On the reproductive capabilities of aneuploid human preimplantation embryos.

In IVF cycles, the application of aneuploidy testing at the blastocyst stage is quickly growing, and the latest reports estimate almost half of cycles in the US undergo preimplantation genetic testing for aneuploidies (PGT-A). Following PGT-A cycles, understanding the predictive value of an aneuploidy result is paramount for making informed decisions about the embryo's fate and utilization. Compelling evidence from non-selection trials strongly supports that embryos diagnosed with a uniform whole-chromosome aneuploidy very rarely result in the live birth of a healthy baby, while their transfer exposes women to significant risks of miscarriage and chromosomally abnormal pregnancy. On the other hand, embryos displaying low range mosaicism for whole chromosomes have shown reproductive capabilities somewhat equivalent to uniformly euploid embryos, and they have comparable clinical outcomes and gestational risks. Therefore, given their clearly distinct biological origin and clinical consequences, careful differentiation between uniform and mosaic aneuploidy is critical in both the clinical setting when counseling individuals and in the research setting when presenting aneuploidy studies in human embryology. Here, we focus on the evidence gathered so far on PGT-A diagnostic predictive values and reproductive outcomes observed across the broad spectrum of whole-chromosome aneuploidies detected at the blastocyst stage to obtain evidence-based conclusions on the clinical management of aneuploid embryos in the quickly growing PGT-A clinical setting.

Does PGT-A affect cumulative live birth rate?

PURPOSE OF REVIEW: Preimplantation genetic testing for the purpose of aneuploidy screening (PGT-A) has increased in use over the last decade. RECENT FINDINGS: Whether PGT-A benefits all of the patients that choose to employ it has been a concern, as recent studies have highlighted a potential decrease in cumulative live birth rate (CLBR) for younger patients undergoing embryo transfer. However, there are limitations to many of these studies and the intended benefit of PGT-A, which is to aid as a selection tool, thus increasing the live birth rate per transfer, must not be ignored. SUMMARY: PGT-A was never intended to increase CLBR. The purpose of PGT-A is to maximize the chance at live birth per transfer while minimizing the risk of clinical miscarriage, ongoing aneuploid pregnancy and futile transfers. However, if it harms CLBR in the process that has to be taken into consideration. This review will discuss PGT-A in terms of its benefits, risks, and how it has been shown to affect the cumulative live birth rate within in-vitro fertilization cycles.

Life-threatening complications among women with severe ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome is a potentially life-threatening clinical condition. OBJECTIVE: The objective of this study was to evaluate risk factors for life-threatening complications for patients with severe ovarian hyperstimulation syndrome in a United States nationwide sample. MATERIALS AND METHODS: Ovarian hyperstimulation syndrome admissions from 2002 to 2011 from the Nationwide Inpatient Sample were included in this study. The association between patient and hospital factors and life-threatening complications (deep vein thrombosis/pulmonary embolism, acute respiratory distress syndrome, acute renal failure, intubation), nonroutine discharge (discharge to skilled nursing facility, transfer hospital), prolonged length of stay, and total hospital charges were analyzed. Survey-adjusted multivariable logistic regression analyses were performed for these outcomes, controlling for risk factors, with adjusted odds ratios with 95% confidence intervals as the measures of effect. RESULTS: A total of 11,562 patients were hospitalized with severe ovarian hyperstimulation syndrome from 2002 to 2011. The majority were white (55.7%), with private insurance (87.7%), aged 25-39 years (84.6%), and hospitalized in an urban location (95%). In all, 19.3% of patients had medical comorbidities including hypertension, diabetes, obesity, hypothyroidism, and anemia. Life-threatening complications occurred in 4.4% of patients (deep vein thrombosis/pulmonary embolism, 2.2%; acute renal failure; acute respiratory distress syndrome, 0.9%; intubation, 0.5%). Patients ≥40 years old (odds ratio, 4.02; 95% confidence interval, 1.37, 11.76), those with comorbidities (odds ratio, 2.29; 95% confidence interval, 1.46, 3.57), and African American patients (odds ratio, 2.15; 95% confidence interval, 1.25, 3.70) were more likely to develop life-threatening conditions. Patients with medical comorbidities (odds ratio, 0.39; 95% confidence interval, 0.24, 0.63) were also less likely to be routinely discharged from the hospital. Adjusting for patient and hospital demographics, patients with comorbidities were more likely to develop deep vein thrombosis/pulmonary embolism (adjusted odds ratio, 2.44; 95% confidence interval, 1.28, 4.65) and acute renal failure (adjusted odds ratio, 2.26; 95% confidence interval, 1.21, 4.23). Patients who developed life-threatening complications had longer hospital length of stay (adjusted odds ratio, 3.72; 95% confidence interval, 2.28, 6.07) and higher hospital costs (adjusted odds ratio, 5.20; 95% confidence interval, 3.22,8.39). CONCLUSION: Patients with common medical comorbidities are at higher risk for life-threatening complications in the setting of severe ovarian hyperstimulation syndrome. Furthermore, these complications are associated with high hospital costs and hospital burden. Given the increasing number of in vitro fertilization patients with medical comorbidities, closer monitoring of at-risk patients may be indicated. As assisted reproductive technology practice changes in recent years with strategies designed to reduce ovarian hyperstimulation syndrome risk, future studies are needed to assess the impact of these changes on hospitalization and complication risk.

