K.M. Upham

Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.

OBJECTIVE To determine whether blastocyst biopsy and rapid quantitative real-time polymerase chain reaction (qPCR)-based comprehensive chromosome screening (CCS) improves in vitro fertilization (IVF) implantation and delivery rates. DESIGN Randomized controlled trial. SETTING Academic reproductive medicine center. PATIENT(S) Infertile couples in whom the female partner (or oocyte donor) is between the ages of 21 and 42 years who are attempting conception through IVF. INTERVENTION(S) Embryonic aneuploidy screening. MAIN OUTCOME MEASURE(S) Sustained implantation and delivery rates. RESULT(S) We transferred 134 blastocysts to 72 patients in the study (CCS) group and 163 blastocysts to 83 patients in the routine care (control) group. Sustained implantation rates (probability that an embryo will implant and progress to delivery) were statistically significantly higher in the CCS group (89 of 134; 66.4%) compared with those from the control group (78 of 163; 47.9%). Delivery rates per cycle were also statistically significantly higher in the CCS group. Sixty one of 72 treatment cycles using CCS led to delivery (84.7%), and 56 of 83 (67.5%) control cycles ultimately delivered. Outcomes were excellent in both groups, but use of CCS clearly improved patient outcomes. CONCLUSION(S) Blastocyst biopsy with rapid qPCR-based comprehensive chromosomal screening results in statistically significantly improved IVF outcomes, as evidenced by meaningful increases in sustained implantation and delivery rates. CLINICAL TRIAL REGISTRATION NUMBER NCT01219283.

Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial.

OBJECTIVE To determine if cleavage- or blastocyst-stage embryo biopsy affects reproductive competence. DESIGN Paired randomized clinical trial. SETTING Academic-assisted reproduction program. PATIENT(S) Attempting conception through IVF. INTERVENTION(S) After selecting two embryos for transfer, one was randomized to biopsy and the other to control. Both were transferred within shortly thereafter. The biopsy was submitted for microarray analysis and single-nucleotide polymorphism (SNP) profiling. Buccal DNA obtained from the neonate after delivery had microarray analysis and SNP profile compared with that of the embryonic DNA. A match confirmed that the biopsied embryo implanted and developed to term, whereas a nonmatch indicated that the control embryo had led to the delivery. MAIN OUTCOME MEASURE(S) Paired analysis of the delivery rates of the transferred embryos. Either twin delivery or failure to deliver represents equivalent outcomes for the biopsied and control embryos. In contrast, singletons were determined to be from the biopsied or the control embryo. RESULT(S) Blastomere biopsy on day 3 of development resulted in a significant reduction in sustained implantation. Only 30% of biopsied embryos had sustained implantation and ultimately developed into live-born infants versus 50% of unbiopsied controls, a relative reduction of 39%. In contrast, sustained implantation rates were equivalent (51% vs. 54%) for biopsied and control blastocysts. CONCLUSION(S) Cleavage-stage biopsy markedly reduced embryonic reproductive potential. In contrast, trophectoderm biopsy had no measurable impact and may be used safely when embryo biopsy is indicated. CLINICAL TRIAL REGISTRATION NUMBER NCT01219504.

The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening

OBJECTIVE To determine the relationship between the age of the female partner and the prevalence and nature of human embryonic aneuploidy. DESIGN Retrospective. SETTING Academic. PATIENT(S) Trophectoderm biopsies. INTERVENTION(S) Comprehensive chromosomal screening performed on patients with blastocysts available for biopsy. MAIN OUTCOME MEASURE(S) Evaluation of the impact of maternal age on the prevalence of aneuploidy, the probability of having no euploid embryos within a cohort, the complexity of aneuploidy as gauged by the number of aneuploid chromosomes, and the trisomy/monosomy ratio. RESULT(S) Aneuploidy increased predictably after 26 years of age. A slightly increased prevalence was noted at younger ages, with >40% aneuploidy in women 23 years and under. The no euploid embryo rate was lowest (2% to 6%) in women aged 26 to 37, was 33% at age 42, and was 53% at age 44. Among the biopsies with aneuploidy, 64% involved a single chromosome, 20% two chromosomes, and 16% three chromosomes, with the proportion of more complex aneuploidy increasing with age. Finally, the trisomy/monosomy ratio approximated 1 and increased minimally with age. CONCLUSION(S) The lowest risk for embryonic aneuploidy was between ages 26 and 30. Both younger and older age groups had higher rates of aneuploidy and an increased risk for more complex aneuploidies. The overall risk did not measurably change after age 43. Trisomies and monosomies are equally prevalent.

