Faculty Bibliography

OBJECTIVE: To compare the euploidy outcome in patients that underwent 2 ovarian stimulation cycles with trophectoderm biopsy. DESIGN: Retrospective repeated-measures cohort study. SETTING: University-based fertility center. PATIENT(S): A total of 116 patients, from 2011 through 2013, that underwent 2 ovarian stimulation cycles followed by trophectoderm biopsy with array comparative genomic hybridization. INTERVENTION(S): Days of stimulation, average diameter of the 2 lead follicles on day of trigger, dose of gonadotropins, type of cycle (gonadotropin-releasing hormone [GnRH] antagonist, GnRH-antagonist plus clomiphene citrate [CC], microdose GnRH agonist). MAIN OUTCOME MEASURE(S): Number of euploid embryos. RESULT(S): Patients were analyzed based on whether they had >/=1 euploid embryos in their first cycle vs. none. There was no increase in the number of euploid embryos with more days of stimulation or increases in the dose of gonadotropins in either group. Significantly more euploid embryos were seen in patients who had no euploid embryo(s) in the first cycle (Group 0) that had CC added to a GnRH-antagonist cycle (1.11 more euploid embryos) or were triggered when follicle sizes were 2 mm larger (0.40 euploid embryos), but these increases were not significant compared with a control group. Patients with euploid embryo(s) in the first cycle (Group 1) had significantly more euploid embryos when daily dose was increased by 75-149 international units, but this relationship was not significant compared with a control group with no increase in daily dose. CONCLUSION(S): No specific intervention increased the number of euploid embryos within the same patient any more than simply repeating a similar stimulation cycle. An attempt was made to control for interpatient variability, but individual patients have considerable intercycle variability.

PURPOSE: In Vitro Fertilization is an effective treatment for infertility; however, it has relatively low success in women of advanced maternal age (>37) who have a high risk of producing aneuploid embryos, resulting in implantation failure, a higher rate of miscarriage or birth of a child with chromosome abnormalities. The purpose of this study was to compare the implantation, miscarriage and live birth rates with and without preimplantation genetic screening (PGS) of embryos from patients aged 40 through 43 years. METHODS: This is a retrospective cohort study, comparing embryos screened for ploidy using trophectoderm biopsy and array comparative genomic hybridization to embryos that were not screened. We compared pregnancy outcomes for traditional fresh IVF cycles with day 5 embryo transfers, Frozen Embryo Transfer (FET) cycles without PGS and PGS-FET (FET of only euploid embryos) cycles of patients with maternal ages ranging from 40 to 43 years, undergoing oocyte retrievals during the period between 1/1/2011 and 12/31/2012. RESULTS: The implantation rate of euploid embryos transferred in FET cycles (50.9 %) was significantly greater than for unscreened embryos transferred in either fresh (23.8 %) or FET (25.4 %) cycles. The incidence of live birth per transferred embryo for PGS-FET (45.5 %) was significantly greater than for No PGS fresh (15.8 %) or No PGS FET (19.0 %) cycles. The incidences of live birth per implanted sac for PGS FET cycles (89.3 %), No PGS fresh cycles (66.7 %) and No PGS FET cycles (75.0 %) were not significantly different. CONCLUSIONS: The present data provides evidence of the benefits of PGS with regard to improved implantation and live birth rate per embryo transferred.

OBJECTIVE: To determine whether an association exists between body mass index (BMI) and embryo ploidy in patients undergoing in vitro fertilization (IVF) with trophectoderm biopsy and 24-chromosome preimplantation genetic screening (PGS). DESIGN: Retrospective cohort study. SETTING: University-based fertility center. PATIENT(S): 279 women aged 20-45 years with documented height and weight from the day of oocyte retrieval who underwent 24-chromosome PGS between 2010 and 2013. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Primary outcomes: number and percentage of euploid embryos. RESULT(S): Patients were grouped by World Health Organization (WHO) BMI class: underweight (<18.5, n = 11), normal weight (18.5-24.9, n = 196), overweight (25-29.9, n = 50), and obese (>/=30, n = 22). Groups were similar by age (mean +/- standard error of the mean: 37.5 +/- 1.2 to 39.2 +/- 0.9), ovarian reserve, and IVF cycle parameters. There was no difference in the number or percentage of euploid embryos by BMI category (<18.5: 27.6% +/- 8.5; 18.5-24.9: 34.5% +/- 2.2; 25-29.9: 32.1% +/- 4.3; >/=30: 30.9% +/- 7.3). Age was inversely related to euploidy, but adjusted multivariate regression models failed to demonstrate a statistically significant relationship between BMI and euploidy in underweight (adjusted odds ratio [AOR] 0.44; 95% confidence interval [CI], 0.09-2.10), overweight (AOR 0.90; 95% CI, 0.43-2.00), or obese (AOR 0.74; 95% CI, 0.25-2.20) patients compared with the normal-weight reference group. CONCLUSION(S): No statistically significant relationship was identified between BMI and euploidy in an otherwise homogenous cohort of patients undergoing IVF with PGS, suggesting that the negative impact of overweight and obesity on IVF and reproductive outcomes may not be related to aneuploidy.

