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PURPOSE: In vitro fertilization (IVF) has been a well-established method for treating infertility for over four decades. The mainstay method of culture of oocytes and embryos has been in gas incubators. More recently, the novel use of a gas-permeable closed vessel to culture oocytes and embryos in the vagina, intravaginal culture (IVC), has been introduced as a viable lower-cost option for infertility patients. Several studies have studied the efficacy of IVC; however, there is no data on the perinatal outcomes of the babies born using this newer technology. METHODS: Our study is a retrospective case series (n = 66) from a single center, uniquely examining the perinatal outcomes of infants born after IVC. RESULTS: There were 50 singleton and 16 twin gestations in this case series. For singleton infants conceived via IVC (n = 50), the mean gestational age at delivery was 38 weeks and 4 days, and the mean birth weight was 3159.1 + / − 501.5 g. Four infants were born with low birth weight, three were born preterm, and one was born macrosomic. The twin pregnancies had a mean gestational age at delivery of 33 weeks 4 days and a mean birth weight of 1992.9 + / − 620.7 g. Twenty-seven infants met the criteria for low birthweight, and twenty-four infants delivered preterm. No twin infants met the criteria for macrosomia. CONCLUSION: This case series provides an initial description of the perinatal outcomes of IVC conceived infants, which shows no concerning trends in adverse birth outcomes for singleton infants. As expected, IVC twin gestations had a high rate of low birth weight and preterm delivery. Continued larger studies are essential to provide more comprehensive data on perinatal outcomes of infants conceived by this new technology.

Background The OncoScan microarray assay (OMA) using highly multiplexed molecular inversion probes for single nucleotide polymorphism (SNP) loci enabled the detection of cytogenomic abnormalities of chromosomal imbalances and pathogenic copy number variants (pCNV). The small size of molecular inversion probes is optimal for SNP genotyping of fragmented DNA from fixed tissues. This retrospective study evaluated the clinical utility of OMA as a uniform platform to detect cytogenomic abnormalities for pregnancy loss from fresh and fixed tissues of products of conception (POC). Results Fresh specimens of POC were routinely subjected to cell culture and then analyzed by karyotyping. POC specimens with a normal karyotype (NK) or culture failure (CF) and from formalin-fixed paraffin-embedded (FFPE) tissues were subjected to DNA extraction for OMA. The abnormality detection rate (ADR) by OMA on 94 cases of POC-NK, 38 cases of POC-CF, and 35 cases of POC-FFPE tissues were 2% (2/94), 26% (10/38), and 57% (20/35), respectively. The detected cytogenomic abnormalities of aneuploidies, triploidies and pCNV accounted for 50%, 40% and 10% in POC-CF and 85%, 10% and 5% in POC-FFPE, respectively. False negative result from cultured maternal cells and maternal cell contamination were each detected in one case. OMA on two cases with unbalanced structural chromosome abnormalities further defined genomic imbalances and breakpoints. Conclusion OMA on POC-CF and POC-FFPE showed a high diagnostic yield of cytogenomic abnormalities. This approach circumvented the obstacles of CF from fresh specimens and fragmented DNA from fixed tissues and provided a reliable and effective platform for detecting cytogenomic abnormalities and monitoring true fetal result from maternal cell contamination.

PURPOSE: To determine the effect of human growth hormone (GH) supplementation during ovarian stimulation in women undergoing IVF/PGT-A cycles, who do not meet the Bologna criteria for poor ovarian response (POR). METHODS: This is a retrospective cohort study of 41 women with suboptimal outcomes in their first cycle of IVF/PGT-A including lower than expected number of MII oocytes, poor blastulation rate, and/or lower than expected number of euploid embryos for their age, who underwent a subsequent IVF/PGT-A cycle with the same fixed dose gonadotropin protocol and adjuvant GH treatment. Daily cotreatment with GH started with first gonadotrophin injection. The IVF cycle outcomes were compared between the control and GH cycle using the Wilcoxon-Signed Rank test. RESULTS: The total number of biopsied blastocysts (mean ± SD; 2.0 ± 1.6 vs 3.5 ± 3.2, p = 0.009) and euploid embryos (0.8 ± 1.0 vs 2.0 ± 2.8, p = 0.004) were significantly increased in the adjuvant GH cycle compared to the control cycle. The total number of MII oocytes also trended to be higher in the GH cycle (10.2 ± 6.3 vs 12.1 ± 8.3, p = 0.061). The overall blastulation and euploidy rate did not differ between the control and treatment cycle. CONCLUSION: Our study uniquely investigated the use of adjuvant GH in IVF/PGT-A cycles in women without POR and without a priori suspicion for poor outcome based on their clinical parameters. Our study presents preliminary evidence that GH supplementation in these women is beneficial and is associated with an increased number of blastocysts for biopsy and greater number of euploid embryos for transfer.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Background: Pregnancy loss is the most common complication of pregnancy and understanding the needs of individuals experiencing pregnancy loss will help the medical team provide patient-centered ca...