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Comprehensive chromosome screening alters traditional morphology-based embryo selection: a prospective study of 100 consecutive cycles of planned fresh euploid blastocyst transfer

OBJECTIVE To determine how often trophectoderm biopsy and rapid, real-time quantitative polymerase chain reaction (PCR)-based comprehensive chromosome screening (CCS) alters clinical management by resulting in the transfer of a different embryo than would have been chosen by traditional day 5 morphology-based criteria. DESIGN Prospective. SETTING Academic center for reproductive medicine. PATIENT(S) Infertile couples (n = 100; mean age 35 ± 4 years) with at least two blastocysts suitable for biopsy on day 5. INTERVENTION(S) Prior to trophectoderm biopsy for CCS the embryologist identified which embryo would have been selected for traditional day 5 elective single ET. MAIN OUTCOME MEASURE(S) The risk of aneuploidy in the embryos that would have been selected on day 5 was calculated and compared with the aneuploidy rate of the cohort of all embryos that underwent CCS testing. The aneuploidy risk was compared between age groups. RESULT(S) After quantitative PCR-based CCS, 22% (95% confidence interval 15%-31%) of the embryos selected by day 5 morphology were aneuploid, which was lower than the 32% aneuploidy rate of the cohort. Patients ≥35 years had a higher risk of an aneuploid blastocyst being selected by morphology than those <35 years old (31% vs. 14%). Among patients who had selection altered by CCS, 74% (14/19) delivered, including 77% (10/13) after elective single ET. Most patients (77%) had an additional euploid blastocyst vitrified for future use. CONCLUSION(S) The CCS results alter embryo selection due to the presence of aneuploidy in embryos with optimal day 5 morphology. Excellent outcomes were obtained when CCS-based selection was different than morphology-based selection.

Blastocyst transfer is not associated with increased rates of monozygotic twins when controlling for embryo cohort quality

OBJECTIVE To compare monozygotic twinning (MZT) rates in patients undergoing blastocyst or cleavage-stage ET. DESIGN Retrospective cohort. SETTING Academic research center. PATIENT(S) Autologous, fresh IVF cycles resulting in a clinical pregnancy from 1999 to 2014. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Monozygotic twin pregnancy in blastocyst-stage transfer vs. cleavage-stage transfer when controlling for patient prognosis and embryo cohort quality factors. RESULT(S) There were a total of 9,969 fresh transfer cycles resulting in a pregnancy during the study period. Of these pregnancies, 234 monozygotic twin pregnancies were identified (2.4%). Of all transfers, 5,191 were cleavage-stage and 4,778 were blastocyst-stage transfers. There were a total of 99 MZT identified in the cleavage-stage group (1.9%) and 135 MZT in the blastocyst ET group (2.4%), which was significant. Multivariable logistic regression revealed that increasing age was associated with a significant reduction in MZT, regardless of transfer order. Embryo cohort quality factors, including the number and proportion of six- to eight-cell embryos and availability of supernumerary embryos, were also significant. When controlling for patient age, time period during which the cycle took place, the number and proportion of six- to eight-cell embryos, and availability of supernumerary embryos, there was no longer a difference in MZT rate between blastocyst and cleavage transfer. CONCLUSION(S) Patient prognosis and embryo cohort quality seem to be major factors in MZT rate in women undergoing blastocyst transfer. Although technology-based effects cannot be excluded, patient and embryo characteristics play an important role.

Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure

PurposeTo characterize each chromosome’s risk for being involved in embryonic aneuploidy.MethodsThis is a retrospective cohort study conducted at a single, academic center. The cohort consisted of 15,169 consecutive trophectoderm biopsies which then underwent comprehensive chromosome screening utilizing validated real-time polymerase chain reaction (RT-PCR) or single nucleotide polymosphism (SNP) array platforms. Analysis was done to determine probability of aneuploidy by chromosome, changes in that risk with increasing maternal age, and in relationship of aneuploidy to chromosomal structure as classified by prior cytogenetic literature.ResultsThe highest prevalence of imbalances leading to aneuploidy was seen for chromosomes 13, 15, 16, 18, 19, 21, and 22. While elevated in all age groups, there was a disproportionate rise in aneuploidy rates for these chromosomes with increasing maternal age. When classic cytogenetic karyotype groups were compared, the overall smaller groups D, E, and G were associated with the highest rates. Similarly, when grouped based upon structure, acrocentric chromosomes exhibited the highest rates of aneuploidy, followed by the metacentric chromosomes, with the lowest prevalence of error in those with submetacentric structures.ConclusionsThe highest rates of chromosomal aneuploidy were found in chromosomes known to be involved in clinically detectable, abnormal pregnancies, not just simply implantation failure. The rate of aneuploidy in these chromosomes rises disproportionately with age when compared to the other chromosomes which may provide information about chromosomal susceptibility to aging. The biological structure groupings did show varied aneuploidy rates which may provide insight into the biology of aneuploidy.

