In virtually all metazoans, oocytes become arrested at the prophase of the first meiotic division. Upon appropriate stimuli, oocytes resume meiosis and initiate maturation in preparation for fertilization. Oocyte maturation is associated with drastic changes in both the nuclear and cytoplasmic compartments and with suppression of transcriptional activity. Consequently, gene expression during oocyte maturation, fertilization, and early embryo development, until zygotic genome activation (ZGA), is mainly regulated by translational activation of maternally-derived mRNAs stored in the oocyte during the first meiotic arrest.
Embryonic poly(A) binding protein (ePAB), identified in Xenopus oocytes (Voeltz et al. Genes & Dev, 2001), is the predominant poly(A) binding protein during early Xenopus development until ZGA. In Xenopus, ePAB is present in both known protein complexes (cytopasmic polyadenylation complex and Pumilio-2/DAZL/ePAB complex) that bind and stabilize dormant mRNAs in immature oocytes. ePAB also appears to be a key factor required for both known pathways (cytoplasmic polyadenylation element-dependent and -independent) that mediate maternal mRNA translational activation upon oocyte maturation.
We hypothesized that ePAB plays a key role in mammalian oocyte and early development and identified mouse (Seli et al. PNAS 2005) and human (Guzeloglu-Kayisli et al. MHR 2008; Guzeloglu-Kayisli et al. MHR 2014) ePAB orthologues expressed in oocytes and early embryos. Similar to that observed in Xenopus, ePAB seems to be the predominant poly(A) binding protein until ZGA in both mouse and human. We then adopted Xenopus, mouse, and human models to investigate the regulation of ePAB function and to understand its role in early vertebrate and mammalian development. Using the Xenopus model, we demonstrated that ePAB is a dynamically modified phosphoprotein and showed by mutation that phosphorylation at a four-residue cluster is required for oocyte maturation. We further demonstrated that these phosphorylations are critical for cytoplasmic polyadenylation but not for ePAB’s inherent ability to promote translation (Friend et al. Biochem J 2012). We also identified novel-ePAB interacting proteins in Xenopus oocytes and are currently characterizing these proteins and their mammalian orthologues as potential regulators of gene expression during early development.
To investigate the role of ePAB in mammalian reproduction, we generated Epab knockout mice by targeted deletion of the Epab gene (Guzeloglu-Kayisli et al. Biochem J 2012). While Epab-/- males and Epab+/- of both sexes were fertile, Epab-/- female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab-/-oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including cyclin B1 and Dazl mRNAs. In addition, late antral follicles in the ovaries of Epab-/- mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding EGF-like growth factors AREG, EREG, and BTC, and their downstream regulators, PTGS2, HAS2, and TNFAIP6. Further studies demonstrated that EPAB is required for chromoatin organization and transcriptional silencing (Lowther and Mehlmann, Biol Reprod, 2015) and for granulosa cell EGF signaling (Yang et al. Endocrinology, 2016).
Our findings indicate that ePAB is necessary for oogenesis, folliculogenesis, and female fertility in mouse. In addition to our studies in animal models, we are actively investigating the role of human ePAB in human infertility and assisted reproduction.
Infertility defined as the inability to conceive after one year of unprotected intercourse is estimated to affect 15% of the reproductive age population. Among the treatment modalities offered to infertile couples, those utilizing Assisted Reproductive Technologies (ART) are associated with the highest success rates. More than 125,000 cycles are started yearly in the United States, where ART accounts for approximately 1% of births.
The high success rates achieved with ART are attained in many cases through the simultaneous transfer of multiple embryos at the risk of multiple pregnancy. In the United States, a mean number of 2.45 embryos were transferred in IVF cycles using fresh non-donor oocytes in 2006, resulting in a 34.3% live birth rate per transfer, of which 32% were multiple-infant live births. Similarly, a mean number of 2.3 embryos were transferred in IVF cycles using fresh donor oocytes, achieving a 52.3% live birth rate per transfer, 40.8% of which were multiple-infant live births. In total, while IVF treatment accounts for only 1% of all births in the United States, 18% of multiple births result from IVF. Moreover, 51% of all IVF neonates are the products of multiple gestations, a frequency 15- to 20-fold greater than with spontaneous conceptions.
