hMG addition affects the change in progesterone level during IVF stimulation and LBR: a retrospective cohort study.

BACKGROUND: Premature progesterone (P) rise during IVF stimulation reduces endometrial receptivity and is associated with lower pregnancy rates following embryo transfer (ET), which can influence provider recommendation for fresh or frozen ET. This study aimed to determine whether change in P level between in IVF baseline and trigger (𝚫P) is predictive of pregnancy outcome following fresh ET, and whether the ratio of gonadotropins influences P rise and, as a result, clinical pregnancy outcomes: clinical pregnancy rate (CPR) and live birth rates (LBR). METHODS: Retrospective cohort study at a single fertility center at an academic institution. The peak P level and 𝚫P were modeled in relation to prediction of CPR and LBR, and the ratios of hMG:rFSH were also modeled in relation to prediction of peak P level on day of trigger, 𝚫P, and CPR/LBR in a total of 291 patients undergoing fresh embryo transfer after controlled ovarian hyperstimulation-IVF (COH-IVF). RESULTS: 𝚫P correlates with CPR, with the most predictive range for success as 𝚫P 0.7-0.85 ng/mL (p = 0.005, 95% CI 0.635, 3.636; predicting CPR of 88.9%). The optimal range for peak P in regard to pregnancy outcome was 0.15-1.349 ng/mL (p = 0.01; 95% CI for coefficient in model 0.48-3.570). A multivariable logistic model for prediction of CPR and LBR using either peak or 𝚫P supported a stronger association between 𝚫P and CPR/LBR as compared to peak P. Furthermore, an hMG:rFSH ratio of > 0.6 was predictive of lowest peak P (p = 0.010, 95% CI 0.035, 0.256) and smallest 𝚫P (p = 0.012, 95% CI 0.030, 0.243) during COH-IVF cycles. Highest CPRs were observed within hMG:rFSH ratios of 0.3-0.4 [75.6% vs. 62.5% within and outside of the range, respectively, (p = 0.023, 95% CI 0.119, 1.618)]. Highest LBRs were seen within the range of 0.3-0.6 hMG:rFSH, [LBR of 55.4% vs. 41.4% (p = 0.010, 95% CI 0.176, 1.311)]. CONCLUSIONS: Our data supports use of 𝚫P to best predict pregnancy rates and therefore can improve clinical decision making as to when fresh ET is most appropriate. Furthermore, we found optimal gonadotropin ratios can be considered to minimize P rise and to optimize CPR/LBR, emphasizing the importance of luteinizing hormone (LH) activity in COH-IVF cycles.

The imprinted H19 lncRNA antagonizes let-7 microRNAs.

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.

Diagnosis and Management of Infertility: A Review.

IMPORTANCE: In the US, approximately 12.7% of reproductive age women seek treatment for infertility each year. This review summarizes current evidence regarding diagnosis and treatment of infertility. OBSERVATIONS: Infertility is defined as the failure to achieve pregnancy after 12 months of regular unprotected sexual intercourse. Approximately 85% of infertile couples have an identifiable cause. The most common causes of infertility are ovulatory dysfunction, male factor infertility, and tubal disease. The remaining 15% of infertile couples have "unexplained infertility." Lifestyle and environmental factors, such as smoking and obesity, can adversely affect fertility. Ovulatory disorders account for approximately 25% of infertility diagnoses; 70% of women with anovulation have polycystic ovary syndrome. Infertility can also be a marker of an underlying chronic disease associated with infertility. Clomiphene citrate, aromatase inhibitors such as letrozole, and gonadotropins are used to induce ovulation or for ovarian stimulation during in vitro fertilization (IVF) cycles. Adverse effects of gonadotropins include multiple pregnancy (up to 36% of cycles, depending on specific therapy) and ovarian hyperstimulation syndrome (1%-5% of cycles), consisting of ascites, electrolyte imbalance, and hypercoagulability. For individuals presenting with anovulation, ovulation induction with timed intercourse is often the appropriate initial treatment choice. For couples with unexplained infertility, endometriosis, or mild male factor infertility, an initial 3 to 4 cycles of ovarian stimulation may be pursued; IVF should be considered if these approaches do not result in pregnancy. Because female fecundity declines with age, this factor should guide decision-making. Immediate IVF may be considered as a first-line treatment strategy in women older than 38 to 40 years. IVF is also indicated in cases of severe male factor infertility or untreated bilateral tubal factor. CONCLUSIONS AND RELEVANCE: Approximately 1 in 8 women aged 15 to 49 years receive infertility services. Although success rates vary by age and diagnosis, accurate diagnosis and effective therapy along with shared decision-making can facilitate achievement of fertility goals in many couples treated for infertility.

