary thrombophilia, and antiphospholipid syndrome. Fetal causes include chromosomal abnormalities and structural malformations both of which are incompatible with life. Nevertheless in more than 50 % of cases no cause can be identified [6], which adds to the distress of the patient.
The prognosis of RM has been reported to be better in secondary than in primary recurrent miscarriage [7]. A descriptive cohort study of 987 women who presented with RM in a tertiary centre showed that approximately two-thirds of women with RM succeeded in having a live birth within 5 years after the first consultation, but a full third did not. There was a significantly decreased chance of at least one subsequent live birth with increasing maternal age and increasing number of miscarriages at first consultation [8].
Recurrent miscarriage is a devastating experience for the patient and a dilemma for the clinician. The emotional and psychological implications for the patient are very significant. There may be feelings of desperation, frustration, guilt, depression, low self-esteem and distrust which may overwhelm the patient. As a result the physicians have never stopped seeking ways of treating RM, especially as these patients are willing to try anything to achieve a live birth.
2 The Role of Progesterone
Progesterone was one of the first female sex hormones discovered. Its role in the successful implantation of an embryo led to progesterone being called ‘the hormone of pregnancy’. It was shown over 40 years ago that surgical removal of the corpus luteum before the eighth week of pregnancy lead to spontaneous miscarriage [9]. All progestogens are placentotrophic and their use was thought to improve trophoblastic proliferation into the spiral arteries. Progestogens are generally thought to be safe, and have become standard treatment for luteal support in assisted reproduction. Worldwide the demand from women for any treatment that provides hope drives clinicians to prescribe progestogens or progesterone. Progestogens have been used for more than half a century for the prevention of RM. Since those early years the use of progestogen preparations has been controversial, many authors have disputed the acclaimed beneficial role [10], and claimed a lack of robust evidence. The debate regarding the efficacy of this treatment is ongoing.
In this chapter we will attempt to present the pathophysiology behind the potential action of progestogens for prevention of RM as well as the best available evidence. The focus will be recurrent miscarriage of unexplained aetiology (also known as idiopathic recurrent miscarriage).
2.1 Potential Mechanisms of Action of Progestogens in Preventing RM
Implantation has been described as a three-stage process. The first stage comprises the apposition of the blastocyst to the endometrium. The second stage involves the adhesion of the embryo to the endometrial epithelium. In this stage the blastocyst can no longer be removed by just being flushed out. The adhesion of the blastocyst on the endometrium is due to cell surface glycoproteins, the specific mechanisms of which, are still largely unknown. The two first stages are mainly mediated by integrins, mucins, trophinins and tastins. The third stage is the invasion and embedding of the trophoblast. The invasive stages consist of two phases: early and deep invasion. Early invasion is an interplay between matrix metalloproteases (MMP) secreted by the embryo and tissue inhibitors of these proteases (TIMMP) secreted by the endometrium. During deep invasion there is an interaction between T-helper (Th) 1 cytokines preventing implantation and Th2 cytokines enhancing implantation [11].
Implantation failure has been thought to be the cause of many cases of recurrent miscarriage, as well as other reproductive sequelae such as recurrent assisted conception failure and pre-eclamptic toxaemia of pregnancy [12]. There are various ways in which progesterone influences and regulates the physiological process of implantation. This is irrespective of the mode of administration, which could be rectal, vaginal or intramuscular.
2.2 Effects of Progesterone on the Uterus and the Endometrial Environment
2.2.1 Endometrial Development and Luteal Phase Deficiency
Oestrogens and progesterone secreted cyclically by the ovaries, control morphological and functional changes in the endometrium, which leading to a favourable endometrial environment for implantation. This favourable environment is commonly referred to as the ‘implantation or nidation window’ a period where endometrial receptivity is optimal [13–15]. The nidation window is approximately 6 days after the LH surge [16] (approximately day 20 in a 28 day cycle). However, there is no consensus as to the duration of such a window [17]. Work by Navot in human embryos suggested that the implantation window lasts 4–5 days synchronous with peak progesterone concentrations [18–21].