Purpose Pregnancy loss ranging from spontaneous abortion (SAB) to stillbirth can result from monogenic causes of Mendelian inheritance. This study evaluated the clinical application of exome sequencing (ES) in identifying the genetic etiology for pregnancy loss. Methods A cohort of 102 specimens from products of conception (POC) with normal karyotype and absence of pathogenic copy-number variants were selected for ES. Abnormality detection rate (ADR) and variants of diagnostic value correlated with SAB and stillbirth were evaluated. Results ES detected 6 pathogenic variants, 16 likely pathogenic variants, and 17 variants of uncertain significance favor pathogenic (VUSfp) from this cohort. The ADR for pathogenic and likely pathogenic variants was 22% and reached 35% with the inclusion of VUSfp. The ADRs of SAB and stillbirth were 36% and 33%, respectively. Affected genes included those associated with multisystem abnormalities, neurodevelopmental disorders, cardiac anomalies, skeletal dysplasia, metabolic disorders, and renal diseases. Conclusion These results supported the clinical utility of ES for detecting monogenic etiology of pregnancy loss. The identification of disease-associated variants provided information for follow-up genetic counseling of recurrence risk and management of subsequent pregnancies. Discovery of novel variants could provide insight for underlying molecular mechanisms causing fetal death.

PurposePregnancy loss ranging from spontaneous abortion (SAB) to stillbirth can result from monogenic causes of Mendelian inheritance. This study evaluated the clinical application of whole exome sequencing (WES) in identifying the genetic etiology for pregnancy loss.MethodsA cohort of 102 specimens from products of conception (POC) with normal karyotype and absence of pathogenic copy number variants were selected for WES. Abnormality detection rate (ADR) and variants of diagnostic value correlated with SAB and stillbirth were evaluated.ResultsWES detected six pathogenic variants, 16 likely pathogenic variants, and 17 variants of uncertain significance favor pathogenic (VUSfp) from this cohort. The ADR for pathogenic and likely pathogenic variants was 22% and reached 35% with the inclusion of VUSfp. The ADRs of SAB and stillbirth were 36% and 33%, respectively. Affected genes included those associated with multi-system abnormalities, neurodevelopmental disorders, cardiac anomalies, skeletal dysplasia, metabolic disorders and renal diseases.ConclusionThese results supported the clinical utility of WES for detecting monogenic etiology of pregnancy loss. The identification of disease associated variants provided information for follow-up genetic counseling of recurrence risk and management of subsequent pregnancies. Discovery of novel variants could provide insight for underlying molecular mechanisms causing fetal death.

Pregnancy loss ranging from spontaneous abortion (SAB) to stillbirth can result from monogenic causes of Mendelian inheritance. This study evaluated the clinical application of exome sequencing (ES) in identifying the genetic etiology for pregnancy loss.A cohort of 102 specimens from products of conception (POC) with normal karyotype and absence of pathogenic copy-number variants were selected for ES. Abnormality detection rate (ADR) and variants of diagnostic value correlated with SAB and stillbirth were evaluated.ES detected 6 pathogenic variants, 16 likely pathogenic variants, and 17 variants of uncertain significance favor pathogenic (VUSfp) from this cohort. The ADR for pathogenic and likely pathogenic variants was 22% and reached 35% with the inclusion of VUSfp. The ADRs of SAB and stillbirth were 36% and 33%, respectively. Affected genes included those associated with multisystem abnormalities, neurodevelopmental disorders, cardiac anomalies, skeletal dysplasia, metabolic disorders, and renal diseases.These results supported the clinical utility of ES for detecting monogenic etiology of pregnancy loss. The identification of disease-associated variants provided information for follow-up genetic counseling of recurrence risk and management of subsequent pregnancies. Discovery of novel variants could provide insight for underlying molecular mechanisms causing fetal death.

