Faculty Bibliography

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.

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

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.

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 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: hr-NGS detects mosaicism in trophectoderm (TE) biopsies when 10%-90% of cells are abnormal. Mosaic TE biopsies may come from embryos that result from euploid, aneuploid or mosaic pregnancies. Mosaic biopsies have higher risk of miscarrying, lower implantation potential but some may go to term. Mosaic embryos should be classified in between euploid and aneuploid in their priority for transfer. This study aims to determine the rate of euploidy, mosaicism and other abnormalities according to maternal age in a large cohort of embryos for purpose of patient counseling. DESIGN: Analysis of PGS procedures involving TE biopsy and hr-NGS performed by two large genetic reference laboratories serving over 250 fertility clinics. MATERIALS AND METHODS: Two laboratories analyzed 4277 and 2303 PGS procedures using TE biopsy by whole genome amplification method (SurePlex), assay for hr-NGS (VeriSeq PGS assay, Illumina), sequencer (MiSeq, Illumina) and software (BlueFuse Multi analysis, Illumina). EMRs (eIVF, practiceHwy and lab's own) used to query the data. Embryos with 1 or 2 aneuploid chromosomes or 1 aneuploid chromosome and 1 mosaic chromosome were called aneuploid. Embryos with 3 aneuploid chromosomes or two aneuploid and 1 or more mosaic chromosomes were complex abnormal. Embryos with a mixture of normal cells and abnormal were called mosaic. RESULTS: Rate of euploidy, mosaicism and complete abnormalities (aneuploidy, complex or polyploidy) was 41% and 47%, 20% and 14%, and 39% and 39%, respectively. The combined results for both datasets shown (Table1). CONCLUSIONS: Being a postmeiotic abnormality, mosaicism does not increase with advancing maternal age (the apparent decrease in the table is due to some mosaics that are also aneuploid being classified as aneuploid). Differences between the two labs were attributed to difference in scoring criteria where the threshold to call an embryo normal or euploid was set (10-90% vs 17-83% or 1/10-9/10 cells vs 1/6-5/6). Regardless, the amount of mosaic embryos, which have lower potential than euploid embryos, is considerable, 11-23% depending on maternal age. (Table Presented)

OBJECTIVE: The recent recognition of increased genetic admixture and advances in technology advances have caused a shift to broader acceptance of pan-ethnic expanded carrier screening (ECS) panels. However, ECS has not been widely accepted across Europe. The resistance is partially due to a belief that the US is genetically diverse from years of admixture while the European population is genetically homogeneous, making pan-ethnic screening unnecessary. We sought to determine if this difference in admixture was present. DESIGN: Retrospective. MATERIALS AND METHODS: Genomic data from an ECS panel was analyzed for 7544 participants, 6297 from US clinics and 1247 from European clinics. For all participants, genetic ancestral origin was predicted by a statistical model based on 672 SNPs validated using samples from the 1000 Genomes Project, and admixture proportions were calculated for 6 ancestral populations (European, Oceania Native, Native American, East Asian, Sub-Saharan African, and South Asian). A comparison of the resulting predicted admixture proportions was made between patients in Europe against patients in the US. Consent to use de-identified genomic data was obtained for all participants. RESULTS: For comparison, the European and American patients were further subdivided into four groups, based upon which ethnicity they self-reported on test requisition forms: European, Mediterranean, Latin American, or African. Across all four comparison groups, our results showed similar average admixture proportions. For example, European patients who self-identified as Mediterranean were genetically predicted to be an average of 86% European, and 5% Sub-Saharan African. American patients who self-identified as Mediterranean were genetically predicted to be an average of 81% European and also 5% Sub-Saharan African. European patients who self-identified as African were predicted to be an average of 78% African and 16% European, which was strikingly similar to the predictions for American patients who self-identified as African (78% African and 12% European). This similarity in admixture proportions was also seen between European and American patients who self-identified as European and Latin American. CONCLUSIONS: These results demonstrate that on a genetic level, both European and American patients demonstrate equal degrees of admixture. The European patient population may not be as genetically homogenous as previously believed. Pan-ethnic carrier screening panels could prove to be effective at identifying carriers in a European population, who would otherwise be missed

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