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.

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.

Irvine Scientific's standard continuous culture media (CSC-Complete) was modified to create a new next generation media (NXC) with reduced lactate to improve metabolic conditions for the embryo. NXC was shown to improve blastocyst development and clinical outcomes in a company sponsored clinical trial. However, transitioning to a new culture media requires significant preclinical evaluation in the IVF laboratory. In addition, the productivity of the media should be analyzed continuously during clinical transition. To test whether the latest continuous-culture media from Irvine Scientific produces a better environment for embryo development. One-cell mouse embryos were thawed from Embryotech (Experiment #1, N=30) and Charles River Laboratories (Experiment #2, N=120). Embryoscope slide dishes were pre-equilibrated with either Control media (CSC-Complete) or Test media (NXC). Embryos were evaluated for up to 135 hours in the time-lapse incubator with regards to embryo developmental time points reached: 1) Time at the initiation of blastocyst hatching; 2) Time at a biopsy ready blastocyst and 3) Time at a fully hatched embryo. Chi-Squared analysis was used to evaluate embryo developmental outcomes immediately before and after the clinical transition to NXC. Mouse embryo developmental and clinical embryological outcomes are shown in Tables 1 and 2, respectively.Table 1Mouse Embryo AnalysisAverage hours at initiation of hatchingAverage hours at biopsy readyAverage hours at fully hatchedExperiment #1CSC-Complete104.4114.4127.0Experiment #1NXC102.4112.8127.1Experiment #2CSC-Complete111.5119.5127.2Experiment #2NXC108.9115.6127.1 Open table in a new tab Table 2Clinical Data AnalysisFertilization RateTotal-Usable BlastocystsGood/Fair Quality Blastocysts on Day 5Immediately before NXC Transition70.7%46.1%41.4%*Immediately after NXC Transition73.6%48.4%46.8%**p<0.05 Open table in a new tab *p<0.05 Both mouse experiments (Table 1) revealed NXC media gave a faster time to initiation of hatching and an overall earlier time to achieve a biopsy-ready embryo. This allows the laboratory to biopsy more embryos on a more optimal day of development, giving a better chance at a successful clinical outcome.

Uncertainty about the fate of mosaic embryos has gained significant attention in preimplantation genetic testing for aneuploidy (PGT-A). Although some physicians advocate for transferring mosaic embryos [1], clinician preferences regarding reporting of mosaicism is largely unknown. Similarly, there are no established standardized cutoff values for reporting of mosaicism, making clinical significance challenging. The objective of our study was to identify the number of clinics requesting mosaicism reporting with the PGT-A results using a single reference genetics laboratory and to outline the classification criteria used for their reporting. Additionally, we analyzed the number of embryos which would be classified as mosaic if certain inclusion/exclusion criteria were applied in the diagnosis of mosaicism. Cross-sectional analysis The number of clinics requesting mosaicism reporting was assessed as a proportion of all clinics utilizing the reference lab for PGT-A. Additionally, we outlined the classification criteria utilized by this laboratory to define embryos as mosaic versus aneuploid. We calculated the proportion of abnormal embryos that would be classified as mosaic applying this criteria. The present sample includes one hundred different IVF centers in the US which utilize a single reference genetics laboratory for PGT-A testing. Only 12 centers (12%) request mosaic reporting, while 88 centers (88%) opt out of mosaic reporting. Embryos are reported as mosaic if the biopsy specimen collected contains >30% but less than <75% aneuploid cells and all the following criteria are met: 1) the embryo can have no other chromosomal abnormalities, 2) the embryo does not contain a partial loss or gain of chromosomal material, and 3) the embryo is not mosaic for chromosomes 13, 16, 18, 21, X or Y. Initial validation data suggested that when these criteria are applied in clinical cases, and an IVF center wishes for mosaic embryo reporting, approximately 2.4% of embryos will be classified as mosaic. Most clinics prefer to be blinded with regards to embryo mosaicism. In these PGT-A cases, the geneticist only offers a diagnosis of euploid or aneuploid. This strategy removes the burden of choice from the clinic/patient regarding the transfer of known genetically anomalous embryos.

