Background PGT-A with Next Generation Sequencing (NGS) is becoming the standard of care, and the number of blastocysts biopsied is increasing. However, there are a small percentage of blastocysts where a result is not obtained from the initial genetic analysis. The immediate desire is to often proceed with reanalysis by warming/rebiopsy/revitrification (WrBrV) for repeat PGT-A analysis. We investigated the efficacy of subjecting previously biopsied blastocysts to WrBrV in a second attempt to obtain genetic results. Objective To determine whether it is laboratory efficient to attempt WrBrV in order to obtain a PGT-A result from a previously biopsied blastocysts. Materials and Methods Previously biopsied and vitrified blastocysts were warmed using our standard protocol (Irvine Scientific Thaw). Assessment of blastocoel cavity expansion was performed ∼2 hours post warming; followed by trophectoderm rebiopsy, where an additional 3-5 cells were excised. The biopsied cells were placed in PCR tubes for analysis and the blastocysts were immediately revitrified. Results From January 2018 through February 2019, PGT-A was performed on trophectoderm cells from 2715 embryos. A ‘No result' was obtained from 33 embryos (1.2%); and all were Good and Fair quality Hatching Blastocyst (HB-G, n=5; HB-F, n=28). Physicians ordered WrBrV for only 12 blastocysts (36%). Two embryos degenerated following warming and were not rebiopsied (17%). The remaining 10 blastocysts underwent biopsy and genetic results were obtained from all. Only two blastocysts were diagnosed as euploid (20%); and a Frozen Embryo Transfer (FET) was performed with a single euploid blastocyst, but resulted in a failed implantation. Conclusions At our center, FETs performed with single euploid blastocysts result in a 69% pregnancy rate. This demonstrates that the biopsy, vitrification and warming techniques as performed by the embryologists at our center do not significantly diminish the potential of euploid embryos to implant. Following an initial PGT-A with no NGS result (1.2%), the process of WrBrV in our laboratory has resulted in an 83% survival rate; however only 20% of embryos have been euploid and 0% have implanted following FET, demonstrating the clinical significance and laboratory efficiency is extremely low. This study, although small in size and in need of further investigation, supports that embryos with no initial NGS result, although of good/fair quality and available for WrBrV, have a very high incidence of aneuploidy and may not warrant additional laboratory resources and reanalysis; especially when other euploid embryos are available.

ObjectiveRecent data suggests that the rate of mosaic diagnosed embryos is increasing following Preimplantation Genetic Testing for Aneuploidy (PGT-A) when using Next Generation Sequencing (NGS). Trophectoderm cell biopsy by laser ablation is proposed as a possible cause of mosaic diagnoses resulting in the subsequent removal of possible genetically normal embryos from availability for embryo transfer. This study investigates whether repeated high intensity laser pulses during trophectoderm laser ablation alters the genetic results or causes increased mosaicism.DesignSeveral trophectoderm biopsied cell samples from each embryo were exposed to either repeated laser pulses or increasingly intense pulses prior to NGS analysis for genetic results.Material and MethodsEmbryos were obtained from patients that consented to discarding their surplus embryos. Two genetically undiagnosed blastocysts were used for this study. Each embryo was subjected to low and high intensity laser pulses for varied pulse durations using the Hamilton Thorne Lykos laser. From both embryos 3-5 biopsied cell samples were obtained for each of the laser test categories. Samples were then exposed to direct and repeated laser pulses for 10 or 20 pulses @ 290µs; or 20 or 40 laser pulses @ 400µs. One additional sample, served as the control, and was exposed only to the standard biopsy procedure of 5 pulses @ 290µs. All samples were then washed through several washing buffer droplets prior to placement in PCR tubes in 2-3µl buffer and frozen at -20°C until NGS analysis was performed.ResultsThis study demonstrated that genetic analysis results obtained from the control and increased intensity samples show no difference, with all providing normal genetic results (46, XY) (Table 1). Importantly, no mosaic genetic results were observed even at the maximum number of pulses or intensity level.ConclusionThis study provides significant evidence that multiple laser pulses, even with increased laser intensities, do not alter the genetic results obtained when performing PGT-A. Additionally, the laser intensity does not cause incidences of mosaicism in the NGS results. To minimize potential damage to the embryo and the biopsied cells, optimal excision of the trophectoderm should be achieved using a limited number of laser pulses at the lowest intensity possible. It is important to note, that extreme repeated laser pulses, with increased intensity, may have a detrimental impact on the actual embryo which could reduce implantation potential of the embryo itself.