A novel, noncoding-RNA-mediated, post-transcriptional mechanism of anti-Mullerian hormone regulation by the H19/let-7 axis.

In reproductive age women, the pool of primordial follicles is continuously depleted through the process of cyclic recruitment. Anti-Mullerian hormone (AMH) both inhibits the initial recruitment of primordial follicles into the growing pool and modulates the sensitivity of growing follicles to follicle stimulating hormone. Thus, AMH may be an important modulator of female infertility and ovarian reserve; however, the mechanisms regulating AMH remain unclear.To evaluate AMH levels in the absence of H19 lncRNA, H19 knockout (H19KO) mice were evaluated for analysis of ovarian AMH gene expression, protein production, and reproductive function, including assessment of follicle numbers and litter size analysis. To further investigate regulation of AMH by the H19/let-7 axis, let-7 binding sites on AMH were predicted, and in vitro studies of the effect of H19 knockdown/overexpression with let-7 rescue were performed. Lastly, response to superovulation was assessed via oocyte counts and estradiol measurements.The H19KO mouse demonstrates subfertility and accelerated follicular recruitment with increased spontaneous development of secondary, preantral, and antral follicles. Ovaries of H19KO mice have decreased AMH mRNA and protein, and AMH mRNA has a functional let-7 binding site, suggesting a plausible ncRNA-mediated mechanism for AMH regulation by H19/let-7. Lastly, in the absence of H19, superovulation results in higher estradiol and more oocytes, suggesting that H19 functions to limit the number of follicles that mature, produce estradiol, and ovulate. Thus, AMH's inhibitory actions are regulated at least in part by H19, likely via let-7, marking this ncRNA pair as important regulators of the establishment and maintenance of the follicular pool.

Aberrant endocrinology and ovarian response to clomiphene citrate during the course of an undiagnosed early intrauterine pregnancy: a case report.

BACKGROUND: This is an unusual case of embryonic exposure to clomiphene citrate (CC) in the setting of an undiagnosed early pregnancy with successful follicular response to CC and progression of pregnancy despite markedly attenuated serum progesterone and estradiol levels and a thin endometrium. A review of literature on the potential of CC for teratogenicity is presented. CASE: A 36-year-old woman underwent 2 ovulation inductions (OIs) with CC. Successful pregnancy followed the second OI cycle. Fetal measurements on transvaginal ultrasound identified the pregnancy to be chronologically advanced and consistent with the first OI treatment cycle. The follicular response to CC during the second OI cycle in the setting of ongoing early pregnancy, and pregnancy progression despite markedly attenuated endometrium, low serum levels of serum progesterone, and estradiol and embryonic exposure to CC, are notable. CONCLUSION: The possibility of inadvertent embryonic exposure to fertility drugs in the event of undiagnosed early pregnancy must be considered in infertile patients pursuing repeat treatment cycles. Serum beta-hCG testing should be considered before repeat treatments.

The Aging Ovary and the Tales Learned Since Fetal Development.

BACKGROUND: While the term "aging" implies a process typically associated with later life, the consequences of ovarian aging are evident by the time a woman reaches her forties, and sometimes earlier. This is due to a gradual decline in the quantity and quality of oocytes which occurs over a woman's reproductive lifespan. Indeed, the reproductive potential of the ovary is established even before birth, as the proper formation and assembly of the ovarian germ cell population during fetal life determines the lifetime endowment of oocytes and follicles. In the ovary, sophisticated molecular processes have been identified that regulate the timing of ovarian aging and these are critical to ensuring follicular maintenance. SUMMARY: The mechanisms thought to contribute to overall aging have been summarized under the term the "hallmarks of aging" and include such processes as DNA damage, mitochondrial dysfunction, telomere attrition, genomic instability, and stem cell exhaustion, among others. Similarly, in the ovary, molecular processes have been identified that regulate the timing of ovarian aging and these are critical to ensuring follicular maintenance. In this review, we outline critical processes involved in ovarian aging, highlight major achievements for treatment of ovarian aging, and discuss ongoing questions and areas of debate. KEY MESSAGES: Ovarian aging is recognized as what may be a complex process in which age, genetics, environment, and many other factors contribute to the size and depletion of the follicle pool. The putative hallmarks of reproductive aging outlined herein include a diversity of plausible processes contributing to the depletion of the ovarian reserve. More research is needed to clarify if and to what extent these putative regulators do in fact govern follicle and oocyte behavior, and how these signals might be integrated in order to control the overall pattern of ovarian aging.