Accumulating evidence has indicated that the genes involved in meiosis are highly correlated with ovarian function. Pumilio 1 (PUM1) is a RNA-binding protein which is involved in the meiotic process. It has been reported that the Pum1 knockout female mice displayed subfertility due to the decrease in primordial follicle pool. The aim of our study is to investigate whether variants of the PUM1 gene are responsible for primary ovarian insufficiency (POI) in Chinese women. We analyzed coding sequence and untranslated regions of the PUM1 gene in 196 Han Chinese women with non-syndromic POI and 192 controls. Seven novel variants were identified, but one of them was synonymous and six were intronic. Besides, seven known single-nucleotide polymorphisms (SNPs) were found, and there were no significant differences in genotype and allele frequencies of the SNPs between patients and controls. The results suggest that the variants in PUM1 may not contribute to POI in Han Chinese women. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-017-1110-4) contains supplementary material, which is available to authorized users.

Adding preimplantation genetic screening to in vitro fertilization has been shown to increase live birth rate in women older than 37. However, preimplantation genetic screening is an expensive procedure. Information on the cost-effectiveness of preimplantation genetic screening can help inform clinical decision making. We constructed a decision analytic model for a hypothetical fresh, autologous in vitro fertilization cycle (with versus without preimplantation genetic screening) for women older than age 37 who had a successful oocyte retrieval and development of at least one blastocyst. The model incorporated probability and cost estimates of relevant clinical events based on data from published literature. Sensitivity analyses were performed to examine the impact of changes in model input parameters. In base-case analysis, IVF-PGS offered a 4.2 percentage point increase in live birth rate for an additional cost of $4509, yielding an incremental cost-effectiveness ratio (ICER) of $105,489 per additional live birth. This ICER was below the expected cost of $145,063 for achieving one live birth with IVF (assuming an average LBR of 13.4% and $19,415 per cycle for this patient population). Sensitivity analysis suggested that ICER improved substantially with decreases in PGS cost and increases in PGS effectiveness. Monte Carlo simulation showed PGS to be cost-effective in 93.9% of iterations at an acceptability cutoff of $145,063. Considering the expected cost of achieving one live birth with IVF, PGS is a cost-effective strategy for women older than 37 undergoing IVF. Additional research on patients’ willingness-to-pay per live birth would further inform our understanding regarding the cost-effectiveness of PGS.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional levels, but not at the post-transcriptional levels. Pumilio (Pum) proteins are among the few known translational regulators required for stem cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double mutants are developmentally delayed at the morula stage and lethal by embryonic day 8.5 (e8.5). Correspondingly, Pum1/2 double mutant ESCs display severely reduced self-renewal and differentiation, revealing the combined function of Pum1 and Pum2 in ESC pluripotency. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, indicating that Pum1 mainly promote differentiation; whereas Pum2-deficient ESCs show decreased expression of pluripotency genes and accelerated differentiation, indicating that Pum2 promotes self-renewal. Thus, Pum1 and Pum2 each uniquely contributes to one of the two complementary aspects of pluripotency. Furthermore, we show that Pum1 and Pum2 achieve ESC functions by forming a negative auto- and inter-regulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation as expected but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal the distinct roles of individual mammalian Pum proteins in ESCs and their collectively essential functions in ESC pluripotency and embryogenesis. Moreover, they demonstrate three novel modes of regulation of Pum proteins towards target mRNAs.SIGNIFICANCE STATEMENTThis report demonstrates the essential functions of mammalian Pumilio (Pum) proteins for embryonic stem cells (ESCs) pluripotency and embryogenesis. Moreover, it reveals the contrasting but complementary function of individual Pum proteins in regulating distinct aspects of ESC pluripotency, despite their largely overlapping expression and extremely high homology. Furthermore, it unravels a complex regulatory network in which Pum1 and Pum2 form a negative auto- and inter-regulatory feedback loop that regulates 1,486 mRNAs not only by translational repression as expected but also by promoting translation and enhancing or reducing stability of different target mRNAs, which reveals novel modes of post-transcriptional regulation mediated by Pum.

