Faculty Bibliography

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

PURPOSE: The purpose of our study was to determine if progesterone (P4) values on day of trigger affect certain cycle outcome parameters, ploidy status of embryos, as well as pregnancy outcomes in the subsequent first frozen embryo transfer cycle. METHODS: Two hundred thirty-eight patients undergoing pre-gestational screening and freeze all protocol at our fertility center from 2013 to 2014 were included. Excluded patients were those whom had cancelled cycles prior to egg retrieval as well as cycles utilizing donor eggs. Once patients were identified as eligible for this study, frozen serum from the day of trigger was identified and analyzed using the Siemens Immulite 2000. Number of eggs retrieved, number of available embryos for biopsy, and number of euploid/aneuploid embryos were analyzed. The first frozen embryo transfer cycle was linked to the initial egg retrieval and outcomes including pregnancy rates, and live birth/ongoing pregnancy rates were calculated and analyzed. A discriminatory P4 value of 1.5 ng/ml was set. Group A had P4 values of less than 1.5 ng/ml and group B had P4 values greater than or equal to 1.5 ng/ml. T tests and chi-squared tests were used for statistical analysis. RESULTS: Group A had an average trigger P4 value of 0.87 +/- 0.3 and group B had an average trigger P4 of 2.1 +/- 0.8. Table 1 shows the baseline characteristics of both group A and group B. The only significant difference between the two groups was total gonadotropin dosage (IU) with a p value of 0.02 and estradiol (pg/ml) at trigger, also with a p value of 0.02 (Table 1). Number of eggs retrieved, number of embryos biopsied, number euploid/aneuploid, and non-diagnosis embryos were all non-significant. Chi-square analysis was used to compare pregnancy rates between the two groups after the first frozen embryo transfer cycle. Group A had a pregnancy rate of 72 % and Group B had a pregnancy rate of 66.7 %, which was not significant. Ongoing pregnancy/live birth rates were 65.6 % in group A and 66.67 % in group B, also not significant (Table 2). CONCLUSIONS: P4 values on day of trigger do not affect number of eggs retrieved and number of chromosomally normal embryos available for transfer in a subsequent embryo transfer cycle. Elevated P4 values (>/=1.5 ng/ml) also do not affect pregnancy rates or live birth/ongoing pregnancy rates in the first subsequent frozen embryo transfer cycle.

STUDY HYPOTHESIS: We wanted to probe the opinions and current practices on preimplantation genetic screening (PGS), and more specifically on PGS in its newest form: PGS 2.0. STUDY FINDING: Consensus is lacking on which patient groups, if any at all, can benefit from PGS 2.0 and, a fortiori, whether all IVF patients should be offered PGS. WHAT IS KNOWN ALREADY: It is clear from all experts that PGS 2.0 can be defined as biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification. Most agree that mosaicism is less of an issue at the blastocyst stage than at the cleavage stage but whether mosaicism is no issue at all at the blastocyst stage is currently called into question. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A questionnaire was developed on the three major aspects of PGS 2.0: the Why, with general questions such as PGS 2.0 indications; the How, specifically on genetic analysis methods; the When, on the ideal method and timing of embryo biopsy. Thirty-five colleagues have been selected to address these questions on the basis of their experience with PGS, and demonstrated by peer-reviewed publications, presentations at meetings and participation in the discussion. The first group of experts who were asked about "The Why" comprised fertility experts, the second group of molecular biologists were asked about "The How" and the third group of embryologists were asked about "The When". Furthermore, the geographical distribution of the experts has been taken into account. Thirty have filled in the questionnaire as well as actively participated in the redaction of the current paper. MAIN RESULTS AND THE ROLE OF CHANCE: The 30 participants were from Europe (Belgium, Germany, Greece, Italy, Netherlands, Spain, UK) and the USA. Array comparative genome hybridization is the most widely used method amongst the participants, but it is slowly being replaced by massive parallel sequencing. Most participants offering PGS 2.0 to their patients prefer blastocyst biopsy. The high efficiency of vitrification of blastocysts has added a layer of complexity to the discussion, and it is not clear whether PGS in combination with vitrification, PGS alone, or vitrification alone, followed by serial thawing and eSET will be the favoured approach. The opinions range from in favour of the introduction of PGS 2.0 for all IVF patients, over the proposal to use PGS as a tool to rank embryos according to their implantation potential, to scepticism towards PGS pending a positive outcome of robust, reliable and large-scale RCTs in distinct patient groups. LIMITATIONS, REASONS FOR CAUTION: Care was taken to obtain a wide spectrum of views from carefully chosen experts. However, not all invited experts agreed to participate, which explains a lack of geographical coverage in some areas, for example China. This paper is a collation of current practices and opinions, and it was outside the scope of this study to bring a scientific, once-and-for-all solution to the ongoing debate. WIDER IMPLICATIONS OF THE FINDINGS: This paper is unique in that it brings together opinions on PGS 2.0 from all different perspectives and gives an overview of currently applied technologies as well as potential future developments. It will be a useful reference for fertility specialists with an expertise outside reproductive genetics.Large scale data: none. STUDY FUNDING AND COMPETING INTERESTS: No specific funding was obtained to conduct this questionnaire.