To compare whether trophectoderm biopsy day or embryo quality have an effect on pregnancy rates after transfer of embryos which underwent pre-implantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing (NGS). Retrospective data analysis of laboratory and clinical outcomes following PGT-A and frozen embryo transfer. Patients who underwent a frozen euploid single embryo transfer between January 2016 and December 2017 at a single laboratory were assessed. Embryos were cultured to the hatching blastocyst stage (HB) and assigned an overall grade of good, fair or poor. Only HB embryos that were of good or fair quality underwent trophectoderm biopsy on day 5 or 6 with subsequent PGT-A using NGS for genetic results. Chi-square was used to compare differences between groups and a multivariate logistic regression was used to adjust for embryo biopsy day, embryo grade as well as oocyte age. P< 0.05 was considered statistically significant. 514 women underwent a total of 581 frozen single euploid embryo transfers. The mean oocyte age was 34.6 years and the overall pregnancy rate was 62.3%. 71% of embryos were biopsied on day 5 and 68% percent of all of the embryos were of good quality at biopsy. A significant overall reduction in pregnancy rates was seen (67.3% down to 50.9%) as the day of biopsy increased and the quality of the embryo declined (p=0.013; Table 1). Although, there was a 6.6% decline in pregnancy rate between day 5/fair and day 6/good embryos, no significant difference was able to be determined due to limited samples in each transfer group (p= 0.43). Additionally, while there was a 9.7% decline in pregnancy rates between day5/fair and day 6 embryos of good or fair quality, this was also not significantly different (p= 0.16). These findings demonstrate that as embryos require longer to grow to a biopsy stage or are of less quality, the overall pregnancy rates significantly decline. Although each step down in embryo quality and advancing day of biopsy results in at least a 5% decrease in pregnancy, a much larger sample size will be required to detect these differences significantly. Additionally, our data suggest that delaying biopsy to potentially improve embryo quality does not result in a significant increased pregnancy rate. Overall, our data can be of use in deciding which embryos to biopsy, on which day of development, as well as guiding patients in setting expectations for transfer outcomes from euploid embryos.Tabled 1Embryo biopsy day and grading characteristics associated with pregnancy outcomesDay of embryo biopsyGrade of embryo biopsyPregnancy (Rate)Mean oocyte age, years (SD)5HB-Good224/333 (67.3%)34.1 (4.9)5HB-Fair48/77 (62.3%)34.6 (5.3)6HB-Good34/61 (55.7%)34.2 (5.3)6HB-Fair56/110 (50.9%)34.9 (5.3) Open table in a new tab

Heated IVF work surfaces, which attempt to emulate physiological temperatures, have greatly improved oocyte and embryo safety. Many manufacturers produce factory calibrated heated surfaces, however, these devices are highly variable and need to be operationally validated and continuously monitored for optimal use. Factory issued digital set points often do not correlate with desired micro-drop temperatures. We analyzed temperature variations of both laminar flow hood and inverted microscope heated surfaces in culture (CUL) and micromanipulation (MM) dishes. Prospective temperature analysis of heated surfaces overtime. CUL dishes with 40μL drops and a 10mL oil overlay were incubated overnight at 37°C. MM dishes with 20μL drops and 4mL of 37°C oil overlay were placed on a laminar flow hood heated surface. Using a certified calibrated thermocouple, temperatures of the drops were measured at various set times ranging from 0 to 70 minutes for the CUL dishes and from 1 to 30 minutes for the MM dishes. Repeated measurements (CUL n=92, MM n=42) with multiple dishes (CUL n=12, MM n=6) were taken over the course of the testing times. To monitor the temperature of a dissecting microscope heated stage in a laminar flow hood, CUL dishes from a 37°C overnight incubator were placed on the heated surface with a digital set point of 41.3°C at a marked spot. To monitor the temperature of a micromanipulation heated stage, MM dishes from a 37°C heated surface, were placed on the micromanipulation stage with a digital set point of 39.9°C at a marked spot. In order to achieve 37°C in the dishes, the laminar flow hood digital set point was required to be 41.3°C and the heated stage set point was required to be 39.9°C. Multiple cycles of heating and cooling occurred on both surfaces. CUL dishes measured a mean temperature of 36.7°C with a standard deviation of 0.22°C (range 35.5-37.5°C). MM dishes measured a mean temperature of 37.2°C with a standard deviation of 0.50°C (range 36.6-38.6°C). The heated surfaces of laminar flow hoods and micromanipulation stages were found to greatly vary in micro-drop temperature compared to their digital set point. The time periods measured can be used to represent common IVF procedures. We believe there can be a reduction in stress on oocytes and embryos through designing clinical protocols and procedures that minimize the exposure times to these varying temperatures that do not correlate with manufacturer digital set points. These changes could include regulating the number of oocytes/embryos undergoing a procedure simultaneously in one dish or having a second embryologist assist during procedures with a large number of oocytes/embryos. Every laboratory should perform their own independent temperature validation prior to using any manufacturer issued set point.

