Clinical Assessment and Management of the Endometrium in Recurrent Early Pregnancy Loss

Bansari G. Patel, M.D.,1 and Bruce A. Lessey, M.D., Ph.D.2

ABSTRACT

Unexplained and recurrent loss of pregnancies is a heartbreaking and frustrating condition. The routine diagnostic workup for pregnancy loss includes hormonal evaluation, screening for genetic or chromosomal defects, immunologic and thrombophilic testing, and evaluation of congenital or acquired Mu llerian defects. In cases of idiopathic pregnancy loss, defects in endometrial receptivity are increasingly being investigated. The role of the endometrium in pregnancy loss has historical roots but remains controversial. Exciting new directions based on microRNAs, proteomics, and epigenetics promises to keep this area of investigation both interesting and complex. With each new diagnostic and therapeutic biomarker identied comes a greater potential for diagnosis and treatment of women. The clinical assessment of the endometrium remains an important part of the investigation of couples with unexplained pregnancy loss.
KEYWORDS: Miscarriage, endometrium, endometriosis, luteal phase defect, hydrosalpinges, pregnancy loss

oss of a single pregnancy is a common event, occurring up to 35% of conceptions.1 Recurrent pregnancy loss (RPL), however, affects only 3 to 5% of women attempting conception.2,3 Factors that increase the risk for RPL include the loss of a chromosomally normal pregnancy, pregnancy loss after the rst trimester, and concomitant infertility. Only half of all miscarriages that occur in the general population can be assigned a cause, including chromosomal abnormalities, immunological, endocrine (luteal phase defect or diabetes), anatomical, thrombophilic, infectious, or iatrogenic causes (smoking and caffeine).3,4 This gap in our diagnostic accuracy for up to 50% of losses3,5 might be lled by an awareness of conditions such as endometriosis or
1 Department of Obstetrics and Gynecology; 2University Medical Group, Greenville Hospital System, Greenville, South Carolina. Address for correspondence and reprint requests: Bruce A. Lessey, M.D., Ph.D., Department of Obstetrics and Gynecology, University Medical Group, Greenville Hospital System, 890 W. Faris Rd., Suite 470, Greenville, SC 29605 (e-mail: blessey@ghs.org). Recurrent Early Pregnancy Loss; Guest Editor, Mary D.

tubal factor that appear to alter endometrial receptivity, leading to implantation failure.6 In fact, many of the factors that render women infertile are a likely cause of unexplained RPL. The efciency of implantation in the human is relatively low compared with other mammals. It is estimated that 10 million couples will seek medical assistance for infertility and/or RPL this year. Up to a third of in vitro fertilization (IVF) pregnancy losses are thought due to poor embryo quality, and it is estimated that up to 60% result from implantation defects. It is safe to say that most women with defects in endometrial receptivity go unrecognized because most couples with unexplained RPL do not have a thorough evaluation of
Stephenson, M.D., M.Sc. Semin Reprod Med 2011;29:491506. Copyright # 2011 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. DOI: http://dx.doi.org/10.1055/s-0031-1293203. ISSN 1526-8004.

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the endometrium. At the same time, despite the availability of specic biomarkers of endometrial receptivity, their use remains low due in part to a lack of randomized controlled clinical trials that validate their usefulness. In addition, a lack of a clear mechanism regarding how endometriosis causes pregnancy loss adds to this lack of consensus regarding the use of endometrial biomarkers. This article reviews the clinical assessment of the endometrium in the setting of RPL; however, much of the literature also extends to studies of unexplained infertility (UI) that we believe is a continuum with unexplained pregnancy loss.

Figure 1 Endometrial dating criteria of Noyes et al.19 This landmark study was published as the rst article in the inaugural issue of Fertility and Sterility. The histological patterns chosen represent an idealized representation of the observed changes in the endometrium throughout the secretory phase of the menstrual cycle.