Endometrial infusion of human chorionic gonadotropin at the time of blastocyst embryo transfer does not impact clinical outcomes: a randomized, double-blind, placebo-controlled trial

OBJECTIVE To determine whether endometrial hCG infusion at the time of human blastocyst transfer impacts implantation rates. DESIGN Randomized double-blinded placebo-controlled trial. SETTING Academic. PATIENT(S) Infertile couples with the female partner less than 43 years old (n = 300) undergoing fresh or frozen ET of one or two blastocysts. INTERVENTION(S) Patients undergoing ET were randomized into either a treatment or a control group. The treatment group received an infusion of 500 IU of hCG diluted in ET media. The control group received a sham infusion of ET media. Infusions were done using a separate catheter less than 3 minutes before actual ET. MAIN OUTCOME MEASURE(S) Sustained implantation rate: ongoing viable gestation (primary outcome) and ongoing pregnancy rate (secondary outcome). RESULT(S) A total of 473 blastocysts were transferred into 300 patients. There were no differences between the two groups in sustained implantation rate (48.1% in the hCG group, 44.2% in the control group) or ongoing pregnancy rate (58.8% in the hCG group, 52.0% in the control group). CONCLUSION(S) Endometrial infusion of hCG at the time of blastocyst ET does not improve sustained implantation rates. CLINICAL TRIAL REGISTRATION NUMBER NCT01643993.

Examining the temperature of embryo culture in in vitro fertilization: a randomized controlled trial comparing traditional core temperature (37°C) to a more physiologic, cooler temperature (36°C)☆

OBJECTIVE To determine whether culture at a more physiologically cooler temperature, as suggested by limited human and animal data, would improve blastulation and pregnancy rates in human clinical IVF. DESIGN Paired randomized controlled trial. SETTING Academic. PATIENT(S) Infertile couples (n=52) with a female partner less than 42 years old with eight or more mature oocytes retrieved. INTERVENTION(S) Mature oocytes obtained from a single cohort of oocytes were randomly divided into two groups; one was cultured at 37°C and the other at 36°C from the time of ICSI to the time of embryo transfer or vitrification. Paired embryo transfers were accomplished by transferring one euploid embryo from each group. DNA fingerprinting was used as needed to determine the outcome for each embryo. MAIN OUTCOME MEASURE(S) Rate of development of expanded blastocysts suitable for transfer or vitrification (primary outcome), fertilization, aneuploidy, and sustained implantation. RESULT(S) A total of 805 mature oocytes were cultured; 399 at 36°C and 406 at 37°C. Paired analysis demonstrated a higher rate of usable blastocyst formation per zygote at 37°C (48.4%) vs. at 36°C (41.2%). Rates of fertilization, aneuploidy, and sustained implantation were equivalent. CONCLUSION(S) IVF culture at 36°C does not improve clinically relevant parameters of embryo development or sustained implantation rates. CLINICAL TRIAL REGISTRATION NUMBER NCT01506089.

Embryology training for Reproductive Endocrine fellows in the clinical human embryology laboratory

ObjectiveTo determine if comprehensive embryology training for clinical Reproductive Endocrinology fellows could be completed to a level of proficiency equivalent to that of experienced embryologists.MethodClinical fellows were integrated into the clinical embryology team and were trained to perform all the various procedures utilized in clinical embryology. The fellows were trained to the same standards as the clinical embryology staff and underwent the same certification and sign off procedures. To determine if inclusion of clinical fellows on the embryology team impacted outcomes, outcomes for individual oocytes/embryos and the clinical cases where the fellows perform embryology procedures were compared to the outcomes of those oocytes/embryos and cases performed by the full time embryology staff.ResultsClinical procedures performed by the fellows included isolation and processing of oocytes following retrieval, loading catheters for embryo transfer, and vitrification (N = 823 cases). Micromanipulation procedures compared included ICSI and assisted hatching (N = 650 cases). For each procedure, the outcomes in those cases performed by the RE fellows were equivalent to those done by the fully trained clinical embryology staff.ConclusionsWhen fellows are trained to perform embryology procedures as an integral part of their fellowship curricula, laboratory efficiencies and clinical outcomes are fully maintained. This experience provides valuable insight into the ART process critical to this subspecialty. It also empowers fellows to fully participate in research relating to the viability of gamete and embryos and optimization of the clinical ART laboratory.