Faculty Bibliography

OBJECTIVE: To assess if SART donor oocyte pregnancy rates are consistent with high percentage of aneuploidy and mosaicism in donor oocyte cycles using Preimplantation Genetic Screening (PGS) with Next Generation Sequencing (NGS). DESIGN: Retrospective study and mathematical analysis. MATERIALS AND METHODS: All trophectoderm biopsy specimens from donor oocyte cycles received by a genetics laboratory were queried for NGS results. The rate of euploidy, aneuploidy, and mosaicism was calculated. A separate analysis was done to find the ongoing pregnancy rate (OPR) from patients' first donor single thawed euploid embryo transfers (STEET) from NGS at a single university fertility center from 2/2015 to 3/2016. Lastly, the 2014 SART National Summary data on non-tested donor embryo transfers and their live birth rates was used to calculate the expected live births from untested donor embryo transfers based on euploidy and mosaicism rates from NGS donor data, applying the the OPR from donor STEET data. The calculated expected live birth rate was then compared to the actual live birth rate of untested donor embryo transfer cycles. RESULTS: 268 donor cycles from 38 IVF centers showed a euploid rate of 48.7%+/-23.8, aneuploid rate of 22.9%+/-20.4 and mosaic rate of 28.4%+/- 21.9 (Table 1). Thirty two patients were included in the donor NGS STEET cycle analysis. The average age at transfer was 42.7 +/-3.9 years with an average donor age of 26.5 +/-2.9 years, for an OPR of 62.2% (n=20/32). The 2014 SART National Summary shows 6070 non-PGS donor egg transfers, averaging 1.7 embryos per transfer, for an estimate of 10319 embryos transferred in total. The reported live birth rate of 53.3% (36.9% singletons, 16.2% twins and 0.2% triplets) gives a cumulative 4242 babies delivered (41.1% per embryo). When the euploid rate of 48.7% is applied, 5025 embryos transferred are estimated to have been euploid. The 62.2% OPR of donor STEETs applied to this gives an expected 3126 singleton deliveries (95% CI 2927-3246). The mosaic rate of 28.4% plus data showing an overall approximate 20%1,2 OPR of mosaic embryos adds 586 (95% CI 421- 524) singleton births. The total expected babies born is 3712 (95% CI 3505-3877) with a live birth rate of 35.9%(95% CI 34.0-37.6) from donor egg transfers. CONCLUSIONS: High rates of aneuploidy and mosaicism in donor oocyte cycles are consistent with the pregnancy rates of untested donor embryo transfers. The use of PGS with NGS can prevent transfer of aneuploid embryos in donor cycles

OBJECTIVE: Mutations in mitochondrial DNA (mtDNA) cause a number of diseases in offspring. Since mtDNA mutations are transmitted exclusively through the oocyte, various manipulations of the oocyte cytoplasm have been proposed to prevent transmission of mtDNA diseases. A method to estimate mtDNA load at the oocyte level would help balance the risk/benefit of oocyte manipulations. We sought to determine whether polar bodies (PB) accurately represent the mitochondrial mutational load in their corresponding oocytes. DESIGN: Translational science research. MATERIALS AND METHODS: IRB approval was obtained for use of human discard material and a total of 10 oocyte-PB pairs were obtained. Immature oocytes received 2 hours post retrieval were placed in an incubator for 48 hours and those that matured were subsequently frozen. Prior to freezing, oocytes were processed with pronase and all excess cumulus cells were removed. The PB was then removed mechanically and frozen separately from the oocyte in PBS solution. A real time PCR assay was developed to measure mitochondrial copy number and the presence or lack there of of the 4977 base pair common deletion (via the presence of the ND4 gene). The absolute mtDNA copy number minus the ND4 gene copy number divided by the absolute copy number yielded the deletion ratio. Paired t-test analysis was used for statistical analysis RESULTS: In all ten oocyte-PB pairs, the mutational load in the PB exceeded that in the associated oocyte. The mean deletion ratio in oocytes was 30% and 61.8% in PBs (p= 0.001). The largest percent difference between the oocyte and PB mtDNA deletion ratio was 76%. Prediction of oocyte deletion ratio based on PB deletion ratio is shown with the equation [Oocyte deletion ratio=0.6064(PB deletion ratio) - 0.0708], however this relationship did not reach statistical significance. CONCLUSIONS: It has been suggested that the PB can be used to estimate mitochondrial mutational load. Here we show that the PB overestimates the mutational load in the oocyte (as high as 76% in some cases). Higher deletion ratios in PBs tended to correlate with higher deletion ratios in associated oocytes. Larger numbers are needed for confirmation, but even from the sample size studied the PB deletion ratio clearly is not representative of its corresponding oocyte. (Table Presented)

