MicroRNA and implantation Daniela Galliano, M.D., Ph.D.,a and Antonio Pellicer, M.D., Ph.D.b,c a Division of Reproductive Medicine, Instituto Valenciano de Infertilidad, Barcelona; b Instituto Valenciano de Infertilidad and Instituto Universitario IVI, and c Department of Obstetrics and Gynecology, School of Medicine, Valencia University, Valencia, Spain

We provide a review of microRNA (miRNA) related to human implantation which shows the potential diagnostic role of miRNAs in impaired endometrial receptivity, altered embryo development, implantation failure after assisted reproduction technology, and in ectopic pregnancy and pregnancies of unknown location. MicroRNAs may be emerging diagnostic markers and potential therapeutic tools for understanding implantation disorders. However, further research is needed before miRNAs can be used in clinical practice for identifying and treating implantation failure. (Fertil Use your smartphone SterilÒ 2014;101:1531–44. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Blastocyst, ectopic pregnancy, endometrial receptivity, implantation, miRNAs, and connect to the PUL Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gallianod-micrornas-implantation/

T

he regulation of numerous key biological processes depends not only on classic transcriptional mechanisms but also on other regulatory phenomena such as epigenetic mechanisms (1). These mechanisms include DNA methylation and the posttranslational modifications of histones, as well as small noncoding RNAs. The latter includes micro RNAs (miRNAs), which are small RNA fragments (18 to 25 nucleotides) that do not encode proteins but rather act as posttranscriptional regulators of many gene targets (1–4). Gene expression is regulated by miRNAs either negatively by mRNA cleavage (5), deadenylation (6), or inhibition of translational repression (7), or positively through the targeting of gene promoters. Some of the relevant biological processes in which miRNAs play a role include cellular differentiation, proliferation, and apoptosis (8). Moreover, the roles exerted by specific miRNAs can be very different from one tissue to another. These basic processes are also involved in embryo formation, early development, and implantation.

To improve the management of infertile patients, many strategies are being developed often in the absence of adequate scientific support. Although progress has been made, we currently have a very limited understanding of implantation both in normal physiological conditions as well as in the physiopathology of failed and abnormal implantation. In this sense, investigating miRNAs as elements that might modify gene function in the reproductive tract is an extraordinarily interesting method for better understanding the basis of health and disease. Moreover, a key characteristic of miRNAs is that they are stable and detectable in blood and other body fluids, thus providing the possibility of their use as biomarkers of specific disease conditions (9). This review will focus on currently existing knowledge of the role of miRNAs in implantation to highlight the extent to which this identification has contributed to our understanding of the physiology of implantation. Additionally, as we will further discuss,

Received January 25, 2014; revised April 2, 2014; accepted April 15, 2014. D.G. has nothing to disclose. A.P. has nothing to disclose. Reprint requests: Daniela Galliano, M.D., Ph.D., Ronda General Mitre, 14, 08017 Barcelona, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. 101, No. 6, June 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.04.023 VOL. 101 NO. 6 / JUNE 2014

discussion forum for this article now.*

* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.

miRNAs have been employed as markers of disease not only for identifying nonviable embryos but also for understanding and modifying failed implantation in women undergoing assisted reproductive technology (ART) cycles as well as in the early diagnosis of ectopic pregnancies (EP) and pregnancies of unknown location (PUL).

miRNAS IN IMPLANTATION AND EARLY STAGES OF GESTATION The Embryo Implantation resulting in a newborn infant is the result of a perfectly orchestrated dialogue between a viable embryo and a receptive endometrium, a process in which many key proteins and growth factors play fundamental roles (10). Micro-RNAs have also been involved in this process, although their exact role in normal embryo formation, endometrial preparation for pregnancy, and in early stages of gestation remains unclear. It has been predicted that up to 50,000 miRNAs may be present in a mammalian cell (11) which may alter the mRNA and protein synthesis of thousands of genes (12, 13). MicroRNAs have been widely associated with mammalian development (14), particularly during the reprogramming 1531

VIEWS AND REVIEWS of the transcriptome and epigenome (15, 16). Literature has shown that miRNAs may target hundreds of critical genes whose expression or repression determines the fate of the embryo. The importance of miRNAs in early embryo development has been identified in a range of species, from Caenorhabditis elegans to mammals (17–22). It has been shown that in these organisms miRNA-mediated gene regulation is essential for normal embryogenesis, as indicated by reduced embryo survival of mutant embryos. For example, the importance of miRNAs has been confirmed by the fact that deletion of the gene Dicer1 (Dcr1) in mice results in arrest of embryo development on day 7.5 due to defects in differentiation (23). Yang et al. (24) have reported similar effects between days 12.5 and 14.5 of gestation due to altered embryonic angiogenesis, which also appeared to be regulated by miRNAs. These investigators demonstrated that, because miRNAs act as posttranscriptional repressors of their target genes, a defect in Dicer leads to defects in miRNAs. This in turn causes an overexpression of target genes, such as Ang-1 and PTEN, in the embryos. Similarly, Medeiros et al. (25) have demonstrated that deficiency of miRNA Mir-290–295 leads to partial embryonic death and germ cell defects. Moreover, they showed that female but not male fertility was compromised in the surviving embryos due to a defect in germ cells which in turn led to premature ovarian failure. Micro-RNAs are also highly expressed in human embryonic stem (hES) cells (26–32). Ren et al. (27) found that miR520 may be closely involved in hES cell function. Other miRNAs, such as the miR-302 and the miR-200 families, have similarly been observed in human (33, 34) and mouse embryonic stem cells (35–37). Rosenbluth et al. (38) studied 754 miRNAs expressed in 14 blastocysts (five female, four male, and five aneuploidy embryos) with array screening. They confirmed with quantitative real-time PCR (qPCR) the manifestation of the most expressed miRNAs, including miR-372, miR-720, and miR- 302c, in 27 blastocysts (seven male, eleven female, and nine aneuploid). These investigators observed that human blastocysts express a large number of miRNAs, which are essential for embryo survival and development as well as for conservation of stem cell pluripotency. Most of these have already been documented in human embryonic stem cells (33, 39), mammalian embryos (40, 41), or primate placentas (42). Moreover, by comparing male versus female embryos, these investigators found that one miRNA, miR-518d-5p, was 5.6 times more expressed in male embryos, suggesting some degree of sexual differentiation even at the blastocyst stage of development. It is interesting that the mRNA Mir-518-d belongs to the C19MC cluster, the largest human cluster in which miRNA genes are organized. In these clusters, miRNA genes are exclusively expressed in the placenta and may be involved in placenta development and implantation. Similarly, miRNA expression has been found to be higher in mouse embryos than in mature mouse tissues, confirming their role during embryo development (43). More recently, Rosenbluth et al. (44) demonstrated that miRNAs can be detected in in vitro fertilization (IVF) culture media, where 1532

they are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome. These results suggest that miRNAs are potential markers for predicting success after IVF.

The Endometrium Endometrial receptivity is a complex event that occurs during the midluteal phase of the menstrual cycle known as the window of implantation (WOI) (45, 46) During this period the endometrium develops characteristics that allow the adhesion and invasion of the embryo to the uterine epithelium (47). In the finite implantation period that spans the 6 to 10 days after ovulation, on days 20 to 24 of a regular menstrual cycle (48) the endometrium becomes receptive. This receptivity is influenced by several molecular mediators, including growth factors, cytokines, chemokines, lipids, and adhesion molecules (49–55), whose expression is regulated by estrogen (E) and progesterone (P). It has been shown that such sex steroid hormones regulate endometrial gene expression, which is essential for the proliferation and receptivity of endometrium (56–58). Many genes have been involved in this complex and redundant phenomenon in mammals (10). Accordingly, if critical genes are not expressed during the WOI, implantation will not happen (59). Indeed, Labarta et al. (60) have described 140 endometrial genes (such as GPX3, PAEP, LIF, CNNI, and others) that are altered in the presence of elevated progesterone levels that have been related with cell adhesion and other developmental processes required for normal endometrial function and receptivity. Several studies have revealed a number of candidate genes concerned with endometrial receptivity (61–71). Only a few of them, however, have been identified as common endometrial receptivity markers (72) and are used in clinical practice. Indeed, although to date no single and clinically relevant marker of endometrial receptivity has been identified, over the past decade new developments in molecular biology technologies along with transcriptomic analysis have allowed the investigation of the genomics of human endometrial development (73–75). Some investigators have examined gene expression patterns in the luteal phase of fertile women (56, 61–64, 70, 76), and others have analyzed endometrial transcriptome in patients with implantation failure (57, 71, 77) or in patients with endometrial pathologies (78, 79). Finally, genomic profiles during controlled ovarian stimulation and hormonal replacement have been studied (66, 80, 81). Collectively, over the past decade these studies have led to the definition of a genomic signature of human endometrial receptivity. In response to these developments, in recent years we have developed a genomic tool, referred to as the endometrial receptivity array (ERA) (82–85), that contains 238 genes and allows the identification of the receptive endometrium within the WOI independently of its histologic appearance. The ERA test has been shown to be more accurate than histologic evaluation for diagnosing personalized endometrial receptivity. This has subsequently led to the VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® new concept of personalized embryo transfer (pET) guided individually by endometrial biomarkers as a therapeutic strategy for patients with implantation failure. In this context, miRNAs have been proposed as candidates for playing a role in endometrial receptivity. They have been described in mouse uterus during embryo implantation, where they regulate uterine gene expression (86–88). Indeed, Hu et al. (86) showed that Reck was the target gene of miR-21, a miRNA highly expressed at implantation sites on day 5 of pregnancy, suggesting that this miRNA plays a key role during embryo implantation. Chakrabarty et al. (87) reported eight differently expressed miRNAs in the mouse uterus between implantation and interimplantation sites. Two of them, mmu-miR-101a and 199a, expressed during implantation, were regulated by cyclooxygenase-2, a gene which is critical for implantation. In humans, different miRNAs have been isolated in recent years in the midsecretory phase endometrium (89–91). These are known to regulate epithelial cell proliferation and differentiation during the endometrial cyclic changes (92–94). For example, Domínguez et al. (95) found a gene associated with human endometrial receptivity, the insulinlike growth factor–binding protein related 1 (IGFBP-rP1), in which miRNA is localized in the glandular epithelium and in endothelial cells. In another study, Kuokkannen et al. isolated endometrial epithelial cells from biopsies taken from women at late proliferative and midsecretory phases of the menstrual cycle and compared the expressed miRNAs with microarrays technology. They described 12 miRNAs increased in the secretory endometrium (miR29B, miR29C, miR30B, miR30D, miR31, miR193A-3P, miR203, miR204, miR200C, miR210, miR582– 5P, and miR345) that down-regulated cell cycle genes, including genes of the MIR family, thus confirming that such miRNAs may suppress cell proliferation (58). Further evidence was provided by Altm€ae et al. (96) who analyzed endometrial biopsy samples taken from prereceptive (LHþ2) and receptive endometrium (LHþ7) of fertile nonstimulated women and found that a subset of miRNAs, hsamiR-30b and hsa-miR-30d, were significantly up-regulated, whereas hsa-miR-494 and hsa-miR-923 were downregulated in receptive endometrium. These findings are in line with previously reported studies where hsa-miR-30b and hsa-miR-30d were up-regulated in nonstimulated LHþ7 versus LHþ2 endometria in infertile patients (97). Micro-RNAs might also be important during the early stages of placenta formation and gestation. Several miRNAs are expressed by the placenta during pregnancy: however, the role of most of these has yet to be fully elucidated (98, 99). A recent study analyzed miRNA from first- and third-trimester placentas and identified 208 miRNAs that promote distinct functions and that were differently expressed in the two trimesters (100). This strongly suggests that miRNAs have important roles in placental development. Moreover, Morales-Prieto et al. (101) analyzed the expression of 762 miRNAs, including miRNAs of first- and third-trimester trophoblast cells. They showed that such miRNAs presented critical differences compared with different trophoblastic cell lines, a finding that may explain their different behavior and characteristics. VOL. 101 NO. 6 / JUNE 2014

