Winifred Mak

Establishment of Histone H3 Methylation on the Inactive X Chromosome Requires Transient Recruitment of Eed-Enx1 Polycomb Group Complexes

Previous studies have implicated the Eed-Enx1 Polycomb group complex in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Here we show that recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure.

Mitotically Stable Association of Polycomb Group Proteins Eed and Enx1 with the Inactive X Chromosome in Trophoblast Stem Cells

X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, which then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Here we show that Eed and a second Polycomb group protein, Enx1, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells.

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFα and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1β). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1α and HIF2α were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.

In Vitro Culture Increases the Frequency of Stochastic Epigenetic Errors at Imprinted Genes in Placental Tissues from Mouse Concepti Produced Through Assisted Reproductive Technologies

ABSTRACT Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (∼20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat−/− mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.

Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation.

In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNA-mediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we describe analysis of a novel targeted mutation, XistINV, designed to test the function of a conserved region located in exon 1 of Xist RNA during X inactivation in mouse. We show that XistINV is a strong hypomorphic allele that is appropriately regulated but compromised in its ability to silence X-linked loci in cis. Inheritance of XistINV on the paternal X chromosome results in embryonic lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting XistINV on the maternal X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the XistINV allele. Analysis of cells that express XistINV RNA demonstrates reduced association of the mutant RNA to the X chromosome, suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.

Polycystic Ovarian Syndrome and the Risk of Cardiovascular Disease and Thrombosis

Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with multiple comorbidities such as diabetes, dyslipidemia, hypertension, and metabolic syndrome, all of which predispose women with PCOS to early atherosclerosis. Women with PCOS also have a higher prevalence of subclinical atherosclerosis, as reflected in dysregulation of endothelial function, increased carotid intimal-medial thickness, and presence of coronary artery calcification. Preliminary data indicate that serum biomarkers of cardiovascular disease such as high-sensitivity C-reactive protein, homocysteine, and adiponectin are abnormal in women with PCOS. There is limited data on abnormalities in the coagulation and fibrinolytic systems, however. The risk of venous thrombosis is unclear in the PCOS population, and further studies are urgently required to address whether first-line treatment for PCOS with oral contraceptive pills is advisable.

National study of factors influencing assisted reproductive technology outcomes with male factor infertility

OBJECTIVE To evaluate the outcomes of assisted reproductive technology (ART) cycles for male factor infertility, and method of sperm collection. DESIGN Historic cohort study. SETTING Clinic-based data. PATIENTS Cycles from the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System database for 2004 to 2008 were limited to three groups: non-intracytoplasmic sperm injection (ICSI) and ICSI cycles for tubal ligation only; non-ICSI and ICSI cycles for male factor infertility only; and all cycles (regardless of infertility diagnosis) using ICSI only. INTERVENTION(S) AND MAIN OUTCOME MEASURE(S) Multivariate logistic regression was used to model the adjusted odds ratio (AOR) of clinical intrauterine gestation (CIG) and live birth (LB) rates for tubal ligation versus male factor infertility only; ICSI versus non-ICSI for male factor infertility only; and ICSI outcomes based on method of sperm collection. RESULT(S) Models for male factor infertility only versus tubal ligation only ICSI cycles had lower CIG (AOR 0.92) but not LB (AOR 0.87). No difference was seen for non-ICSI cycles. Within male factor infertility only cycles, ICSI had a worse outcome than non-ICSI for CIG (AOR 0.93) but not for LB (AOR 0.94). For all ICSI cycles with no male factor infertility and ejaculated sperm as the reference group, models showed better rates of CIG with male factor infertility ejaculated sperm (AOR 1.07) and with male factor infertility aspirated sperm (AOR 1.09). The LB rate was higher with male factor infertility ejaculated sperm only (AOR 1.04). CONCLUSION(S) The ICSI and sperm source influence CIG and LB rates in male factor infertility cases.

