Paternal aging impacts expression and epigenetic markers as early as the first embryonic tissue lineage differentiation.

BACKGROUND: Advanced paternal age (APA) is associated with adverse outcomes to offspring health, including increased risk for neurodevelopmental disorders. The aim of this study was to investigate the methylome and transcriptome of the first two early embryonic tissue lineages, the inner cell mass (ICM) and the trophectoderm (TE), from human blastocysts in association with paternal age and disease risk. High quality human blastocysts were donated with patient consent from donor oocyte IVF cycles from either APA (≥ 50 years) or young fathers. Blastocysts were mechanically separated into ICM and TE lineage samples for both methylome and transcriptome analyses. RESULTS: Significant differential methylation and transcription was observed concurrently in ICM and TE lineages of APA-derived blastocysts compared to those from young fathers. The methylome revealed significant enrichment for neuronal signaling pathways, as well as an association with neurodevelopmental disorders and imprinted genes, largely overlapping within both the ICM and TE lineages. Significant enrichment of neurodevelopmental signaling pathways was also observed for differentially expressed genes, but only in the ICM. In stark contrast, no significant signaling pathways or gene ontology terms were identified in the trophectoderm. Despite normal semen parameters in aged fathers, these significant molecular alterations can adversely contribute to downstream impacts on offspring health, in particular neurodevelopmental disorders like autism spectrum disorder and schizophrenia. CONCLUSIONS: An increased risk for neurodevelopmental disorders is well described in children conceived by aged fathers. Using blastocysts derived from donor oocyte IVF cycles to strategically control for maternal age, our data reveals evidence of methylation dysregulation in both tissue lineages, as well as transcription dysregulation in neurodevelopmental signaling pathways associated with APA fathers. This data also reveals that embryos derived from APA fathers do not appear to be compromised for initial implantation potential with no significant pathway signaling disruption in trophectoderm transcription. Collectively, our work provides insights into the complex molecular mechanisms that occur upon paternal aging during the first lineage differentiation in the preimplantation embryo. Early expression and epigenetic markers of APA-derived preimplantation embryos highlight the susceptibility of the future fetus to adverse health outcomes.

In Vitro Culture Alters Cell Lineage Composition and Cellular Metabolism of Bovine Blastocyst.

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.

Impact of aneuploidy on reproductive success in young infertile women: prospective analysis.

RESEARCH QUESTION: What is the clinical outcome of the first attempt at conception between two embryo selection methods, blastocyst morphology and preimplantation genetic testing for aneuploidies (PGT-A), chosen at the initial physician IVF consultation? DESIGN: In this prospective analysis, a clinical decision regarding embryo selection, blastocyst morphology (group A) or PGT-A (group B) was made during initial physician IVF consultation. Female infertility patients were matched based on maternal age (mean 32.6 ± 3.6 years; range 25-43 years) and a similar time frame of oocyte retrieval. The primary outcome was live birth rate from the initial consultation to the first conception attempt for all female patients and for a subset analysis of patients aged <35 years. RESULTS: The inclusion of PGT-A (group B) for embryo selection during the initial physician IVF consultation resulted in 23 additional women out of the total 100 achieving a healthy live birth following the first conception attempt in this maternally age-matched infertile population (group B = 72.0% versus group A = 49.0%; P = 0.0014). This same benefit was observed for age-matched, younger infertility patients (<35 years), with live birth rates from the initial consultation being significantly higher when the upfront clinical decision included PGT-A for embryo selection (group B = 76.7% versus group A = 53.4%; P = 0.0052). Interestingly, 17 women from group B would have received an aneuploid embryo transfer if selection had been determined by blastocyst morphology alone, as their best-grade embryo was aneuploid. CONCLUSIONS: This prospective analysis from the initial physician IVF consultation revealed that euploid embryo selection significantly improved live birth potential with the first conception attempt, even for younger women with infertility.

Control of mitochondrial integrity influences oocyte quality during reproductive aging.