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THE ENDOMETRIUM The endometrium is a steroid target tissue7,8 that forms the inner mucosal lining of the uterus. It is composed of epithelial and stromal cells, and together with the myometrium is derived from the paramesonephric ducts that fuse during early embryogenesis. Like the kidney, the endometrium establishes its epithelial compartment from mesenchymal transformation, rather than preexisting embryonic epithelium.9 Adult endometrium consists of a basal and functional layer made up of columnar epithelial cells, supported by stromal and vascular elements. In lieu of pregnancy, the cycling endometrium is shed monthly and renews itself from both the exposed

stromal and glandular fragments. A 2009 study using hysteroscopic examination combined with scanning electron microscopy suggested that reestablishment of the luminal epithelium over the basal layer occurs through mesenchymal-epithelial transformation, reminiscent of its embryological origins.10 In addition to epithelial and stromal components, up to 50% of the endometrium is comprised of leukocytes. The immune system can be divided into innate immunity including monocytes, macrophages, dendritic cells, neutrophils, basophils, and mast cells, and uterine natural killer cells (uNKs) from the lymphoid lineage. The adaptive immune system includes the T and B cells, which require cells from innate immunity to establish an immunological memory.11 Bone marrow derived cells are known to trafc to the endometrium, through steroid-regulated chemokine production,12,13 that may have implications for pregnancy loss related to systemic inammation.1418 Given these dynamic cyclic changes along with easy access to the endometrium by ultrasound, hysteroscopy, or endometrial sampling, there are many ways to evaluate endometrial function. Histological changes of the endometrium were classied by the landmark studies of Noyes and colleagues, who established the original dating criteria for assess of endometrial development (Fig. 1).19 Simultaneously, Georgina Seegar Jones in

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194920 developed the concept of what is now called luteal phase defect (LPD). The diagnosis of LPD remains one of the most studied areas of endometrial assessment and even today is of uncertain clinical importance.2124

Figure 2 (A) Schematic of the stages of implantation starting at ovulation and extending until the time of embryo apposition, adhesion, and invasion. (B) image from the Carnegie series showing stage 5a embryo that has completed its transition through the luminal epithelium and is becoming invested in the endometrial stroma (decidua).

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THE IMPLANTATION WINDOW AND DEFECTS IN UTERINE RECEPTIVITY RPL, like UI, can be attributed to implantation failure.6,2527 The success of an early pregnancy requires synchronous interactions between the endometrium, corpus luteum (CL), and embryo; conversely, delayed implantation contributes to pregnancy loss.28,29 Hemochorial placentation in human and the higher primates occurs in stages (Fig. 2A) but requires apposition (initial interaction) between the embryo and the endometrial surface.30,31 The luminal surface epithelium is the primary barrier to embryo implantation throughout the menstrual cycle, except during a narrow window of implantation (WOI). Endometrial receptivity is acquired during the midsecretory phase in the

normal fertile women 5 to 7 days after ovulation, reecting the expression of a vast repertoire of genes that regulate multiple biochemical pathways facilitating embryouterine adhesion, embryonic survival, and subsequent invasion of the nascent embryo (Fig. 2B).3234 The concept of a receptor-mediated mechanism of implantation is now well established.3538 Based on numerous studies, multiple mechanisms for the initial embryoendometrial attachment phase have been envisioned (Fig. 3). Apposition is a brief event, followed quickly by intrusive probing by the embryonic trophoblast between the weakened epithelialepithelial lateral plane.39 Lateral adhesion allows cytotrophoblast invasion, digesting through the basement membrane into the underlying stroma (decidua).40,41 Once within stroma, vascular, immunological, and stromal contributions both stimulate and limit invasion. These complex interactions and communications provide a wealth of potential biomarkers for the evaluation of endometrial receptivity. Indeed, much of our current understanding of endometrial physiology is based on individual investigations of these many proteins that orchestrate implantation.

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Figure 3 Models of embryoendometrial attachment (used with permission from Seminars in Reproductive Medicine, 2007).6 Adhesion molecules serve many functions throughout the body. Endometrialembryo interactions likely involve one or more of these mechanisms. As shown, many different combinations of ligands and receptors have been proposed, as outlined in the text. Interactions can involve integrins, extracellular matrix molecules such as bronectin, tastin, and trophinin, lectins, and selectins. Although not mutually exclusive, the primary receptor for the embryo is likely a loose attachment as proposed for L-selectin and its ligand that allows a transient attachment phase that precedes invasion. Other mechanisms as depicted may have other roles including immune modulation and cell signaling at the time of implantation. Used with permission from Thieme Medical Publishers, Inc.6