PURPOSE: Preimplantation genetic screening (PGS) and diagnosis (PGD) with euploid embryo transfer is associated with improved implantation and live birth rates as compared to routine in vitro fertilization. However, misdiagnosis of the embryo is a potential risk. The purpose of this study was to investigate the clinical discrepant diagnosis rate associated with transfer of trophectoderm-biopsied blastocysts deemed to be euploid via array comparative genomic hybridization (aCGH). METHODS: This is a retrospective cohort study including cycles utilizing PGS or PGD with trophectoderm biopsy, aCGH, and euploid embryo transfer at a large university-based fertility center with known birth outcomes from November 2010 through July 2014 (n = 520). RESULTS: There were 520 embryo transfers of 579 euploid embryos as designated by aCGH. Five discrepant diagnoses were identified. Error rate per embryo transfer cycle was 1.0 %, 0.9 % per embryo transferred, and 1.5 % per pregnancy with a sac. The live birth (LB) error rate was 0.7 % (both sex chromosome errors), and the spontaneous abortion (SAB) error rate was 17.6 % (3/17 products of conception tested, but could range from 3/42 to 7/42). No single gene disorders were mistakenly selected for in any known cases. CONCLUSIONS: Although aCGH has been shown to be a highly sensitive method of comprehensive chromosome screening, several possible sources of error still exist. While the overall error rate is low, these findings have implications for counseling couples that are contemplating PGS and PGD with aCGH.

Study question: Does undetected aneuploidy or mosaicism contribute to pregnancy loss after transfer of euploid embryos by array comparative genomic hybridization (aCGH)? Summary answer: NGS detects more cases of mosaicism and triploidy than aCGH, and mosaicism rates are significantly higher among pregnancies resulting in miscarriage than live birth. What is known already: Array CGH is widely used for pre-implantation genetic screening (PGS). NGS is capable of detecting more cases of mosaicism and triploidy (69XXY), which may assist in reducing the incidence of spontaneous abortion and increase ongoing pregnancy rates. Study design, size, duration: Retrospective study of 183 patients undergoing PGS by aCGH between 8/2012 and 5/2015 at New York University Fertility Center and Oregon Reproductive Medicine. Participants/materials, setting, methods: Saved amplified DNA samples from the 183 blastocyst trophectoderm (TE) biopsies previously diagnosed as euploid by aCGH were re-analyzed using the miSeq NGS platform (Illumina, USA) and VeriSeq NGS technology (Illumina, USA). 44 embryos resulting in a biochemical pregnancy, 62 resulting in miscarriage, and 77 resulting in live birth were available for re-analysis. Main results and the role of chance: 25% (11/44) of embryos resulting in biochemical pregnancies were mosaic, and one embryo was found to be triploid (69, XXY) by NGS. 33.9% (21/62) and 3.2% (2/62) of embryos resulting in miscarriage were mosaic and triploid by NGS, respectively. In contrast, the mosaicism rate among embryos resulting in live birth was only 13% (10/77), which was significantly lower than the rate of mosaicism among miscarriages (p = 0.0062, RR 1.78 with 95% CI 1.23-2.5). Limitations, reasons for caution: This study was limited by its retrospective design. Up to 10% of DNA samples that were undergoing re-analysis were excluded due to degraded DNA, although the frequency was similar in all three groups. Wider implications of the findings: Undetected mosaicism may increase the risk of first trimester pregnancy loss. NGS is more sensitive at picking up mosaicism and triploidy than aCGH. Mosaic embryos can be considered for transfer after genetic counseling and informed consent, but they have a higher miscarriage rate as well as unknown post-natal genetic effects