Reduced quality in oocytes from women of advanced maternal age (AMA) is associated with dysfunctional mitochondria. The objective of this study was to investigate the mechanisms controlling mitochondrial quality during maternal aging in mouse and human oocytes. We first evaluated the expression of proteins involved in the mitochondrial unfolded protein response (UPRmt) and mitophagy in in vivo matured metaphase II (MII) oocytes collected from young and aged mice. Expression of UPRmt proteins, HSPD1 and LONP1, and mitophagy proteins, total-PRKN and phosphorylated-PRKN, was significantly decreased in aged compared to young oocytes. Treatment of aged oocytes during in vitro maturation with the mitochondrially targeted antioxidant mitoquinone (MQ) specifically restored total-PRKN and phosphorylated-PRKN expression to levels seen in young oocytes. We next investigated whether maturing young oocytes under a high-oxygen environment would mimic the effects observed in oocytes from aged females. Phosphorylated-PRKN expression in oxidatively stressed young oocytes was reduced compared to that in oocytes matured under normal oxygen levels, and the mitochondrial DNA (mtDNA) copy number was increased. Treating oxidatively challenged young oocytes with MQ restored the phosphorylated-PRKN expression and mtDNA copy numbers. Treatment of oxidatively challenged oocytes with MQ also increased the co-localization of mitochondria and lysosomes, suggesting increased mitophagy. These data correlated with the developmental potential of the oocytes, as blastocyst development and hatching of oxidatively stressed oocytes were reduced, while treatment with MQ resulted in a significant increase in blastocyst development and hatching, and in the percentage of inner cell mass. Consistent with our results in mice, MII oocytes from women of AMA exhibited a significant decrease in phosphorylated-PKRN and total-PRKN compared to those of young women. Our findings suggest that the protein machinery to control the health of the mitochondria via UPRmt and mitophagy may be compromised in oocytes from aged females, which may result in inefficient clearance of dysfunctional mitochondria and reduced oocyte quality.

Estrogen signaling encourages blastocyst development and implantation potential.

PURPOSE: Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. METHODS: Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8 nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. RESULTS: ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. CONCLUSION: Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.

Euploid day 7 blastocysts of infertility patients with only slow embryo development have reduced implantation potential.

RESEARCH QUESTION: What is the reproductive potential of embryos that achieve blastulation on day 7 followed by preimplantation genetic testing for aneuploidies (PGT-A) for infertility patients with slow embryo development? DESIGN: This was a retrospective cohort study in a private IVF clinic of consecutive female infertility patients (n = 2966) aged 24-48 (36.3 ± 3.8) years who underwent frozen embryo transfer (FET) of a single euploid blastocyst. RESULTS: The women underwent single euploid FET of an embryo that achieved blastulation on day 5 (n = 1880), day 6 (n = 986) or day 7 (n = 100). Day 7 embryos resulted in lower implantation and live birth rates compared with both day 5 and day 6 embryos (P < 0.001). The day 5, day 6 and day 7 groups had 68.5%, 55.2% and 36.0% live birth rates, respectively. The day 7 group was older than the day 5 group (P < 0.001); comparing age-matched cohorts, the day 7 group still had lower implantation and live birth rates (P < 0.0001 and P < 0.001, respectively). Embryo grade was unrelated to live birth rates. Day 7 embryos of expansion grade 5 or 6 or trophectoderm grade A were more likely to be euploid compared with expansion grade 3 or trophectoderm grade B. CONCLUSIONS: Euploid day 7 embryos represented reduced implantation potential, even when controlling for maternal age. Of all day 7 embryos that underwent PGT-A, euploidy was associated with expansion grade 5 or 6 and trophectoderm grade A. These results can help providers manage patient expectations in cases where infertile women have slow embryo development.

Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Forecasting early onset diminished ovarian reserve for young reproductive age women.

PURPOSE: To investigate the biological networks associated with DOR in young women and the subsequent molecular impact on preimplantation embryos. METHODS: Whole peripheral blood was collected from patients: young women presenting with diminished ovarian reserve (DOR) and age-matched young women with normal ovarian reserve. Maternal exome sequencing was performed on the NovaSEQ 6000 and sequencing validation was completed using Taqman® SNP Genotyping Assays. Blastocyst global methylome and transcriptome sequencing were also analyzed. RESULTS: Exome sequencing revealed 730 significant DNA variants observed exclusively in the young DOR patients. Bioinformatic analysis revealed a significant impact to the Glucocorticoid receptor (GR) signaling pathway and each young DOR female had an average of 6.2 deleterious DNA variants within this pathway. Additional stratification based on patient age resulted in a cut-off at 31 years for young DOR discrimination. Embryonic global methylome sequencing resulted in only a very small number of total CpG sites with methylation alterations (1,775; 0.015% of total) in the DOR group. Additionally, there was no co-localization between these limited number of altered CpG sites and significant variants, genes, or pathways. RNA sequencing also resulted in no biologically significant transcription changes between DOR blastocysts and controls. CONCLUSION: GR signaling DNA variants were observed in women with early-onset DOR potentially compromising oocyte production and quality. However, no significant downstream effects on biological processes appear to impact the resulting blastocyst. The ability to forecast premature DOR for young women may allow for earlier identification and clinical intervention for this patient population.