Based on numerous studies, it is estimated that the WOI extends from postovulatory days 6 to 10 (corresponding to cycle day 20 to 24). Wilcox and colleagues conrmed the timing of this window in normal fertile women and demonstrated that delayed implantation results in a higher risk of miscarriage.29 Based on a concept of asynchrony between the endometrium, ovary, and embryo, histology delay in the endometrium would logically move the WOI, providing a mechanism for pregnancy loss in women with LPD. The ovarian CL, like the endometrium, has an ephemeral window of receptivity, and delayed implantation likely occurs when the CL is less able to respond and protect the nascent embryo from menstruation. Endometriosis appears to play a role in unexplained RPL, as it does in UI.5,42 This disease of retrograde menstruation and inammation is an estrogen-dependent condition found in up to 70% of women with pain and 25 to 40% of infertile women.43,44 Its role in RPL has not been as well studied but could account for the bulk of idiopathic pregnancy loss. The presence of endometrial cells outside the uterine cavity results in

biochemical alterations in the eutopic endometrium leading to elevated estrogen sensitivity45 and progesterone resistance,46,47 resulting in absent or delayed expression of those proteins essential for establishment or maintenance of pregnancy.48,49 The remainder of this article reviews the established and new opportunities for the clinical assessment of the endometrium in women with RPL.

CLINICAL ASSESSMENT OF THE ENDOMETRIUM Endometrial Histology Noyes and colleagues rst described the criteria for endometrial dating in 1950. This landmark study was published in the rst issue of Fertility and Sterility, underscoring the perceived importance of endometrial receptivity early on.19,50 The study consisted of cataloging histological changes in endometrial biopsies from infertile women that were therefore abnormal by denition. After 60 years, this study was recently

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repeated and validated using only fertile controls.23 The histological criteria established for endometrial dating provide a helpful idealization of endometrial progression during the secretory phase; however, they appear to lack the precision and reproducibility required for individual patients. Excessive variability in these criteria exists between both subjects and observers, rendering accurate prospective chronological assignment through the menstrual cycle a challenge.23 Despite their shortcomings, these criteria remain a primary method for the clinical assessment of the endometrium.23,24

Hormonal Assessment The state of endometrial receptivity in RPL could be assessed indirectly by measuring the endocrine milieu during the menstrual cycle. Serum levels of thyroidstimulating hormone and prolactin should always be considered, along with follicle-stimulating hormone and estradiol on day 3 of the cycle as a measure of ovarian reserve as well as ruling out thyroid disease and hyperprolactinemia. Patients with polycystic ovary syndrome (PCOS), who generally have a poor reproductive performance and increased risk of miscarriage,51 may require additional hormonal testing. Abnormalities in the endocrine milieu of PCOS can disrupt hypothalamic-pituitary-ovarian function leading to alterations in the endometrium contributing to implantation failure.52,53 Assessment of insulin and androgen levels is obligatory in these women because adverse effects of hyperinsulinemia or hyperandrogenemia may lead to a nonreceptive endometrium.51,5457 Furthermore, hyperandrogenemia and hyperinsulinemia have also been associated with LPD and alterations in reproductive function. Progesterone is the sentinel marker of the luteal phase and essential for pregnancy success.58 Serum measurements of progesterone are a clinical tool for assessment of ovulation and a surrogate marker for endometrial response.5961 The use of progesterone as a measure of reproductive competence is not without concerns. A low serum progesterone level may result from suboptimal follicular development leading to RPL or infertility.62 The activity of the CL depends on a tonic level of luteinizing hormone (LH) secretion from the pituitary. Strott and colleagues studied women with a short luteal phase (dened as <10 days in duration) and reported a relative deciency in gonadotropin levels, as well as LH surges of reduced magnitude.63 A gonadotropin deciency was created as a model for LPD, using antibodies to LH, noting a prompt decline in circulating progesterone levels following treatment.64 As expected, administration of gonadotropin-stimulating hormone antagonists produces a similar decrease in progesterone levels65 because progesterone is secreted in a pulsatile manner, corresponding to LH pulse frequency.66 Any