and NGS without mosaicism reporting (woM) were used.NGS with mosaicism reporting (wM) began in 2016.Euploid embryo rates and clinical pregnancy rates within the two groups, woM and wM, were stratified by age of the donor (21-29 years) at the time of retrieval.Clinical pregnancy was defined by the number of fetal heartbeats at 6-7 weeks gestation by the total number of embryos transferred.Descriptive data was analyzed as percentages and means with standard deviations.Statistical differences between groups were determined using Fisher's exact and/or chi-square with a p-value <0.05 considered significant.RESULTS: 925 donor oocyte IVF/PGT-A cycles were performed, resulting in 9325 trophectoderm biopsies, 3414 in the woM group and 5911 in the wM group.The euploid rate in the woM group was a bell curve with lowest euploid rate in the extreme age groups (ages 21 and 29, 69% & 73%).In the wM group, the euploid rate remained similar across age groups (mean 63%) and was 10% lower than the woM group.Despite this difference, the percentage of cycles without a euploid embryo for transfer was the same between groups (0.09%).There was no significant difference in clinical pregnancy rate per embryo transferred between the PGT-A technologies.CONCLUSIONS: This large donor oocyte/PGT-A cohort verifies previous data regarding the effect of age on the euploid rate when utilizing techniques that do not report mosaicism.In contrast, when mosaicism is reported, there is a similar euploid rate across age groups.Despite a lower number of euploid embryos in the wM group, there was no difference in the percentage of cycles that had no embryos available for transfer or clinical pregnancy rate.This study supports utilizing donors (age 21-29) including the ends of the standard age range with new PGT-A technology with mosaicism reporting.

Currently, there are a multitude of oocyte warming protocols ranging from large volumes to micro-droplets being used in different laboratories. There are also different procedural steps ranging from use of 3 to 5 droplets with variable timing. The objective of this study was to assess whether laboratory outcomes are affected based on volume of warming media at the time of oocyte thaw. We directly compared the use of 50μl droplets to use of 200μl droplets during our standard 5 step dilution oocyte warming protocol. Retrospective study comparing laboratory outcomes using two oocyte warming micro-droplet volumes. Oocyte warming consists of a five step protocol using Irvine Scientific Vitrification Thaw media (90137, CA). We compared the laboratory outcomes of 13 cases with use of 50μl droplets to 14 cases with use of 200μl droplets. For both study arms, all oocytes were originally frozen using vitrification. The initial thawing solution used was a 2ml volume droplet at 37°C and the timing for warming was exactly the same in both groups. Micro-drop warming was performed through 5 room-temperature micro-drop dilutions of TS 1min; TS:DS 2min; DS 2min; DS:WS 2min and WS for 5min. Oocyte recovery, initial survival, 3hr survival, 2PN fertilization, cleavage and usable blastocyst rates were determined. Analysis was performed by Chi-square. Tabled 1Droplet SizeCases# Thawed% Recovered% Initial Survival% 3hr Survival% 2PN% Cleaved% Usable Blastocysts50μl1316298.274.271.763.280.738200μl1417597.791.887.77274.732.1P-value>0.05<0.0001<0.001>0.05>0.05>0.05 Open table in a new tab In the recent years there has been nearly a 5 fold increase in rate of elective fertility preservation for women. Success rates both in blastocyst formation in the laboratories as well as pregnancy outcomes has improved with the use of vitrification techniques for oocyte cryopreservation and warming. In this study we assessed whether the oocyte warming thaw protocol could affect laboratory outcomes. We demonstrated that initial survival (<0.0001) and survival after 3 hours (<0.001) were significantly improved with the 200μl droplet size. However, the rate of embryo development of the surviving oocytes in either study arm were not different for day 3 cleavage or overall usable blastocysts. These findings suggest that if an oocyte survives the warming and initial 3hr culture, they have the ability to develop equally. Size does matter, oocytes favor a larger droplet during the 5 step warming process. The 200μl study group provided the oocytes an optimal environment to recover and develop into normal embryos.