ObjectiveMitochondria are cellular power houses acting as the principal site for ATP production. Mitochondrial function, mtDNA gene expression, and energy are required to undergo necessary cellular divisions in the formation of a high quality embryo. Unlike other cellular organelles, mitochondria contain their own DNA (mtDNA). It is known that mitochondrial functions are imperative during preimplantation development, however the quantity of mtDNA an embryo requires is unknown. Our current best practices for embryo selection include day of development, morphology grade and preimplantation genetic testing for aneuploidy (PGT-A). In this study we compared clinical pregnancy rates of our current protocols for embryo selection and any relationship to the Mitoscore ranking.DesignRetrospective analysis of clinical outcomes as they associate with laboratory characteristics for embryo development, embryo quality and Mitoscore ranking.Materials & MethodsDuring days 5-7 of embryo development, trophectoderm biopsy was performed on blastocysts by an embryologist using a laser to remove 3-5 trophectoderm cells for PGT-A. The biopsied cells are washed through buffer media, loaded into PCR tubes and frozen in a -20°C freezer until analysis. Whole genome amplification and next generation sequencing (NGS) using the Ion ReproSeq PGS Kit for 24 chromosome aneuploidy screening using the Ion Reporter software was performed. The genetic analysis uses complex algorithms to delineate mtDNA scores (Mitoscore). Frozen single euploid embryo transfer decisions were based on embryo developmental day and overall embryo quality grading criteria but did not include Mitoscore ranking. Pregnancy outcomes were analyzed and assessed as they associated with the Mitoscore number and its rank in the cohort.ResultsFrom this study we learned that using our current embryo selection criteria, excluding Mitoscores, resulted in only a 64.4% overall pregnancy rate. However, in embryos with Mitoscore values <25 we had a significantly improved pregnancy rate at 85.2% (p<0.001) and achieved the highest implantation at 90.9% with Mitoscores <20 (trending toward significance with a p=0.067) (Table 1).ConclusionsCorrelation with the Mitoscore data demonstrated that <20 provided the best overall pregnancy rate and that <25 significantly predicted pregnancy success (p<0.001). This analysis confirmed that lower Mitoscore values can be used as biomarkers for embryo quality and can serve as a complimentary selection tool for achieving higher clinical pregnancy rates.

Although PGT-A with Next Generation Sequencing (NGS) is becoming the standard of care, there are a large number of blastocysts that were frozen prior to having embryo biopsy and genetic analysis. As a result, there are an increasing number of patients requesting genetic information of their previously undiagnosed frozen blastocysts. It is important to understand how these embryos perform following warming (thaw), biopsy and revitrification (refreeze) for PGT-A analysis; and their ability to initiate pregnancies following re-warming. To determine whether it is clinically beneficial to attempt warm/biopsy/revitrify (WBRV) to obtain a PGT-A result from a previously untested frozen blastocyst. Previously untested frozen blastocysts were warmed using our standard protocol (Irvine Scientific Thaw). Assessment of blastocoel cavity expansion was performed 2 hours post warming, or following overnight incubation; followed by trophectoderm biopsy, where 3-5 cells were excised using a Lykos Laser. The biopsied cells were placed in PCR tubes for analysis and the blastocysts were immediately revitrified. Warmed blastocysts (n=61) were biopsied 90% of the time (n=55) with genetic results obtained from all. All Hatching Blastocysts (HB), Expanded Blastocysts (EXPB) and Early Blastocysts (EB) of Good, Fair and Poor quality are listed on Table 1; with their associated biopsy and ploidy status following WBRV. A 67% pregnancy rate (n=6) was obtained when good and fair quality euploid blastocysts (n=7) were transferred.Table 1Results of Blastocysts following WBRVDevelopmental Stage and GradeNo. Of Embryos ThawedNo. BiopsiedPercentage BiopsiedNormal Genetic ResultsPercentage Normal ResultsHB-Good1818100.00%1372.22%HB-Fair1616100.00%956.25%HB-Poor3133.33%00.00%EXPB- Good1616100.00%850.00%EXPB- Fair7342.86%133.33%EB-Fair11100.00%1100.00%Total615590.16%3258.18% Open table in a new tab A blastocysts' grading appears to be the best predictor for an embryo to be biopsied and euploid, as the majority of poor quality embryos were unable to be biopsied, even with an extra day in culture, or were aneuploid. This study demonstrates that IVF Programs should strive to only vitrify good quality blastocysts, in order to provide the best opportunity for the highest success for patients' outcomes. Euploid good and fair quality embryos that undergo WBRV prior to FET were found to have a similar pregnancy rate (67%), when compared to transferring euploid embryos warmed only once at our center (69%). As a result, it appears that it is not detrimental to good quality euploid blastocysts that have been vitrified to undergo WBRV. This should alleviate concerns associated with subjecting embryos to 2 possible vitrifications and warmings. Other IVF programs should investigate this process to determine their rates for WBRV in order to assist physicians in counseling patients regarding the percentage of embryos that could be available for biopsy; along with the expected euploidy and pregnancy rates.