The high multiple pregnancy rates associated with IVF have significant public health consequences as the increased rate of preterm delivery in multiple infant pregnancies compromises the survival of neonates and increases their risk of lifelong disability. Indeed, while multiple births constitute approximately 1/33 of all births in the United States, they account for 1/8 of preterm births (<37 weeks), and 1/4 of very low birth weight infants (<2000g). Largely due to the increased incidence of prematurity, cerebral palsy is increased 8-fold in twins and 47-fold in triplets, while infant deaths (birth to 1 year) are increased 6-fold in twins, and 17-fold in triplets and higher order gestations. Recently, we estimated that preterm births that result from IVF-related multiple pregnancies yearly account for approximately $1 billion of cost to the society in the United States (Bromer and Seli, Cur Opin Obstet Gynecol 2011). These estimations do not include maternal complications associated with multiple gestations, including a 2 to 4-fold increase in pregnancy induced hypertension and postpartum hemorrhage.
The sentinel issue surrounding multiple gestations following ART is the inability to precisely estimate the reproductive potential of individual embryos. Soon after the report of the first successful pregnancy after in vitro fertilization, and development of controlled ovarian stimulation in order to obtain more than one egg in a given cycle, it became apparent that morphology and cleavage rate of embryos correlate with their implantation potential. Thereafter, grading systems based on embryo cleavage rate and morphology were developed leading to significant improvements in implantation and pregnancy rates and reductions in multiple gestation rates. Unfortunately, their precision is still insufficient to compel most patients and clinicians to reduce the number of embryos transferred to a point where twins are uncommon and high order multiple gestations are rare or eliminated.
The limitations of morphologic evaluation of embryos have led many investigators to pursue adjunctive technologies for the assessment of the reproductive potential of a given embryo. Several metabolic parameters of developing embryos have been measured using a number of non-invasive techniques (reviewed in Bromer and Seli, Cur Opin Obstet Gynecol 2008). While these studies suggest the presence of metabolic differences between embryos with different reproductive potential, the application of these technologies to a clinical setting has been limited for a variety of reasons. Many of these technologies are expensive, require dedicated equipment and technical staff which would be cost prohibitive in most embryology laboratories, and frequently do not produce results quickly enough to allow the information be used clinically in the limited window of time acceptable for embryo transfer. Moreover, none of the technologies has ever been validated using culture media evaluated in a blinded fashion and shown to correlate with the implantation potential of embryos that have been transferred. Therefore, the need for a technology that predicts reproductive potential of embryos through a rapid, non-invasive, and clinically applicable platform remains.
We have asked whether metabolomic profile of spent embryo culture media could be used to predict embryo viability in IVF. The complete array of small-molecule metabolites that are found within a biological system constitutes the metabolome and reflects the functional phenotype. Metabolomics, is the systematic study of this dynamic inventory of metabolites, as small molecular biomarkers representing the functional phenotype in a biological system. Using various forms of spectral and analytical approaches, metabolomics attempts to determine metabolites associated with physiologic and pathologic states.
In 2007, we reported the results of a proof-of-concept study where day 3 spent culture media of individually cultured embryos with known pregnancy outcome were analyzed using near infrared (NIR) and Raman spectroscopies (Seli et al. Fertil Steril 2007). Using a multivariate analysis approach, we compared spectral profiles between embryos that resulted in live birth and embryos that failed to implant. Spectral regions that discriminated between the two study populations and were most predictive of pregnancy outcome were identified as markers of oxidative stress, including vibrational modes of –CH, –NH and –OH groups. These spectral regions were then quantified in a multi-linear regression algorithm and expressed as a viability score of each individual embryo’s reproductive potential.