Although the current review is not intended to assess the potential contribution of miRNAs beyond early pregnancies, findings suggest that these placental miRNAs are also associated with preeclampsia through their alteration of placenta development (102–105). Hence, it is likely that these dysregulated miRNAs could serve as noninvasive biomarkers for defective placentation and preeclamptic pregnancies. In this context, a recent study by Hossain et al. (106) investigated the expression of 377 miRNAs in bovine pregnancies that were established after transfer of cloned or in vitro produced embryos and found a massive deregulation of miRNAs in the bovine placentas. This is likely caused by reprogramming failure during trophoblast differentiation, which leads to placental abnormalities and thus to embryonic losses. This study and others in bovines (107, 108) highlight the role of aberrant miRNAs as regulators of genetic and epigenetic modifications in abnormal placentogenesis. The family of Let-7 is a group of 11 related miRNAs, encoded by four gene clusters. It is among the most highly expressed in mammals (109), participating in the control of cellular pluripotency, proliferation, and differentiation, and also acting as a tumor suppressor (110, 111). LIN28 is an RNA-binding protein capable of binding the precursors of Let-7 miRNAs. By doing so, it blocks their capacity to interact with the miRNA enzyme, Dicer, and prevents them from developing into mature miRNAs (112). In turn, Let-7 miRNAs participate in the regulation of LIN28 expression, which is also controlled by other upstream elements. These include myc and, according to bioinformatics predictions, other miRNAs such as mir-132 and mir-145, which together form a complex regulatory hub that is involved in a variety of processes (113). The expression of Let-7 miRNAs and LIN28 has been reported in human gestational tissues at term (114). Lozoya et al. (115) recently studied embryonic tissues of 43 women with normal ongoing gestations who voluntarily terminated their pregnancies. They showed that the expression of LIN28B mRNA was detectable at low levels in embryonic tissue from early stages of gestation and abruptly increased thereafter until it plateaued between gestational weeks 7 and 9. In contrast, expression levels of Let-7, mir-132, and mir-145 were high in embryonic tissue from early gestations (%6 weeks) and sharply declined thereafter, especially for Let-7. Exactly the opposite trend was observed for mir-3233p. These data in normal pregnancies were compared with EPs and are described herein. In summary, evidence thus far strongly highlights the importance of miRNA-mediated regulation of gene expression during the development of a viable embryo. It further suggests that endometrium miRNAs are likely to regulate genes involved in the implantation process, early stages of placenta formation, and gestation, although their exact role is not yet understood (Fig. 1).

MICRO-RNAS AND ABNORMAL EMBRYO DEVELOPMENT Over the past 30 years, improvements in biotechnology have greatly improved outcomes in the IVF laboratory. These include 1533

VIEWS AND REVIEWS

FIGURE 1

Diagram showing the action mechanisms of miRNA in the implantation process. miRNAs produce a dual and simultaneous effect on embryonic and endometrial cells. Specific miRNAs synthesized by the embryo or endometrial cells bind to their complementary mRNAs, repressing their translation and modulating cellular events and pathways involved in embryo implantation. It is already known that in some pathologies, such as polycystic ovary syndrome or male factor infertility, miRNA synthesis might be altered. The same seems to be true for patients with repeated implantation failure or those having abnormal ovarian stimulation with elevated progesterone levels. Galliano. MiRNA and implantation. Fertil Steril 2014.

the use of intracytoplasmic sperm injection (ICSI) (116, 117), improved culture media (118–120), new oocytes and embryo freezing techniques (121–126), and the introduction of preimplantation genetic diagnosis (PGD) or screening (PGS) (127–132). Indeed, it is well known that one of the most frequent causes of IVF failure results from chromosomal abnormalities, which lead to abnormal embryos and consequently to poor clinical outcomes (133–137). A randomized study by Yang et al. (138) showed that blastocysts selected only based on morphology presented a 44.9% aneuploidy rate, highlighting the importance of genetic tools for embryo screening. Moreover, if PGS cannot be performed, in the case of patients with good prognosis or due to legal or economic reasons, embryo selection through time-lapse technology (139) currently represents a 1534

noninvasive alternative for selecting chromosomally normal embryos (140). In addition, increased knowledge of embryo development, along with the use of new technologies including proteomics, metabolomics, and secretomics (141–148) have led to the identification of potential new biomarkers of good quality embryos, with the aim of improving the clinical outcomes in ART. In this context, miRNAs appear to be noninvasive biomarkers for embryo development. Although some studies have revealed that miRNA plays a critical role in embryo development and stem cell differentiation in a number of species, only a few studies have analyzed the expression of miRNAs in human embryos (38, 149). McCallie et al. (149) performed qPCR in human vitrified-thawed blastocysts for a VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® set of 12 miRNAs previously found in either mouse embryos (14, 150, 151) or human embryonic stem cells (34) (RNU48, hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7g, hsa-miR-19a, hsa- miR-19b, hsa-miR-21, hsa-miR-24, hsa-miR-34b, hsamiR-92, and hsa-miR-93). These investigators observed for the first time that morphologically similar blastocysts derived from patients with male factor infertility and polycystic ovaries (PCO) presented abnormal miRNA profiles. More specifically, compared with fertile control blastocysts, patients with male factor infertility showed a significant decrease in the expression of two miRNAs (hsa-let-7a and hsa-miR-24), whereas patients with PCO showed reduced expression in six miRNAs (hsa-let-7a, hsa-miR-19a, hsa-miR-19b, hsamiR-24, hsa-miR-92, and hsa-miR-93). These findings suggest that similar quality embryos might lead to different clinical outcomes, which may be due to aberrant miRNA profiles expressed in the embryos contributing to implantation failure. Gene targets of these differentially expressed miRNAs are associated with gene ontology biologic processes and molecular functions, such as cell growth and transcription, or nucleic acid binding. For example, gene targets of hsa-miR-19a and hsa-miR-19b showed negative regulation of SOCS1, which is involved in the inhibition of interleukin-6 (IL-6) growth signaling. As previously noted, miRNAs may be negative or positive regulators of gene expression. A decrease in miRNA expression may lead to an increase or decrease in a target gene expression, depending on the particular mechanisms involved in the regulation. In the study by McCallie et al. (149), the expression of three predicted miRNA target genes, ARIH2, KHSRP, and NFAT5, were shown to be altered in embryos derived from either male factor infertility or PCO patients compared with those of fertile patients. This was due to the altered miRNA expression profile, where the expression of ARIH2 and KHSRP increased and that of NFAT5 decreased in such blastocysts, thus suggesting that miRNA aberration could result in altered expression of miRNA target genes. As mentioned above, Rosenbluth et al. (38) studied seven miRNAs expressed in female and male embryos as well as euploid and aneuploidy embryos. They observed that some miRNA, including miR-141, miR-27b, miR-339-3p, and miR345, were differently expressed in euploid versus aneuploid embryos. This suggests that miRNAs might be an early indicator of the prognosis of human embryos from the very beginning of conception, as early as day 5 of development. Moreover, it has recently been demonstrated that differential expression of miRNAs in IVF culture media may serve as a potential marker of IVI success. Indeed, it was observed that secreted miRNAs in IVF media samples correlate with embryonic aneuploidy, which may help to determine chromosomal status before embryo transfer, thus improving pregnancy outcomes (44). The targets of embryonic miRNAs at this stage are essential for regulating development (35), cell proliferation (152), and differentiation (34). These data indicating the expression of different miRNAs based on sex or ploidy status confirm the need to further investigate miRNAs as promising markers of a human embryo's potential. Another potentially important avenue is a better understanding of the role of miRNAs in gene networks with estabVOL. 101 NO. 6 / JUNE 2014

lished essential functions in embryo development and endometrial receptivity. Moreover, knowing the crossregulation between miRNAs and coding genes may help to identify profiles specific to certain clinical conditions or prognostics. Along these lines, a study by Tan et al. (153) highlighted that miRNAs are pluripotency regulators that have critical functions in the preimplantation mouse embryo. Indeed, the investigators showed that the miRN-290–295 cluster of miRNAs, which are directly controlled by Oct4 and Nanog, regulated DNA methylation by targeting the retinoblastoma-like 2 (Rbl2) mRNA and through Rbl2 regulation of Dnmt3b transcription in the preimplantation embryo.