change in pulse generator could theoretically alter progesterone secretion, leading to LPD, but given the wide range of progesterone levels between its peak and trough can give a sixfold difference in measurements, raising questions about the usefulness of a single measurement. Thus the use of progesterone levels in the assessment of LPD would likely lead to more questions than answers.49,67 Progesterone is known to have a direct and indirect stimulatory action on key endometrial proteins, hence rendering it critical to the success of an early pregnancy.6881 Progesterone downregulates endometrial steroid receptors (estrogen receptors and progesterone receptors) during the midsecretory phase. The phenomena of inadequate progesterone production or secondary progesterone response (progesterone resistance),49 may be responsible for alterations in steroid receptor downregulation noted in the endometrium of some women with RPL. Persistent estrogen or progesterone receptors during the midluteal phase may alter that balance of estrogen and progesterone-mediated effects, and alter the paracrine dynamics that underlie the acquisition of endometrial receptivity.75,81 Thus both qualitative and quantitative study of steroid receptors, as well as the enzymes that metabolize these steroids, may serve as a useful tool for the assessment of endometrial function.45,75,82,83 Because progesterone is essential for the success of pregnancy, low progesterone action would logically lead to delayed endometrial maturation62 or differentiated function.49,84 The study by Usadi and colleagues in 2008, however, demonstrated signicant plasticity in the response to articially lowered progesterone levels during a mock luteal phase. In normal fertile women, they failed to demonstrate observable effects on endometrial histology.22 Functional studies on these samples using DNA microarray do, however, reveal dose-dependent differences in gene expression, suggesting that histology may be a blunt tool to study the true effect of progesterone on endometrial function.85

Endometrial Ultrastructure
PINOPODS

In 1972, during a series of scanning electron microscopic studies of receptive endometrium, morel-like apical projections from the rat uterine luminal surface were observed coincident with the implantation window. Enders and Nelson in 1973 determined that these projections from the surface cells were involved in the nonspecic uptake of uid from the uterine lumen. The term pinopods (translated from drinking foot; also known as pinopodes, uterodomes) described these bleb-like projections (Fig. 4AC).86 Pinopods have been extensively described in the human endometrium and suggested to be markers

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Figure 4 Endometrial pinopods (A) are present in the secretory phase on the luminal surface and appear to be the same structures decorated with luminal biomarkers, such as (B) anb3 integrin and its presumed ligand (C) osteopontin (OPN).

Endometrial Imaging Imaging techniques are readily available and commonly used for the clinical evaluation of the uterus and endometrium.116119 Transvaginal ultrasound is now available to most practitioners and offers a convenient method to assess endometrial thickness, contour, and pelvic anatomy. Ultrasound is a sensitive method to identify abnormalities that could contribute to RPL including a uterine septum or acquired defects such as uterine broids or polyps. Compared with laparoscopy, ultrasound has been reported to have a sensitivity of 86%, a specicity of 98%, a positive predictive value of 99%, and a negative predictive value of 79% for the detection of pathology.119 During the proliferative phase the endometrium grows to a thickened strip with a trilaminar appearance. Some investigators found that a thin endometrium is associated with a poor outcome or reduced pregnancy rates,120124 but others found no predictive value of endometrial thickness.125127 A thicker endometrium was associated with positive pregnancy outcome in early

SONOHYSTEROGRAPHY

Two-dimensional (2D) and three-dimensional (3D) ultrasound are increasingly performed with uid instillation

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of uterine receptivity8794 in both fertile and infertile women.93102 Although similar in structural appearance to those described in the rat, pinopods in the human endometrium do not appear to possess pinocytic activity.103 Pinopods are expressed in endometrial culture and might be a preferred site for embryo attachment.104,105 These expanded blebs of plasma membrane appear to be a site of various endometrial proteins including integrins,97 leukemia inhibitory factor (LIF), glycodelin, osteopontin and galectin-9 and galectin-3, L-selectin ligands, and decay accelerating factor.106112 Two prospective randomized studies did not conrm the expected pattern of pinopod expression in cycling women, drawing into question their signicance.113,114 Quinn et al found that pinopodes can be detected in the progesterone-exposed endometrium for an extended period of time, thus casting doubt on their role as markers for the implantation window in the human endometrium.115