Study question: Can quantification of mitochondrial DNA (mtDNA) in trophectoderm biopsy specimens provide information concerning embryo viability, potentially enhancing embryo selection and improving IVF treatment outcomes? Summary answer: This study demonstrates that mtDNA levels are highly predictive of embryo potential. Euploid embryos of good morphology, but with high mtDNA levels fail to implant. What is known already: Better methods of embryo selection are highly desirable in order to improve IVF treatment efficiency. Even the transfer of chromosomally normal embryos of high morphological grade cannot guarantee that a pregnancy will follow. Recently, the quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability-higher levels of mtDNA associated with reduced implantation potential. However, to date no prospective blinded studies have been undertaken to confirm this possibility. The current investigation involves the first evaluation of the predictive power of mtDNA quantification in a prospective, blinded, non-selection setting. Study design, size, duration: mtDNA was quantified in 280 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic screening (PGS). These were generated by 143 couples (average female age 37.2 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection 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. Participants/materials, setting, methods: Embryos were biopsied at the blastocyst stage. The trophectoderm samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis using a next generation sequencing strategy (NGS). The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After embryo transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with blastocyst implantation. Main results and the role of chance: mtDNA analysis of the 280 blastocysts revealed that 15 (5.4%) contained unusually high levels of mtDNA. At the time of writing, outcome data was available for 111 of the blastocysts, transferred after PGS results had been obtained but before mtDNA levels were known. All transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 78 (70%) led to ongoing pregnancies, and all (100%) had mtDNA levels considered to be normal/low. The remaining 33 (30%) blastocysts failed to implant. Among these non-viable embryos there were 7 (21%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 76% (78/103). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/7 (0%). This difference was highly statistically significant (P < 0.0001). Limitations, reasons for caution: This study provides strong evidence that mtDNA quantification can serve as a valuable tool to improve the identification of viable blastocysts. However, to determine the true extent of any clinical benefits a randomised clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion. Wider implications of the findings: 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 implant, supporting its use as a viability biomarker. 76% of euploid embryos with normal/low mtDNA implanted vs. 70% for the cohort as a whole

Study question: What is the overall value of mitochondrial DNA (mtDNA) assessment in a clinical setting, and how can it help improve IVF outcomes? Summary answer: Retrospective analysis demonstrates that mtDNA quantification has a high negative predictive value and can be used as an independent biomarker in determining embryo viability. What is known already: Chromosomal abnormalities are a major cause of implantation failure. Recently our group has validated the relationship between mtDNA quantification and IVF outcomes in a non-selection study. In particular we have been able to establish a threshold above which the probability of implantation is greatly reduced. While further randomized testing is being conducted, we took this opportunity to retrospectively analyze the overall value of the established mtDNA threshold in previously completed clinical cases. Study design, size, duration: Retrospective study in which mtDNA was assessed in a total of 572 euploid blastocysts obtained from 328 couples (average maternal age 34.95 +/- 0.27 years) undergoing preimplantation genetic screening (PGS). Outcome data was collected from 6 different IVF centers for routine follow up. Implantation outcomes were then utilized to determine the validity of the established threshold. Participants/materials, setting, methods: DNA from blastocyst biopsy samples was amplified (Sureplex, Illumina, USA) and then subjected to aneuploidy analysis using next generation sequencing (NGS, Veriseq protocol, Illumina, USA). Only those embryos classified as chromosomally normal had their mtDNA levels assessed using MitoGrade (Reprogenetics). mtDNA levels were then compared to the pregnancy outcomes to confirm implantation predictions. All embryos were single embryo transfers (SET). Main results and the role of chance: Nearly 14% (80/572) of all blastocysts analyzed contained mtDNA levels above the established threshold and were predicted to have lower chances of implantation. To date, 246 euploid embryos were replaced in SET with a pregnancy rate of 62.3% (153/246). Retrospective assessment of mtDNA levels revealed 216 embryos to contain normal mtDNA levels. Therefore the pregnancy rate post mtDNA quantification was 71% (153/216) in the normal level mtDNA group. Furthermore of the 30 embryos with elevated mtDNA levels, only one led to a successful pregnancy. Therefore the negative predictive value of mtDNA quantification was 96.7% (29/30). This highly significant (p < 0.001) difference between implanting and non-implanting embryos validates the clinical applicability of mtDNA quantification. More importantly, the mtDNA threshold retained its validity across six different IVF centers and was unaffected by maternal age. Limitations, reasons for caution: The study was retrospective and the number of cycles was not enough to show a difference between no selection (62% pregnancy rate) and selection against MitoGrade elevated embryos (71% pregnancy rate). It was however large enough to show a significant difference in implantation rates between elevated and normal MitoGrade embryos. Wider implications of the findings: This study demonstrates the validity of MitoGrade as an independent variable in predicting embryonic implantation potential of euploid embryos. Further research involving the biological significance of mtDNA levels and implantation rates would be invaluable