MICRO-RNAS IN WOMEN WITH IMPLANTATION FAILURE AFTER ART Throughout a natural menstrual cycle, receptivity is well timed as the embryo passes through the fallopian tubes toward the uterus. However, in ART, the transfer of the blastocyst into the uterus is based on its stage of development and not on the synchronicity between maternal endometrium and the embryo. Moreover, in ART cycles, genes relevant to the WOI are altered due to the abnormal hormonal environment to which the endometrium is exposed after hormonal controlled ovarian hyperstimulation (COH) for ART cycles (154–159). Frozen-thawed embryos transferred into natural cycles have been reported to improve endometrial receptivity and increase pregnancy rates (160–162). Defects in implantation and trophoblast invasion are considered possible reasons for the failure of ART (47, 163, 164). Although improvements in embryo quality have been achieved, due in part to the better hormone protocols used in stimulated IVF cycles, pregnancy rates in ART remain relatively low (165). Therefore, at present the major challenge is to improve endometrial receptivity for successful implantation and pregnancy (166). Endometrial morphology and thickness have traditionally been used to predict endometrial receptivity, but with restricted positive predictive value (167, 168). Similarly, histologic endometrial evaluation has been used as a predictor of endometrial receptivity (169, 170), but doubts have been raised as to its clinical relevance in both prospective (171–175) and randomized studies (176, 177). As stated previously, a variety of genes have been associated with endometrial receptivity during the WOI, although less is known about the molecular mechanisms that regulate their expression (82–85, 178). Moreover, many studies assessing endometrial gene expression have included only a small number of patients, and thus the endometrial samples analyzed have been limited and the conclusions that can be drawn are restricted (56, 68, 75, 179). Thus, new markers of endometrial receptivity are sought and, in this context, miRNA have been also studied in humans. Sha et al. (97) investigated the expression of 626 known miRNAs and discovered 17 novel miRNAs in the endometrium of patients undergoing IVF in natural and stimulated cycles. The investigators studied miRNA expression in endometrial biopsy samples taken from early (LHþ2) and middle (LHþ7) secretory phases of a natural cycle and compared 1535

VIEWS AND REVIEWS them with those on the days of human chorionic gonadotropin administration hCGþ4 (equivalent to early secretory) and hCGþ7 (equivalent to midsecretory) in stimulated cycles. When they were comparing the data of the hCGþ7 samples with those of natural cycles on LHþ7, they found 22 dysregulated miRNAs, indicating that these miRNAs were responding to the ovarian hormones. Moreover, miRNA expression profile on hCGþ4 was more similar to that on LHþ7 than it was to hCGþ7, suggesting a displacement in endometrial maturation due to ovarian stimulation (97). Indeed, other studies have demonstrated that on the day of oocyte retrieval (36 hours after hCG administration), the advanced maturation status of the endometrium after COH (180) correlates with altered gene expression (80, 181). Together, these data suggest that miRNA expression could help to diagnose endometrial maturation phase during ovarian stimulation, which might subsequently alter the WOI. Li et al. (182) also highlighted the importance of hsa-miR30b in normal receptive endometrium in a study demonstrating its up-regulation in endometrial biopsy samples from patients with normal P concentrations versus women with elevated P levels on the day of hCG administration in IVF cycles. The investigators found dissimilar endometrial receptivity, abnormal expressions of cytokines including osteopontin, and decreased vascular endothelial growth factor in patients with high P levels on the day of hCG administration, thus demonstrating an association between reduced endometrial receptivity and poor pregnancy rates. Data from this study may explain the reduced pregnancy rate in patients with elevated P and further suggest that miRNAs could serve as new markers for enhanced treatment in patients undergoing IVF cycles with elevated P levels. Revel et al. (179) demonstrated that the secretory endometrium of patients with repeated implantation failure (RIF) is characterized by a miRNA expression that differs from that of fertile women. In the study, they identified 13 differentially expressed miRNAs that regulated the expression of 3,800 genes. Ten of these miRNAs were overexpressed (including miR 145, 23b and 99a) and only three were underexpressed (hsa-miR-32, 628-5p and 874). It is interesting that miR 145, 23b and 99a, were previously shown to be associated with other endometrial dysfunctions such as endometriosis (183, 184). Micro-RNA-regulated molecular pathways such as adherens junctions, cell adhesion molecules, Wntsignaling, and cell cycle pathways are crucial for implantation but were lower in RIF patients (179). Accordingly, these RIF-associated miRNAs might possibly be employed for diagnosis and treatment of embryo implantation failures. However, not all of the studies presented have achieved the same results, nor have the specific and sensitive miRNAs relevant for prediciting the status of receptive endometrium been identified. Therefore, the specific role played by miRNAs during embryo implantation remains unclear and requires further investigation. This will be an essential step before miRNAs can be used as guidelines for identifying and treating impaired endometrial receptivity. To summarize, Figure 1 shows the potential mechanisms of action of miRNA in the implantation process, which induce a dual and simultaneous effect on embryonic and endometrial 1536

cells. Specific miRNAs synthesized by the embryo or endometrial cells bind to their complementary mRNAs, repressing their translation and modulating cellular events and pathways involved in embryo implantation. This could alter embryo growth and survival, and it is already known that in some pathologies, such as PCOS or male factor infertility, miRNA synthesis might be altered. The same seems to be true for patients with RIF or those who have an abnormal ovarian stimulation with elevated P levels.

MICRO-RNAS IN EARLY ABNORMAL PREGNANCIES Ectopic pregnancy (EP), defined as the implantation of a fertilized ovum outside the uterus, presents an incidence of approximately 2% in the general population (185–187) and up to 16% in pregnant women in emergency rooms for abdominal symptoms and vaginal bleeding (188, 189). This condition can lead to tubal rupture and is responsible for approximately 6% of all pregnancy-related deaths in the first trimester of pregnancy (190–192), often because the early signs and symptoms of the condition are not identified. Over 98% of EPs implant in the fallopian tube, particularly in the ampulla (193–197). The pathogenesis of EP remains relatively unclear, but it seems that an abnormal transport of the embryo through the tube along with an impaired tubal enviroment and inflammation may lead to ectopic implantation (198–202). It has also been hypothesized that a high P concentration may cause a dysfunction in cilia motility and thus raise the risk of EP in women using a progestin-only pill (203). This may occur, for example, after failure of pregnancy contraception with Levonorgestrel (204). It has been shown that risk factors for EP include infection, especially Chlamydia trachomatis, surgery, smoking, and IVF (205–214). Patients with previous EP have an augmented risk of infertility (215–218) and/or future EP (213, 219). A pregnancy of unknown location (PUL) can be defined as a situation in which a woman has a positive pregnancy test but where no intrauterine or extrauterine pregnancy is visualized on transvaginal sonographic (TVS) or with laparoscopy (220). A PUL may have four possible final outcomes, including disappearance of the pregnancy (44%–69%), normal intrauterine pregnancy (34%–40%), EP (8%–14%), and persisting PUL (2%) (221–224). Altogether, a PUL represents a potentially dangerous condition that requires careful follow-up observation with hormone measurements, repeated TVS, and possible laparoscopy until a diagnosis is confirmed due to the possible risk of being an EP (225, 226). Consequently, the multiple visits and tests necessary to achieve a final diagnosis represent a great cost for the health-care system (218, 227). As stated earlier, the potential benefit of studying miRNAs in different clinical situations is to gain clues about the origin of disease. Some serum-free (or other body fluid-free) miRNAs may also be employed as biomarkers to improve diagnosis. With regard to the former, Lozoya et al. (115) recently included in a prospective study 17 patients suffering from tubal EP and 43 women with normal ongoing gestation that VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® desired voluntary termination of pregnancy. They analyzed the expression of gene LIN28B and the related miRNAs Let7a, mir-132 and mir-145, in early stages of placentation. They found that levels of LIN28B miRNA were higher in the EP group than in normal gestation at %6 weeks. In contrast, Let-7a and mir-323-3p expression were lower in EP, while miR-132 and miR-145 levels were similar in normal pregnancies and EP. Together, this provides further evidence of the involvement of miRNAs in the pathogenesis of EP, although their exact function is still unknown. Regarding diagnosis of abnormal gestations, several diagnostic algorithms have been published for the management of patients with PUL and the diagnosis of EP (228–230). Moreover, a recent study involving 1,962 pregnancies has suggested a protocol to safely minimize follow-up evaluations in low-risk PULs. This consequently reduces the number of visits, scans, and biochemical tests, and leads to a more efficient use of resources for pregnancies at a higher risk of being ectopic (231). This suggests the great need to identify possible tests that integrate accuracy with reproducibility and simplicity to predict the outcome of PUL and reduce follow-up evaluations. When TVS does not show an extrauterine gestational sac, as occurs in 8% to 31% of patients attending specialists (232), biochemical or surgical investigation is needed to diagnose EP. The use of TVS, as well as serum hCG and progesterone (233–236), has been shown to have low clinical utility for identifying women with EP, due to high false-positive and false-negative rates and difficulties differentiating between EP and spontaneous abortion (SA). Indeed, a single determination of hCG is only useful for indicating pregnancy viability rather than the location of the gestational sac. An increase in the hCG level of 53% in 48 hours is associated with viable intrauterine pregnancy, as suggested by the American College of Obstetricians and Gynecologists (237). Serial quantitative serum hCG concentrations are used to diagnose potential viable gestation, SA, or EP (238–241). However, during the 48-hour wait for the next hCG measurement, women are at risk for possible complications, particularly tubal rupture (242). To avoid this situation, and subsequently to reduce health-care costs with earlier diagnosis (224, 243), several serum markers have been studied over the decades but with uncertain clinical utility (244–248). Arguments to limit the use of serum markers stem from the fact that serum concentrations vary across pregnancies, and that they can often distinguish EP from a viable intrauterine pregnancy, but not from SA (244). On the contrary, an ideal biomarker would be one that does not change throughout gestational age and which also allows the discrimination of EP from both a viable intrauterine pregnancy and SA. For instance, Rausch et al. (247) showed increased diagnostic utility for diagnosing EP with an algorithm combining four markers, including P, vascular endothelial growth factor, inhibin A, and activin A. This study, however, did not include patients with SA, so it is unclear how far the utility of these markers extends. In this context, miRNAs have been proposed as new and noninvasive serum tests to detect EP earlier with high sensitivity and specificity, thereby improving an often problematic VOL. 101 NO. 6 / JUNE 2014