Objective: It is not standard of care to screen IVF patients for translocations due to their low existence in the general population at ~0.2%. However, the percentage of carriers of balanced translocations may be much higher in the infertile population and they are most likely unaware of their condition. We describe the clinical and laboratory outcomes of an advanced maternal age (AMA) couple seeking infertility care, and the inadvertent discovery of a male partner translocation. Design: We describe a single case study of a rare preimplantation genetic screening (PGS) result, which could have been avoided with an update to genetic pre-screening for any patients initiating expensive IVF cycles. Materials and Methods: A couple's case files were reviewed and are described to demonstrate the discovery of a male translocation carrier following a donor oocyte IVF/PGS cycle. Results: The couple: 40-year old, G1, P0 (SAB) female with unremarkable hormonal/ultrasonic findings; and a low teratozoospermic, otherwise unremarkable male. The male had a normal genetic carrier screening panel. The couple was counseled about AMA and underwent an autologous IVF cycle with PGS. Two blastocysts developed, they were biopsied and the genetic results were: Monosomy 16/Trisomy 21 and Trisomy 3/Monosomy 16. The couple decided to proceed with donor oocytes for a subsequent cycle with PGS to transfer only a single normal embryo. In the donor cycle, using partner sperm, 20 blastocysts developed, they were biopsied and the PGS results showed 6 normal embryos (30%); 11 embryos (55%) with abnormalities always involving chromosomes 3 and/or 16 (monosomies, trisomies and complex abnormal). Very low numbers of normal embryos from an oocyte donor and repeated abnormalities in chromosomes 3 and 16 from both the autologous and donor cycles resulted in the male being karyotyped, discovering a balanced Reciprocal Translocation: 46 XY; t(3;16)(q22; q22). Conclusions: Although standard pre-screening for an AMA couple seeking infertility treatment was followed, along with completing an autologous IVF cycle with PGS demonstrating findings that would be considered standard in an AMA couple, we discovered a male translocation following a donor oocyte/PGS cycle. The very low cost of a standard karyotype may be warranted prior to initiating donor oocyte cycles. It may even be preferable to change the standard of care to karyotype all couples prior to any infertility treatments to increase the likelihood success. Disclosures: None. Funding: None.