Luteal phase deficiency or luteal phase defect (LPD) is a term that was introduced in 1949 by Georgianna-Segar Jones [22]. It has been used to describe a decrease in the amount or the duration of progesterone secretion from the corpus luteum or lack of an adequate endometrial response to ovarian steroids [22–24]. The gold standard for the diagnosis of LPD has traditionally been the morphological examination of a precisely timed luteal phase endometrial sample according to the Noyes’ criteria [25]. The possible presence of two types of LPD has also been suggested: one related to immature follicles, and one where the follicles are mature [26]. Regardless of the type, progesterone supplementation aids the creation of a more receptive endometrial environment. Over the years, there have been several reports of significant variability in the histological evaluation of human endometrium dating [27, 28]. The need and usefulness of histological dating of the endometrium itself has been questioned [29], and although it has served its cause, it is nowadays considered outdated.
In recent years scientists have been seeking novel approaches to characterise the endometrium. They have utilised the ability to describe morphological changes using scanning electron microscopy (SEM), and the development of techniques that focus on molecular aspects of endometrial development. Using SEM on the uterine epithelium, both Martel et al. [30], and Nikasand et al. [31] have shown the existence of specialised cell surface structures called pinopodes (oruterodomes). The development of pinopodes has been associated with the adhesion of blastocysts to the luminal epithelium [32] and have thus been considered as markers of receptivity. Progesterone stimulates the appearance of pinopodes, whereas oestrogens cause their regression [30]. Supraphysiological levels of oestradiol such as those achieved during controlled ovarian stimulation have been associated with impairment of uterine receptivity [33]. However, other studies have failed to demonstrate a reliable pattern of pinopod expression [34, 35], and their significance as markers of endometrial receptivity remains a matter of much debate.
Despite these uncertainties, the role of progesterone in successful implantation is not disputed. The failure to synchronize the complex mechanisms involved in the crosstalk between the endometrium and the embryo results in failure of implantation. Of the numerous parameters of this synchronization process, the role of steroid hormones has been extensively studied. It is generally accepted that progesterone deficiency could contribute to the pathophysiology of recurrent pregnancy loss by delaying endometrial development. Low progesterone levels have been found in recurrent miscarriage with delayed endometrial ripening compared to normal endometrial ripening [36–38].
An increase in the secretion of oestradiol precedes ovulation and promotes the proliferation and differentiation of uterine epithelial cells. It is then followed by the secretion of progesterone, which induces stromal cell development [39]. Progesterone acts on the endometrium via specific progesterone receptors (PR) or by changing the isoforms ratio and possibly their expression level. Receptor synthesis is controlled by oestrogens through oestrogen receptors during the proliferative phase. By down-regulating oestrogen receptors, progesterone leads to a fall of both oestrogen and progesterone receptors [40].
Polymorphisms of progesterone receptors (PROGINS) have been reported to act as a risk-modulating factor in women with RM. Receptor polymorphism may cause an alteration in the biological function of PR and can be associated with an individual susceptibility to pregnancy loss, though this concept has not been confirmed in a recent meta-analysis [41, 42]. It appears that inappropriate endometrial development can occur even with sufficient progesterone levels [43], possibly due to genetic variation of progesterone receptors. The concept of absolute or relative progesterone deficiency in the pathophysiology of recurrent miscarriage, could explain why progesterone treatment may benefit some but not all women with unexplained RM.
2.2.2 Induction of Uterine Quiescence
In animal models, progesterone has been recognised as one of the major causes of inhibition of myometrial contractility. Withdrawal of progesterone is responsible for the initiation of labour. In humans, there is no detectable progesterone withdrawal, but biochemical events suggest ‘functional progesterone withdrawal’. Potential mechanisms include changes in receptor isoforms and decreased myometrial sensitivity to progesterone. Nitric oxide (NO) generated in the pregnant uterus has been shown to maintain uterine relaxation [44]. Several studies have shown that progesterone enhances NO production in the endometrium [45–48]. Pharmacological withdrawal of progesterone by administration of 3-beta-hydroxysteroid dehydrogenase inhibitors or mifepristone (RU486) is associated with the onset of labour, and has been widely used to terminate early pregnancy by competitively blocking the PR [49].