Objective: Cryopreservation, extended embryo culture and preimplantation genetic screening (PGS) has given patients increased reproductive potential. Over the years, cryopreservation has changed from slow-freezing with ~40% survival to vitrification with significantly higher survival. Extended embryo culture to the blastocyst stage has become routine. PGS has changed from cleavage stage biopsies with FISH on limited chromosomes to trophectoderm biopsies with Next Generation Sequencing (NGS) of all 24 chromosomes. This case report describes a patient who after having one child in 2002 returned 14.3 years later and requested thawing, blastocyst biopsy and NGS evaluation of her previously cryopreserved Day 2 embryos.

Preimplantation genetic screening (PGS) has enabled our ability to screen for euploid embryos, and led to improved implantation and decreased miscarriage rates per embryo transferred. With improved embryo selection and pregnancy outcomes, the average number of embryos transferred using autologous oocytes in women <35 years old has decreased by 57% from 2004 -20141. Unfortunately, in 2014 the average number of embryos transferred was still 1.6, leading to 20-25% of the live births still being multiple gestations. Maternal and neonatal risks of twin pregnancies include but are not limited to a higher rate of spontaneous abortions, low birth weight, maternal hypertensive diseases, and a premature delivery rate over 50% leading to increased neonatal morbidity and mortality2-4. The purpose of this study was to evaluate if double embryo transfer of euploid embryos had significantly higher implantation and clinical pregnancy rates in order to justify the increased adverse obstetrical risk associated with multifetal gestations. To determine whether there is a difference in pregnancy outcomes between single embryo transfer (SET) and double embryo transfer (DET) with euploid embryos screened by array comparative genomic hybridization (aCGH). Retrospective cohort study of all euploid embryo transfers tested with aCGH from January 2014 to December 2015 at a single, high-volume fertility practice. We compared a total of 272 SET's to 120 DET's. Data was analyzed for pregnancy rate (+ hCG/transfer), implantation rate (gestational sac/embryo transferred), and clinical pregnancy rate (+ FHR/transfer). Fisher's exact test (two-tailed) was used for statistical analysis. When comparing the two groups, there was no significant difference in initial PR, although there was a trend between DET (65.8%) and SET (56%), p=0.075 (Table 1). There was also no significant difference in implantation rate between DET (38%) and SET (43%), p=0.24. However, clinical pregnancy rate was found to be significantly higher for DET (51%) compared to SET (39%), p<0.04. The twin pregnancy rate was 1.9% for SET and 31% for DET. When comparing pregnancy outcomes between single and double euploid embryo transfers, an additional embryo does not improve the implantation rate. Although there was a significantly increased clinical pregnancy rate with DET, this increase was only 12%. However, there was a significant increase in the twin pregnancy rate from 1.9% in the SET group to 31% in the DET group. Therefore, for the slightly increased overall pregnancy rate with DET, the higher risk of multiple gestations and potential complications does not warrant a DET with PGS. Further studies are needed to access outcomes of these pregnancy, in particular adverse outcomes of twin gestations.

Preimplantation genetic screening (PGS) improves implantation and decreases miscarriage rates per embryo transferred1. PGS has evolved from fluorescence in-situ hybridization (FISH) to array comparative genomic hybridization (aCGH), and recently to next-generation sequencing (NGS). Compared to aCGH, various NGS protocols using amplified DNA have been shown to have a sensitivity and specificity as high as 100% for detecting aneuploidy in blastocysts2,3. Some NGS protocols may be able to detect partial chromosomal gains and losses and identify more cases of mosaicism within trophectoderm cells than other techniques4-6. The impact of mosaicism detected at the embryo level on pregnancy rates (PRs) is unclear, but it has been associated with lower implantation rates and higher miscarriage rates7. The purpose of this study was to evaluate whether transfer of a euploid embryo screened by NGS resulted in an improved pregnancy rate compared to aCGH. To determine whether there is a difference in pregnancy rates after transfer of a euploid embryo that has undergone preimplantation genetic screening with NGS compared to aCGH. Retrospective cohort study of all euploid embryo transfers screened with either aCGH or NGS from January 2014 to December 2015 at a single, high-volume fertility practice. We compared a total of 317 women in the aCGH group who underwent a total of 390 transfers with 506 embryos to a total of 36 patients in the NGS group who underwent a total of 38 transfers with 45 embryos (Table 1). Data was analyzed for pregnancy rate (+ hCG/transfer), presence of gestation sac/transfer, implantation rate (gestational sac/embryo transferred), and clinical pregnancy rate (+ FHR/transfer). Fisher's exact test (two-tailed) was used for statistical analysis. Of the 390 transfers in the aCGH group and 38 transfers in the NGS group, the average number of embryos per transfer was 1.29 and 1.18, respectively. When comparing the two groups, the pregnancy rate was significantly higher for NGS (78.9%) than aCGH (59%), p<0.03 (Table 2). The presence of a gestational sac/transfer was significantly higher with NGS (65.7%) than aCGH (47%). Implantation rate was significantly higher for NGS (57.8%) than aCGH (41.6%), p<0.05. Clinical pregnancy rate was significantly higher for NGS (65.7%) than for aCGH (44%), p<0.02. When comparing pregnancy outcomes after transfer of NGS screened embryos to aCGH screened embryos, we found that transfer of NGS screened embryos resulted in significantly higher rates of a positive pregnancy test, presence of a gestational sac, implantation rate, and clinical pregnancy rate. Overall, this translates into a 38% increase in the chance of implantation and a 47% increase in the chance of a clinical pregnancy with transfer of a euploid embryo screened by NGS. This may be due to an increased diagnosis of mosaicism and improved detection of partial chromosomal gains and losses. Our study was limited by its retrospective analysis, small numbers in the NGS group, and live birth rates not currently available. However, it appears that PGS with NGS may be a more sensitive screening tool for chromosomal abnormalities leading to increased pregnancy rates.