diagnosis. In addition to preventing the potentially associated complications of EP, such as maternal morbidity and pregnancy-related first-trimester deaths, pregnancyassociated circulating miRNAs might also prevent unnecessary surgical or medical interventions as well as treatment delays for EP and PUL. Indeed, when detected early, EP may be treated with systemic methotrexate (MTX) (249) or conservative laparoscopy surgery (250), reducing both the morbidity and mortality of this condition. Zhao et al. (242) studied the clinical and diagnostic utility of several miRNAs for detecting EP and found that one of them in particular, miR-323-3p, improved the diagnostic accuracy for EP when incorporated into a model including hCG and P. Previous studies have shown that miR-323-3p is a pregnancy-associated miRNA that is quickly cleared from maternal plasma after delivery and does not change between the first and third trimesters (251). In agreement with these findings, Zhao et al. (242) found that miR-323-3p serum concentrations do not change with gestational age and were significantly higher in the EP group of patients compared with the viable intrauterine pregnancy and SA groups. Given the above results, these two characteristics of miR-323-3p might make it an ideal marker for detecting EP. Although the precise mechanisms through which miRNAs lead to the clinical manifestations of EP are not completely clear, it is known that they are exported into circulation where their concentrations may reflect pathologic states of the tissues (252). In fact, Luo et al. (253) have proposed that circulating miRNAs released from syncytiotrophoblasts into the maternal circulation via exosomes may be used as diagnostic tests for pregnancy status. Moreover, Zhao et al. (242) observed that when trophoblasts invaded and eroded the tubal wall in the case of tubal EP, miRNAs were released into the maternal circulation. This resulted in increased serum concentrations, probably due to tubal damage, tubal implantation, or peritoneal inflammation. Patients with tubal ET present high levels of serum IL-6, which has been implicated in the regulation of ovarian steroid production, folliculogenesis, and implantation (214, 254, 255). The effects of IL-6 are mediated by its receptors IL6Ra and glycoprotein 130 (Gp130). Various miRNAs are involved in the molecular regulation of trophoblast apoptosis and the inhibition of IL-6 miRNA. A recent study first compared the IL-6Ra and Gp130 miRNA expression in the fallopian tubes of nonpregnant women, women with normal pregnancies, and patients with EP (256). The investigators reported higher IL-6Ra miRNA expression and lower Gp130 expression in the fallopian tubes of patients with EP compared with that found in nonpregnant women, suggesting that these miRNAs may play a role in EP (256). In contrast, other data have shown no difference between IL-6Ra expression in the fallopian tubes of patients with EP and the normal group (257). These inconsistencies may be due to the internal bias of such studies or other methodologic differences. Recent data have highlighted the role of miRNAs released into the extracellular space. Of particular interest are those from the largest gene cluster of human miRNAs, the chromosome 19 miRNA cluster (C19MC), which are exclusively expressed in the placenta and in undifferentiated cells 1537

VIEWS AND REVIEWS (258, 259). Tests for such placental miRNAs in maternal circulation may be used to distinguish EP from intrauterine pregnancies. This has been shown in a study that found lower levels of placental miRNA, including hPL mRNA and b-hCG mRNA, in the maternal blood of 12 women with EP compared with 13 women who had intrauterine pregnancies (260). This is possibly due to a reduced blood supply, caused by the lack of trophoblast formation within the tube, which could lead to a reduction of mRNAs in the maternal circulation of women with EP. It has also been shown that the gene encoding the miR323-3p, an epigenetic biomarker associated with EP, is present in a miRNA cluster. A recent review has confirmed its role as a promising therapeutic target for this disease, although the exact mechanism implicated remains unknown (261). Even with the accumulated and informative evidence reported so far, no standard procedure currently exists for the routine use of miRNAs as biomarkers in the diagnosis of EP or for predicting the outcome of a PUL. Therefore, further work should be aimed at the development of appropriate miRNA assays for clinical use in the near future.

10.

11.

12.

13. 14.

15.

16.

17. 18. 19.

CONCLUSION This review has presented the published evidence regarding miRNAs as potential gene expression regulators during implantation. We have shown the role of previously characterized miRNAs implicated in impaired endometrial receptivity, altered embryo development, implantation failure after ART, and in ectopic pregnancy and pregnancies of unknown location. In fact, there is currently great interest in identifying miRNAs that can be used as markers for the early detection of implantation disorders. However, the implications of the findings described throughout this review need to be confirmed in large clinical trials before such noninvasive miRNAs become a real approach in daily clinical practice for assessing embryonic and endometrial health, and thereby increasing pregnancy rates per transfer after IVF.

20.

REFERENCES

26.

1. 2. 3. 4. 5.

6. 7. 8.

9.

1538

Wilkins-Haug L. Epigenetics and assisted reproduction. Curr Opin Obstet Gynecol 2009;21:201–6. Ambros V. MicroRNAs: tiny regulators with great potential. Cell 2001;107: 823–6. Maccani MA, Marsit CJ. Epigenetics in the placenta. Am J Reprod Immunol 2009;62:78–89. Prieto DM, Markert UR. MicroRNAs in pregnancy. J Reprod Immunol 2011; 88:106–11. Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 2005; 122:553–63. Wu L, Fan J, Belasco JG. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci USA 2006;103:4034–9. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. Kotlabova K, Doucha J, Hromadnikova I. Placental-specific microRNA in maternal circulation-identification of appropriate pregnancy-associated microRNAs with diagnostic potential. J Reprod Immunol 2011;89:185–91. Scholer N, Langer C, Dohner H, Buske C, Kuchenbauer F. Serum microRNAs as a novel class of biomarkers: a comprehensive review of the literature. Exp Hematol 2010;38:1126–30.

21.

22.

23. 24.

25.

27.

28.

29.

30.

31.

32.

33.

n C. Molecular interplay in successful implanCha J, Vilella F, Dey SK, Simo tation. In: Sanders S, editor. Ten critical topics in reproductive medicine. Washington, DC: Science/AAAS; 2013:44–8. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 2005;120:21–4. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by micro-RNAs. Nature 2008;455:58–63. Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008;455:64–71. Tang F, Kaneda M, O'Carroll D, Hajkova P, Barton SC, Sun YA, et al. Maternal microRNAs are essential for mouse zygotic development. Genes Dev 2007;21:644–8. Hemberger M, Dean W, Reik W. Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal. Nat Rev Mol Cell Biol 2009;10:526–37. Suh N, Baehner L, Moltzahn F, Melton C, Shenoy A, Chen J, et al. MicroRNA function is globally suppressed in mouse oocytes and early embryos. Curr Biol 2010;20:271–7. Suh N, Blelloch R. Small RNAs in early mammalian development: from gametes to gastrulation. Development 2011;138:1653–61. Blakaj A, Lin H. Piecing together the mosaic of early mammalian development through microRNAs. J Biol Chem 2008;283:9505–8. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000;403:901–6. Laurent LC. MicroRNAs in embryonic stem cells and early embryonic development. J Cell Mol Med 2008;12:2181–8. Foshay KM, Gallicano GI. MiR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation. Dev Biol 2009;326:431–43. Wang R, Hu Y, Song G, Hao CJ, Cui Y, Xia HF, et al. MiR-206 regulates neural cells proliferation and apoptosis via Otx2. Cell Physiol Biochem 2012;29: 381–90. Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, et al. Dicer is essential for mouse development. Nat Genet 2003;35:215–7. Yang WJ, Yang DD, Na S, Sandusky GE, Zhang Q, Zhao GJ. Dicer is required for embryonic angiogenesis during mouse development. J Biol Chem 2005;280:9330–5. Medeiros LA, Dennis LM, Gill ME, Houbaviy H, Markoulaki S, Fu D, et al. Mir-290–295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects. Proc Natl Acad Sci USA 2011;108: 14163–8. Gunaratne PH. Embryonic stem cell microRNAs: defining factors in induced pluripotent (iPS) and cancer (CSC) stem cells? Curr Stem Cell Res Ther 2009;4:168–77. Ren J, Jin P, Wang E, Marincola FM, Stroncek DF. MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells. J Transl Med 2009;7:20. Zovoilis A, Nolte J, Drusenheimer N, Zechner U, Hada H, Guan K, et al. Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles. Mol Hum Reprod 2008;14:521–9. De Felici M, Farini D, Dolci S. In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells. Curr Stem Cell Res Ther 2009;4:87–97. Ciaudo C, Servant N, Cognat V, Sarazin A, Kieffer E, Viville S, et al. Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation. PLoS Genet 2009;5:e1000620. Mallanna SK, Rizzino A. Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol 2010;344:16–25. Li SS, Yu SL, Kao LP, Tsai ZY, Singh S, Chen BZ, et al. Target identification of microRNAs expressed highly in human embryonic stem cells. J Cell Biochem 2009;106:1020–30. Suh MR, Lee Y, Kim JY, Kim SK, Moon SH, Lee JY, et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol 2004;270:488–98.

VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® 34.

35. 36. 37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47. 48. 49. 50. 51.

52.

53.

54.

55.

56.

Lakshmipathy U, Love B, Goff LA, Jornsten R, Graichen R, Hart RP, et al. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev 2007;16:1003–16. Tang F, Hajkova P, Barton SC, Lao K, Surani MA. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 2006;34:e9. Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev Cell 2003;5:351–8. Chen C, Ridzon D, Lee CT, Blake J, Sun Y, Strauss WM. Defining embryonic stem cell identity using differentiation-related microRNAs and their potential targets. Mamm Genome 2007;18:316–27. Rosenbluth EM, Shelton DN, Sparks AE, Devor E, Christenson L, Van Voorhis BJ. MicroRNA expression in the human blastocyst. Fertil Steril 2013;99:855–61. Tzur G, Levy A, Meiri E, Barad O, Spector Y, Bentwich Z, et al. MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells. PloS One 2008;3:e3726. Yang Y, Kai G, Pu XD, Qing K, Guo XR, Zhou XY. Expression profile of micro-RNAs in fetal lung development of Sprague-Dawley rats. Int J Mol Med 2012;29:393–402. Hand NJ, Master ZR, Eauclaire SF, Weinblatt DE, Matthews RP, Friedman JR. The microRNA-30 family is required for vertebrate hepatobiliary development. Gastroenterology 2009;136:1081–90. Noguer-Dance M, Abu-Amero S, Al-Khtib M, Lefevre A, Coullin P, Moore GE, et al. The primate-specific microRNA gene cluster (C19MC) is imprinted in the placenta. Hum Mol Genet 2010;19:3566–82. Yu Z, Jian Z, Shen SH, Purisima E, Wang E. Global analysis of micro-RNA target gene expression reveals that miRNA targets are lower expressed in mature mouse and Drosophila tissues than in the embryos. Nucleic Acids Res 2007;35:152–64. Rosenbluth EM, Shelton DN, Wells LM, Sparks AET, Van Voorhis BJ. Human embryos secrete microRNAs into culture media—a potential biomarker for implantation. Fertil Steril. Published online March 12, 2014. Harper MJ. The implantation window. Baillieres Clin Obstet Gynaecol 1992;6:351–71. Salamonsen LA, Nie G, Hannan NJ, Dimitriadis E. Society for Reproductive Biology Founders' Lecture 2009. Preparing fertile soil: the importance of endometrial receptivity. Reprod Fertil Dev 2009;21:923–34. Edwards RG. Physiological and molecular aspects of human implantation. Hum Reprod 1995;10:60–6. Navot D, Bergh P. Preparation of the human endometrium for implantation. Ann NY Acad Sci 1991;622:212–9. Kayisli UA, Guzeloglu-Kayisli O, Arici A. Endocrine-immune interactions in human endometrium. Ann NY Acad Sci 2004:50–63. Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update 2006;6:731–46. Boomsma CM, Kavelaars A, Eijkemans MJ, Amarouchi K, Teklenburg G, Macklon NS. Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity. Reprod Biomed Online 2009;18:85–94. Boomsma CM, Kavelaars A, Eijkemans MJ, Lentjes EG, Fauser BC, Heijnen CJ, et al. Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF. Hum Reprod 2009;24:1427–35. Boomsma CM, Kavelaars A, Eijkemans MJ, Fauser BC, Heijnen CJ, Macklon NS. Ovarian stimulation for in vitro fertilization alters the intrauterine cytokine, chemokine, and growth factor milieu encountered by the embryo. Fertil Steril 2010;94:1764–8. Perrier d'Hauterive S, Charlet-Renard C, Berndt S, Dubois M, Munaut C, Goffin F, et al. Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium. Hum Reprod 2004;19:2633–43. Srivastava A, Sengupta J, Kriplani A, Roy KK, Ghosh D. Profiles of cytokines secreted by isolated human endometrial cells under the influence of chorionic gonadotropin during the window of embryo implantation. Reprod Biol Endocrinol 2013;11:116. Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillips SC, et al. Determination of the transcript profile of human endometrium. Mol Hum Reprod 2003;1:19–33.

