A workflow for simultaneous DNA copy number and methylome analysis of inner cell mass and trophectoderm cells from human blastocysts.

OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.

Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.

Study question: What is the predictive value of trophectoderm mitochondrial DNA (mtDNA) quantity for blastocyst reproductive potential? Summary answer: This study demonstrates that, within a given cohort, mtDNA quantitation does not distinguish between embryos that implant and embryos that do not implant after double embryo transfer (DET). What is already known: An association between implantation failure and increased quantities of mtDNA has been observed in two studies but not in a third. Study design, size and duration: A total of 187 patients (nine who received donor oocytes) with DET of one male and one female euploid blastocyst were included in this retrospective study, with 69 singleton deliveries providing the primary dataset to evaluate the predictive value of mtDNA for reproductive potential between January 2010 and July 2016. Participants/materials, setting and method: MtDNA was quantified in cell lines to validate the quantitative PCR assay on limited quantities of starting material and then applied to 374 blastocyst biopsies. Pregnancies resulting in a singleton outcome were analyzed and newborn gender was utilized as a means to identify the implanted embryo. MtDNA quantity was then compared between implanted and non-implanted embryos in order to define the predictive value of mtDNA content for reproductive potential in this subset of patients. Main results and the role of chance: An initial comparison of mtDNA levels between all successful and unsuccessful embryos revealed no significant differences. In order to control for patient-specific variables, gender was subsequently used to identify the implanted embryo in DETs resulting in a singleton (n = 69). No systematic difference in relative mtDNA quantity was detected between implanted and non-implanted embryos. Limitations, reasons for caution: This study was conducted at a single center and did not evaluate the entire cohort of embryos from each patient to evaluate cohort specific variation in mtDNA quantity. Although the largest of its kind so far, the sample size of DETs leading to a singleton was relatively small. Wider implications of the findings: These data highlight the importance of control over patient-specific variables when evaluating candidate biomarkers of reproductive potential. All current available data suggest that mtDNA quantification needs further study before its clinical use to augment embryo selection. Study funding/competing interests: The authors have no potential conflict of interest to declare. No external funding was obtained for this study. Trial registration number: Not applicable.

Embryonic aneuploidy does not differ among genetic ancestry according to continental origin as determined by ancestry informative markers.

STUDY QUESTION: Is embryonic aneuploidy, as determined by comprehensive chromosome screening (CCS), related to genetic ancestry, as determined by ancestry informative markers (AIMs)? SUMMARY ANSWER: In this study, when determining continental ancestry utilizing AIMs, genetic ancestry does not have an impact on embryonic aneuploidy. WHAT IS KNOWN ALREADY: Aneuploidy is one of the best-characterized barriers to ART success and little information exists regarding ethnicity and whole chromosome aneuploidy in IVF. Classifying continental ancestry utilizing genetic profiles from a selected group of single nucleotide polymorphisms, termed AIMs, can determine ancestral origin with more accuracy than self-reported data. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study of patients undergoing their first cycle of IVF with CCS at a single center from 2008 to 2014. There were 2328 patients identified whom had undergone IVF/CCS and AIM genotyping. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent IVF/ICSI and CCS after trophectoderm biopsy. Patients' serum was genotyped using 32 custom AIMs to identify continental origin. Admixture proportions were determined using Bayesian clustering algorithms. Patients were assigned to the population (European, African, East Asian or Central/South Asian) corresponding to their greatest admixture proportion. MAIN RESULTS AND THE ROLE OF CHANCE: The mean number of embryos tested was 5.3 (range = 1-40) and the mode was 1. Patients' ethnic classifications revealed European (n = 1698), African (n = 103), East Asian (n = 206) or Central/South Asian (n = 321). When controlling for age and BMI, aneuploidy rate did not differ by genetic ancestry (P = 0.28). LIMITATIONS, REASONS FOR CAUTION: The study type (retrospective) and the ability to classify patients by continental rather than sub-continental origin as well as the predominantly European patient mix may impact generalizability. Post hoc power calculation revealed power to detect a 16.8% difference in embryonic aneuploidy between the two smallest sample size groups. WIDER IMPLICATIONS OF THE FINDINGS: These data do not support differences in embryonic aneuploidy among various genetic ancestry groups in patients undergoing IVF/CCS. We used a novel approach of determining continental origin using a validated panel of AIMs as opposed to patient self-reported ethnicities. It does not appear that specific recommendations for aneuploidy screening should be made based upon continental heritage. STUDY FUNDING/COMPETING INTERESTS: None.

Genotypically determined ancestry across an infertile population: ovarian reserve and response parameters are not influenced by continental origin.