INTRODUCTION: PI3K/AKT/mTOR up-regulation results in accelerated activation of the primordial follicle (PMF) pool and has been implicated in cyclophosphamide (CY)-induced 'follicular exhaustion'. We compared follicular dynamics in murine models of CY-induced POI and age-related DOR, hypothesizing that 'follicular exhaustion' may occur similarly in both groups. METHODS: C57BL/6 female mice aged 8 wks (n=25) housed in identical conditions were assigned to 5 groups: 150mg/kg CY intraperitoneal (IP) x1, 75mg/kg CY IP weekly x3, control (8 wks and 11 wks), and DOR (13 months). Blood was extracted by terminal cardiac puncture for anti-mullerian hormone (AMH). Ovaries were paraffin-embedded, sectioned, and H&E stained; blinded follicle counts were confirmed by two reviewers. Follicle counts are presented as follicles/section area (mm2); data presented as mean+/-SEM. One-way ANOVA and student's t-test were used for statistical analysis (p<0.05). 'Follicular exhaustion' was calculated as the ratio of total growing follicles (primary, secondary, antral) to PMFs. RESULTS: A DOR murine model was confirmed by low AMH in 13-mth-old mice compared to controls (7.2+/-0.1 vs. 12.1+/-0.5, p<0.05) and fewer PMFs/mm2 (0.6+/-0.2) compared to 8-wk (7.7+/-1.2) and 11 wk controls (6.0+/-1) (p<0.05). A CY-induced POI model was confirmed by lower AMH in 150mg/kg CY-exposed mice compared to age-matched controls (9.6+/-0.9 vs. 12.1+/-0.5) and fewer PMFs/mm2 (2.5+/-0.4 vs. 7.7+/-1.2, p<0.05). CY-treated mice had more primary & secondary follicles/mm2 compared to controls (p<0.05), suggesting increased follicle activation. PMF counts were similar in POI [75mg/kg CY (2.4 +/- 0.5) and 150mg/ kg CY (1.9+/-0.3)] compared to DOR (0.6+/-0.2) despite an 11-mth age difference. Importantly, similar rates of 'follicular exhaustion' were seen in CY-exposed POI mice (75mg/kg CY ratio 1.9; 150mg/kg CY ratio 2.6) and DOR mice (ratio 2.4). In contrast, low 'follicular exhaustion' ratios were seen in both control groups (8-wks ratio 0.64; 11-wks ratio 0.89) signifying maintenance of follicles as PMFs. CONCLUSIONS: Accelerated PMF activation and subsequent follicular exhaustion occur similarly in murine models of CY-induced POI and age-related DOR. Future studies should investigate if PI3K/AKT/mTOR up-regulation is also involved in DOR, suggesting possible implications for prevention