VOL. 101 NO. 6 / JUNE 2014

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73. 74. 75.

76.

Koler M, Achache H, Tsafrir A, Smith Y, Revel A, Reich R. Disrupted gene pattern in patients with repeated in vitro fertilization (IVF) failure. Hum Reprod 2009;10:2541–8. Kuokkanen S, Chen B, Ojalvo L, Benard L, Santoro N, Pollard JW. Genomic profiling of microRNAs and messenger RNAs reveals hormonal regulation in microRNA expression in human endometrium. Biol Reprod 2010;82: 791–801. Nabilsi NH, Broaddus RR, McCampbell AS, Lu KH, Lynch HT, Chen LM, et al. Sex hormone regulation of survivin gene expression. J Endocrinol 2010;207:237–43. Labarta E, Martínez-Conejero JA, Alama P, Horcajadas JA, Pellicer A, n C, et al. Endometrial receptivity is affected in women with high Simo circulating progesterone levels at the end of the follicular phase: a functional genomics analysis. Hum Reprod 2011;26:1813–25. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach-Carson MC, Vernon M, et al. Changes in gene expression during the early to midluteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 2002;8:871–9. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, et al. Global gene profiling in human endometrium during the window of implantation. Endocrinology 2002;143:2119–38. Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, et al. Gene expression profiling of human endometrial receptivity on days LH þ 2 versus LH þ 7 by micro array technology. Mol Hum Reprod 2003;9: 253–64. Horcajadas JA, Riesewijk A, Martin J, Cervero A, Mosselman S, Pellicer A, et al. Global gene expression profiling of human endometrial receptivity. J Reprod Immunol 2004;63:41–9. Mirkin S, Arslan M, Churikov D, Corica A, Diaz JI, Williams S, et al. In search of candidate genes critically expressed in the human endometrium during the window of implantation. Hum Reprod 2005;20:2104–17. Simon C, Oberye J, Bellver J, Vidal C, Bosch E, Horcajadas JA, et al. Similar endometrial development in oocyte donors treated with either high- or standard-dose GnRH antagonist compared to treatment with a GnRH agonist or in natural cycles. Hum Reprod 2005;20:3318–27. Talbi S, Hamilton AE, Vo KC, Tulac S, Overgaard MT, Dosiou C, et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 2006;147:1097–121. Feroze-Zaidi F, Fusi L, Takano M, Higham J, Salker MS, Goto T, et al. Role and regulation of the serum- and glucocorticoid-regulated kinase 1 in fertile and infertile human endometrium. Endocrinology 2007;148: 5020–9. Haouzi D, Assou S, Mahmoud K, Tondeur S, Reme T, Hedon B, et al. Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients. Hum Reprod 2009;24: 1436–44. Haouzi D, Mahmoud K, Fourar M, Bendhaou K, Dechaud H, De Vos J, et al. Identification of new biomarkers of human endometrial receptivity in the natural cycle. Hum Reprod 2009;24:198–205. n C, Horcajadas JA, Altm€ae S, Martínez-Conejero JA, Salumets A, Simo Stavreus-Evers A. Endometrial gene expression analysis at the time of embryo implantation in women with unexplained infertility. Mol Hum Reprod 2010;16:178–87. Horcajadas JA, Pellicer A, Simon C. Wide genomic analysis of human endometrial receptivity: new times, new opportunities. Hum Reprod Update 2007;13:77–86. Martín J, Domínguez F, Avila S, Castrillo JL, Remohí J, Pellicer A, et al. Human endometrial receptivity: gene regulation. J Reprod Immunol 2002;55:131–9. n C. Human endometrial recepDomínguez F, Remohí J, Pellicer A, Simo tivity: a genomic approach. Reprod Biomed Online 2003;6:332–8. n C. Horcajadas JA, Riesewijk A, Domínguez F, Cervero A, Pellicer A, Simo Determinants of endometrial receptivity. Ann NY Acad Sci 2004;1034: 166–75. Ponnampalam AP, Weston GC, Trajstman AC, Susil B, Rogers PA. Molecular classification of human endometrial cycle stages by transcriptional profiling. Mol Hum Reprod 2004;10:879–93.

1539

VIEWS AND REVIEWS 77.

78. 79.

80.

81.

82.

83.

84.

85.

86.

87.

88. 89.

90.

91.

92.

93.

94. 95.

96.

97.

1540

Tapia A, Vilos C, Marín JC, Croxatto HB, Devoto L. Bioinformatic detection of E47, E2F1 and SREBP1 transcription factors as potential regulators of genes associated to acquisition of endometrial receptivity. Reprod Biol Endocrinol 2011;27:9–14. Matsuzaki S. DNA microarray analysis in endometriosis for development of more effective targeted therapies. Front Biosci 2011;3:1139–53. Habermann JK, Bundgen NK, Gemoll T, Hautaniemi S, Lundgren C, Wangsa D, et al. Genomic instability influences the transcriptome and proteome in endometrial cancer subtypes. Mol Cancer 2011;10:132. Horcajadas JA, Minguez P, Dopazo J, Esteban FJ, Dominguez F, Giudice LC, et al. Controlled ovarian stimulation induces a functional genomics delay of the endometrium with potential clinical implications. J Clin Endocrinol Metab 2008;93:4500–10. Mirkin S, Nikas G, Hsiu JG, Diaz J, Oehninger S. Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 2004;89:5742–52. Díaz-Gimeno P, Horcajadas JA, Martínez-Conejero JA. A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril 2011;95:50–60. Díaz-Gimeno P, Ruiz-Alonso M, Blesa D, Bosch N, Martínez-Conejero J, Alam a P, et al. The accuracy and reproducibility of the endometrial receptivity array is superior to histology as a diagnostic method for endometrial receptivity. Fertil Steril 2013;99:508–17. mez T, Ruiz-Alonso M, Blesa D, Diaz-Gimeno P, Vilella F, Garrido-Go n C. Profiling the gene signature of endometrial receptivity: clinical reSimo sults. Fertil Steril 2013;99:1078–85. mez E, Fernandez-Sanchez M, Ruiz-Alonso M, Blesa D, Díaz-Gimeno P, Go Carranza F, et al. The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure. Fertil Steril 2013;100:818–24. Hu SJ, Ren G, Liu JL, Zhao ZA, Yu YS, Su RW, et al. MicroRNA expression and regulation in mouse uterus during embryo implantation. J Biol Chem 2008;283:23473–84. Chakrabarty A, Tranguch S, Daikoku T, Jensen K, Furneaux H, Dey SK. MicroRNA regulation of cyclooxygenase-2 during embryo implantation. Proc Natl Acad Sci USA 2007;104:15144–9. Gonzalez G, Behringer RR. Dicer is required for female reproductive tract development and fertility in the mouse. Mol Reprod Dev 2009;76:678–88. n C, McNoe LA, Evans GE, Martínez-Conejero JA, Phillipson GT, Simo Sykes PH, et al. Gene and protein expression signature of endometrial glandular and stromal compartments during the window of implantation. Fertil Steril 2012;97:1365–73. Creighton CJ, Benham AL, Zhu H, Khan MF, Reid JG, Nagaraja AK, et al. Discovery of novel microRNAs in female reproductive tract using next generation sequencing. PLoS One 2010;5:e9637. Boren T, Xiong Y, Hakam A, Wenham R, Apte S, Wei Z, et al. MicroRNAs and their target messenger RNA associated with endometrial carcinogenesis. Gynecol Oncol 2008;110:206–15. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;5:593–601. Korpal M, Lee ES, Hu G, Kang Y. The miR-200 family inhibits epithelialmesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem 2008; 22:14910–4. Lam EW, Shah K, Brosens JJ. The role of microRNAs and FOXO transcription factors in cycling endometrium and cancer. J Endocrinol 2012;212:13–25. Domínguez F, Avila S, Cervero A, Martín J, Pellicer A, Castrillo JL, et al. A combined approach for gene discovery identifies insulin-like growth factor-binding protein-related protein 1 as a new gene implicated in human endometrial receptivity. J Clin Endocrinol Metab 2003;88:1849–57. Altm€ ae S, Martinez-Conejero JA, Esteban FJ, Ruiz-Alonso M, StavreusEvers A, Horcajadas JA, et al. MicroRNAs miR-30b, miR-30d, and miR494 regulate human endometrial receptivity. Reprod Sci 2013;20:308–17. Sha AG, Liu JL, Jiang XM, Ren JZ, Ma CH, Lei W, et al. Genome-wide identification of micro-ribonucleic acids associated with human endometrial