To assess rates of mosaic (MOS) and abnormal (ABN) embryos across physicians practicing within an in vitro fertilization (IVF) center (internal physicians) compared to physicians practicing outside the center (external physicians), all using the same IVF laboratory. Retrospective data analysis. Preimplantation genetic screening (PGS) reports from blastocysts biopsied on days 5, 6 or 7 and subjected to next generation sequencing (NGS) at a single genetic testing laboratory (Reprogenetics, CA) were analyzed from 1/1-12/31, 2016. Rates of normal euploid, simple ABN (aneuploidy of 1-2 chromosomes), simple MOS (1-2 chromosomes), complex ABN (aneuploidy of >2 chromosomes), and complex MOS (>2 chromosomes) embryos were assessed. Rates were calculated across the group of internal practicing physicians (n = 5) and external physicians (n = 14) who utilize the same IVF laboratory. Chi-squared and student t tests were used for statistics. 4,608 embryos were analyzed from patients of average ages 28.0 - 42.5 years. Of these, 1,830 (39.7%) were normal, 1,292 (28%) simple ABN, 539 (11.7%) complex ABN, 759 (16.5%) simple MOS, and 188 (4.1%) complex MOS. There were no statistically significant differences in rates of MOS, ABN, or normal euploid embryos when comparing internal and external physicians (Table 1). In this single laboratory study, our findings demonstrate consistent rates of MOS and ABN embryos despite heterogeneous physician practice patterns and locations. Consistent PGS results from internal vs. external physicians demonstrate that, in our IVF laboratory, laboratory conditions and embryo manipulation do not have a role in the diagnosis rate of MOS or ABN embryos.Tabled 1Table 1. PGS SummaryExternal Physicians (n=14)Internal Physicians (n=5)P valueAverage Patient Age (years)36.436.4--% Normal Euploid40.640.40.86% Simple Abnormal26.627.40.63% Simple Mosaic16.016.90.58% Complex Abnormal11.011.30.60% Complex Mosaic4.44.10.70% All Mosaic (Simple + Complex)20.320.90.75 Open table in a new tab