OBJECTIVE: Single thawed euploid embryo transfer (STEET) decreases the risk of maternal and neonatal morbidity associated with multiple gestation. [1] If transfer of a euploid embryo fails to progress to a clinical pregnancy, we sought to determine if there are identifiable risk factors for a failed second STEET. DESIGN: Retrospective case-control. MATERIALS AND METHODS: All single thawed transfers of embryos designated as euploid by Next Generation Sequencing (NGS) were identified between April 2015 and February 2016 (n=263 transfers, 232 patients). Only patients with a first failed STEET and a subsequent second attempt at elective STEET were included (n=25 patients). Cycle characteristics were compared between those with and without a successful second transfer. Analyses were performed using t-tests and chi square analyses. RESULTS: Clinical pregnancy (fetal heart rate) after NGS diagnosis with STEET is 62.3% in our patient population. After one failed STEET, clinical pregnancy occurred in 56% of patients in the second STEET cycle (p=0.54). Endometrial thickness was significantly increased in patients that achieved clinical pregnancy on the second transfer as compared to those that did not (p=0.01). A lower average age at transfer and higher number of euploid blastocysts remaining was seen in the group achieving pregnancy, but statistical significance was not reached (Table 1). (Table presented) CONCLUSIONS: Lower pregnancy rates might be expected after an initial failed transfer cycle, as the number of good quality blastocysts available is reduced. However, clinical pregnancy rate with a second STEET following an initial failed cycle was similar to the pregnancy rate seen for all first transfers in our population, providing hope for couples. A thicker endometrial echo and younger maternal age at transfer were associated with increased ability to achieve clinical pregnancy in a second STEET after previous failure, indicating a possible underlying uterine or embryologic factor. Further research is needed to identify other potential contributions to pregnancy failure with STEET in order to increase utilization of single embryo transfer

OBJECTIVE: PGS is used clinically to enhance embryo selection in patients of advanced maternal age (AMA) and those with recurrent pregnancy loss (RPL) or recurrent IVF failure. We sought to understand if patients' motivations for pursuing PGS are consistent with these established indications. DESIGN: Anonymous quantitative and qualitative survey. MATERIALS AND METHODS: Anonymous survey emailed confidentially to all patients who underwent their first cycle of IVF with PGS between 1/2014 and 3/2015 (n=395). Responses are reported as percentage (%). RESULTS: 80 patients completed the survey; 7 respondents underwent PGD/PGS for single gene disorders and were excluded. The majority identified as Caucasian (77%) or Asian (19%). 26% had no insurance coverage and 18% had < 50% of expenses covered. The majority of patients identified with the following religions: Christianity (25%), Judaism (19%), Catholicism (15%) or none (16%). 86% were married. The majority were AMA (18% ages 35-37y, 32% ages 38-40, 15% ages 41-42 and 16% over age 42), but nearly 20% were <35y. The vast majority (64%) had not heard of PGS prior to their fertility consultation, 23% were referred from an outside physician, and 7% from a friend. A minority of patients pursued PGS for the indications of recurrent IVF failure (12% with > 2 prior IVF cycles) or RPL (26% had > 2 SAB). 64% of patients had not done a previous IVF cycle and 17% had been trying to conceive for under one year. 51% had zero prior miscarriages, 23% only 1 miscarriage and 33% already had 1 living child. The most common infertility diagnosis was unexplained infertility (36%). When asked the primary motivation for PGS, the most common response was "to maximize IVF efficiency and have a baby sooner" (36%). Only 26% cited their primary indication as previous miscarriage, 12% wanted to decrease the chance of miscarriage but had not yet had a miscarriage, 11% reported multiple failed attempts at IVF, and 14%chose PGS electively and were young, undergoing their first IVF cycle, and without priormiscarriage. 15% (n =11) reported 'other,' with reasons including family balancing and 'to reduce the number of unknowns.' 27% of patients agreed that they may be more likely to pursue pregnancy with donor eggs if unable to conceive from IVF with PGS. Overall, 94% of patients were happy they pursued PGS, regardless of their outcome, as the information they obtained was deemed valuable. CONCLUSIONS: Beyond the standard indications of advanced maternal age, recurrent IVF failure, and recurrent pregnancy loss, an increasing number of patients are using PGS as part of routine IVF to improve efficiency, reduce miscarriage and decrease the time to pregnancy. Understanding these motivations will help providers deliver appropriate support and counseling