receptivity in natural and stimulated cycles by deep sequencing. Fertil Steril 2011;96:150–155.e5. 98. Mouillet JF, Chu T, Sadovsky Y. Expression patterns of placental microRNAs. Birth Defects Res A Clin Mol Teratol 2011;91:737–43. 99. Buckberry S, Bianco-Miotto T, Roberts CT. Imprinted and X-linked noncoding RNAs as potential regulators of human placental function. Epigenetics 2014;9:81–9. 100. Gu Y, Sun J, Groome LJ, Wang Y. Differential miRNA expression profiles between the first and third trimester human placentas. Am J Physiol Endocrinol Metab 2013;304:E836–43. 101. Morales-Prieto DM, Chaiwangyen W, Ospina-Prieto S, Schneider U, Herrmann J, Gruhn B, et al. MicroRNA expression profiles of trophoblastic cells. Placenta 2012;33:725–34. 102. Chen DB, Wang W. Human placental microRNAs and preeclampsia. Biol Reprod 2013;88:130. 103. Fu G, Brkic J, Hayder H, Peng C. MicroRNAs in human placental development and pregnancy complications. Int J Mol Sci 2013;14:5519–44. 104. Morales-Prieto DM, Ospina-Prieto S, Schmidt A, Chaiwangyen W, Markert UR. Elsevier Trophoblast Research Award Lecture: Origin, evolution and future of placenta miRNAs. Placenta 2014;35(Suppl):S39–45. 105. Wu L, Zhou H, Lin H, Qi J, Zhu C, Gao Z, et al. Circulating microRNAs are elevated in plasma from severe preeclamptic pregnancies. Reproduction 2012;143:389–97. €ll MJ, Rings F, et al. 106. Hossain MM, Tesfaye D, Salilew-Wondim D, Held E, Pro Massive deregulation of miRNAs from nuclear reprogramming errors during trophoblast differentiation for placentogenesis in cloned pregnancy. BMC Genomics 2014;15:43. 107. Salilew-Wondim D, Tesfaye D, Hossain M, Held E, Rings F, Tholen E, et al. Aberrant placenta gene expression pattern in bovine pregnancies established after transfer of cloned or in vitro produced embryos. Physiol Genomics 2013;45:28–46. 108. Betsha S, Hoelker M, Salilew-Wondim D, Held E, Rings F, GrosseBrinkhause C, et al. Transcriptome profile of bovine elongated conceptus obtained from SCNT and IVP pregnancies. Mol Reprod Dev 2013;80: 315–33. 109. Roush S, Slack FJ. The let-7 family of microRNAs. Trends Cell Biol 2008;18: 505–16. 110. Boyerinas B, Park SM, Hau A, Murmann AE, Peter ME. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 2010;17:F19–36. 111. Bussing I, Slack FJ, Grosshans H. Let-7 microRNAs in development, stem cells and cancer. Trends Mol Med 2008;14:400–9. 112. Viswanathan SR, Daley GQ. Lin28: A microRNA regulator with a macro role. Cell 2010;140:445–9. 113. Sangiao-Alvarellos S, Manfredi-Lozano M, Ruiz-Pino F, Navarro VM, Sanchez-Garrido MA, Leon S, et al. Changes in hypothalamic expression of the Lin28/let-7 system and related microRNAs 361 during postnatal maturation and after experimental manipulations of puberty. Endocrinology 2013;154:942–55. 114. Chan HW, Lappas M, Yee SW, Vaswani K, Mitchell MD, Rice GE. The expression of the let-7 miRNAs and Lin28 signalling pathway in human term gestational tissues. Placenta 2013;34:443–8. 115. Lozoya T, Dominguez F, Romero-Ruiz A, Steffani L, Martinez S, Monterde M, et al. The Lin28/Let-7 system in early human embryonic tissue and ectopic pregnancy. PloS One 2014;9:e87698. 116. Van Steirteghem A, Nagy Z, Liu J, Joris H, Verheyen G, Smitz J, et al. Intracytoplasmic sperm injection. Baillieres Clin Obstet Gynaecol 1994;8:85–93. 117. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995;63:1231–40. 118. Ciray HN, Aksoy T, Goktas C, Ozturk B, Bahceci M. Time-lapse evaluation of human embryo development in single versus sequential culture media-a sibling oocyte study. J Assist Reprod Genet 2012;29:891–900. 119. Pool TB, Schoolfield J, Han D. Human embryo culture media comparisons. Methods Mol Biol 2012;912:367–86. 120. Rato ML, Gouveia-Oliveira A, Plancha CE. Influence of post-thaw culture on the developmental potential of human frozen embryos. J Assist Reprod Genet 2012;29:789–95.

VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® 121.

122.

123.

124. 125.

126.

127.

128.

129.

130. 131.

132.

133.

134.

135.

136. 137.

138.

139.

140.

141.

Martínez-Burgos M, Herrero L, Megías D, Salvanes R, Montoya MC, Cobo AC, et al. Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril 2011;95:374–7. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011; 96:277–85.  D, Gamiz P, Campos P, Remohí J. OutCobo A, de los Santos MJ, Castello comes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril 2012;98:1138–1146.e1. Cobo A. Oocyte vitrification: a watershed in ART. Fertil Steril 2012;98: 600–1.  D, Vallejo B, Albert C, de los Santos JM, Remohí J. Cobo A, Castello Outcome of cryotransfer of embryos developed from vitrified oocytes: double vitrification has no impact on delivery rates. Fertil Steril 2013;99: 1623–30. n C, Cobo A. Effect of vitrificaDominguez F, Castello D, Remohí J, Simo tion on human oocytes: a metabolic profiling study. Fertil Steril 2013;99: 565–72. Handyside AH, Pattinson JK, Penketh RJ, Delhanty JD, Winston RM, Tuddenham EG. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989;1:347–9. Mir P, Rodrigo L, Mateu E, Peinado V, Milan M, Mercader A, et al. Improving FISH diagnosis for preimplantation genetic aneuploidy screening. Hum Reprod 2010;25:1812–7. Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril 2013;100:624–30. Scott KL, Hong KH, Scott RT Jr. Selecting the optimal time to perform biopsy for preimplantation genetic testing. Fertil Steril 2013;100:608–14. Schoolcraft WB, Katz-Jaffe MG. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 2013;100: 615–9. Rubio C, Rodrigo L, Mir P, Mateu E, Peinado V, Milan M, et al. Use of array comparative genomic hybridization (array-CGH) for embryo assessment: clinical results. Fertil Steril 2013;99:1044–8. Munne S, Sandalinas M, Magli C, Gianaroli L, Cohen J, Warburton D. Increased rate of aneuploid embryos in young women with previous aneuploidy conceptions. Prenat Diagn 2004;24:638–43. Munne S, Chen S, Colls P, Garrisi J, Zheng X, Cekleniak N, et al. Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage-stage embryos. Reprod Biomed Online 2007;14:628–34. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med 2009;15:577–83. Kuliev A, Cieslak J, Verlinsky Y. Frequency and distribution of chromosome abnormalities in human oocytes. Cytogenet Genome Res 2005;111:193–8. Magli MC, Gianaroli L, Ferraretti AP, Lappi M, Ruberti A, Farfalli V. Embryo morphology and development are dependent on the chromosomal complement. Fertil Steril 2007;87:534–41. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5:24. Meseguer M, Rubio I, Cruz M, Basile N, Marcos J, Requena A. Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study. Fertil Steril 2012;98:1481–1489.e10. Basile N, Nogales MD, Bronet F, Florensa M, Riqueiros M, Rodrigo L, et al. Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis. Fertil Steril 2014;101:699–704. n C. Domínguez F, Gadea B, Esteban FJ, Horcajadas JA, Pellicer A, Simo Comparative protein-profile analysis of implanted versus non-implanted human blastocysts. Hum Reprod 2008;23:1993–2000.

VOL. 101 NO. 6 / JUNE 2014

142.

143.

144.

145.

146.

147.

148. 149.

150.

151.

152. 153.

154.

155.

156. 157. 158.

159.

160.

161.

Seli E, Vergouw CG, Morita H, Botros L, Roos P, Lambalk CB, et al. Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer. Fertil Steril 2010;94:535–42. Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2008;90:2183–9. Vergouw CG, Botros LL, Roos P, Lens JW, Schats R, Hompes PG, et al. Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection. Hum Reprod 2008;23:1499–504. n C. EmDominguez F, Gadea B, Mercader A, Esteban FJ, Pellicer A, Simo bryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system. Fertil Steril 2010;93:774–782.e1. Vergouw CG, Kieslinger DC, Kostelijk EH, Botros LL, Schats R, Hompes PG, et al. Day 3 embryo selection by metabolomic profiling of culture medium with near-infrared spectroscopy as an adjunct to morphology: a randomized controlled trial. Hum Reprod 2012;27:2304–11. €nsson T, Sanchez-Ribas I, Riqueros M, Vime P, Puchades-Carrasco L, Jo Pineda-Lucena A, et al. Differential metabolic profiling of non-pure trisomy 21 human preimplantation embryos. Fertil Steril 2012;98:1157– 1164.e1–2. Katz-Jaffe MG, McReynolds S. Embryology in the era of proteomics. Fertil Steril 2013;99:1073–7. McCallie B, Schoolcraft WB, Katz-Jaffe MG. Aberration of blastocyst microRNA expression is associated with human infertility. Fertil Steril 2010;93: 2374–82. Yang Y, Bai W, Zhang L, Yin G, Wang X, Wang J, et al. Determination of microRNAs in mouse preimplantation embryos by microarray. Dev Dyn 2008;237:2315–27. Mineno J, Okamoto S, Ando T, Sato M, Chono H, Izu H, et al. The expression profile of microRNAs in mouse embryos. Nucleic Acids Res 2006;34: 1765–71. Carleton M, Cleary MA, Linsley PS. MicroRNAs and cell cycle regulation. Cell Cycle 2007;6:2127–32. Tan MH, Au KF, Leong DE, Foygel K, Wong WH, Yao MW. An Oct4-Sall4Nanog network controls developmental progression in the preimplantation mouse embryo. Mol Syst Biol 2013;9:632. Bosch E, Labarta E, Crespo J, Simon C, Remohi J, Jenkins J, et al. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum Reprod 2010;25:2092–100. Xu B, Li Z, Zhang H, Jin L, Li Y, Ai J, et al. Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,000 cycles. Fertil Steril 2012;97: 1321–1327.e1–4. Rackow BW, Kliman HJ, Taylor HS. GnRH antagonists may affect endometrial receptivity. Fertil Steril 2008;89:1234–9. Bourgain C, Devroey P. The endometrium in stimulated cycles for IVF. Hum Reprod Update 2003;9:515–22. Kolibianakis EM, Bourgain C, Platteau P, Albano C, Van Steirteghem AC, Devroey P. Abnormal endometrial development occurs during the luteal phase of nonsupplemented donor cycles treated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil Steril 2003;80:464–6. Evans J, Hannan NJ, Hincks C, Rombauts LJ, Salamonsen LA. Defective soil for a fertile seed? Altered endometrial development is detrimental to pregnancy success. PLoS One 2012;7:e53098. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders. Fertil Steril 2011; 96:344–8. Fatemi HM, Kyrou D, Bourgain C, Van den Abbeel E, Griesinger G, Devroey P. Cryopreserved-thawed human embryo transfer: spontaneous

1541

VIEWS AND REVIEWS

162.