To assess whether rates of mosaic (MOS) embryos significantly vary or are linked across 5 physicians using a single IVF laboratory. Retrospective data analysis from a large, private fertility clinic. Preimplantation genetic screening (PGS) reports from blastocysts biopsied on days 5, 6 or 7 and subjected to next generation sequencing (NGS) at a single genetic testing laboratory (Reprogenetics, CA) were analyzed from 1/1-12/31, 2016. Rates of normal euploid, simple abnormal (aneuploidy of 1-2 chromosomes), simple MOS (1-2 chromosomes), complex abnormal (>2 chromosomes), and complex MOS (>2 chromosomes) embryos were assessed. Rates were analyzed across 5 physicians using Chi-squared, student t tests, and Pearson's coefficient. 3,908 embryos were analyzed from patients of average ages 34.2 - 38.0 years. Of these embryos, 1,552 (39.7%) were normal, 1,112 (28.5%) simple abnormal, 429 (11%) complex abnormal, 657 (16.8%) simple MOS, and 158 (4%) complex MOS. Age did not correlate with MOS rate (r=-0.02, n=5, p =0.97). For results by individual physician, see Table 1. The number of abnormal embryos correlated with the number of complex MOS embryos (r=0.96, n=5, p=0.0086). Comparing across 5 physicians, the rate of all MOS embryos (simple MOS + complex MOS) was significantly different between each physician (p<0.05). The rate of normal euploid embryos also significantly differed among physicians (p<0.05), but the euploid rate did not correlate in a linked pattern with MOS (simple MOS: r=0.62, n=5, p=0.27; all MOS: r=0.81, n= 5, p=0.10). To our knowledge, this is the first study to show a difference in normal euploid and MOS rates of biopsied embryos when comparing different physicians from the same group practice using the same IVF laboratory. As all laboratory treatments and conditions are constant, this suggests that individual physician practice patterns may influence PGS results. Additionally, we demonstrate a correlation between meiotic aneuploidy errors and mitotic complex MOS embryos.Table 1PGS Summary by Physician (*p < 0.05)PhysicianABCDEAverage Patient Age (years)34.237.436.638.035.9% Normal Euploid*44.737.737.142.340.2% Simple Abnormal23.529.629.524.829.5% Simple Mosaic18.516.816.018.514.5% Complex Abnormal9.013.314.09.211.1% Complex Mosaic*4.22.63.45.44.8% All Mosaic (Simple + Complex)*22.819.419.423.919.2 Open table in a new tab

analyzed. Results: Among the embryos, 38 resulted in livebirth. Demographics and analytic parameters are shown in Table 1. In addition, C/S score vs morphological grade on Day 3 (P = 0.049) and day 5 ICM (P = 0.006) by McNemar’s test indicate increased sensitivity of C/S scores and that Day 5 ICM scores are more important than day 5 TE scores. Conclusions: While morphological grading is still necessary in the process of embryo selection for transfer, mathematical C&S shows a higher sensitivity as a diagnostic tool for pregnancy outcome. Disclosures: None. Funding: None.

aCGH is an established chromosome screening technique in use since 2010. All chromosomes are studied, with resolution as low as 6 MB. Over 91679 embryos and 44801 cycles have been screened using this method. In this study we present follow-up of these cycles. Follow-up data was requested from 202 centers for embryos tested via aCGH from 2011-2016. Day 3 and day 5 were reported separately. From 11/2010-4/2016, aCGH was performed on 2141 blastomere cycles (16862 embryos) and 42660 blastocyst cycles (74817 embryos) for a total of 91679 embryos. Each sample was amplified and tested via aCGH (24sure, Illumina). Follow-up was requested from IVF centers on 18,046 cases for patients with at least one euploid embryo. For this study, a pregnancy includes biochemical, ectopic, and pregnancies that ended in spontaneous abortion, as well ongoing or delivered. aCGH data was obtained on 1353 blastomere cycles (1434 embryos) and 6010 blastocyst cycles (6944 embryos). Overall implantation rate for blastomere and blastocyst was 41.7% and 63.9% respectively. Pregnancy rate per cycle was 43.2% and 49.6% respectively, with pregnancy rate per transfer at 59.1% for blastomere and 60.5% for blastocyst. Miscarriage rate was 9.7% and 10.7% respectively, and ongoing pregnancy rate was 51.7% and 55.9% for blastomere and blastocyst samples. For blastocyst biopsy we further stratify the data by age in the following table: For blastocyst biopsy cases, implantation rate across entire maternal age (MA) range was constant. This indicates that once an euploid embryo is replaced it implants well at any MA and further solidifies that loss of implantation with advancing MA is due to chromosome abnormalities. There are patients with euploid embryos that have not yet had a transfer, diluting the pregnancy rate per cycle, however, the pregnancy rate per transfer was calculated based on patients who have had transfers and is a better representation of overall success. For ongoing pregnancy rate, there is a significant difference (p.<001) between blastomere and blastocyst samples reflecting the expected improved outcome of a blastocyst biopsy.Tabled 1Egg Donor< 3535-3738-4041-42>42Implantation42%41%47%38%43%41%Pregnancy Rate/Transfer66%62%50%58%58%51%Miscarriage Rate9%8%9%13%12%16%Ongoing Pregnancy Rate67%57%51%53%53%43% Open table in a new tab

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.