Minimizing mosaicism: assessing the impact of fertilization method on rate of mosaicism after next-generation sequencing (NGS) preimplantation genetic testing for aneuploidy (PGT-A).

PURPOSE: Advances in preimplantation genetic testing (PGT) have led to practice changes in assisted reproductive technologies (ART), enabling fertility centers to transfer single embryos while maintaining excellent ongoing pregnancy rates, reducing miscarriage rates, and dramatically reducing ART-associated multiple pregnancies. The introduction of next-generation sequencing (NGS) allows PGT laboratories to assess for embryo mosaicism-although the true incidence and reproductive potential of predicted mosaic embryos are controversial. Due to concern for genetic contamination from other spermatozoa, most reference laboratories require use of intracytoplasmic sperm injection (ICSI) for single gene preimplantation genetic diagnosis (PGT-M). However, in PGT for aneuploidy (PGT-A), conventional insemination (IVF) is typically permissible. The purpose of this study was to evaluate rates of euploid, aneuploid, and mosaic in trophectoderm biopsy samples from embryos in IVF versus ICSI PGT-A cycles. Secondary aims were to assess sex ratio, and subtypes of aneuploidy and mosaicism in IVF versus ICSI PGT-A cycles. METHODS: We performed a retrospective review of women undergoing PGT-A at a single academic fertility center from July 1, 2015, to September 1, 2017. In all cycles, PGT-A was performed via trophectoderm biopsy on day 5 or 6 and analyzed using NGS at a single reference lab. We collected and compared patient demographics, fertility testing, cycle characteristics, and PGT-A outcomes between IVF and ICSI cycles. RESULTS: Three hundred two PGT-A cycles were included for analysis: 75 IVF and 227 ICSI cycles, resulting in 251 IVF and 724 ICSI biopsied blastocysts. Mean oocyte age of included cycles was 38.6 years (IVF) and 38.5 years (ICSI), p = 0.85. Baseline characteristics of IVF and ICSI PGT-A cycles were similar with the exception of semen parameters: IVF cycles had higher sperm concentration and total motility compared to ICSI cycles. PGT-A outcomes did not differ between IVF and ICSI cycles: euploid 27.9% (IVF) versus 30% (ICSI); aneuploid 45.4% (IVF) versus 43.1% (ICSI); no result 4.4% (IVF) versus 6.2% (ICSI). Though not significant, we identified a trend toward higher rate of mosaicism in IVF (25.9%) versus ICSI (20.9%). Among mosaic embryos, a lower percentage of simple mosaic embryos resulted from IVF (53.8%) versus ICSI (70.2%). Among aneuploid embryos, a non-significant higher percentage of complex aneuploidy resulted from IVF (16.3%) versus ICSI (9%). IVF resulted in a non-significant higher proportion of cycles with no transferrable embryos (42.7%) versus ICSI (36.6%). Numerical and sex chromosome involvement in mosaicism and aneuploidy were similar between IVF and ICSI cycles. CONCLUSION: IVF and ICSI NGS PGT-A have similar rates of euploid, aneuploid, and no result embryos, though IVF may result in higher rates of mosaicism and demonstrates differences in proportions of mosaic and aneuploid subtypes compared to ICSI. ICSI may be preferable to conventional insemination to minimize the rate of mosaic results in NGS PGT-A cycles.