IVF studies.128130 In cycles where conception occurred, endometrial thickness was reported to increase compared with nonconception cycles.131 Endometrial thickness is measured at the endometrialmyometrial interface through the sagittal plane of the uterine body, 1 cm from the fundus.132 Errors in endometrial thickness measurements can occur if an oblique section of the uterus is captured or if the uterus is not imaged along its entire length to achieve maximal fundal endometrial thickness. Despite the dependence on steroid hormones, studies suggested that endometrial growth does not always correlate well with hormone levels,124 and that neither thickness nor echogenic pattern correlated well with endometrial histology.133 Doppler ow studies are noninvasive ultrasound methods used to evaluate the blood ow to the uterus and endometrium. The extent to which blood ow and Doppler ndings predict the rate of implantation remains an active area of investigation. Blood ow changes during the menstrual cycle have been reported134,135 and correlate with serum progesterone levels.136 Uterine artery blood ow resistance has been reported to be predictive of implantation potential.137140 One report suggested that Doppler ndings also correlated with immunohistochemical biomarkers of uterine receptivity.141 Elevated uterine impedance was reported to be abnormal in RPL as well.136 A recent report found that women with an end-diastolic blood ow, an endometrial-subendometrial blood ow, and a multilayered endometrium were more likely to have a successful pregnancy than women without one or more of these signs.117 Schild, in contrast, found that blood ow in both the uterine and spiral arteries measured by power Doppler showed no correlation with implantation rates.142 There is presently no consensus about the predictive potential of measurements of ow or resistance in the endometrial blood vessels.119

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into the uterine cavity (sonohysterography) to allow evaluation of the endometrium in women with RPL as well as those with infertility or abnormal bleeding.143147 These techniques are highly sensitive at detecting intrauterine pathology.116,144,147149 Compared with normal sonohysterograms, polyps or broids are easily detected when saline is introduced into the cavity and can be well displayed using 2D and 3D technology. A uterine septum may predispose to RPL and can be routinely detected by sonohysterography. Although patients with signicant pathology will ultimately undergo more invasive procedures such as hysteroscopy and/or endometrial dilation and curettage to diagnose and remove the abnormality, sonohysterogram is an essential screening tool to assess the uterine cavity and aid in the clinical assessment of the endometrium.

Biomarkers of Endometrial Receptivity A biomarker has been dened as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Emerging evidence indicates that the endometrium can be biochemically defective without a histological delay.48,49,150 Derangements in the endometrium in otherwise normally developed endometrium have greatly expanded the possibilities for the use of alternative methods to assess endometrial receptivity as a predictor of future reproductive success.151 The extracellular matrix (ECM) along with its associated cell adhesion molecules (CAMs) maintain tissue integrity and hormonal responsiveness within tissues.152 Changes in the organization and composition of ECM in the human endometrium during the menstrual cycle and early pregnancy was assessed by immnunouorescence,153,154 showing cycle-specic changes in ECM components and CAMS that in turn suggested a role in embryoendometrial interactions.155,156 Integrins are a family of CAMs that function as both cell substratum and cellcell adhesion receptors. They interact with ECM ligands and other CAMs as well as matrix metalloproteinases to participate in a wide variety of physiological processes.157 Dynamic changes in integrin expression occur during the menstrual cycle and into pregnancy.67,154,158160 The three amino acid motif arggly-asp (RGD) was implicated in the process of implantation by several investigators.161163 RGD is present on many ECM ligands in the receptive endometrium, including osteopontin, tenascin, insulinlike growth factor-binding protein 1, and bronectin.164167 A functional blockade using RGD peptides and anti-RGD snake venom components was shown to effectively block implantation or attachment of embryos, suggesting a critical role of integrins and related ligands in the cascade of events leading to successful implantation.162,166,168,169

SELECTINS/CADHERINS

The search for a true receptor that mediates embryo attachment has been a goal for many implantation researchers35,36 and has led to numerous candidates,158,176179 including growth factor/receptor pairs, CAMS, ECM, and members of the cell adhesion families. Recent evidence supports L-selectin, a member of the selectin family, as a key mediator of the interaction between the uterus and embryo during the initial attachment.180 The presumed endometrial ligand for L-selectin appears to be a sialyl glycoprotein associated with the Lewis-X family that is recognized by the monoclonal antibody MECA-79. This factor has now been studied in normal cycling women during the menstrual cycle.181 Additionally, ligands of L-selectin have been suggested to be clinically useful markers of endometrial receptivity and may be absent in women with RPL.111
CYTOKINES