Reproductive and Fertility Knowledge and Attitudes Among Transgender and Gender-Expansive Youth: A Replication and Extension.

Purpose: This study sought to replicate and expand a previous pilot investigation of reproductive knowledge, attitudes toward fertility and parenthood, and sources of information on these topics among transgender and gender-expansive (TGE) youth. Methods: The Yale Pediatric Gender Program (YPGP) Reproductive Knowledge and Experiences Survey (YPGP-RKES) was administered to 70 TGE adolescents receiving care at an interdisciplinary clinic providing gender-affirming health care at an academic medical center. Data gathered included sources of information on reproduction and fertility, concerns about future parenthood and reproduction, and interest in different types of parenthood. Results: Over a third (39.1%) of participants reported it was important to them to have a child one day, while only a small proportion (23.2%) reported an interest in biological parenthood. A plurality of participants (37.3%) reported at least one concern about future fertility. The number of reproductive concerns did not differ by age or treatment (puberty blockers or gender-affirming hormones vs. no treatment) status. With respect to needs for more information and sources of information, most (56.5%) participants received information about fertility issues before this study, with the most cited source of information being online research. Conclusions: The current study replicated and extended previous findings on the reproductive attitudes and knowledge of TGE adolescents. Understanding the informational needs and priorities of adolescent TGE patients presenting for medical treatment will allow providers to give more robust patient education. This will, in turn, facilitate patients' ability to provide fully informed consent for treatment that aligns with their fertility and reproductive priorities and goals.

Expression and T cell regulatory action of the PD-1 immune checkpoint in the ovary and fallopian tube.

PROBLEM: Immune cell trafficking and surveillance within the ovary and fallopian tube are thought to impact fertility and also tumorigenesis in those organs. However, little is known of how native cells of the ovary and fallopian tube interact with resident immune cells. Interaction of the Programmed Cell Death Protein-1 (PD-1/PDCD-1/CD279) checkpoint with PD-L1 is associated with downregulated immune response. We have begun to address the question of whether PD-1 ligand or its receptors (PD-L1/-L2) can regulate immune cell function in these tissues of the female reproductive tract. METHOD OF STUDY: PD-1 and ligand protein expression was evaluated in human ovary and fallopian tube specimens, the latter of which included stages of tubal cell transformation and early tumorigenesis. Ovarian expression analysis included the determination of the proteins in human follicular fluid (HFF) specimens collected during in vitro fertilization procedures. Finally, checkpoint bioactivity of HFF was determined by treatment of separately-isolated human T cells and the measurement of interferon gamma (IFNγ). RESULTS: We show that membrane bound and soluble variants of PD-1 and ligands are expressed by permanent constituent cell types of the human ovary and fallopian tube, including granulosa cells and oocytes. PD-1 and soluble ligands were present in HFF at bioactive levels that control T cell PD-1 activation and IFNγ production; full-length checkpoint proteins were found to be highly enriched in HFF exosome fractions. CONCLUSION: The detection of PD-1 checkpoint proteins in the human ovary and fallopian tube suggests that the pathway is involved in immunomodulation during folliculogenesis, the window of ovulation, and subsequent egg and embryo immune-privilege. Immunomodulatory action of receptor and ligands in HFF exosomes is suggestive of an acute checkpoint role during ovulation. This is the first study in the role of PD-1 checkpoint proteins in human tubo-ovarian specimens and the first examination of its potential regulatory action in the contexts of normal and assisted reproduction.

Aberrant H19 Expression Disrupts Ovarian Cyp17 and Testosterone Production and Is Associated with Polycystic Ovary Syndrome in Women.