BackgroundThe advent of effective blastocyst culture and vitrification has allowed significantly increased implantation rates in frozen embryo transfer (FET) cycles. In fact, recent evidence suggests that FET cycles may have high implantation potential due to increased uterine receptivity. However, not all embryos reach the same quality at the time of vitrification (overall grade range: A/Great, B/Good or C/Fair). Embryologists spend years developing and refining the skills necessary to classify blastocysts, allowing for affective decisions about which blastocysts to vitrify.ObjectiveOur retrospective data analysis at a single large private fertility center was devised to evaluate the pregnancy rates in FET cycles based specifically on the overall blastocyst grade at the time it was vitrified.Materials and MethodsFET cycles from 2006-2013 (n=3854) were analyzed for the grade of the blastocysts at the time of vitrification, with grade A being high quality, B being good quality and C being fair quality. Only FET cycles in which all of the embryos transferred were of the same grade were included (A=1554, B=2127, C=173) allowing for a "pure" analysis of pregnancy rates for each embryo grade. Analysis for significance was performed by χ2 with Yates correction.Result(s)Pregnancy rates significantly declined in FET cycles as the grade of the blastocysts at the time of vitrification declined: 51.7% for Grade A, 40.0% for Grade B and 29.5% for Grade C (p<0.0001).Conclusion(s)Blastocysts that do not reach the overall high quality/good grades of A and B at the time of vitrification still have the potential to create a pregnancy in subsequent FET cycles. Our findings demonstrate that when pregnancy can be analyzed in "pure" FET cycles that blastocyst grading does significantly impact the outcome. The ability of embryologist to appropriately classify the grade of embryos demonstrates that it is still worth vitrifying grade C blastocysts. Physicians and patients can now be counselled that although they may only have embryos vitrified with an overall grade C quality, that they still have a 29.5% pregnancy potential in an FET cycle with these fair quality blastocysts.Financial SupportNone. BackgroundThe advent of effective blastocyst culture and vitrification has allowed significantly increased implantation rates in frozen embryo transfer (FET) cycles. In fact, recent evidence suggests that FET cycles may have high implantation potential due to increased uterine receptivity. However, not all embryos reach the same quality at the time of vitrification (overall grade range: A/Great, B/Good or C/Fair). Embryologists spend years developing and refining the skills necessary to classify blastocysts, allowing for affective decisions about which blastocysts to vitrify. The advent of effective blastocyst culture and vitrification has allowed significantly increased implantation rates in frozen embryo transfer (FET) cycles. In fact, recent evidence suggests that FET cycles may have high implantation potential due to increased uterine receptivity. However, not all embryos reach the same quality at the time of vitrification (overall grade range: A/Great, B/Good or C/Fair). Embryologists spend years developing and refining the skills necessary to classify blastocysts, allowing for affective decisions about which blastocysts to vitrify. ObjectiveOur retrospective data analysis at a single large private fertility center was devised to evaluate the pregnancy rates in FET cycles based specifically on the overall blastocyst grade at the time it was vitrified. Our retrospective data analysis at a single large private fertility center was devised to evaluate the pregnancy rates in FET cycles based specifically on the overall blastocyst grade at the time it was vitrified. Materials and MethodsFET cycles from 2006-2013 (n=3854) were analyzed for the grade of the blastocysts at the time of vitrification, with grade A being high quality, B being good quality and C being fair quality. Only FET cycles in which all of the embryos transferred were of the same grade were included (A=1554, B=2127, C=173) allowing for a "pure" analysis of pregnancy rates for each embryo grade. Analysis for significance was performed by χ2 with Yates correction. FET cycles from 2006-2013 (n=3854) were analyzed for the grade of the blastocysts at the time of vitrification, with grade A being high quality, B being good quality and C being fair quality. Only FET cycles in which all of the embryos transferred were of the same grade were included (A=1554, B=2127, C=173) allowing for a "pure" analysis of pregnancy rates for each embryo grade. Analysis for significance was performed by χ2 with Yates correction. Result(s)Pregnancy rates significantly declined in FET cycles as the grade of the blastocysts at the time of vitrification declined: 51.7% for Grade A, 40.0% for Grade B and 29.5% for Grade C (p<0.0001). Pregnancy rates significantly declined in FET cycles as the grade of the blastocysts at the time of vitrification declined: 51.7% for Grade A, 40.0% for Grade B and 29.5% for Grade C (p<0.0001). Conclusion(s)Blastocysts that do not reach the overall high quality/good grades of A and B at the time of vitrification still have the potential to create a pregnancy in subsequent FET cycles. Our findings demonstrate that when pregnancy can be analyzed in "pure" FET cycles that blastocyst grading does significantly impact the outcome. The ability of embryologist to appropriately classify the grade of embryos demonstrates that it is still worth vitrifying grade C blastocysts. Physicians and patients can now be counselled that although they may only have embryos vitrified with an overall grade C quality, that they still have a 29.5% pregnancy potential in an FET cycle with these fair quality blastocysts. Blastocysts that do not reach the overall high quality/good grades of A and B at the time of vitrification still have the potential to create a pregnancy in subsequent FET cycles. Our findings demonstrate that when pregnancy can be analyzed in "pure" FET cycles that blastocyst grading does significantly impact the outcome. The ability of embryologist to appropriately classify the grade of embryos demonstrates that it is still worth vitrifying grade C blastocysts. Physicians and patients can now be counselled that although they may only have embryos vitrified with an overall grade C quality, that they still have a 29.5% pregnancy potential in an FET cycle with these fair quality blastocysts.