OBJECTIVE: To evaluate the relationship between genetic ethnicity using ancestry informative markers (AIMs) and ovarian reserve and response parameters as evidenced by FSH, antimüllerian hormone (AMH), basal antral follicle count (BAFC), and total oocyte yield in IVF. DESIGN: Retrospective. SETTING: Academic medical center. PATIENTS(S): A total of 2,508 infertile patients undergoing IVF at a single center. INTERVENTION(S): Patients were genotyped for 32 AIMs and analyzed for differences in allele frequencies. A validated Bayesian clustering algorithm was then used to assign individuals into one of four ethnic populations: European, African, Central/South Asian, or East Asian. MAIN OUTCOME MEASURE(S): FSH, AMH, BAFC, and oocyte yield variation. RESULT(S): After controlling for age and body mass index, genetic ethnicity had no impact on AMH, BAFC, and oocyte yield. FSH was found to be lower in patients of Central/South Asian ancestry (6.46 ng/mL vs. 6.97 ng/mL); however, the absolute difference is of little clinical significance. Subgroup analyses of 1,327 patients restricted to those with limited genetic admixture as determined by AIMs indicated that FSH, AMH, BAFC, and oocyte yield were equivalent. CONCLUSION(S): When determining ethnicity using AIMs, ethnic background does not have an impact on markers of ovarian reserve or ovarian response. Specifically, no differences were found in AMH, BAFC, or oocyte yield relative to genotypic ethnicity. Using AIMs rather than self-reported ethnicity allows for elimination of reporting biases and nonreporting of ethnicity, which can confound data. Based upon these data, specific recommendations for ovarian reserve testing should thus be made based on other factors besides ethnic background.

Expanded carrier screening in an infertile population: how often is clinical decision making affected?

PURPOSE: Options for preconception genetic screening have grown dramatically. Expanded carrier screening (ECS) now allows for determining carrier status for hundreds of genetic mutations by using a single sample, and some recommend ECS prior to in vitro fertilization. This study seeks to evaluate how often ECS alters clinical management when patients present for infertility care. METHODS: All patients tested with ECS at a single infertility care center from 2011 to 2014 were evaluated. The overall rate of positive ECS results and the number of couples who were carriers of the same genetic disorder were evaluated. RESULTS: A total of 6,643 individuals were tested, representing 3,738 couples; 1,666 (25.1%) of the individuals had a positive test result for at least one disorder. In 8 of the 3,738 couples, both members of the couple were positive for the same genetic disorder or had a test result that placed them at risk of having an affected child. Three of eight cases were cystic fibrosis. In this cohort, ECS affected clinical care eight times after 6,643 tests (0.12%, confidence interval: 0.05-0.24%) in 3,738 couples (0.21%, confidence interval: 0.09-0.42%). CONCLUSIONS: ECS is becoming more widespread. In a large case series, ECS affected clinical decision making for patients presenting for infertility care in 0.21% of cases. This information must be weighed when utilizing these tests and may be a helpful part of patient counseling.Genet Med 18 11, 1097-1101.

Effect of administration of caffeine or green tea on the mutation profile in the p53 gene in early mutant p53-positive patches of epidermal cells induced by chronic UVB-irradiation of hairless SKH-1 mice.

Irradiation of SKH-1 mice with UVB light for 20 weeks resulted in a large number of patches of epidermal cells, which was visualized with an antibody that recognizes mutated p53 protein. Oral treatment of mice with caffeine (0.4 mg/ml) or green tea (6 mg tea solids/ml) as the drinking fluid during UVB irradiation decreased the number of patches by approximately 40%. Sequencing analysis of the p53 gene (exons 3 to 9) detected 88, 82 or 39 point mutations in 67, 70 or 29 patches from water, caffeine or tea treated mice, respectively. A major hotspot at codon 270 (Arg-->Cys) accounted for 47.7% (water), 70.7% (caffeine) or 46.2% (tea) of all mutations. Patches from caffeine treated mice had fewer types of mutations than patches from mice treated with water or tea. Administration of caffeine or tea during 20 weeks of UVB irradiation eliminated mutations at codons 149 (Pro-->Ser) and 210 (Arg-->Cys) but increased the frequency of mutations at codon 238 (Ser-->Phe). Topical applications of caffeine (1.2 mg in 100 microl acetone) once a day, five times a week for 6 weeks after stopping UVB decreased the number of patches by 63% when compared with mice treated with acetone. DNA sequencing analysis detected 63 and 68 mutations in 48 and 57 patches from acetone or caffeine treated mice, respectively. Although no differences in the frequency, position or types of mutations were observed, the caffeine group harbored less homozygous mutations (12.3% of the total) than the acetone group (31.3% of the total, P = 0.029). In summary, oral treatment of mice with caffeine or green tea during chronic UVB irradiation changed the mutation profile of the p53 gene in early mutant p53 positive epidermal patches, and topical applications of caffeine after discontinuation of chronic UVB irradiation specifically eliminated patches harboring homozygous p53 mutations.