Jacqueline R. Ho

Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos

OBJECTIVE To determine the accuracy of cell-free DNA (cfDNA) in spent embryo medium (SEM) for ploidy and sex detection at the cleavage and blastocyst stages. To determine if assisted hatching (AH) and morphologic grade influence cfDNA concentration and accuracy. DESIGN Prospective cohort. SETTING Academic fertility center. PATIENT(S) Nine patients undergoing IVF; 41 donated two-pronuclei embryos and 20 embryos from patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). INTERVENTIONS(S) In a donated embryo arm, SEM was collected on days 3 and 5, with one-half of the embryos undergoing AH before and one-half after. In a clinical arm, SEM was collected on day 5 before trophectoderm (TE) biopsy. Samples underwent PGT-A with the use of next-generation sequencing. cfDNA results were compared with corresponding whole embryos and TE biopsies. MAIN OUTCOME MEASURE(S) Concordance rates, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for ploidy and sex detection with the use of cfDNA. RESULT(S) Of 141 samples, cfDNA was amplified in 39% and 80.4% of days 3 and 5 SEM, respectively. Concordances for ploidy and sex, respectively, were 56.3% and 81.3% between day 3 cfDNA and whole embryos, and 65% and 70% between day 5 cfDNA and TE biopsies. Day 5 cfDNA sensitivity and specificity for aneuploidy were 0.8 and 0.61, respectively. PPV and NPV were 0.47 and 0.88, respectively. Timing of AH and morphology did not influence cfDNA concentration or accuracy. CONCLUSION(S) cfDNA is detectable on days 3 and 5, but more accurate on day 5. Although our data suggest moderate concordance rates, PGT-A with the use of cfDNA must be further optimized before clinical implementation.

Blastulation timing is associated with differential mitochondrial content in euploid embryos

PurposePreimplantation genetic screening (PGS) and assessment of mitochondrial content (MC) are current methods for selection of the best embryos for transfer. Studies suggest that time-lapse morphokinetics (TLM) may also be helpful for selecting embryos more likely to implant. In our study, we sought to examine the relationship between TLM parameters and MC to determine if they could be used adjunctively in embryo selection. We also examined the relationship between MC with ploidy and blastulation.MethodsCryopreserved human embryos at the zygote stage were thawed and cultured in a time-lapse system. Blastomere and trophectoderm biopsies were performed on days 3 and 6. Biopsied cells and all whole embryos from day 6 were analyzed for MC (ratio of mitochondrial to nuclear DNA) and ploidy using next-generation sequencing.ResultsIn embryos, MC per cell declined between day 3 and day 6. While early cleavage parameters did not predict MC, embryos with longer blastulation timing had higher MC on day 6. Day 6 MC was lower in euploid vs. aneuploid embryos and lower in blastocysts vs. arrested embryos.ConclusionsA lower MC at the blastocyst stage was associated with euploid status and blastocyst formation, indicating better embryo quality compared to those with a higher MC. Higher MC in aneuploid and arrested embryos may be explained by slower cell division or degradation of genomic DNA over time. Blastulation timing may be helpful for selection of higher quality embryos. Combining blastulation timing and MC along with morphologic grading and euploid status may offer a new direction in embryo selection.

Clomiphene Stair-Step Protocol for Women With Polycystic Ovary Syndrome

OBJECTIVE To compare time to ovulation, ovulation rates, and side effect profile of traditional and the stair-step protocol for ovulation induction using clomiphene citrate in women with polycystic ovary syndrome (PCOS). METHODS We performed a retrospective study of women seeking care for infertility with a diagnosis of PCOS at a university-based infertility clinic from July 2012 to July 2014. We included patients who were resistant to the initial starting dose of 50 mg clomiphene. The primary outcome was time to ovulation. Secondary outcomes included ovulation rates, clinical pregnancy rates, and mild and moderate-to-severe side effects based on dose. For the traditional protocol, higher doses of clomiphene were used each subsequent month if no ovulation occurred. For the stair-step protocol, higher doses of clomiphene were given 7 days after the last dose if no dominant follicles were seen on ultrasonography. Our study had 80% power to detect a 20% difference in ovulation. RESULTS One hundred nine patients were included in the analysis with 66 (60.6%) in the traditional and 43 (39.4%) in the stair-step protocol. Age and body mass index were similar between groups. The time to ovulation was decreased in the stair-step protocol group compared with the traditional protocol group (23.1±0.9 days vs 47.5±6.3 days). Ovulation rates were increased in the stair-step group compared with the traditional group at 150 mg (16 [37%] vs 8 [12%], P=.004) and at 200 mg (9 [21%] vs 3 [5%], P=.01). Pregnancy rates were similar between groups once ovulation was achieved (12 [18.1%] vs 7 [16.3%], P=.08). The stair-step protocol had an increased incidence of mild side effects (vasomotor flushes, headaches, gastrointestinal disturbance, mastalgia, changes in mood; 18 [41%] vs 8 [12%]), but there was no difference in the incidence of severe side effects (headaches, visual disturbances). CONCLUSION For women with PCOS, the stair-step clomiphene protocol is associated with decreased time to ovulation and increased ovulation rates at higher doses when compared with the traditional protocol.