2.3 Immunological Role of Progestogens
The survival of the semi-allogeneic fetus has been described as an immunological paradox. Maternal recognition of fetal antigens does not appear to compromise pregnancy. On the contrary it induces functional modifications that allow the conceptus to survive and develop. Maternal immunetolerance is established in the decidua, in a specific area known as the feto-maternal interface. It is recognised and accepted that progesterone exhibits immunomodulating properties. Indeed, there is increasing evidence that progesterone facilitates an immune environment conducive to the early development of pregnancy.
2.3.1 Involvement of Progesterone in Maternal Cytokine Production
Progesterone-induced blocking factor (PIBF) is a protein synthesised by lymphocytes of pregnant women in the presence of progesterone. Progesterone receptors on lymphocytes are moderated by the immunological recognition of pregnancy [50]. PIBF is associated with both the immunomodulatory [51] and anti-abortive [52–54] properties of progesterone. Lymphocytes from healthy pregnant women produce significantly more PIBF than those of women with pathological pregnancies.
In pregnancy there is a physiological shift in the decidual cytokine pattern from a Th1 response to a Th2 response. The cytokine shift may be modulated by PIBF [55]. Th1-type pro-inflammatory cytokines (TFN-α, IFN-γ, IL-2) support allograft rejection and are thought to be detrimental to pregnancy. TNF-, α activates natural killer (NK) cells, promotes apoptosis of the trophoblast and initiates coagulation, at least in mice [56]. Inteferon-γ can induce expression of major histocompatibility antigens on the trophoblast, where they are not normally expressed. Th2- cytokines (TGF-β2, IL-3, IL-4, IL-5, IL-10) inhibit pro-inflammatory Th1 responses, and seem to benefit pregnancy maintenance [57]. TGF-β2 induces trophoblast proliferation, IL-4 and IL-10 inhibit prothrombinase.
The activation of peripheral blood mononuclear cells (PBMC) by trophoblast antigens confirmed that women with RM have a Th1-type cytokine profile [58]. Increased production by PBMC of Th1-type cytokines and decreased levels of Th2-type cytokines have been demonstrated in non-pregnant women with recurrent early pregnancy losses [59]. Bates et al. [56] failed to demonstrate the proposed defect in the shift fromTh1 to Th2 cytokines in women with RM. Instead, increased production of IL-4 and IL-10 was shown in such women, along with reduced IFN-γ in pregnant women [56]. Nevertheless the case for a possible association between maternal Th1 dominance and recurrent miscarriage is strong. Researchers are therefore faced with the challenge of determining the optimal Th1/Th2 cytokine balance and trying to manipulate it towards an immune favourable environment.
Vaginal micronised progesterone has been reported to be associated with a decrease in IFNγ (Th-1) and increase in IL-10 (Th-2) in endocervical fluid [60]. In addition, progesterone up-regulates Leukemia inhibitory factor (LIF) mRNA expression in vitro [61]. LIF is essential for implantation in muridae.
Dydrogesterone is the most commonly used progestogen to support early pregnancy. Dydrogesterone can be administered orally and has high affinity with the PR, resembling endogenous progesterone in its pharmacology and biochemistry. Raghupathy et al., investigated the effects of dydrogesterone therapy on Th1 and Th2 cytokine production in RM. Down-regulation of Th1 cytokines (TNFα and IFNγ) and stimulation of Th2 cytokines (IL-4 and IL-6), and induction of PIBF production was reported [62]. Other researchers have suggested that the induction of PIBF production in humans could be the indirect mechanism by which dydrogesterone improves pregnancy outcome [63].
2.3.2 Involvement of Progesterone in Maternal Natural Killer (NK) Cells
There has been a considerable amount of research into the association between NK cells and recurrent miscarriage. The number of peripheral NK cells (pNK) have been associated with RM [64, 65]. However, the number or killing activity of pNK cells may not reflect the condition in the endometrium where implantation occurs; hence, the potential role of pNK cells in the pathophysiology of miscarriage remains uncertain. However, large numbers of uterine NKcells (uNK) cells may be associated with RM, but the mechanism is still unclear. High numbers of uNK cells have been found in luteal-phase endometrial biopsies of women with recurrent miscarriage compared to the pre-implantation endometrium in normal pregnancies [66]. Interestingly a recent meta-analysis that evaluated uNK cells expressed as percentage of the stromal cells failed to demonstrate a significant difference between these two groups [67]. However, the studies included in the meta-analytical pooling appeared to suffer from significant heterogeneity.