As one of the most common endocrine disorders affecting women, polycystic ovary syndrome (PCOS) is associated with serious conditions including anovulation, endometrial cancer, infertility, hyperandrogenemia, and an increased risk for obesity and metabolic derangements. One contributing etiology to the pathophysiology of hyperandrogenemia associated with PCOS is an intrinsic alteration in ovarian steroidogenesis, leading to enhanced synthesis of androgens including testosterone. Studies have suggested that the increased testosterone synthesis seen in PCOS is driven in part by increased activity of CYP17A1, the rate-limiting enzyme for the formation of androgens in the gonads and adrenal cortex, which represents a critical factor driving enhanced testosterone secretion in PCOS. In this work, we evaluated the hypothesis that dysregulation of the noncoding RNA H19 results in aberrant CYP17 and testosterone production. To achieve this, we measured Cyp17 in ovarian tissues of H19 knockout mice, and quantified serum testosterone levels, in comparison with wild-type controls. We also evaluated circulating and ovarian H19 expression and correlated results with the presence or absence of PCOS in a group of women undergoing evaluation and treatment for infertility. We found that the loss of H19 in a mouse model results in decreased ovarian Cyp17, along with decreased serum testosterone in female mice. Moreover, utilizing serum samples and cumulus cells from women with PCOS, we showed that circulating and ovarian levels of H19 are increased in women with PCOS compared to controls. Findings from our multimodal experimental strategy, involving both a mouse model of dysregulated H19 expression and clinical serum and ovarian cellular samples from women with PCOS, suggest that the loss of H19 may disrupt androgen production via a Cyp17-mediated mechanism. Conversely, excess H19 may play a role in the pathogenesis of PCOS-associated hyperandrogenemia.

Cardiovascular disease and ovarian function.

PURPOSE OF REVIEW: Coronary heart disease (CHD) is the leading cause of death in the aging female population in the developed world. Ovarian endocrinology plays an important role in modulating a woman's CHD risk. We herein present an overview of our current understanding of CHD risk profile in the context of ovarian physiology and senescence. RECENT FINDINGS: Endogenous ovarian estrogen has long been recognized to offer cardiac benefit and vascular protection against atherosclerosis. Existing data, however, do not allow for an extrapolation of the recognized cardioprotective implications of the reproductive-age endogenous estrogenic milieu to the use of exogenous estrogen in postmenopausal women. Ongoing efforts are targeting the concept that when reintroduced proximate to onset of ovarian senescence, exogenous estrogen may retard the process of atherogenesis. Until this hypothesis is substantiated, cardioprotection must not be an indication for initiating hormone therapy in menopausal women. SUMMARY: Ovarian hormones modulate the processes of atherosclerosis and the mechanisms underlying CHD. The female reproductive hormones offer a cardioprotective milieu that is rapidly attenuated with the cessation of ovarian function (be it following natural menopause or after medical or surgical ovarian extirpation). The role of exogenous hormone therapy, and the nuances of timing and duration of exposure, are still being elucidated.

Updates on molecular and environmental determinants of luteal progesterone production.

Progesterone, a critical hormone in reproduction, is a key sex steroid in the establishment and maintenance of early pregnancy and serves as an intermediary for synthesis of other steroid hormones. Progesterone production from the corpus luteum is a tightly regulated process which is stimulated and maintained by multiple factors, both systemic and local. Multiple regulatory systems, including classic mediators of gonadotropin stimulation such as the cAMP/PKA pathway and TGFß-mediated signaling pathways, as well as local production of hormonal factors, exist to promote granulosa cell function and physiological fine-tuning of progesterone levels. In this manuscript, we provide an updated narrative review of the known mediators of human luteal progesterone and highlight new observations regarding this important process, focusing on studies published within the last five years. We will also review recent evidence suggesting that this complex system of progesterone production is sensitive to disruption by exogenous environmental chemicals that can mimic or interfere with the activities of endogenous hormones.

Emerging roles for noncoding RNAs in female sex steroids and reproductive disease.

The "central dogma" of molecular biology, that is, that DNA blueprints encode messenger RNAs which are destined for translation into protein, has been challenged in recent decades. In actuality, a significant portion of the genome encodes transcripts that are transcribed into functional RNA. These noncoding RNAs (ncRNAs), which are not transcribed into protein, play critical roles in a wide variety of biological processes. A growing body of evidence derived from mouse models and human data demonstrates that ncRNAs are dysregulated in various reproductive pathologies, and that their expression is essential for female gametogenesis and fertility. Yet in many instances it is unclear how dysregulation of ncRNA expression leads to a disease process. In this review, we highlight new observations regarding the roles of ncRNAs in the pathogenesis of disordered female steroid hormone production and disease, with an emphasis on long noncoding RNAs (lncRNAs) and microRNAs (miRNAs). We will focus our discussion in the context of three ovarian disorders which are characterized in part by altered steroid hormone biology - diminished ovarian reserve, premature ovarian insufficiency, and polycystic ovary syndrome. We will also discuss the limitations and challenges faced in studying noncoding RNAs and sex steroid hormone production. An enhanced understanding of the role of ncRNAs in sex hormone regulatory networks is essential in order to advance the development of potential diagnostic markers and therapeutic targets for diseases, including those in reproductive health. Our deepened understanding of ncRNAs has the potential to uncover new applications and therapies; however, in many cases, the next steps will involve distinguishing critical ncRNAs from those which are merely changing in response to a particular disease state, or which are altogether unrelated to disease pathophysiology.