OBJECTIVE: To document and report the relevance and implications of incidental findings identified during PGD in an effort to bring awareness and emphasize the need for implementing a standard operating protocol to report such findings to patients and their physicians. DESIGN: Incidental findings identified in couples undergoing PGD for gene disorders were recorded and reported from July 2015 to March 2016. MATERIALS AND METHODS: During the 9 month period, 269 cases were prepared through utilization of single nucleotide polymorphism (SNP) arrays (Karyomapping; Illumina, USA). DNA samples were obtained from couples and family members for test preparation purposes. During evaluation of sample quality, review of the SNP array profiles was completed. All incidental findings were reported to the physician and patient and recommendation for follow-up microarray testing was provided. RESULTS: Incidental findings were identified in 10/269 PGD test preparations, affecting chromosomes 4, 14, 15, 16, 22, and X. Nine were microduplications and 1 was a microdeletion. The size of microduplications detected ranged from 0.18 megabases (Mb) to 3.5 Mb. Interestingly, three of the microduplications detected (4q35.2, 15q11.2, and 16q23.3) were each seen in 2 separate cases, accounting for 6/10 cases. The microdeletion identified (located on chromosome X) was determined to be of considerable size at 28Mb, potentially associated with health implications in the carrier female. Follow-up microarray analysis was pursued in 7 of the cases, as 3 of the patients declined further evaluation. Three of the incidental findings were reported as variants of uncertain significance (VUS), and 3 were reported as normal population variants. Results are still pending on 1 case. One patient elected to pursue PGD for the microduplication. Results from 2 PGD cycles for this patient with a total of 9 embryos, revealed 3 embryos free of the microduplication, but a total of 1 embryo available for transfer as the other 2 were affected with the single gene disorder and/or aneuploidy. CONCLUSIONS: With the implementation of new and advanced methodologies in clinical practice, it is becoming progressively more common to incidentally obtain information that is additional to the requested test but may have significant health implications to the patient, other family members and/or future children. It is vital to acknowledge and address this issue in the clinical laboratory setting and a standard operating procedure has to be in place to handle such situations

OBJECTIVE: Recent reports have suggested that the quantity of mtDNA in embryonic cells may serve as an indicator of embryo viability, higher levels being associated with reduced implantation potential. The current investigation represents the first evaluation of the predictive potential of mtDNA quantification in a prospective, blinded, non-selection setting. DESIGN: Prospective blinded clinical study. MATERIALS AND METHODS: mtDNA was assessed in trophectoderm (TE) samples biopsied from 337 blastocysts that had been shown to be chromosomally normal. These were generated by 195 couples (average female age 36.7 years). All patients underwent IVF in a single clinic. Cytogenetic assessment occurred via next generation sequencing, whereas mtDNA quantification utilized quantitative PCR. The study took place in a blinded, nonselection manner - i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured. RESULTS: mtDNA assessment showed that 25/336 (7.4%) of embryos contained elevated mtDNA levels. At the time of writing, 159 of the blastocysts have been transferred. All transfers involved a single chromosomally normal blastocyst of good morphology. mtDNA amounts were not known at the time of transfer. 109 (69%) of these led to ongoing pregnancies, and all (100%) had mtDNA levels in the normal range. The remaining 50 (31%) blastocysts failed to implant. 8 (16%) of these non-viable embryos were found to have elevated quantities of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with elevated mtDNA levels, was 0/8 (0%), a highly significant difference compared to the 72% pregnancy rate observed for similar embryos with normal levels of mtDNA (P<0.0001). CONCLUSIONS: This is the first study to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely if ever implant, providing support for its use as a viability biomarker. 72% of euploid embryos with normal quantities of mtDNA implanted vs. 69% for the cohort as a whole. No embryos with elevated mtDNA levels implanted