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 (

Previous studies show that aberrant tryptophan catabolism reduces maternal immune tolerance and adversely impacts pregnancy outcomes. Tryptophan depletion in pregnancy is facilitated by increased activity of tryptophan-depleting enzymes [i.e. the indolamine-2,3 dioxygenase (IDO)1 and IDO2) in the placenta. In mice, inhibition of IDO1 activity during pregnancy results in fetal loss; however, despite its important role, regulation of Ido1 gene transcription is unknown. The current study shows that the Ido1 and Ido2 genes are imprinted and maternally expressed in mouse placentas. DNA methylation analysis demonstrates that nine CpG sites at the Ido1 promoter constitute a differentially methylated region that is highly methylated in sperm but unmethylated in oocytes. Bisulfite cloning sequencing analysis shows that the paternal allele is hypermethylated while the maternal allele shows low levels of methylation in E9.5 placenta. Further study in E9.5 placentas from the CBA/J X DBA/2 spontaneous abortion mouse model reveals that aberrant methylation of Ido1 is linked to pregnancy loss. DNA methylation analysis in humans shows that IDO1 is hypermethylated in human sperm but partially methylated in placentas, suggesting similar methylation patterns to mouse. Importantly, analysis in euploid placentas from first trimester pregnancy loss reveals that IDO1 methylation significantly differs between the two placenta cohorts, with most CpG sites showing increased percent of methylation in miscarriage placentas. Our study suggests that DNA methylation is linked to regulation of Ido1/IDO1 expression and altered Ido1/IDO1 DNA methylation can adversely influence pregnancy outcomes.

Background RNA binding proteins play a pivotal role during the oocyte-to-embryo transition and maternal phase of embryogenesis in invertebrates, but their function in these processes in mammalian systems remain largely understudied. Results Here we report that a member of the Pumilio/FBF family of RNA binding proteins in mice, Pumilio 1 (Pum1), is a maternal effect gene. The absence of maternal PUM1 in the oocyte does not affect meiotic maturation but leads to abnormal preimplantation development. Furthermore, genome-wide transcriptome analysis of oocytes and embryos revealed that there is a concomitant perturbation of the mRNA milieu. Of note, putative PUM1 mRNA targets were equally perturbed as non-direct targets, which indicates that PUM1 regulates the stability of maternal mRNAs both directly and indirectly. We show Cdk1 mRNA, a known PUM1 target essential for meiosis and preimplantation development, is not degraded appropriately during meiosis, leading to an increase in CDK1 protein in mature oocytes, which indicates that PUM1 post-transcriptionally regulates Cdk1 mRNA; this could partially explain the observed abnormal preimplantation development. Furthermore, our results show that maternal and zygotic PUM1 are required for postnatal survival. Conclusions These findings indicate that PUM1 is essential in the process of cytoplasmic maturation and developmental competence of the oocyte. These results reveal an important function of maternal PUM1 as a post-transcriptional regulator during mammalian embryogenesis. Electronic supplementary material The online version of this article (10.1186/s13578-018-0251-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

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 [1]. 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 [2–4]. 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 [5]. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp [6]. This process is also Xist dependent [7]. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice [8]. 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.

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.

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.

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., Author Summary During oocyte growth and maturation, vital proteins and enzymes are produced to ensure that, when fertilized, a healthy embryo will arise. When this natural process is interrupted, one or more of these essential elements can fail to be produced thus compromising the health of the future embryo. We are using a mouse model, lacking an enzyme (DNMT1o) produced in the oocyte and only required post-fertilization in the early embryo for the maintenance of inherited DNA methylation marks. Here, we reveal that oocytes lacking DNMT1o, when fertilized, generated conceptuses with a wide variety of placental abnormalities. These placental abnormalities were more frequent and severe in females, and showed specific genomic regions constantly deprived of their normal methylation marks. The affected genomic regions were concentrated on the X chromosome. Interestingly, we also found that a region important for the regulation of the X chromosome inactivation process was hypomethylated in female blastocysts and was associated with sex-specific abnormalities in the placenta, relaxation of imprinted X chromosome inactivation, and disruption of DNA methylation later in development. Our findings provide a novel unanticipated role for DNA methylation events taking place within the first few days of life specifically in female preimplantation embryos.

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.