163.

164.

165.

166.

167. 168.

169. 170. 171.

172. 173.

174.

175.

176.

177.

178.

179. 180.

181.

182.

183.

1542

natural cycle is superior to human chorionic gonadotropin-induced natural cycle. Fertil Steril 2010;94:2054–8. Xiao Z, Zhou X, Xu W, Yang J, Xie Q. Natural cycle is superior to hormone replacement therapy cycle for vitrificated-preserved frozen-thawed embryo transfer. Syst Biol Reprod Med 2012;58:107–12. Macklon NS, Stouffer RL, Giudice LC, Fauser BC. The science behind 25 years of ovarian stimulation for in vitro fertilization. Endocr Rev 2006;27: 170–207. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod 2007;22:1506–12. Ferraretti AP, Goossens V, de Mouzon J, Bhattacharya S, Castilla JA, Korsak V, et al. Assisted reproductive technology in Europe, 2008: results generated from European registers by ESHRE. Hum Reprod 2012;27: 2571–84. Herrler A, Von Rango U, Beier HM. Embryo-maternal signalling: how the embryo starts talking to its mother to accomplish implantation. Reprod Biomed 2003;6:244–56. Pierson RA. Imaging the endometrium: are there predictors of uterine receptivity? J Obstet Gynaecol Can 2003;25:360–8. Sher G, Herbert C, Maassarani G, Jacobs MH. Assessment of the late proliferative phase endometrium by ultrasonography in patients undergoing in-vitro fertilization. Hum Reprod 1991;6:232–7. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3–25. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122:262–3. Balasch J, Vanrell JA, Creus M, Marquez M, Gonzalez-Merlo J. The endometrial biopsy for diagnosis of luteal phase deficiency. Fertil Steril 1985; 44:699–701. Balasch J, Fabregues F, Creus M, Vanrell JA. The usefulness of endometrial biopsy for luteal phase evaluation in infertility. Hum Reprod 1992;7:973–7. Scott RT, Snyder RR, Strickland DM, Tyburski CC, Bagnall JA, Reed KR, et al. The effect of interobserver variation in dating endometrial histology on the diagnosis of luteal phase defects. Fertil Steril 1988;50:888–92. Scott RT, Snyder RR, Bagnall JW, Reed KD, Adair CF, Hensley SD. Evaluation of the impact of intraobserver variability on endometrial dating and the diagnosis of luteal phase defects. Fertil Steril 1993;60:652–7. Gibson M, Badger GJ, Byrn F, Lee KR, Korson R, Trainer TD. Error in histologic dating of secretory endometrium: variance component analysis. Fertil Steril 1991;56:242–7. Coutifaris C, Myers ER, Guzick DS, Diamond MP, Carson SA, Legro RS, et al. Histological dating of timed endometrial biopsy tissue is not related to fertility status. Fertil Steril 2004;82:1264–72. Murray MJ, Meyer WR, Zaino RJ, Lessey BA, Novotny DB, Ireland K, et al. A critical analysis of the accuracy, reproducibility, and clinical utility of histologic endometrial dating in fertile women. Fertil Steril 2004;81:1333–43. Devroey P, Bourgain C, Macklon NS, Fauser BC. Reproductive biology and IVF: ovarian stimulation and endometrial receptivity. Trends Endocrinol Metab 2004;15:84–90. Revel A, Achache H, Stevens J, Smith Y, Reich R. MicroRNAs are associated with human embryo implantation defects. Hum Reprod 2011;26:2830–40. Zapantis G, Szmyga MJ, Rybak EA, Meier UT. Premature formation of nucleolar channel systems indicates advanced endometrial maturation following controlled ovarian hyperstimulation. Hum Reprod 2013;28: 3292–300. Van Vaerenbergh I, Van Lommel L, Ghislain V, In'tVeld P, Schuit F, Fatemi HM, et al. GnRH antagonist/rec-FSH stimulated cycles, advanced endometrial maturation on the day of oocyte retrieval correlates with altered gene expression. Hum Reprod 2009;24:1085–91. Li R, Qiao J, Wang L, Li L, Zhen X, Liu P, et al. MicroRNA array and microarray evaluation of endometrial receptivity in patients with high serum progesterone levels on the day of hCG administration. Reprod Biol Endocrinol 2011;9:29. Burney RO, Hamilton AE, Aghajanova L, Vo KC, Nezhat CN, Lessey BA, et al. MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis. Mol Hum Reprod 2009;10:625–31.

184.

185. 186. 187.

188. 189.

190. 191. 192.

193. 194. 195. 196.

197.

198.

199.

200.

201. 202.

203.

204.

205.

206.

207. 208. 209. 210.

Ohlsson Teague EM, Van der Hoek KH, Van der Hoek MB, Perry N, Wagaarachchi P, Robertson SA, et al. MicroRNA-regulated pathways associated with endometriosis. Mol Endocrinol 2009;23:265–75. Marion LL, Meeks GR. Ectopic pregnancy: history, incidence, epidemiology, and risk factors. Clin Obstet Gynecol 2012;55:376–86. Barnhart KT. Ectopic pregnancy. N Engl J Med 2009;361:379–87. Bouyer J, Coste J, Fernandez H, Pouly JL, Job-Spira N. Sites of ectopic pregnancy: a 10 year population-based study of 1800 cases. Hum Reprod 2002; 17:3224–30. Murray H, Baakdah H, Bardell T, Tulandi T. Diagnosis and treatment of ectopic pregnancy. CMAJ 2005;173:905–12. Bottomley C, Van Belle V, Mukri F, Kirk E, Van Huffel S, Timmerman D, et al. The optimal timing of an ultrasound scan to assess the location and viability of an early pregnancy. Hum Reprod 2009;24:1811–7. Farquhar CM. Ectopic pregnancy. Lancet 2005;366:583–91. Leke RJ, Goyaux N, Matsuda T, Thonneau PF. Ectopic pregnancy in Africa: a population-based study. Obstet Gynecol 2004;103:692–7. Hoover KW, Tao G, Kent CK. Trends in the diagnosis and treatment of ectopic pregnancy in the United States. Obstet Gynecol 2010;115: 495–502. Walker JJ. Ectopic pregnancy. Clin Obstet Gynecol 2007;50:89–99. Varma R, Gupta J. Tubal ectopic pregnancy. Clin Evid (Online) 2009;2009. Shaw JL, Horne AW. The paracrinology of tubal ectopic pregnancy. Mol Cell Endocrinol 2012;358:216–22. Shaw JL, Dey SK, Critchley HO, Horne AW. Current knowledge of the aetiology of human tubal ectopic pregnancy. Hum Reprod Update 2010;16: 432–44. Kiran G, Kiran H, Ertopcu K, Kilinc M, Ekerbicer HC, Vardar MA. Tuba uterina leukemia inhibitory factor concentration does not increase in tubal pregnancy: a preliminary study. Fertil Steril 2005;83:484–6. Li P, Zhu WJ, Ma ZL, Wang G, Peng H, Chen Y, et al. Enhanced beta-catenin expression and inflammation are associated with human ectopic tubal pregnancy. Hum Reprod 2013;28:2363–71. Al-Azemi M, Refaat B, Amer S, Ola B, Chapman N, Ledger W. The expression of inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in ectopic pregnancy. Fertil Steril 2010;94:833–40. Shao R, Nutu M, Karlsson-Lindahl L, Benrick A, Weijdeg ard B, Lager S, et al. Downregulation of cilia-localized Il-6R a by 17b-estradiol in mouse and human fallopian tubes. Am J Physiol Cell Physiol 2009;297:C140–51. Jabbour HN, Sales KJ, Catalano RD, Norman JE. Inflammatory pathways in female reproductive health and disease. Reproduction 2009;138:903–19. Gebeh AK, Willets JM, Marczylo EL, Taylor AH, Konje JC. Ectopic pregnancy is associated with high anandamide levels and aberrant expression of FAAH and CB1 in fallopian tubes. J Clin Endocrinol Metab 2012;97: 2827–35. Paltieli Y, Eibschitz I, Ziskind G, Ohel G, Silbermann M, Weichselbaum A. High progesterone levels and ciliary dysfunction—a possible cause of ectopic pregnancy. J Assist Reprod Genet 2000;17:103–6. Huang C, Zhang M, Meng C, Shi W, Sun L, Zhang J. Expressions of candidate molecules in the human fallopian tube and chorionic villi of tubal pregnancy exposed to levonorgestrel emergency contraception. Reprod Biol Endocrinol 2013;11:46. Bjartling C, Osser S, Persson K. Deoxyribonucleic acid of Chlamydia trachomatis in fresh tissue from the fallopian tubes of patients with ectopic pregnancy. Eur J Obstet Gynecol Reprod Biol 2007;134:95–100. Waylen AL, Metwally M, Jones GL, Wilkinson AJ, Ledger WL. Effects of cigarette smoking upon clinical outcomes of assisted reproduction: a meta-analysis. Hum Reprod Update 2009;15:31–44. Talbot P, Riveles K. Smoking and reproduction: the oviduct as a target of cigarette smoke. Reprod Biol Endocrinol 2005;3:52. Pisarska MD, Carson SA, Buster JE. Ectopic pregnancy. Lancet 1998;351: 1115–20. Tay JI, Moore J, Walker JJ. Ectopic pregnancy. BMJ 2000;320:916–9. €m M, Stener-Victorin E, et al. Shao R, Zou S, Wang X, Feng Y, Br€annstro Revealing the hidden mechanisms of smoke-induced fallopian tubal implantation. Biol Reprod 2012;86:131.

VOL. 101 NO. 6 / JUNE 2014

Fertility and Sterility® 211.

212.

213. 214.

215.

216.

217.

218.

219.

220.

221.

222.

223.

224.

225.

226.

227. 228. 229.

230.

231.

232.

233.