OBJECTIVE: To compare the rates of euploid embryos in FRESH vs. OOT cycles. DESIGN: Retrospective cohort study of all FRESH vs. OOT cycles, including donors, undergoing preimplantation genetic screening (PGS) with microarray-based comparative genomic hybridization (aCGH) or next-generation sequencing (NGS) from 2011-2015 at a single, high-volume university-based fertility center. MATERIALS AND METHODS: Embryos biopsied for PGS derived from FRESH were compared to those from OOTon a per-cycle basis (n=1643 and 111, respectively). PGS was performed by aCGH or NGS for both FRESH (n=1149 and 504, respectively) and OOT (n=64 and 47, respectively). Age was controlled for by linear regression analysis of euploidy rate vs age. Euploid embryos were divided into no. euploid per egg and no. euploid per embryo biopsied and by method of PGS performed; comparison was made by examining residuals from the regression line for FRESH. SEM and t test were used. RESULTS: The average no. of euploid blasts per FRESH cycle was 1.77+/-2.26 and 1.19+/-1.61 for aCGH and NGS respectively; the average no. of eggs was 13.8+/-8.0 and 13.8+/-8.4. The average no. of euploid blasts per OOT cycle was 1.24+/-1.65 and 0.85+/-1.01 for aCGH and NGS respectively; the average no. of eggs per thaw was 14.72+/-8.8 and 13.69+/-5.71. Euploidy rate per egg was significantly lower in the OOT than in the FRESH for both aCGH and NGS when analyzing the mean residuals from the regression line. However, there was no difference in euploidy rate per biopsied blast in either group, irrespective of PGS method. CONCLUSIONS: Previous research at our facility has shown that the blast formation rate is significantly lower in OOT vs FRESH [1]. After controlling for the age-effect on aneuploidy, we observed that the euploidy rate per egg was lower in the OOT vs the FRESH for both aCGH and NGS; this difference disappears once the embryos make it to blastocyst stage, as there was no difference in no. of euploid per embryos biopsied. The difference in average no. eggs in FRESH vs OOT is insignificant and a misleading comparison as all oocytes retrieved were not thawed in totality for each OOT. The data suggest that attrition in the OOT group may be secondary to the cryopreservation technique, although attrition occurs regardless of ploidy. Further improvements in cryopreservation technique are necessary to optimize patient outcomes