Preimplantation genetic screening (PGS) using Array CGH, qPCR or SNP arrays provided very high levels of diagnostic accuracy, however, very little ability to recognize low-level mosaicism. The newest Next Generation Sequencing (NGS) technique provides the same level of diagnostic accuracy and allows for an increased chance to detect chromosomal mosaics, down to as low as 20%. We set out to determine the presence, quantity and the types of genetic mosaicism in trophectoderm cells following embryo biopsy in clinical PGS cases using NGS analysis. Retrospective data analysis from a single, large, private fertility clinic. Sixteen blastocyst biopsy PGS cycles using NGS were analyzed. No ovum donor cycles were included. The mean female age was 36. Trophectoderm biopsy was performed on 128 blastocysts cultured 5, 6 and/or 7 days. All embryos were vitrified and the biopsied cells were sent to a genetic testing laboratory (Reprogenetics, Los Angeles, CA). Results were compiled, including the genetic diagnosis rate, the quantity of mosaics, the types of mosaics, and the number of occurrences for each chromosome involved in the mosaicism. Fisher's Exact Test was used for statistical analysis. Genetic results were obtained in 124 of 128 (96.9%) blastocysts, with 1 failure to amplify DNA and 3 chaotic profiles not allowing diagnoses. Mosaicism was reported in 17 (13.7%) samples, as compared with our previous experience with aCGH of 6728 blastocysts reporting only 10 (0.14%) mosaics (p<0.0001). NGS detected partial mosaicism of a single chromosome twice (1.6%), and evidence of mosaicism of two chromosomes in a single sample was found 3 times (2.4%). Fourteen chromosomes were affected, with 5 chromosomes affected twice. Overall, both monosomy and trisomy mosaics were equally represented 10 times. Finally, neither the day of embryo biopsy nor the morphological classification were found to correlate with the presence or type of mosaicism. Trophectoderm biopsy with aCGH, qPCR and SNP arrays provided results with very little ability to recognize the presence of mosaics. As a result, and based on our aCGH outcomes, it was generally believed that mosaicism in biopsied trophectoderm cells was a rare occurrence. Our initial NGS data finds the occurrence of mosaicism at 13.7%, significantly higher than originally estimated from the aCGH results. This level of previously undetected mosaicism could be a contributing factor to implantation failure, biochemical pregnancies and pregnancies that were not carried to full term following the older technologies. Mosaic detection in trophectoderm cells screened by NGS could allow for selection of normal embryos with a higher implantation potential, thereby increasing success rates and decreasing less desirable outcomes.

The human embryo is known to undergo Zona-Pellucida (ZP) hardening due both to extended culture to the blastocyst and as a consequence of vitrification. This may affect the blastocysts ability to escape the ZP post-warming, especially within an optimal window of endometrial receptivity. We set out to determine if the percentage of blastocysts that were able to fully escape the ZP post-warming was different if laser assisted hatching (LAH) of ∼33% of the ZP was performed immediately after warming compared to embryos that were not LAH. Prospective analysis of donated vitrified/warmed embryos placed in an Embryoscope with and without LAH. Overall, 24 research good/fair quality blastocysts vitrified on Day 5 and 6 were used. The mean age was 38.3 with a range of 23-46. Warming of embryos was performed per established protocol on the same day, at the same time, under the same incubation conditions. Embryos were randomized to either a group that had ∼33% of the ZP ablated using LAH on a Zilos laser at 400 μs (Group 1) or no LAH (Group 2). All embryos were incubated for 48 hours in an Embryoscope time lapse incubator in GTL medium (Vitrolife). We recorded if the embryo escaped the ZP, the time it took to fully hatch from the ZP and the number of expansions each embryo underwent to escape the ZP. Assessment was performed to determine if and when embryos hatched out. In Group 1, 11 of 14 embryos (78.6%) achieved complete hatching within 24 hours, with an average of 17.0 hours elapsed to fully hatch and an average of 2.1 expansions. In Group 2, none of the 10 embryos achieved complete hatching within 24 hours, after having undergone an average of 2.6 expansions. Two of the Group 2 embryos (20%) achieved complete hatching, at 37 and 48 hours with an average of 5 expansions. Group 1 vs. Group 2 complete hatching within 24 hours was significantly different (p<0.01). Time lapse video evidence provides compelling information regarding the hatching process of blastocysts post-warming. A significantly higher percentage of warmed embryos were capable of completing the hatching process when ∼33% of the ZP was ablated as compared to those that were not LAH. Importantly, the average time for the LAH embryos to completely escape the ZP was within the optimal time range of endometrial receptivity. The LAH embryos also demonstrated that the expenditure of energy to complete the hatching process, through repeated expansions, is minimized, thus ensuring the energy stores are not depleted prior to implantation. Conversely, an overwhelming percentage of warmed embryos that do not have LAH prior to embryo transfer are unsuccessful in completing the hatching process, even within 48 hours, which is outside optimal endometrial receptivity. Overall, LAH of ∼33% of the ZP post-warming is highly beneficial to the embryo's escape and could significantly increase implantation and success rates.