Early detection of cryostorage tank failure using a weight-based monitoring system.

PURPOSE: To study the relationship between liquid nitrogen loss and temperature in cryostorage dewars and develop an early-warning alarm for impending tank failure. METHODS: Cryostorage dewars were placed on custom-engineered scales, and weight and temperature data were continuously monitored in the setting of slow, medium, and fast rate-loss of LN2 to simulate three scenarios of tank failure. RESULTS: LN2 Tank weights and temperatures were continuously monitored and recorded, with a calculated alarm trigger set at 10% weight loss and temperature of - 185 °C. With an intact tank, a 10% loss in LN2 occurred in 4.2-4.9 days. Warming to - 185 °C occurred in 37.8-43.7 days, over 30 days after the weight-based alarm was triggered. Full evaporation of LN2 required ~ 36.8 days. For the medium rate-loss simulation, a 10% loss in LN2 occurred in 0.8 h. Warming to - 185 °C occurred in 3.7-4.8 h, approximately 3 h after the weight-based alarm was triggered. For the fast rate-loss simulation, a 10% weight loss occurred within 15 s, and tanks were depleted in under 3 min. Tank temperatures began to rise immediately and at a relatively constant rate of 43.9 °C/h and 51.6 °C/h. Temperature alarms would have sounded within 0.37 and 0.06 h after the breech. CONCLUSIONS: This study demonstrates that a weight-based alarm system can detect tank failures prior to a temperature-based system. Weight-based monitoring could serve as a redundant safety mechanism for added protection of cryopreserved reproductive tissues.

Expanded carrier screening in an infertile population: how often is clinical decision making affected?

PURPOSE: Options for preconception genetic screening have grown dramatically. Expanded carrier screening (ECS) now allows for determining carrier status for hundreds of genetic mutations by using a single sample, and some recommend ECS prior to in vitro fertilization. This study seeks to evaluate how often ECS alters clinical management when patients present for infertility care. METHODS: All patients tested with ECS at a single infertility care center from 2011 to 2014 were evaluated. The overall rate of positive ECS results and the number of couples who were carriers of the same genetic disorder were evaluated. RESULTS: A total of 6,643 individuals were tested, representing 3,738 couples; 1,666 (25.1%) of the individuals had a positive test result for at least one disorder. In 8 of the 3,738 couples, both members of the couple were positive for the same genetic disorder or had a test result that placed them at risk of having an affected child. Three of eight cases were cystic fibrosis. In this cohort, ECS affected clinical care eight times after 6,643 tests (0.12%, confidence interval: 0.05-0.24%) in 3,738 couples (0.21%, confidence interval: 0.09-0.42%). CONCLUSIONS: ECS is becoming more widespread. In a large case series, ECS affected clinical decision making for patients presenting for infertility care in 0.21% of cases. This information must be weighed when utilizing these tests and may be a helpful part of patient counseling.Genet Med 18 11, 1097-1101.

Dehydroepiandrosterone (DHEA) supplementation results in supraphysiologic DHEA-S serum levels and progesterone assay interference that may impact clinical management in IVF.