In this initial study, using both NIR and Raman spectroscopy, the mean viability score of embryos that implanted and resulted in a live birth was significantly higher compared to the mean viability score of embryos that failed to implant. Raman and NIR spectroscopies predicted pregnancy/delivery with a sensitivity and specificity of > 75%. Moreover, the test was rapid (less than 1 minute per sample) and required a very small sample volume (less than 15ml).
Subsequently, the regression algorithm for Raman spectra described above was tested in a blinded trial (Scott et al. Fertil Steril, 2008) analyzing day 3 and day 5 spent media collected at a different IVF center, where embryos were cultured in a different volume and type of culture medium. In this study, the previously developed algorithm was applied to Raman spectra of embryo culture media, and successfully predicted the pregnancy outcome for embryos transferred on day 3 and day 5.
The initial studies described above were followed by studies with larger sample size from our group (Seli et al. Fertil Steril 2010) and others (Vergouw et al. Hum Reprod 2008; Ahlstrom et al. Reprod Biomed Online 2011) using samples collected in IVF centers that routinely perform single embryo transfer (SET). In these studies, spent culture media was analyzed by NIR spectroscopy as previously described, and independent regression algorithms for NIR spectra were developed for embryos that underwent SET on day 2, 3 and 5. Consistent with the findings of the initial studies, embryos that resulted in a pregnancy with fetal heart activity had a higher mean viability score compared to those that failed to implant. In addition, metabolomic profiling of embryo culture media was independent of morphology, providing an independent parameter in embryo viability assessment. This technology using NIR spectroscopy has been developed by a private company for clinical use as an “on-site test”, with limited success. Two randomized prospective trials showed that adding the commercial instrument to morphologic grading does not improve pregnancy outcome in women undergoing IVF (Hardarson et al. Hum Reprod 2012; Vergouw et al. Hum Reprod 2012).
In addition to NIR and Raman spectroscopies, we applied proton nuclear magnetic resonance metabolomics (1H NMR) to determine components of embryo culture media associated with viability and identified glutamate as a biomarker significantly elevated in the culture media of embryos that result in a pregnancy (Seli et al. Fertil Steril 2008).
More recently, we expanded our research of embryo viability assessment to include other potential parameters and/or species. Currently we are studying differential expression of specific mRNAs and microRNAs in cumulus cells that surround oocytes as potential biomarkers of oocyte viability and fertilization potential (Karakaya et al. Fertil Steril 2015); metabolomic profiling of embryo culture media to determine viability and sex of bovine embryos (Munoz et al. Biomed Res Int 2014); and the role of embryo aneuploidy assessment to identify viable embryos and increase success rates in women undergoing IVF treatment.
The life expectancy of a 15-year old female who survived infancy and early childhood in the 16th century United Kingdom was only 48 years. For the same girl, life expectancy would be 56 in the 18th century, 65 in the 19th century, and 75 by mid-20th century. As a result of the significant reduction in infant and teen mortality, as well as improvements in access to healthcare over the course of the 20th century, life expectancy at birth for females in the United States reached 81.2 years in 2012.
Parallel with the increase in life expectancy, and promoted by the social changes that followed industrialization, many contemporary women choose to delay parenthood. Indeed, the average age of first-time mothers increased from 21.4 in 1970 to 25.0 in 2006 in the United States, where the number of women delivering their first child after the age of 35 increased from 1/100 to 1/12 in the same time period.
However, despite the significant increase in longevity, studies show that the median age of menopause is currently around 50 in Western industrialized societies, with little apparent change over the past century. Consequently, today, at a time when women live longer and delay parenthood without a tangible change in the timing of their reproductive ageing, assisted reproduction has become a beacon of reproductive hope.
