Impaired endometrial function and unexplained recurrent pregnancy loss

Abstract

Couples with unexplained recurrent pregnancy loss (RPL) cannot achieve a live birth due to repeated sporadic abortions or undetected causes of RPL on routine examinations. Adverse intrauterine circumstances and endometrial decidualization are candidate risk factors for unexplained RPL. During peri-implantation and decidualization, endometrial stromal cells are transformed into decidual cells through acute inflammatory reactions, followed by an anti-inflammatory state, via reprogramming of the corticosteroid and retinoid signaling pathways. Inappropriate inflammation in mid-luteal endometrial stromal cells, such as chronic endometritis, is associated with aberrantly elevated uNK cell density, abnormal angiogenesis, and impaired endometrial decidualization, leading to repeated reproductive failure and complications of pregnancy such as hypertensive disorders. To the best of our knowledge, no efficient treatment for unexplained RPL has yet been established. The optimization of intrauterine circumstances and endometrial decidualization may be key to treating unexplained RPL.

Introduction

Approximately 10–15% of all human clinical pregnancies end up in pregnancy failure. Recurrent pregnancy loss (RPL) is defined as two or more pregnancy losses and includes biochemical pregnancy loss.¹⁾ The relative frequencies of two and three repeated pregnancy losses have been calculated to be 1–3% and 0.1–0.3%, respectively, which are significantly lower than the actual rates of 4.2% and 0.9%, respectively.²⁾ This suggests that factors associated with miscarriage are likely to contribute to increased pregnancy loss. Risk factors for RPL include uterine malformation, thrombophilia, endocrine abnormality, and chromosomal anomaly. Yet, >50% of women suffering from RPL show no specific problems in routine examinations.³⁾ Thus, even after testing negative for RPL, some might experience repeated sporadic abortions, or fail to achieve live birth due to undetected factors. Possible risk factors for unexplained RPL include adverse intrauterine circumstances and endometrial decidualization.⁴⁾ This review highlights the importance of optimal endometrial decidualization for maintaining early pregnancy and the relationship between impaired endometrial function and unexplained RPL.

Decidual transformation of the uterine endometrium for implantation and early pregnancy

In embryo implantation, human endometrial stromal cells (HESCs) differentiate—a process called decidualization—via the stimulation of progesterone (P4) secretion from the corpus luteum after ovulation and local cyclic adenosine monophosphate (AMP) production.⁵⁾ Decidualization plays important roles in regulating trophoblast invasion, protecting against oxidative stress, and modulating specialized uterine natural killer (uNK) cells (CD56^bright and CD16^dim).⁵⁾ uNK cells regulate inflammation, angiogenesis, and vascular remodeling via interleukin (IL)-11 and IL-15 secretion.^6,7,8,9) During decidualization, HESCs are transformed into decidual cells through acute inflammatory reactions, followed by an anti-inflammatory state. These inflammatory reactions synchronize the window of implantation.

Glucocorticoids exert potent anti-inflammatory and immunosuppressive effects. Endogenous glucocorticoids play important roles in embryo implantation, placentation, and fetal growth during early pregnancy. Glucocorticoids include active cortisol and inert cortisone that are mutually catalyzed by 11β-hydroxysteroid dehydrogenase (11βHSD). The NADPH-dependent 11βHSD isoform type 1 converts cortisone to cortisol, which is activated and binds to the glucocorticoid receptor (GR). However, cortisol also has a high binding affinity for the mineralocorticoid receptor (MR).¹⁰⁾ On the other hand, 11βHSD type 2 inactivates cortisol. During the transformation from HESCs to decidual cells, 11βHSD1, but not 11βHSD2, is highly induced. This induction is driven by P4 secretion, leading to the local production of active cortisol.¹¹⁾ Predominant cortisol-binding receptors alter GR to MR in undifferentiated cells in decidualizing HESCs.¹¹⁾ Balanced corticosteroid signaling is thus important for regulating inflammatory reactions to promote implantation and maintain early pregnancy in the decidualizing endometrium.

Glucocorticoid treatment can help modulate uNK cell count via GR, a surface nuclear receptor on uNK cells.¹²⁾ Moreover, 11βHSD1/GR signaling regulates inflammatory cytokines and prostaglandin and NF-κB signaling via GR.