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice.

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.

Egg cylinder development during in vitro extended embryo culture predicts the post transfer developmental potential of mouse blastocysts.

PURPOSE: To establish parameters during mouse extended embryo culture that accurately predict fetal developmental potential of a blastocyst without performing embryo transfer. METHODS: Embryos of three varying qualities were produced: poor quality embryos produced from in vitro matured oocytes (IVM), intermediate quality embryos produced from in vivo matured oocytes followed by in vitro fertilization and embryo culture (IVF); high quality embryos developed in vivo (VIVO). Embryonic day (E) 3.5 embryos from each group with similar morphologies were used for surgical embryo transfer to assess implantation and fetal developmental potential, in addition to placing these embryos into extended culture until E 8.5 to examine outgrowth area, egg cylinder volume, epiblast cell number, and outgrowth morphologies by immunofluorescence and 3D confocal microscopy. RESULTS: The proportional differences in epiblast cell number are strikingly similar to fetal development following embryo transfer, suggesting that this parameter may be indicative of the potential of an embryo to successfully develop into a fetus. CONCLUSION: Extended embryo culture provides more accurate information regarding developmental potential than blastocyst morphological assessment. Specifically, epiblast cell number is an accurate and valuable predictor of fetal developmental potential. This work sets the stage for routine evaluation of embryo quality past the time embryos would normally be transferred. The ability to determine post implantation potential without embryo transfer may greatly improve efforts to culture higher quality embryos in vitro for human IVF, as well as reducing animal use and eliminating confounding maternal factors associated with embryo transfer experiments in research.

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans†.

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

Compromised global embryonic transcriptome associated with advanced maternal age.

PURPOSE: To investigate the global transcriptome and associated embryonic molecular networks impacted with advanced maternal age (AMA). METHODS: Blastocysts derived from donor oocyte IVF cycles with no male factor infertility (< 30 years of age) and AMA blastocysts (≥ 42 years) with no other significant female factor infertility or male factor infertility were collected with informed patient consent. RNA sequencing libraries were prepared using the SMARTer® Ultra® Low Kit (Clontech Laboratories) and sequenced on the Illumina HiSEQ 4000. Bioinformatics included Ingenuity® Pathway Analysis (Qiagen) with ViiA™ 7 qPCR utilized for gene expression validation (Applied Biosystems). RESULTS: A total of 2688 significant differentially expressed transcripts were identified to distinguish the AMA blastocysts from young, donor controls. 2551 (95%) of these displayed decreased transcription in the blastocysts from older women. Pathway analysis revealed three altered molecular signaling networks known to be critical for embryo and fetal development: CREBBP, ESR1, and SP1. Validation of genes within these networks confirmed the global decreased transcription observed in AMA blastocysts (P < 0.05). CONCLUSIONS: A significant, overall decreased global transcriptome was observed in blastocysts from AMA women. The ESR1/SP1/CREBBP pathway, in particular, was found to be a highly significant upstream regulator impacting biological processes that are vital during embryonic patterning and pre-implantation development. These results provide evidence that AMA embryos are compromised on a cell signaling level which can repress the embryo's ability to proliferate and implant, contributing to a deterioration of reproductive outcomes.

A carnivore embryo's perspective on essential amino acids and ammonium in culture medium: effects on the development of feline embryos.

Carnivores are an interesting model for studies of embryonic amino acid metabolism and ammonium (NH4+) toxicity given the high-protein content of their diets. Our objectives were to examine concentration- and stage-specific effects of essential amino acids (EAA; 0×, 0.125×, 0.25×, 0.5×, or 1.0× the concentrations in Minimum Essential Medium) and NH4+ (0, 300, or 600 µM) on the development and metabolism of feline embryos. The presence of EAA, regardless of concentration, during days 3-7 of culture increased (P < 0.01) the proportion of embryos that initiated hatching (>14.3%) and the total number of cells per blastocyst (>148.3 cells) compared to embryos cultured without EAA (0.0% and 113.2 ± 3.7 cells, respectively). The presence of EAA during days 1-3 (0.25×) and 3-7 (1.0×) of culture increased (P < 0.01) the proportions of embryos that formed blastocysts (82.9 ± 4.2%) and initiated hatching (32.9 ± 5.2%), and the number of cells per blastocyst (247.9 ± 12.1 cells), compared to control embryos (60.0 ± 5.3%, 0.0%, 123.2 ± 8.1 cells, respectively). The presence of NH4+ in the medium did not affect (P > 0.05) development of feline embryos. The addition of EAA or NH4+ during culture did not affect (P > 0.05) the production of Gln by feline embryos, but decreased (P < 0.05) production of Ala and increased (P < 0.05) production of urea. Additional work is needed to determine if our observations are unique to feline embryos or reflect an adaptation to a high-protein diet that is conserved in other carnivores.