An Important Role of Pumilio 1 in Regulating the Development of the Mammalian Female Germline

ABSTRACT Pumilio/FBF (PUF) proteins are a highly conserved family of translational regulators. The Drosophila PUF protein, Pumilio, is crucial for germline establishment and fertility. In mammals, primordial folliculogenesis is a key process that establishes the initial cohort of female mammalian germ cells prior to birth, and this primordial follicle pool is a prerequisite for female reproductive competence. We sought to understand whether PUF proteins have a conserved role in mammals during primordial folliculogenesis and female reproductive competency. In mammals, two homologs of Pumilio exist: Pumilio 1 (Pum1) and Pum2. Here, we report that PUMILIO (PUM) 1 plays an important role in the establishment of the primordial follicle pool, meiosis, and female reproductive competency, whereas PUM2 does not have a detectable function in these processes. Furthermore, we show that PUM1 facilitates the transition of the late meiotic prophase I oocyte from pachytene to diplotene stage by regulating SYCP1 protein. Our study reveals an important role of translational regulation in mammalian female germ cell development.

Natural cycle IVF reduces the risk of low birthweight infants compared with conventional stimulated IVF

STUDY QUESTION Are perinatal outcomes improved in singleton pregnancies resulting from fresh embryo transfers performed following unstimulated/natural cycle IVF (NCIVF) compared with stimulated IVF? SUMMARY ANSWER Infants conceived by unstimulated/NCIVF have a lower risk of being low birthweight than infants conceived by stimulated IVF; however, this risk did not remain significant after adjusting for gestation age. WHAT IS ALREADY KNOWN Previous studies have shown that infants born after modified NCIVF have a higher average birthweight and are less likely to be low birthweight than those infants conceived with conventional stimulated IVF. STUDY DESIGN, SIZE AND DURATION Retrospective cohort study of singleton live births in non-smoking women undergoing fresh IVF-embryo transfer cycles from 2007 to 2013 in a single IVF center. The women were stratified by stimulated (n = 174) or unstimulated (n = 190) IVF exposure status. Unstimulated/NCIVF is defined as IVF without the use of exogenous gonadotrophins, and only includes the use of HCG to time oocyte retrieval. PARTICIPANTS/MATERIALS, SETTING, METHODS Demographic data including maternal age, BMI, infertility diagnosis and IVF cycle characteristics were collected. The perinatal outcomes used for comparison between the two study groups were length of gestation, birthweight, preterm delivery, very preterm delivery, low birthweight, small for gestational age and large for gestational age. MAIN RESULTS AND ROLE OF CHANCE Although women in the NCIVF group were older than those in the stimulated group (35.0 versus 34.2 years, P < 0.05), parity and history of prior ART cycles were comparable between the groups. The mean birthweight was significantly higher in the NCIVF group by 163 g than in the stimulated group (3436 ± 420 g versus 3273 ± 574 g, P < 0.05). Consistent with this finding, there were also less low birthweight (<2500 g) infants in the NCIVF group versus stimulated group (1 versus 8.6%, P < 0.005). The reduction in risk for low birthweight in the NCIVF group remained significant after adjustment for maternal age, infertility diagnosis, ICSI, number of embryos transferred and blastocyst transfer (odds ratio (OR) 0.07; 95% CI 0.014-0.35). As NCIVF group had less preterm infants, additional adjustment for gestational age was performed and this showed a tendency towards lower risk of low birthweight in NCIVF (OR 0.11; 95% CI 0.01-1.0). While gestational age at delivery was comparable between the groups, both preterm births (<37 weeks gestation) (31 versus 42%, P < 0.05) and very preterm births (<32 weeks gestation) (0.52 versus 6.3%, P < 0.005) were significantly reduced in the NCIVF group. However, after adjustment for potential confounders, the reduction in risk of preterm and very preterm delivery associated with the NCIVF group was no longer significant (OR 1.1; 95% CI 0.48-2.5). LIMITATIONS, REASONS FOR CAUTION Limitations of this study are the retrospective nature of the data collection and the lack of information about parental characteristics associated with birthweight. WIDER IMPLICATIONS OF THE FINDINGS The improved perinatal outcomes following successful unstimulated/NCIVF suggest that this treatment should be considered as a viable option for infertile couples. NCIVF could reduce potential adverse perinatal outcomes such as low birthweight related to fresh embryo transfers performed following ovarian stimulation. The etiology of the improved perinatal outcomes following NCIVF needs to be explored further to determine if the improvement is derived from endometrial factors versus follicular/oocyte factors. STUDY FUNDING/COMPETING INTERESTS The study was supported by the following grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD K12HD047018 (W.M.), NICHD K12HD001271 (L.A.K.). The authors have no competing interests.