MR is a predominant cortisol-binding receptor in decidualizing HESCs.¹¹⁾ HESCs with high uNK cell density are associated with 11βHSD1 and MR expression, but not GR expression;¹³⁾ therefore, MR and its gene network, including retinoid metabolism signaling, play essential roles in implantation and decidualization.¹¹⁾ Retinoids are versatile vitamin A (retinol) derivatives that regulate various cellular functions and bestow immune tolerance.¹⁴⁾ Retinol is transported to target cells, converted to retinal, and finally converted to all-trans-retinoic acid (RA), which is the most active retinoid metabolite^14,15) (Figure 1). Depending on the activation of specific pathways, RA regulates the following two opposing cell fate decisions: differentiation and apoptosis or senescence. RA bound to cellular RA-binding proteins (CRABP2 and FABP5) activates the RA receptor (RAR), leading to cell senescence or apoptosis. In contrast, RA-dependent activation of peroxisome proliferator-activated receptor (PPAR) β/δ promotes cellular differentiation¹⁶⁾ (Figure 1). The human endometrium is decidualized through changes in the expression of retinoid-related genes, including: (1) induction of PPARβ/δ involved in cellular differentiation; (2) suppression of the proapoptotic CRABP2/RAR pathway; (3) upregulation of enzymes (DHRS3 and RDH12) associated with retinol conversion; and (4) induction of transporter RBP4 expression, accounting for the marked decrease in cellular RA and retinal levels upon HESC differentiation into decidual cells.¹⁷⁾ Decidual transformation is involved in a wholesale reprogramming of the retinoid signaling pathway.

Figure 1.

Retinoid metabolism signaling.

Retinol (vitamin A), which is hydrolyzed from retinyl ester, is transformed to retinal and generates retinoic acid (RA) following two-step oxidation. RA binds to CRABP2 and activates the RA receptor (RAR), leading to apoptosis or senescence. In contrast, RA-dependent activation of PPAR β/δ is mediated through FABP5. Decidualization of human endometrial stromal cells decreases the expression of CRABP2 and FABP5 and downregulates and upregulates RAR and PPARβ/δ, respectively. In addition, decidualization is also associated with upregulation of retinol-binding protein 4 (RBP4), cytochrome P450 26A1 (CYP26A1), RDH12, and DHRS3, suggesting that decidualization suppresses RA signaling by decreasing key cytoplasmic-binding proteins and upregulating retinoid metabolism. Up- and down-arrows: upregulation and down-regulation upon decidualization.

Impaired endometrial decidualization and unexplained RPL

Decidualized HESCs have recently been reported to serve as biosensors of embryo quality upon implantation.^18,19,20,21) Developmentally inappropriate human embryos may suppress the secretion of implantation regulators, including inflammatory cytokines from decidualizing HESCs.¹⁸⁾ Upon implantation, the endometrium of normal fertile women can migrate toward high-quality blastocysts, but not toward low-quality embryos. However, the endometrium of women with a history of unexplained RPL exhibits high migratory activity toward any embryo regardless of quality; this may hamper the ability to avoid low-quality embryos.¹⁹⁾ In fact, Sugiura–Ogasawara et al.²²⁾ reported that unexplained RPL was mainly caused by abnormal embryonic karyotypes; however, an endometrium with impaired selective ability may cause repeated miscarriage. Furthermore, several studies have reported a relationship between unexplained RPL and aberrantly elevated uNK cell density in the mid-luteal endometrium.^{7,23,24,25,26)} uNK cells are the most abundant immune cells in the pre-implantation endometrium and early pregnancy decidua.²⁷⁾ The aberrantly high uNK cell density is associated with poor induction of 11βHSD1 and decidual marker genes, such as prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP1), in decidualizing HESCs.¹³⁾ Decreased 11βHSD1 expression in decidualizing HESCs may lead to insufficient production of cortisol and impair the regulation of GR gene networks, including that of uNK cell density at local implantation sites in women with a history of unexplained RPL (Figure 2).¹¹⁾ Perturbed inflammatory regulators, such as IL33/ST2 in the IL1 superfamily, in decidualizing HESCs are associated with unexplained RPL.²⁸⁾ From a genetic viewpoint, NLRP2 and NLRP7, which regulate IL1β production, are also reportedly associated with unexplained RPL.²⁹⁾ Therefore, the obvious evidence suggests an intimate interaction between unexplained RPL and inappropriate inflammation, which leads to abnormal angiogenesis in mid-luteal HESCs during peri-implantation and decidualization.

Figure 2.

Regulation of corticosteroid and retinoid signaling pathways upon endometrial decidualization.

Corticosteroid signaling via 11βHSD1 induction regulates glucocorticoid receptor (GR)-specific gene networks including inflammation and angiogenesis at the implantation site. Furthermore, retinoid signaling is controlled via 11βHSD1/mineralocorticoid receptor (MR) signaling, thereby achieving an optimal balance between cell differentiation and apoptosis or senescence, such as decidualization of endometrial stromal cells.