The impact of infertility diagnosis on embryo-endometrial dialogue.

Initial stages of implantation involve bi-directional molecular crosstalk between the blastocyst and endometrium. This study investigated an association between infertility etiologies, specifically advanced maternal age (AMA) and endometriosis, on the embryo-endometrial molecular dialogue prior to implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts (n = 41 ≥ Grade 3BB) donated from patients presenting with AMA or endometriosis, compared to fertile donor oocyte controls. Extracellular vesicles isolated from co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Specifically, AMA resulted in 16 miRNAs with increased expression (P ≤ 0.05) and strong evidence for negative regulation toward 206 target genes. VEGFA, a known activator of cell adhesion, displayed decreased expression (P ≤ 0.05), validating negative regulation by 4 of these increased miRNAs: miR-126; 150; 29a; 29b (P ≤ 0.05). In endometriosis patients, a total of 10 significantly altered miRNAs displayed increased expression compared to controls (miR-7b; 9; 24; 34b; 106a; 191; 200b; 200c; 342-3p; 484) (P ≤ 0.05), targeting 1014 strong evidence-based genes. Three target genes of miR-106a (CDKN1A, E2F1 and RUNX1) were independently validated. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation (P ≤ 0.05). These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue and may be prospective biomarkers of implantation success. One of the limitations of this study is that it was a stimulated, in vitro model and therefore may not accurately reflect the in-vivo environment.

Exogenous growth factors do not affect the development of individually cultured murine embryos.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 µl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P < 0.05) effect of growth factors was a decrease in the proportion of embryos that formed blastocysts by day 5 in CF1 IVM embryos. The presence of growth factors also did not affect blastocyst cell numbers. For CF1 IVO embryos, the presence of growth factors during culture did not affect the proportion of embryos that attached to fibronectin-coated dishes, the size of the resulting outgrowths, or in vivo development following transfer. CONCLUSION: Combinations of paf, EGF, GM-CSF, and IGF-1 did not improve development of murine embryos cultured individually in a sequential medium containing a defined protein source.

Infertility diagnosis has a significant impact on the transcriptome of developing blastocysts.

STUDY QUESTION: Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)? SUMMARY ANSWER: The global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis. WHAT IS KNOWN ALREADY: Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development. STUDY DESIGN, SIZE, DURATION: Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control). PARTICIPANTS/MATERIALS, SETTING, METHODS: Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were <38 years old with male patients <40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR). MAIN RESULTS AND THE ROLE OF CHANCE: Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9) and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold). LARGE SCALE DATA: Not available due to participant privacy. LIMITATIONS, REASONS FOR CAUTION: Blastocyst samples for microarray analysis required pooling. While this allows for an overall average in each infertility etiology group and can reduce noise from sample-to-sample variation, it cannot give a detailed analysis of each blastocyst within the group. WIDER IMPLICATIONS OF THE FINDINGS: Underlying patient infertility diagnosis has an impact on the blastocyst transcriptome, modifying gene expression associated with developmental competence and implantation potential. STUDY FUNDING AND COMPETING INTEREST(S): No conflict of interest or outside funding provided.

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

STUDY QUESTION: Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? SUMMARY ANSWER: A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. WHAT IS KNOWN ALREADY: Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. LIMITATIONS, REASONS FOR CAUTION: Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. WIDER IMPLICATIONS OF THE FINDINGS: IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. LARGE SCALE DATA: N/A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles.