Study question Does ICSI improve outcomes in ART cycles without male factor, specifically in couples with a history of tubal ligation as their infertility diagnosis? Summary answer The use of ICSI showed no significant improvement in fertilization rate and resulted in lower pregnancy and live birth (LB) rates for women with the diagnosis of tubal ligation and no male factor. What is known already Prior studies have suggested that ICSI use does not improve fertilization, pregnancy or LB rates in couples with non-male factor infertility. However, it is unknown whether couples with tubal ligation only diagnosis and therefore iatrogenic infertility could benefit from the use of ICSI during their ART cycles. Study design, size, duration Longitudinal cohort of nationally reported cycles in the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS) of ART cycles performed in the USA between 2004 and 2012. Participants/materials, setting, methods There was a total of 8102 first autologous fresh ART cycles from women with the diagnosis of tubal ligation only and no reported male factor in the SART database. Of these, 957 were canceled cycles and were excluded from the final analysis. The remaining cycles were categorized by the use of conventional IVF (IVF, n = 3956 cycles) or ICSI (n = 3189 cycles). The odds of fertilization, clinical intrauterine gestation (CIG) and LB were calculated by logistic regression modeling, and the adjusted odds ratios (AORs) with 95% confidence intervals were calculated by adjusting for the confounders of year of treatment, maternal age, race and ethnicity, gravidity, number of oocytes retrieved, day of embryo transfer and number of embryos transferred. Main results and the role of chance The main outcome measures of the study were odds of fertilization (2PN/total oocytes), clinical intrauterine gestation (CIG/cycle) and live birth (LB/cycle). The fertilization rate was higher in the ICSI versus IVF group (57.5% vs 49.1%); however, after adjustment this trend was no longer significant (AOR 1.14, 0.97-1.35). Interestingly, both odds of CIG (AOR 0.78, 0.70-0.86), and odds of LB were lower (AOR 0.77, 0.69-0.85) in the ICSI group. Plurality at birth, mean length of gestation and birth weight did not differ between the two groups. Limitations, reasons for caution This was a retrospective study, therefore only the available parameters could be included, with parameters of interest such as smoking status not available for inclusion. Smoking status may have led practitioners to use ICSI to improve pregnancy and LB outcomes. Wider implications of the findings Studies have shown that in the USA there is an increasing usage of ICSI for non-male factor infertility despite a lack of evidence-based benefit. Our study corroborates this increasing use over the last 8 years, specifically in the tubal ligation only patient population. Even after adjusting for multiple confounders, the patients who underwent ICSI had no statistically significant improvement in fertilization rate and actually had a lower likelihood of achieving a clinical pregnancy and LB. Therefore, our data suggest that the use of ICSI in tubal ligation patients has no overall benefit. This study contributes to the body of evidence that the use of ICSI for non-male factor diagnosis does not improve ART outcomes over conventional IVF. Study funding/competing interests None.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). 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, HIF1alpha and HIF2alpha 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.

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.

BACKGROUND: The aim of this study was to determine whether surgical intervention is effective for women with tubal disease who are due to undergo treatment with IVF. METHODS: A systematic review employing the principles of the Cochrane Menstrual Disorders and Subfertility Group was undertaken. Three randomized controlled trials were included, the population of women in all three studies having hydrosalpinges. RESULTS: The odds of pregnancy [odds ratio (OR) = 1.75, 95% confidence interval (CI) 1.07-2.86] and of ongoing pregnancy and live birth (OR = 2.13, 95% CI 1.24-3.65) were increased with laparoscopic salpingectomy for hydrosalpinges prior to IVF. There were no significant differences in the odds of embryo implantation (OR = 1.34, 95% CI 0.87-2.05), ectopic pregnancy (OR = 0.42, 95% CI 0.08-2.14), miscarriage (OR = 0.49, 95% CI 0.16-1.52) or treatment complications (OR = 5.80, 95% CI 0.35-96.79). No data were available concerning the odds of multiple pregnancy or the proportion of IVF cycles resulting in embryo transfer. CONCLUSION: Laparoscopic salpingectomy should be considered for all women with hydrosalpinges due to undergo IVF; further research is required to assess other pre-IVF surgical interventions (such as needle aspiration of hydrosalpinx fluid, laparoscopic proximal tubal occlusion and laparoscopic salpingostomy) for women with hydrosalpinges.