Within the context of assisted reproduction, ovarian aging is often used to refer to the declining potential of ovaries to produce oocytes in adequate number or quality in response to controlled ovarian stimulation. Patients who present with ovarian ageing are commonly diagnosed with diminished ovarian reserve (DOR), and offered in vitro fertilization (IVF) using either their own eggs or donated eggs or embryos. The number of women undergoing IVF for DOR has steadily increased over the past decade in the United States, where DOR accounted for 10% of ART cycles in 2003, 14% in 2008, and 18% in 2013, and the number of fresh IVF cycles using non-donor oocytes performed for women with DOR increased from 6,947 in 2003 to 13,219 in 2013. Similarly, the number of women undergoing oocyte donation in the United States increased from 11,627 in 2003 to 19,320 in 2013. These numbers and many other analyses support the view that ovarian ageing is an increasingly important reproductive health issue.
As science makes strides toward treating cardiovascular and metabolic disorders, infections, and malignancies, almost doubling life expectancy over a period of 200 years, understanding the mechanisms of ageing and developing strategies to prevent or delay ageing attract an increasing amount of attention from the scientists and lay people alike. However, despite the recent focus in ageing research, many interesting questions on ovarian ageing remain to be answered: What is the mechanism of ovarian ageing? Is it the same or different from mechanism(s) that regulate ageing in other tissues? Can ovarian ageing be stopped or delayed? Can it be reversed? What can we offer women afflicted with ovarian ageing while we wait for miraculous solutions? Our laboratory is investigating these questions, using mouse model, and with special emphasis on mitochondrial function and metabolic challenges.
The presence of endometrial stomal and epithelial cells outside of the uterus represents a pathologic condition termed endometriosis. Endometriosis most commonly presents with pelvic pain and/or infertility and affects 7-10% of women in the general population, leading to significant morbidity and financial burden for individuals and for society. Multiple theories have been proposed to explain the pathogenesis of endometriosis. Among these, the most commonly accepted remains Sampson's theory of retrograde menstruation from the uterus, through the fallopian tubes, and into the pelvic cavity. Because retrograde menstruation occurs in 76-90% of women wiht patent fallopian tubes, it is recognized that additional factors must play a role in determining susceptibility to endometriosis.
A host of immunologic abnormalities have been observed in women with endometriosis, suggesting tht an altered immune reponse may lead to inadequate removal of refluxed menstrual debris and/or promote the growth of ectopic endometrium in susceptible women. Several cytokines and growth factors are elevated int the peritoneal fluid of women with endometriosis and have been implicated in promoting the establishment and growth of ectopic endometrium by inducing cellular proliferation and angiogenesis.
Gene expression for many cytokines and growth factors is regulated post-transcriptionally, at the level of mRNA stability. Much of this regulation occurs by the binding and stabilizing or destabilizing of these mRNAs by proteins that recognize Adenosine- and Uridine-rich elements (i.e. AU-rich elements or AREs) in 3’ untranslated regions of target transcripts. ARE-binding proteins (ARE-BPs) alter the stability of their target mRNAs and/or regulate target mRNA translation by binding to core and flanking sequences of these AU-rich elements and then interacting with protein complexes essential for mRNA degradation or translation.
We have found that two prototypical ARE-BPs, HuR/ELAVL1 (mRNA stabilizing) and TIA-1 (mRNA destabilizing) are expressed in eutopic endometrial cells and that their expression is altered in ectopic endometrium (Karipcin et al. Reprod Sci 2011; Karalok et al. J Clin Endocrinol Metab 2014). Currently we are investigating whether 1) the net cytokine and growth factor production from eutopic and ectopic endometrial stromal and epithelial cells is regulated by the balance between mRNA-stabilizing and -destabilizing ARE-BPs, and 2) altered ARE-BP expression contributes to the pathogenesis of endometriosis by influencing local cytokine and growth factor production from ectopic endometrial cells.
Professor of Obstetrics, Gynecology & Reproductive Sciences
Endometriosis; Infertility; Menstruation Disturbances; Polycystic Ovary Syndrome
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