PURPOSE: To determine if day of embryo transfer (ET) affects gestational age (GA) and/or birth weight (BW) at a single university fertility center that primarily performs day 5/6 ET. METHODS: Retrospective cohort study of 2392 singleton live births resulting from IVF/ICSI at a single large university fertility center from 2003 to 2012. Patients were stratified by day 3 or day 5/6 ET. Outcome variables included patient age, gravidity, prior miscarriages, prior assisted reproduction technology cycles, number of embryos transferred, number of single ET, infertility diagnosis, neonatal sex, GA at birth, and BW. Subanalyses were performed on subgroups of preterm infants. A comparison was made between the study data and the Society of Assisted Reproductive Technologies (SART) published data. RESULTS: There was no difference in GA at birth (39 +/- 2.1 weeks for day 3 ET, 39 +/- 1.9 weeks for day 5/6 ET) or BW between ET groups (3308 +/- 568 g for day 3 ET, 3268 +/- 543 g for day 5/6 ET). There was also no difference in the number of preterm deliveries (8.5 % for day 3 ET vs. 10.8 % for day 5/6 ET). The day 5/6 ET study data had significantly fewer pre-term deliveries than the SART day 5/6 ET data. CONCLUSION: In contrast to published SART data, GA and BW were not influenced by day of ET. Data may be more uniform at a single institution. Day 5/6 ET continues to offer improved pregnancy rates without compromising birth outcomes.

OBJECTIVE: To determine if long-term cryopreservation of human oocytes affects oocyte developmental competence, blastocyst euploidy, or live-birth rates. DESIGN: Retrospective cohort study. SETTING: University-based fertility center. PATIENT(S): A total of 33 patients with cryopreserved oocytes underwent oocyte thaw, blastocyst culture, trophectoderm biopsy, and 24-chromosome preimplantation genetic screening (PGS) with array comparative genomic hybridization between December 2011 and July 2014; subjects were compared with 2:1 age-matched controls with fresh oocytes whose embryos underwent trophectoderm biopsy and PGS during the same period. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rates of fertilization, blastulation, euploidy, implantation, and live birth. RESULT(S): Thirty-three patients (mean age 36.2 +/- 3.8 y) thawed 475 oocytes that had been cryopreserved for a median of 3.5 years. Compared with 66 age-matched controls who underwent in vitro fertilization and PGS with fresh oocytes, embryos derived from cryopreserved oocytes demonstrated compromised blastocyst formation (54.5% vs. 66.2%) despite no impairment in fertilization (72.8% vs. 73.2%). Results showed no difference in the number of euploid blastocysts (1.7 +/- 1.9 vs. 2 +/- 2.5), percentage of euploid blastocysts (44.5% vs. 47.6%), rate of implantation (65% vs. 65%), or rate of live birth and ongoing pregnancy (62.5% vs. 55%) after 24-chromosome PGS with cryopreserved or fresh oocytes. CONCLUSION(S): Embryos derived from cryopreserved oocytes demonstrate impaired blastulation but equivalent rates of euploidy, implantation, and live birth compared with blastocysts derived from fresh oocytes, supporting the safety and efficacy of oocyte cryopreservation.

BACKGROUND:The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. METHODS:Embryo biopsy, whole genome amplification and semiconductor sequencing. RESULTS:A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. CONCLUSIONS:This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability.

The chief cause for failure of in vitro fertilization (IVF)-assisted reproductive treatments may be the high frequency of aneuploid preimplantation embryos, especially among women of advanced reproductive age. It has been hypothesized that pregnancy loss with advancing maternal age can be prevented by selective transfer of euploid embryos. Preimplantation genetic diagnosis (PGD) for aneuploidy was first attempted more than 20 years ago. It was hoped that screening embryos for aneuploidy and transferring only those found to be euploid would increase implantation and pregnancy rates and reduce pregnancy loss rates. Initial attempts to detect aneuploidy used fluorescence in situ hybridization analysis (first-generation PGD). However, several randomized controlled trials found no benefit for first-generation PGD or even a negative impact on implantation, pregnancy, or loss rates. A more accurate version of PGD was needed. Array comparative genomic hybridization (aCGH) is a second-generation PGD. Use of this technique in randomized controlled trials improved pregnancy rates. The major drawback of aCGH is the need for at least 3 full days for an analysis to be completed. With the introduction of vitrification (freezing), safe cryopreservation of embryos that underwent biopsy became possible, allowing delay of embryo biopsy from 3 days (gastrula stage) to 5 to 6 days (blastocyst stage), which is less detrimental to embryo development. This multicenter retrospective study assessed the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. The aim of the study was to determine whether aCGH followed by selective transfer of euploid embryos would mitigate the age-related decline in implantation rates observed in IVF cycles. Preimplantation chromosome screening was performed in women undergoing IVF at a number of fertility clinics in the United States. Implantation rates across different maternal ages were examined with embryo biopsy on day 3 or day 5/6 followed by aCGH. The primary outcome measures were aneuploidy, implantation, pregnancy, and loss rates. Aneuploidy rates increased with advancing maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for day 5/6 biopsies. Implantation rates for euploid embryos for ages 35 to 42 years were maintained after PGD; rates ranged from 44% to 32% for day 3 biopsies and 51% to 40% for day 5 biopsies. Ongoing pregnancy rates per transfer remained stable for maternal ages younger than 42 years, ranging from 48.5% to 38.1% for day 3 biopsies and 64.4% to 54.5% for day 5 biopsies. For patients 42 years or older, implantation rates were 23.3% with day 3 biopsies and 27.7% with day 5/6, and the ongoing pregnancy rate was 9.3% for day 3 biopsies and 10.3% for day 5. These data show no significant difference in implantation and pregnancy rates after selective transfer of euploid embryos between reproductively younger and older patients up to 42 years old. The enhanced embryo selection afforded by methods such as aCGH cannot improve pregnancy rates in patients who fail to produce at least 1 euploid embryo, a situation increasingly common with advancing maternal age. These findings and mounting data from other studies suggest that aneuploidy is the primary reason for the marked decline in IVF treatment success rates in women of advanced reproductive age when PGD is not used.