Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

PURPOSE/STUDY QUESTION: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? METHODS: Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). RESULTS: Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). CONCLUSION: The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Identifying parental and cell-division origins of aneuploidy in the human blastocyst.

Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%-100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n = 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.

Accurate detection and frequency of abnormal ploidy in the human blastocyst.

OBJECTIVE: To validate the detection of abnormal ploidy in preimplantation embryos and evaluate its frequency in transferrable blastocysts. DESIGN: A high-throughput genome-wide single nucleotide polymorphism microarray-based preimplantation genetic testing (PGT) platform was validated using multiple positive controls, including cell lines of known haploid and triploid karyotypes and rebiopsies of embryos with initial abnormal ploidy results. This platform was then tested on all trophectoderm biopsies in a single PGT laboratory to calculate the frequency of abnormal ploidy and the parental and cell division origins of error. SETTING: Preimplantation genetic testing laboratory. PATIENT(S): The embryos from in vitro fertilization patients who elected for PGT were evaluated. Any patients who provided saliva samples were further analyzed for the parental and cell division origins of abnormal ploidy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Evaluable positive controls showed 100% concordance with original karyotypes. The overall frequency of abnormal ploidy within a single PGT laboratory cohort was 1.43%. RESULT(S): All cell lines showed 100% concordance with the expected karyotype. Additionally, all evaluable rebiopsies showed 100% concordance with the original abnormal ploidy karyotype. The frequency of abnormal ploidy was 1.43%, with 29% of those being haploid or uniparental isodiploid, 2.5% uniparental heterodiploid, 68% triploid, and 0.4% tetraploid. Twelve haploid embryos contained maternal deoxyribonucleic acid, and 3 contained paternal deoxyribonucleic acid. Thirty-four triploid embryos were of maternal origin, and 2 were of paternal origin. Thirty-five triploid embryos had a meiotic origin of error, and 1 was of mitotic error. Of those 35 embryos, 5 originated from meiosis I, 22 originated from meiosis II, and 8 were deemed inconclusive. On the basis of specific abnormal ploidy karyotypes, 41.2% of embryos would be falsely classified as euploid, and 22.7% would be false-positive mosaics with the use of the conventional next-generation sequencing-based PGT methods. CONCLUSION(S): This study demonstrates the validity of a high-throughput genome-wide single nucleotide polymorphism microarray-based PGT platform to accurately detect abnormal ploidy karyotypes and predict the parental and cell division origins of error of evaluable embryos. This unique method improves the sensitivity of detection for abnormal karyotypes, which can reduce the chances of adverse pregnancy outcomes.

Long-term mental health treatment on campus; a report from the University of Southern California.

The demand for mental health treatment on university campuses continues to increase. This report will outline sources of increased demand and how traditional campus-based models have managed it. It will then describe the University of Southern California's work mobilizing resources to develop a unique model for insurance based long-term care emphasizing the importance of collaboration between long-term and short-term resources. Finally, this report will suggest interventions that may help to ease the burden of increased demand for mental health treatment on campuses.