Control of Murine Primordial Follicle Growth Activation by IκB/NFκB Signaling.

The transcription factor NFκB has been associated with the timing of menopause in a large human genome-wide association study. Furthermore, preclinical studies demonstrate that loss of Tumor necrosis factor alpha (Tnfα) or its receptor Tnfr2 slows primordial follicle growth activation (PFGA). Although Tnfα:receptor signaling stimulates NFκB and may mechanistically link these findings, very little is known about NFκB signaling in PFGA. Because signaling downstream of Tnfα/Tnfr2 ligand/receptor interaction has not been interrogated as relates to PFGA, we evaluated the expression of key NFκB signaling proteins in primordial and growing follicles, as well as during ovarian aging. We show that key members of the NFκB pathway, including subunits, activating kinases, and inhibitory proteins, are expressed in the murine ovary. Furthermore, the subunits p65 and p50, and the cytosolic inhibitory proteins IκBα and IκBß, are present in ovarian follicles, including at the primordial stage. Finally, we assessed PFGA in genetically modified mice (AKBI) previously demonstrated to be resistant to inflammatory stress-induced NFκB activation due to overexpression of the NFκB inhibitory protein IκBß. Consistent with the hypothesis that NFκB plays a key role in PFGA, AKBI mice exhibit slower PGFA than wild-type (WT) controls, and their ovaries contain nearly twice the number of primordial follicles as WT both at early and late reproductive ages. These data provide mechanistic insight on the control of PFGA and suggest that targeting NFκB at the level of IκB proteins may be a tractable route to slowing the rate of PFGA in women faced with early ovarian demise.

Absence of the long noncoding RNA H19 results in aberrant ovarian STAR and progesterone production.

The steroidogenic acute regulatory protein (STAR) governs the rate-limiting step in steroidogenesis, and its expression varies depending on the needs of the specific tissue. It is well known that tight control of steroid production is essential for multiple processes involved in reproduction. We recently showed that Star is regulated at the posttranscriptional level in vitro by H19 and let-7. Here we demonstrate that this novel regulatory mechanism is functional in vivo, regulated by cAMP, and that loss of H19 not only disrupts ovarian STAR but also results in altered progesterone production in an H19KO mouse model. This work further strengthens the possibility that noncoding-RNA-mediated regulation of STAR may play an important role in the regulation of steroid hormone production, and contributes further to our understanding of the many ways in which this important gene is regulated.

H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas.

Uterine leiomyomas or fibroids (UFs) are benign tumors characterized by hyperplastic smooth muscle cells and excessive deposition of extracellular matrix (ECM). Afflicting ~80% of women, and symptomatic in 25%, UFs bring tremendous suffering and are an economic burden worldwide; they cause severe pain and bleeding, and are the leading cause of hysterectomy. Yet, UFs are severely understudied with few effective treatment options available; those that are available frequently have significant side effects such as menopausal symptoms. Recently, integrated genome-scale studies have revealed mutations and fibroid subtype-specific expression changes in key driver genes, with MED12 and HMGA2 together contributing to nearly 90% of all UFs, but their regulation of expression is poorly characterized. Here we report that the expression of H19 long noncoding RNA (lncRNA) is aberrantly increased in UFs. Using cell culture and genome-wide transcriptome and methylation profiling analyses, we demonstrate that H19 promotes expression of MED12, HMGA2, and key ECM-remodeling genes via multiple mechanisms including a new class of epigenetic modification by TET3. Our results mark the first example of an evolutionarily conserved lncRNA in pathogenesis of UFs and regulation of TET expression. Given the link between a H19 single-nucleotide polymorphism (SNP) and increased risk and tumor size of UFs, and the existence of multiple fibroid subtypes driven by key pathway genes regulated by H19, we propose a unifying mechanism for pathogenesis of uterine fibroids mediated by H19 and identify a pathway for future exploration of novel target therapies for uterine leiomyomas.

The Steroidogenic Acute Regulatory Protein (StAR) Is Regulated by the H19/let-7 Axis.