Natural killer cells, which are present in the endometrium in the luteal phase of the menstrual cycle become decidual NK cells in pregnancy. They increase in number in the first trimester [68] and are thought to control trophoblastic invasion through the production of immunoregulatory cytokines and angiogenic factors [69, 70]. Although decidual NK cells do not express progesterone receptors both the number and function of these cells are influenced indirectly by progesterone [71]. Szekeres-Bartho et al., have demonstrated that a low proportion of PIBF-positive lymphocytes is inversely related to NK cell activity and pregnancy loss [72]. PIBF is thought to contribute to the suppression of decidual NKcells cytolytic activity [73].
The role of progesterone on maternal NK cells is still being evaluated. When interpreting studies on NK cells, one should take account of the compartment (peripheral blood, endometrial, decidua etc.) in which the cells or other prognostic markers are investigated.
3 The Evidence for Progesterone Use in Recurrent Miscarriage
There have been several meta-analyses evaluating the use of progesterone for the prevention of subsequent miscarriage after RM. A recent meta-analysis in the Cochrane database showed a statistically significant reduction in miscarriage in favour of those patients randomized to the progestogen group (OR 0.39; 95 % CI 0.21–0.72) [74]. A similar outcome was reported by the same authors 2008 [75] and by two earlier meta-analyses in 2003 [76] and 1989 [36], respectively. The results of these metaanalyses do not differ as all the metaanalyses summarise the same three studies [77–79], which are now more than 50 years old and suffer from inadequate design and methodology. There have also been issues in the meta-analytical pooling. The three papers included in the earlier metaanalyses, did not account for the confounding factor of embryonic aneuploidy, nor did they stratify for the number of miscarriages. Additionally, three different progestogens were analysed, medroxy-progesterone acetate, 17-hydroxyprogesterone acetate and an implant. One study of dydrogesterone [80] was excluded from the earlier metaanalyses due to quasi-randomization rather than true randomization. However, El-Zibdeh et al. [80] is the largest to date including 180 women younger than 35 years and with a history of three or more consecutive idiopathic miscarriages in their trial. Women were randomized according to the day of the week in which they presented to receive either dydrogesterone (10 mg orally twice daily), human chorionic gonadotropin (hCG, 5,000 IU intramuscularly once every 4 days) or no treatment until week-12 of gestation. Miscarriages were significantly less common in the dydrogesterone group (13.4 %) than in the control group (29 %). Furthermore, there were no differences across the groups in respect to pregnancy complications or congenital anomalies. El-Zibdeh et al.’s [80] study, was included in the most recent meta-analysis [69], although neither placebo-controlled nor blinded. Three [77–80] of the included studies appeared to have had selection biases, and apparently only two of these studies [79, 80] enrolled women who had suffered three or more consecutive miscarriages. Two other studies [77, 78] recruited women with two or more miscarriages and provided separate pregnancy outcome data by the number of previous consecutive pregnancy losses. There was also significant variability with regard to the age of the subjects, previous successful pregnancy outcome, the investigations used to exclude other causes of miscarriage, as well as to the duration, dosage, time of initiation of treatment and choice of progestogen regimes. At present, a large randomised trial is underway (PROMISE) to assess micronised progesterone in RM. The results are eagerly awaited.
4 Safety of Progestogen Treatment for Prevention of Recurrent Miscarriage
4.1 Safety of the Mother
Worldwide millions of women have used progesterone or progestogens, and 39 million women have been assumed to have been exposed to dydrogesterone alone [81]. Few studies have accounted on the maternal adverse effects of progesterone use to prevent miscarriage [82]. However, few side effects have been reported. Typical maternal side effects of progestogens such as nausea, bloating, dizziness, breast tenderness, mood changes, cephalalgia may occur, but can also be attributed to the physiological changes occurring in early pregnancy.