OBJECTIVE: The impact of obesity on poor reproductive outcomes, especially miscarriage, is well documented. Our group previously reported that obesity does not increase embryonic aneuploidy, suggesting other factors may be involved in obesity-related subfertility, infertility, and miscarriage. We sought to determine if BMI affects pregnancy rates in patients undergoing euploid FET . DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Patients who underwent in vitro fertilization with pre-implantation genetic screening by trophectoderm biopsy and array comparative genomic hybridization were identified. Height/weight were recorded upon oocyte retrieval. Those who underwent euploid FET within 6 months of retrieval were analyzed. Patients were grouped by BMI according to WHO class. Pertinent parameters from controlled ovarian hyper- stimulation (COH) and FET cycles were collected including day 2 FSH/E2, endometrial thickness/E2 prior to FET, and no. of the following: oocytes retrieved, M2, 2PN, embryos biopsied, euploid embryos, percentage euploid embryos, and embryos transferred. Demographics and pregnancy outcomes were collected. Implantation rate (IR) was defined by gestational sac(s) per embryo(s) transferred, and clinical pregnancy rate (CPR) by fetal cardiac activity (FCA). Miscarriage (SAB) was defined by pregnancy loss following FCA. Data analysis was performed using the Shapiro-Wilk test for normality, the Kruskal-Wallis test to compare means, and Fisher's exact test for dichotomous variables (mean +/- SD, p<0.05, GraphPad Prism). For dichotomous variables, each BMI cohort was compared to the normal weight reference group. RESULTS: 291 patients met inclusion criteria. There were no differences between groups when comparing COH and FET cycle parameters, although underweight group was younger (p=0.005). Following euploid FET, there were no differences in IR, CPR, or live birth rate (LBR) between groups. The miscarriage rate was > 2-fold higher in the obese group compared to normal-weight (18.2% vs. 7.4%) and to all other BMI cohorts, but this did not reach statistical significance. CONCLUSIONS: The miscarriage rate was more than 2-fold higher in the obese group following euploid FET compared to the normal-weight group, although this finding did not reach significance. The trends in our findings support further study into the relationship between obesity, the intrauterine milieu, and pregnancy loss, particularly after the transfer of a highly-competent euploid embryo

OBJECTIVE: To determine whether undetected aneuploidy contributes to pregnancy loss after transfer of euploid embryos that have undergone array comparative genomic hybridization (aCGH). DESIGN: Case-control study. SETTING: University-based fertility center. PATIENT(S): Cases included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in miscarriage. Controls included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in a live birth. INTERVENTION(S): Next-generation sequencing (NGS) protocols were internally validated. Saved amplified DNA samples from the blastocyst trophectoderm biopsies previously diagnosed as euploid by aCGH were reanalyzed using NGS. Cytogenetic reports of the products of conception for 20 of the pregnancies resulting in miscarriage were available for comparison. MAIN OUTCOME MEASURE(S): The incidence of aneuploidy and mosaicism using NGS within embryos resulting in miscarriage and live birth. RESULT(S): Of euploid embryos analyzed by aCGH resulting in miscarriage, 31.6% were mosaic and 5.2% were polyploid by NGS. The rate of chromosomal abnormalities was significantly higher in embryos resulting in miscarriage (36.8%) than in those resulting in live births (15.8%). The rate of mosaicism was twice as high among embryos resulting in miscarriage than those resulting in live birth, but this was not statistically significant. Next-generation sequencing detected more cases of mosaicism than cytogenetic analysis of products of conception. CONCLUSION(S): Undetected aneuploidy may increase the risk of first trimester pregnancy loss. Next-generation sequencing may detect mosaicism and triploidy more frequently than aCGH, which could help to identify embryos at high risk of miscarriage. Mosaic embryos, however, should not be discarded as some can result in live births.

PURPOSE OF REVIEW: Elective oocyte cryopreservation for deferred childbearing has gained popularity worldwide, commensurate with increased knowledge regarding age-related fertility decline. The purpose of this review is to summarize recent data regarding trends in delayed childbearing, review recent findings surrounding age-related fertility decline, acknowledge significant gaps in knowledge among patients and providers regarding fertility decline and review outcomes following elective oocyte cryopreservation. RECENT FINDINGS: Despite an inevitable decline in fertility and increase in miscarriage with increasing female age, there is a growing worldwide trend to delay childbearing. Patients and providers alike demonstrate large gaps in knowledge surrounding age-related fertility decline. Oocyte cryopreservation is clinically approved for medically indicated fertility preservation, but a growing number of women are using oocyte cryopreservation to defer childbearing and maintain reproductive autonomy. Mounting data support the efficacy and safety of oocyte cryopreservation when used to electively defer childbearing, with recent studies demonstrating rates of euploidy, implantation and live birth rates equivalent to in-vitro fertilization (IVF) with fresh oocytes. SUMMARY: Oocyte cryopreservation provides women with an option to defer childbearing and maintain reproductive autonomy, with IVF success rates on par with fresh IVF. However, it is critical that patients understand the limitations of oocyte cryopreservation. Greater education regarding age-related fertility decline should be geared toward patients and providers to prevent unintended childlessness.