11βHSD1/MR signaling and its MR gene network, including the retinoid metabolism pathway, play essential roles in endometrial decidualization (Figure 2).¹¹⁾ During pregnancy, retinoid synthesis is necessary for embryogenesis and fetal development; however, excessive levels of retinoid are toxic to the embryo and fetus.^30,31) In HESCs, treatment of decidualizing cells with RA selectively upregulates the expression of proapoptotic CRABP2 and RAR, but not FABP5 or PPARβ/δ, leading to decreased decidual marker gene expression. Additional RA exposure is toxic for decidualizing HESCs and induces a response that may lead to implantation failure or early pregnancy loss.¹⁷⁾

Chronic endometritis and pregnancy loss

Chronic endometritis (CE) is observed in the uterine endometrium of 50–70% of women with a history of RPL.^32,33) CE is asymptomatic and undetectable in general fertility and RPL testing. Moreover, it is characterized by persistent inflammation of the endometrium with the presence of plasma cells.³³⁾ Although the relationship between CE and RPL remains unknown, CE is strongly associated with implantation failure and pregnancy loss. The mechanism underlying the increased risk of implantation failure and pregnancy loss is believed to involve perturbed intrauterine circumstance due to CE, leading to the altered secretion of endometrial cytokines and paracrine factors and impaired endometrial decidualization.^34,35) Thus, CE should be diagnosed and adequately treated in women with RPL.

CE is primarily caused by intrauterine infection with a wide variety of microorganisms, including Gardnerella vaginalis, Streptococcus spp., Escherichia coli, Enterococcus faecalis, Mycoplasma spp., and Chlamydia trachomatis.³⁶⁾ Yet, responsible microorganisms are not detected in some women with CE.³⁶⁾ Therefore, histopathologic confirmation of plasmacytes using CD138 immunostaining as a plasma cell marker (≥5 plasma cells on 10 nonoverlapping random stromal areas) is the gold standard for the diagnosis of CE.³³⁾ The recommended treatment regimen for CE is broad-spectrum antibiotics that target a wide range of microorganisms. Oral doxycycline (100 mg) twice daily for two weeks is the first-choice treatment,^33,37) whereas a combination of ciprofloxacin (500 mg) and metronidazole (500 mg) twice daily for two weeks is the second.³⁷⁾ This treatment protocol provides a high recovery rate, leading to improvements in clinical pregnancy outcome. However, no clinical, randomized controlled trial has been conducted in women with RPL with CE.

Previous trials using hormonal treatment for unexplained RPL

Optimization of HESC decidualization through corticosteroid and retinoid metabolism signaling is a target in the treatment of unexplained RPL. Tang et al.³⁸⁾ performed a pilot randomized controlled trial of prednisolone treatment (20 mg for six weeks, 10 mg for one week, 5 mg for one week after pregnancy and randomization) for women with RPL with elevated uNK cell density (>5%). The live birth rate was higher in the prednisolone group than in the placebo group (60.0%, 12/20 vs. 40.0%, 8/20 cases, respectively), but with no significant difference. Implantation and decidualization require an appropriate inflammatory process via local secretion of pro-inflammatory cytokines and prostaglandins from the decidual endometrium.^39,40,41,42) Thus, appropriate prednisolone administration for regulating corticosteroid signaling during implantation and decidualization may be difficult.

Corticosteroid signaling is highly induced via 11βHSD1 induction in response to P4 treatment in decidualizing HESCs.¹¹⁾ Therefore, P4 treatment can also control uNK cells via 11βHSD1/GR signaling. Furthermore, P4 can modulate uNK cells directly via GR, regardless of whether uNK cells express GR, but not PR, as a surface nuclear receptor.^43,44) P4 has favorable effects on pregnancy, such as inhibition of uterine contraction, production of prostaglandins, and regulation of helper T-cell cytokines, leading to maternal uterine receptivity for an embryo.⁴⁵⁾ Therefore, P4 is expected to be therapeutically beneficial for unexplained RPL. Coomarasamy et al.⁴⁶⁾ performed a multicenter randomized controlled trial (PROMISE study) of vaginal P4 suppositories (400 mg twice daily) for women with a history of three or more pregnancy losses. Among 836 women with RPL, the live birth rate was 65.8% (262/398 women) in the P4 group, which was comparable to 63.3% (271/428 women) in the placebo group. According to a systematic review, dydrogesterone, a synthetic P4, is effective for unexplained RPL.^47,48) In particular, Kumar et al.⁴⁹⁾ showed the efficacy of dydrogesterone treatment (10 mg twice daily) on unexplained RPL with a decrease and increase in helper T-cell type 1 and 2 cytokines, respectively. Systemic synthetic P4 treatment may be effective for maintaining early pregnancy via optimization of maternal immune tolerance without adverse effects on implantation and the fetus.

Closing comments

To the best of our knowledge, no efficient treatment for unexplained RPL currently exists. In this regard, the optimization of intrauterine circumstances and endometrial decidualization may be key to treating unexplained RPL. Furthermore, given that dysregulated decidualization is associated with complications during pregnancy, such as hypertensive disorders,⁵⁰⁾ and unexplained RPL is involved in adverse obstetric events,⁵¹⁾ the optimization of endometrial decidualization may also help reduce the risk of complications during pregnancy. The roles of endometrial functions in unexplained RPL warrant further investigation.

Acknowledgements

This study was funded by JSPS KAKENHI Grant Number 18K09273.

Conflict of interest

There are no conflicts of interest to report.

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