The steroidogenic acute regulatory protein (StAR) governs the rate-limiting step in steroidogenesis, and its expression varies depending on the needs of the specific tissue. Tight control of steroid production is essential for multiple processes involved in reproduction, including follicular development, ovulation, and endometrial synchronization. Recently, there has been a growing interest in the role of noncoding RNAs in the regulation of reproduction. Here we demonstrate that StAR is a novel target of the microRNA let-7, which itself is regulated by the long noncoding RNA (lncRNA) H19. Using human and murine cell lines, we show that overexpression of H19 stimulates StAR expression by antagonizing let-7, which inhibits StAR at the post-transcriptional level. Our results uncover a novel mechanism underlying the regulation of StAR expression and represent the first example of lncRNA-mediated control of the rate-limiting step of steroidogenesis. This work thus adds to the body of literature describing the multiple roles in oncogenesis, cellular growth, glucose metabolism, and now regulation of steroidogenesis, of this complex lncRNA.

Poor ovarian response in women undergoing in vitro fertilization is associated with altered microRNA expression in cumulus cells.

OBJECTIVE: To analyze the association of micro-ribonucleic acid (miRNA) expression with the number of oocytes retrieved, in women undergoing in vitro fertilization (IVF). DESIGN: Experimental study. SETTING: Academic medical center. PATIENT(S): A total of 189 women undergoing IVF-intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Pooled cumulus cells were collected. MAIN OUTCOME MEASURE(S): Poor responders were identified as patients who produced fewer oocytes than the 25th percentile of their respective age group. MicroRNAs were extracted from cumulus cells, and an miRNA microarray was performed, comparing poor responders (n = 3) to non-poor responders (n = 3). Expression of miR-21-5p (active strand of miR-21) and miR-21-3p was tested in poor responders (n = 21) and non-poor responders (n = 29), using reverse transcription real-time polymerase chain reaction (qRT-PCR). Regulation of miR-21-5p and miR-21-3p, in human granulosa-like tumor (KGN) cells, by estradiol (E2), was tested in vitro. RESULT(S): MicroRNA microarray analysis showed up-regulation of 16 miRNAs and down-regulation of 88 miRNAs in poor responders. Notably, miR-21 was significantly up-regulated 5-fold in poor-responder samples. Analysis using qRT-PCR confirmed that miR-21-5p expression was significantly up-regulated in poor responders, whereas miR-21-3p expression was significantly lower, suggesting that elevated miR-21-5p expression in cumulus cells is not regulated at the pre-miR-21 level in poor responders. Both miR-21-5p and miR-21-3p were increased in KGN cells in response to higher doses of E2; their expression was not affected at lower E2 concentrations. CONCLUSION(S): We found that poor response to IVF is associated with altered miRNA expression in cumulus cells, specifically with elevated expression of miR-21-5p, and that this elevated expression is independent of lower serum E2 levels in poor responders.

HOXA10 Regulates Expression of Cytokeratin 15 in Endometrial Epithelial Cytoskeletal Remodeling.

OBJECTIVE: The mammalian cytoskeleton is composed in part from keratin filaments which form a complex, highly dynamic intracellular network. We investigate the expression of cytokeratin 15 (CK15) in human endometrium and its regulation by HOXA10 in the human endometrial cell lines. METHODS: Endometrial biopsies from throughout the menstrual cycle (N = 32) were evaluated for CK15 protein expression by immunohistochemistry using a mouse monoclonal antibody. The human endometrial epithelial cell line (Ishikawa) was transfected with pcDNA/HOXA10. Total RNA was isolated and quantitative real-time polymerase chain reaction was performed to determine expression levels of CK15. RESULTS: In the peri-implantation window (days 16 through 23) CK15 protein expression in glandular epithelium of human endometrium decreased to 50% of proliferative phase expression levels. Expression of CK15 messenger RNA decreased by 99% (P < .05) after pcDNA/HOXA10 transfection of Ishikawa cells. The CK15 expression corresponded to the time of maximal secretory epithelial remodeling. CONCLUSION: Gene expression of CK15 is decreased in a HOXA10-dependent fashion in human endometrial epithelial cells. Expression decreases in the peri-implantation period concurrent with maximal HOXA10 expression. Dramatic changes in cellular architecture are necessary to achieve the secretory changes in the endometrial epithelium that bring about the implantation window. Alterations in CK15 likely facilitate these cytoskeletal changes, ultimately promoting endometrial receptivity.