The expression of cytokines and growth factors along with their receptors during the menstrual cycle has been implicated in both endometrial development and implantation. LIF was one of the rst glycoproteins demonstrated to be critical for implantation. Null mutation of the LIF gene in female mice resulted in complete lack of implantation and exhibited decidualization failure.182184 Interestingly, this phenotype could be rescued by administration of exogenous LIF. Interleukin (IL)-11, another member of this IL-6 family, has also been implicated in decidualization. Both LIF and IL-11 signal through the

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Integrins are perhaps the best characterized of the endometrial markers. In 1992, constitutive and cycledependent patterns of integrin expression were rst described.158,170 Certain integrin moieties appeared to be regulated within the cycling endometrium, and disruption of their expression was reported to have adverse effects on uterine receptivity and fertility. Three integrins were noted to be coexpressed and receptive to endometrium only during the putative window of implantation (Fig. 5).67 The anb3 integrin appears on apical endometrial epithelium at the opening of the window of implantation around cycle day 20 or 21 and is present into pregnancy. Daftary and colleagues also showed that b3-integrin subunit expression in the endometrium is regulated by HOXA10.171 This factor was more recently shown to be reduced in women with endometriosis, hydrosalpinges, and broids.172 Studies on integrins in RPL in 2006 found no difference in expression in women with unexplained RPL,173 but more recently, increased expression of a 1 and a 4 integrins was seen in some women with miscarriage.174 A polymorphism (A1/A2) in the b3integrin subunit was found to be signicantly associated with fetal loss before 10 weeks of gestation.175

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Figure 5 Endometrial integrins throughout the menstrual cycle. Endometrial integrins undergo changes in expression throughout the menstrual cycle. The expression pattern of three integrins including a1b1, anb3, and a4b1 appear to frame the window of implantation that was later dened by Wilcox and colleagues.29 The anb3 integrin is located on the endometrial luminal surface appearing at the time of implantation and is absent in some women with implantation defects. This protein also predicts in vitro fertilization success and can be used to identify women with endometriosis who have otherwise unexplained infertility. Used with permission from the American Society for Reproductive Medicine and Elsevier.67

gp130 receptor. Numerous studies have imputed reductions in LIF and related proteins in women with implantation failure and infertility.185189 We reported decreased LIF expression in the endometrium of PCOS women190 that could account for their more frequent miscarriages compared with normal women. LIF remains one of the most validated biomarkers for endometrial receptivity.
DNA MICROARRAY

In the future, assessment of RPL will undoubted use panels of biomarkers that dene different and distinct types of endometrial defects. The process of biomarker discovery has been dramatically accelerated by highthroughput techniques such as DNA microarray, using both normal and pathological endometrium.34,80,191205 The advent of DNA microarrays has enabled expeditious identication of new pathways, signaling paradigms, and metabolic processes. The characterization of the endometrium throughout the normal menstrual cycle34 has set the stage for a much better understanding of the

factors involved in endometrial development and menstruation.192 This technique is not only enabling researchers to rediscover known biomarkers but also allows discovery of new pathways and unsuspected proteins.192,195,206 Development of these emerging techniques in conjunction with bioinformatics and proteomics will likely revolutionize our current understanding of RPL. Studies in infertility patients have already resulted in the new concept of progesterone resistance,49 with clear relevance to the study of RPL.46 MicroRNAs (miRNAs) are small nonprotein coding RNAs that regulate specic posttranscriptional messenger RNAs targeting them for degradation. Research on these molecules has recently exploded and will likely lead to the next class of biomarkers of endometrial dysfunction. Genes coding for miRNA represent 1 to 4% of the genes in higher organisms and are highly processed sequences derived from larger RNA transcripts. These small precursor miRNAs form a single stranded miRNA that binds to complementary sequences on target mRNA preventing their transcription.207209 Recent

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characterization of miRNAs has added to our understanding of the regulation of gene expression in the normal and diseased endometrium.210214
IMMUNE CELLS OF THE ENDOMETRIUM