warmed oocytes during assisted reproductive technology (ART).The HOPE Registry was open to all US ART clinics offering oocyte cryopreservation.OBJECTIVE: A post hoc analysis of HOPE Registry data was conducted to compare outcomes in subjects using autologous oocytes with those using donor oocytes and using vitrification as the method for cryopreservation.MATERIALS AND METHODS: Women, aged 18-50 years, undergoing ART aiming to achieve pregnancy using autologous or donor oocytes following cryopreservation, were recruited from June 2008 until enrollment closed in September 2010.Enrolled subjects were then followed until May 2012.A post hoc analysis of data in women who received vitrified oocytes was performed; six centers (involving 85/145 [58%] subjects) were audited and regular monitoring across all centers ensured clean patient data.RESULTS: Sixteen centers participated and recruited 240 women.The analysis of vitrified oocytes included 145 subjects: 50 in the autologous group and 95 in the donor group.Oocyte age (the subject's or donor's age at the time oocytes were obtained), number of oocytes warmed and outcomes are shown in Table 1.Significantly fewer oocytes per cycle were warmed in the donor group.The mean number of fertilized oocytes (2PN) was not significantly different between groups.Most embryo transfers in the autologous group occurred on Day 3, whereas most in the donor group occurred on Day 5.The mean number of embryos transferred was significantly less in the donor oocyte group.Significantly more cycles in the donor group resulted in embryo cryopreservation, but there was no significant difference in mean number of embryos cryopreserved.Oocyte survival, implantation, clinical pregnancy and live birth rates were significantly higher among women receiving donor oocytes compared with those receiving autologous oocytes.CONCLUSIONS: In this post hoc analysis of HOPE registry observational data, among women using vitrified/warmed oocytes, significantly better outcomes, including clinical pregnancy and live birth rates, were achieved in cycles using donor oocytes than in those using autologous oocytes.Oocyte age was significantly lower in the donor oocyte group than in the autologous oocyte group.

To determine the effect of maternal age on implantation and pregnancy rate per transfer after PGS. Retrospective Analysis. A total of 1776 PGS cycles performed via array CGH, and with follow up information obtained through EMR eIVF (PracticeHwy, TX) or by direct contact with center, were referred to us by fertility centers from 7/22/2010 to 3/17/2015. They were categorized into SART maternal age groups. The results are shown in the table below:Tabled 1Implantation Rates Per Age Group.EGD35-37 Years38-40 Years41-42 Years>42 YearsAverage Euploid Embryos6.44.63.62.51.91.1Implantation Rate (Sacs/Embryos Replaced)61.9%73.2%75.3%71.9%83.1%59.1%Total Patients75542359371226203% Pregnant68.63%66.30%64.42%63.27%64.73%53.69% Open table in a new tab Pregnancy rates per transfer were constant up to age 42, and then showed a tendency to decrease but the differences were not statistically significant. The implantation rate instead decreased significantly past 42 years of age (p<1.0). Provided that euploid embryos are present in a cycle, pregnancy rates are mostly constant at any maternal age up to 42 years of age. However, the number of euploid blastocysts available per transfer decreases significantly with age, resulting in more cycles without a transfer. Fewer euploid embryos to select for best morphology may also result in lower implantation rate after 42.