STUDY QUESTION: Is there a distinct sperm histone-retained epigenetic signature in unexplained male factor infertility patients resulting in compromised blastocyst development? SUMMARY ANSWER: Using only donor oocyte IVF cycles, sperm DNA methylation patterns and miRNA profiles were significantly altered in normozoospermic patients resulting in poor blastocyst development, reflecting a subset of unexplained male factor infertility. WHAT IS KNOWN ALREADY: Aberrant sperm DNA methylation has been associated with known male factor infertility, particularly noted in oligozoospermic patients. Unexplained male factor infertility remains a significant proportion of in vitro fertilization failures having unknown underlying physiology. STUDY DESIGN, SIZE, DURATION: Sperm samples (n = 40) and blastocysts (n = 48) were obtained during fertile donor oocyte IVF cycles with normozoospermic parameters, thereby excluding known female and male infertility factors. Samples were divided into two groups based on blastocyst development (Good Group = ≥20% embryos with D5 grade 'AA' blastocysts, and ≥60% embryos of transferable quality on D5 and D6; Poor Group = ≤10% embryos with D5 grade 'AA' blastocysts, and ≤40% embryos of transferable quality on D5 and D6). PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were obtained from patients undergoing IVF treatments with informed consent and institutional review board approval. The Infinium HumanMethylation450 BeadChip microarray was used to identify histone-retained CpG island genes and genomic regions showing differences in sperm DNA methylation between the Good Group and the Poor Group. Pathway and gene network analysis for the significantly altered genes was performed, and targeted DNA methylation validation was completed for 23 genes and two imprinting control regions. Sperm miRNA profiles were assessed using the TaqMan® Human MicroRNA Array Card, with corresponding blastocyst mRNA gene expression examined by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Our study is the first to investigate unexplained male factor infertility while significantly eliminating confounding female factors from our sample population by using only young fertile donor oocytes. We identified 1634 CpG sites located at retained histone CpG island regions that had significant sperm DNA methylation differentials between the two embryogenesis groups (P < 0.05). A largely hypermethylated profile was evident in the Good Group, with a small but distinct and statistically significant shift (P < 0.05) observed in the Poor Group. Genes involved in embryonic development were highly represented among histone-retained CpG sites with decreased methylation in the Poor Group (P < 0.05). Ten significantly altered sperm miRNAs (P < 0.05), correlated with altered target gene mRNA expression in the blastocysts from the Poor Group (P < 0.05). Taken together, significantly impacted sperm miRNA and target transcript levels in blastocysts from the Poor Group may contribute alongside aberrant sperm DNA methylation to the compromised blastocyst development observed. LIMITATIONS, REASONS FOR CAUTION: Our examination of CpG island regions restricted to retained histones represents only a small part of the sperm epigenome. The results observed are descriptive and further studies are needed to elucidate the functional effects of differential sperm DNA methylation on unexplained male factor infertility and blastocyst development. WIDER IMPLICATIONS OF THE FINDINGS: Slight epigenetic changes in sperm may have a cumulative effect on fertility and embryonic developmental competence. Knowledge of sperm epigenetics and inheritance has important implications for future generations, while providing evidence for potential causes of unexplained male factor infertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. None of the authors have any competing interest.

GnRH agonist administration prior to embryo transfer in freeze-all cycles of patients with endometriosis or aberrant endometrial integrin expression.

Prolonged gonadotrophin-releasing hormone agonist (GnRHa) administration before IVF with fresh embryo transfer to patients with endometriosis or aberrant endometrial integrin expression (-integrin) improves outcomes but may suppress ovarian response and prevents elective cryopreservation of all embryos. This retrospective cohort pilot study evaluates freeze-all cycles with subsequent prolonged GnRHa before embryo transfer in these populations. Patients from 2010 to 2015 who met inclusion criteria and received a long-acting GnRHa every 28 days twice before FET were evaluated. A subset underwent comprehensive chromosomal screening (CCS) after trophectoderm biopsy. Three groups were identified: Group 1: + CCS, +endometriosis (20 patients, 20 transfers); Group 2: +CCS, -integrin (12 patients, 13 transfers); Group 3: no CCS, +endometriosis or -integrin (10 patients, 12 transfers); Group 4: all transfers after CCS for descriptive comparison only (n = 2809). Baseline characteristics were similar among Groups 1-3 except that the mean surgery to oocyte aspiration interval was longer for Group 1 than Group 3. Implantation and ongoing pregnancy rates were statistically similar among the three groups and compared favourably to Group 4. A non-significant trend towards improved outcomes was noted in Group 1. Prolonged GnRHa after freeze-all in these patients avoids excessive ovarian suppression and results in excellent outcomes.