MANAGEMENT OF ENDOMETRIAL RECEPTIVITY DEFECTS IN RECURRENT PREGNANCY LOSS Only 50% of RPLs are explained. Many of these cases exhibit signs of mild ovulatory dysfunction and/or biochemical progesterone resistance including women with endometriosis and PCOS.46,190 After endocrine abnormalities (diabetes mellitus, hyperprolactinemia, and thyroid disease) are diagnosed and treated and mechanical causes involving the uterus (uterine septae, broids, polyps, Ashermans syndrome) are ruled out or corrected, one should consider other underlying causes of endometrial receptivity including PCOS, endometriosis, and subtle tubal disease.6 PCOS is a known risk factor for miscarriage.228230 Treatment of PCOS and associated hyperandrogenism and hyperinsulinemia has been the subject of several recent reviews.231234 Aromatase inhibitors in particular appear to be superior compared with clomiphene citrate in this condition,235237 although controlled trials for RPL are limited. In ovulatory women with PCOS, endometriosis can be a cofactor because many women with heavy irregular bleeding would be at risk for development of endometriosis. Elevated body mass index is also a risk factor for endometriosis.238

SUMMARY AND CONCLUSIONS Implantation failure and defects in endometrial receptivity are associated with a third to a half of all RPLs. Consensus for a systematic clinical assessment of the endometrium is lacking, although many opportunities exist but remain investigational. Endometrial histology appears to be the weakest index of function, given the variation seen between observers and between patients and the lack of correlation between histology and gene expression. The use of biomarkers for endometrial assessment is rapidly evolving, with the recent advancements using DNA microarray techniques. As new biomarkers become available as a test for endometriosis, the relationship between subtle endometriosis and RPL may become better established. The next frontier in the diagnosis and potentially treatment of RPL will likely be based on a study of the epigenetic changes in the cycling endometrium.244 Therapeutics directed at underlying dysfunction will likely provide better treatment options for women with this distressing condition.

REFERENCES
1. Gilchrist DM, Livingston JE, Hurlburt JA, Wilson RD. Recurrent spontaneous pregnancy loss. Investigation and reproductive follow-up. J Reprod Med 1991;36(3):184188 2. Katz VL, Kuller JA. Recurrent miscarriage. Am J Perinatol 1994;11(6):386397

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Successful embryo implantation requires a complex and coordinated series of biochemical changes in the endometrium that allows for a controlled invasion of the fetal allograft at the interaction of maternalfetal compartments.35 Loss of the immunotolerant regulatory T cells and their associated interactions appear to account for both infertility and RPL observed in women with endometriosis or other inammatory diseases.215222 Altered T cells in the normal midsecretory phase could also affect the number of uNK cells. Studies report that the number of CD56bright NK cells are decreased in women with unexplained miscarriage223,224 and unexplained IVF failure.225 Further, monocytes can directly alter endometrial receptivity in the mouse model.226 Endometrial immune cells, their cytokines and chemokines, and the stromal paracrine signals such as IL-15 are all potential biomarkers that could be exploited to understand aberrant endometrium associated with unexplained recurrent pregnancy loss (uRPL).216,227 Many of these immunological cells and messages are potential biomarkers for the assessment of endometrial competence but will have to await further characterization in this rapidly expanding area of research.

Prior miscarriage is a risk factor for endometriosis,239 and endometriosis has been associated with RPL.5,42,240,241 Laparoscopy is the mainstay for diagnosis and treatment of endometriosis and should be considered for uRPL, especially when symptoms of dysmenorrhea are present. Medical management, at least for infertility associated with endometriosis, has not been proven efcacious.242 In our experience, women with mild endometriosis and RPL can be effectively treated without surgery. Because women with uRPL often conceive easily, the pregnancy losses likely occur as a result of disrupted endometrial/CL synchrony.28 Delayed implantation, due to a shift in the WOI, leads to inadequate CL response to rising human chorionic gonadotropin (hCG) levels. In a past systematic review, hCG was shown to be superior to luteal progesterone therapy for treatment of pregnancy loss, 243 perhaps because of this mechanism of loss. A single intramuscular injection of 5000 IU of hCG given 1 week after ovulation is an effective treatment for uRPL. We have achieved an overall 80% success rate using this simple method for women with otherwise unexplained RPL (unpublished results).

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