Theoretically there could be concerns that progesterone may delay spontaneous miscarriage, by promotion of uterine quiescence or even aid retention of chromosomally abnormal embryos. However, it seems that the mechanism in immunologically mediated abortion may be different to that in abortion due to aneuploidy. In the latter event implantation fails altogether, whereas in immunologically mediated abortion there is adequate implantation and subsequently an immunologically mediated process of vasculitis, inflammation leading to thrombus formation. Progestogens only influence chromosomally normal embryos by regulating immunomodulation [83, 84].
4.2 Safety of the Fetus
There have been reports suggesting an association between intrauterine exposure to progestogens in the first trimester of pregnancy and genital abnormalities in both male and female fetuses. Some progestogens such as ethisterone have been thought to induce mild virilisation of the external genitalia in the female fetus [85, 86]. However, virilisation has only been seen in rat fetuses. The lack of evidence in humans has made it impossible to quantify the risk. Carmichael et al. [87] reported that maternal intake of progestins in early pregnancy is associated with an increased risk of hypospadias in the male fetus due to an anti-androgenic effect (OR 3.7, 95 % CI 2.3, 6.0) [81]. Other studies do not indicate an increased risk with exposure to progestins. Interestingly dydrogesterone has less of an antiandrogenic effect than progesterone itself. The urogenital groove is fused by 16 weeks of gestation, in order to avoid the antiandrogenic effect, some authors have recommended that progesterone containing medications should be avoided in the first trimester of pregnancy [88].
5 Future Research
The lack of robust data has generated the need for well-conducted studies to assess the validity of intervention with progesterone supplementation in RM. In a small unpublished study it was reported that 90 % of obstetricians and gynaecologists called for a definitive placebo controlled randomised trial [89]. The potential pitfalls for such a study are many, and careful design is of paramount importance. Matching age and number of miscarriages is one component. Stratification for primary versus secondary miscarriage is another factor which has to be taken into account. Unfortunately heterogeneity in human populations is unavoidable even in patients with exactly the same clinical characteristics. All pregnancies should have been conceived with the same partner, which is again almost impossible to elicit. The exclusion of other causes of miscarriage is also difficult to ascertain, as there are subjective variables such as the presence of congenital uterine anomalies. One randomized study is underway and the results are eagerly awaited. The study will hopefully shed more light in the use of progestogens for RM. The ‘Progesterone in recurrent miscarriage (PROMISE) study’ (ISRCTN92644181) is a randomised, double blind, placebo controlled multicentre trial based in UK and the Netherlands studying the effect of progesterone treatment given in the first trimester of pregnancy in women with a history of unexplained recurrent miscarriages, who conceive spontaneously. The intervention being assessed is vaginal micronised progesterone pessaries 400 mg twice daily starting as soon after a positive pregnancy test as possible (but no later than 6 weeks of gestation) and continued up to 12 weeks of gestation. The primary outcome is live birth beyond 24 completed weeks. The results are awaited in March 2015 [89].
6 Conclusions
Progesterone is a ‘pro-gestational’ agent that maintains the pregnant state. The concept of the association of RM with retarded endometrial development in the peri-implantation period or luteal phase defect is well recognised. The immunomodulatory function of progesterone appears to be decisive in early pregnancy. It is therefore quite possible that there may be a role for its use in women with unexplained RM. The paucity of good quality evidence about the efficacy of progesterone in women with a history of recurrent early pregnancy loss is responsible for contradictory and ever-changing views amongst clinicians.
Research into RM is ongoing and focused on several areas, mostly at the bio-molecular level. Factors such as circulating pro-coagulant microparticles, glycoproteins (for example, hCG and glycodelin), leptin receptors and TNF-α inhibitors are being investigated. As our understanding of the pathophysiology of recurrent miscarriage improves new causes are likely to be identified. This will lead to individualisation of treatment for RM.
The role of progesterone treatment for unexplained recurrent miscarriage will be re-evaluated after new data from ongoing studies becomes available. However, even if these studies fail to demonstrate evidence of benefit, maybe in the future the validity of the results could be questioned as the criteria for diagnosis of unexplained RM in this high-risk population may change dramatically in the light of novel basic science research findings.
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