Shao R. Understanding the mechanisms of human tubal ectopic pregnancies: new evidence from knockout mouse models. Hum Reprod 2010;25: 584–7. Smith LP, Oskowitz SP, Dodge LE, Hacker MR. Risk of ectopic pregnancy following day-5 embryo transfer compared with day-3 transfer. Reprod Biomed Online 2013;27:407–13. Oron G, Tulandi T. A pragmatic and evidence-based management of ectopic pregnancy. J Minim Invasive Gynecol 2013;20:446–54. Papathanasiou A, Djahanbakhch O, Saridogan E, Lyons RA. The effect of interleukin-6 on ciliary beat frequency in the human fallopian tube. Fertil Steril 2008;90:391–4. Boots CE, Gustofson RL, Feinberg EC. Does methotrexate administration for ectopic pregnancy after in vitro fertilization impact ovarian reserve or ovarian responsiveness? Fertil Steril 2013;100:1590–3. Beall S, De Cherney AH. Management of tubal ectopic pregnancy: methotrexate and salpingostomy are preferred to preserve fertility. Fertil Steril 2012;98:1118–20. de Bennetot M, Rabischong B, Aublet-Cuvelier B, Belard F, Fernandez H, Bouyer J, et al. Fertility after tubal ectopic pregnancy: results of a population-based study. Fertil Steril 2012;98:1271–6. Uyar I, Yucel OU, Gezer C, Gulhan I, Karis B, Hanhan HM, et al. Effect of single-dose methotrexate on ovarian reserve in women with ectopic pregnancy. Fertil Steril 2013;100:1310–3. Fernandez H, Capmas P, Lucot JP, Resch B, Panel P, Bouyer J. GROG. Fertility after ectopic pregnancy: the DEMETER randomized trial. Hum Reprod 2013;28:1247–53. Barnhart K, van Mello NM, Bourne T, Kirk E, Van Calster B, Bottomley C, et al. Pregnancy of unknown location: a consensus statement of nomenclature, definitions, and outcome. Fertil Steril 2011;95:857–66. Banerjee S, Aslam N, Woelfer B, Lawrence A, Elson J, Jurkovic D. Expectant management of early pregnancies of unknown location: a prospective evaluation of methods to predict spontaneous resolution of pregnancy. BJOG 2001;108:158–63. Condous G, Okaro E, Khalid A, Lu C, Van Huffel S, Timmerman D, et al. A prospective evaluation of a single-visit strategy to manage pregnancies of unknown location. Hum Reprod 2005;20:1398–403. Condous G, Kirk E, Van Calster B, Van Huffel S, Timmerman D, Bourne T. Failing pregnancies of unknown location: a prospective evaluation of the human chorionic gonadotrophin ratio. BJOG 2006;113:521–7. Reid S, Condous G. Is there a need to definitively diagnose the location of a pregnancy of unknown location? The case for ‘‘no.’’ Fertil Steril 2012;98: 1085–90. Condous G, Timmerman D, Goldstein S, Valentin L, Jurkovic D, Bourne T. Pregnancies of unknown location: consensus statement. Ultrasound Obstet Gynecol 2006;28:121–2. Condous G, Okaro E, Khalid A, Timmerman D, Lu C, Zhou Y, et al. The use of a new logistic regression model for predicting the outcome of pregnancies of unknown location. Hum Reprod 2004;19:1900–10. Sagili H, Mohamed K. Pregnancy of unknown location: an evidence-based approach to management. Obstetrician Gynaecol 2008;10:224–30. Gracia CR, Barnhart KT. Diagnosing ectopic pregnancy: decision analysis comparing six strategies. Obstet Gynecol 2001;97:464–70. Mertz HL, Yalcinkaya TM. Early diagnosis of ectopic pregnancy: does use of a strict algorithm decrease the incidence of tubal rupture? J Reprod Med 2001;46:29–33. Ankum WM, Van der Veen F, Hamerlynck JV, Lammes FB. Suspected ectopic pregnancy. What to do when human chorionic gonadotropin levels are below the discriminatory zone. J Reprod Med 1995;40:525–8. Van Calster B, Abdallah Y, Guha S, Kirk E, Van Hoorde K, Condous G, et al. Rationalizing the management of pregnancies of unknown location: temporal and external validation of a risk prediction model on 1962 pregnancies. Hum Reprod 2013;28:609–16. Shaunik A, Kulp J, Appleby DH, Sammel MD, Barnhart KT. Utility of dilation and curettage in the diagnosis of pregnancy of unknown location. Am J Obstet Gynecol 2011;204:130.e1–6. Stovall TG, Ling FW, Andersen RN, Buster JE. Improved sensitivity and specificity of a single measurement of serum progesterone over serial quantita-

VOL. 101 NO. 6 / JUNE 2014

234.

235.

236.

237.

238.

239.

240.

241.

242.

243.

244.

245. 246.

247.

248. 249.

250.

251.

252. 253.

254.

255.

tive beta-human chorionic gonadotrophin in screening for ectopic pregnancy. Hum Reprod 1992;7:723–5. El Bishry G, Ganta S. The role of single serum progesterone measurement in conjunction with beta hCG in the management of suspected ectopic pregnancy. J Obstet Gynaecol 2008;28:413–7. Mol BW, Lijmer JG, Ankum WM, van der Veen F, Bossuyt PM. The accuracy of single serum progesterone measurement in the diagnosis of ectopic pregnancy: a meta-analysis. Hum Reprod 1998;13:3220–7. Day A, Sawyer E, Mavrelos D, Tailor A, Helmy S, Jurkovic D. Use of serum progesterone measurements to reduce need for follow-up in women with pregnancies of unknown location. Ultrasound Obstet Gynecol 2009;33: 704–10. American College of Obstetricians Gynecologists. ACOG Practice Bulletin No. 94: medical management of ectopic pregnancy. Obstet Gynecol 2008;111:1479–85. Barnhart K, Sammel MD, Chung K, Zhou L, Hummel AC, Guo W. Decline of serum human chorionic gonadotropin and spontaneous complete abortion: defining the normal curve. Obstet Gynecol 2004;104:975–81. Seeber BE, Sammel MD, Guo W, Zhou L, Hummel A, Barnhart KT. Application of redefined human chorionic gonadotropin curves for the diagnosis of women at risk for ectopic pregnancy. Fertil Steril 2006;86: 454–9. Barnhart KT, Sammel MD, Rinaudo PF, Zhou L, Hummel AC, Guo W. Symptomatic patients with an early viable intrauterine pregnancy: hCG curves redefined. Obstet Gynecol 2004;104:50–5. Butts SF, Guo W, Cary MS, Chung K, Takacs P, Sammel MD, et al. Predicting the decline in human chorionic gonadotropin in a resolving pregnancy of unknown location. Obstet Gynecol 2013;122:337–43. Zhao Z, Zhao Q, Warrick J, Lockwood CM, Woodworth A, Moley KH, et al. Circulating microRNA miR-323-3p as a biomarker of ectopic pregnancy. Clin Chem 2012;58:896–905. Hidlebaugh D, O'Mara P. Clinical and financial analyses of ectopic pregnancy management at a large health plan. J Am Assoc Gynecol Laparosc 1997;4:207–13. Cartwright J, Duncan WC, Critchley HOD, Horne AW. Serum biomarkers of tubal ectopic pregnancy: current candidates and future possibilities. Reproduction 2009;138:9–22. Reid S, Casikar I, Barnhart K, Condous G. Serum biomarkers for ectopic pregnancy diagnosis. Expert Opin Med Diagn 2012;6:153–65. Skubisz MM, Brown JK, Tong S, Kaitu'u-Lino T, Horne AW. Maternal serum macrophage inhibitory cytokine-1 as a biomarker for ectopic pregnancy in women with a pregnancy of unknown location. PLoS One 2013;8:e66339. Rausch ME, Sammel MD, Takacs P, Chung K, Shaunik A, Barnhart KT. Development of a multiple marker test for ectopic pregnancy. Obstet Gynecol 2011;117:573–82. Rausch ME, Barnhart KT. Serum biomarkers for detecting ectopic pregnancy. Clin Obstet Gynecol 2012;55:418–23. Practice Committee of American Society for Reproductive Medicine. Medical treatment of ectopic pregnancy: a committee opinion. Fertil Steril 2013;100:638–44. Hajenius PJ, Mol F, Mol BW, Bossuyt PM, Ankum WM, van der Veen F. Interventions for tubal ectopic pregnancy. Cochrane Database Syst Rev 2007:CD000324. Miura K, Miura S, Yamasaki K, Higashijima A, Kinoshita A, Yoshiura K, et al. Identification of pregnancy-associated microRNAs in maternal plasma. Clin Chem 2010;56:1767–71. Wittmann J, J€ack HM. Serum microRNAs as powerful cancer biomarkers. Biochim Biophys Acta 2010;1806:200–7. Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, et al. Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes. Biol Reprod 2009;81:717–29. Dimitriadis E, White CA, Jones RL, Salamonsen LA. Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update 2005;11:613–30. Machelon V, Emilie D, Lefevre A, Nome F, Durand-Gasselin I, Testart J. Interleukin-6 biosynthesis in human preovulatory follicles: some of its potential roles at ovulation. J Clin Endocrinol Metab 1994;79:633–42.

1543

VIEWS AND REVIEWS 256.

257.

258.

1544

Yousefian E, Novin MG, Fathabadi FF, Farahani RM, Kachouei EY. The expression of IL-6Ra and Gp130 in fallopian tubes bearing an ectopic pregnancy. Anat Cell Biol 2013;46:177–82. Balasubramaniam ES, Van Noorden S, El-Bahrawy M. The expression of interleukin (IL)-6, IL-8, and their receptors in fallopian tubes with ectopic tubal gestation. Fertil Steril 2012;98:898–904. Ouyang Y, Mouillet JF, Coyne CB, Sadovsky Y. Review: placenta-specific microRNAs in exosomes—good things come in nano-packages. Placenta 2014;35(Suppl):S69–73.

259.

260.

261.

Donker RB, Mouillet JF, Chu T, Hubel CA, Stolz DB, Morelli AE, et al. The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes. Mol Hum Reprod 2012;18:417–24. Takacs P, Jaramillo S, Datar R, Williams A, Olczyk J, Barnhart K. Placental mRNA in maternal plasma as a predictor of ectopic pregnancy. Int J Gynaecol Obstet 2012;117:131–3. Xu T, Li L, Huang C, Li X, Peng Y, Li J. MicroRNA-323-3p with clinical potential in rheumatoid arthritis, Alzheimer's disease and ectopic pregnancy. Expert Opin Ther Targets 2014;18:153–8.

VOL. 101 NO. 6 / JUNE 2014