PURPOSE: Dehydroepiandrosterone (DHEA) is often prescribed for poor responders in IVF in an effort to improve response to ovarian stimulation. The effect of DHEA supplementation and resultant supraphysiologic DHEA-S serum levels on sex steroid assays has not been evaluated in this population. This study seeks to determine the relationship between DHEA supplementation and progesterone measurements to characterize the degree of interference with particular immunoassays. METHODS: Characterization was accomplished in two phases. First, DHEA-S standard control reagents with no progesterone present were assayed for both DHEA-S and progesterone levels. Second, serum pools from 60 unique IVF patients' serum were used to create six pooled serum samples: three from patients on DHEA supplementation and three from patients not on DHEA supplementation. The three pools were composed of patients whose serum fell into low, medium, and high progesterone ranges. Baseline DHEA-S and progesterone were measured, and the mean level of DHEA-S in the mid-range progesterone pool was used as the mid-point for addition of DHEA-S standard to the serum pools from patients without DHEA supplementation. Progesterone from these pools was then measured on three commercially available immunoassay systems. RESULTS: The first experiment revealed a linear increase in progesterone when analyzing the DHEA-S standard ranging from 0.5 µg/dL [corrected] in the blank control (no DHEA-S) to up to 2.0 µg/dL [corrected] in the high control (DHEA-S >700 µg/dL), [corrected] indicating that the DHEA-S cross-reacts with the progesterone assays. In the second experiment, patients' serum DHEA-S and progesterone were measured from pooled serum samples of those taking DHEA and those not taking DHEA. Adding DHEA-S to the pooled serum of those not taking DHEA resulted in a linear increase in progesterone levels on two of three commercially available immunoassays (p < 0.05). CONCLUSIONS: DHEA-S can interfere with standard progesterone immunoassays used in clinical ART programs, and thus serum progesterone levels in IVF patients on DHEA supplementation may not reflect truly bioactive progesterone.

Multiple thrombophilic single nucleotide polymorphisms lack a significant effect on outcomes in fresh IVF cycles: an analysis of 1717 patients.

PURPOSE: The aim of the study is to determine if thrombophilic single nucleotide polymorphisms (SNPs) affect outcomes in fresh in vitro fertilization (IVF) cycles in a large general infertility population. METHODS: A prospective cohort analysis was performed at a university-affiliated private IVF center of female patients undergoing fresh non-donor IVF cycles. The effect of the following thrombophilic SNPs on IVF outcomes were explored: factor V (Leiden and H1299R), prothrombin (G20210A), factor XIII (V34L), ß-fibrinogen (-455G → A), plasminogen activator inhibitor-1 (4G/5G), human platelet antigen-1 (a/b9L33P), and methylenetetrahydrofolate reductase (C677T and A1298C). The main outcome measures included positive pregnancy test, clinical pregnancy, embryo implantation, live birth, and pregnancy loss. RESULTS: Patients (1717) were enrolled in the study, and a total of 4169 embryos were transferred. There were no statistically significant differences in positive pregnancy test, clinical pregnancy, embryo implantation, live birth, or pregnancy loss in the analysis of 1717 patients attempting their first cycle of IVF. Receiver operator characteristics and logistic regression analyses showed that outcomes cannot be predicted by the cumulative number of thrombophilic mutations present in the patient. CONCLUSIONS: Individual and cumulative thrombophilic SNPs do not affect IVF outcomes. Therefore, initial screening for these SNPs is not indicated.

SNP array-based analyses of unbalanced embryos as a reference to distinguish between balanced translocation carrier and normal blastocysts.

PURPOSE: The purpose of the study is to validate a method that provides the opportunity to distinguish a balanced translocation carrier embryo from a truly normal embryo in parallel with comprehensive chromosome screening (CCS). METHODS: A series of translocation carrier couples that underwent IVF with single nucleotide polymorphism (SNP) array-based CCS on 148 embryos were included. Predictions of balanced or normal status of each embryo were made based upon embryonic SNP genotypes. In one case, microdeletion status was used to designate whether embryos were balanced or normal. In 10 additional cases, conventional karyotyping was performed on newborns in order to establish the true genetic status (balanced or normal) of the original transferred embryo. Finally, implantation potential of balanced or normal embryos was compared. RESULTS: Phasing SNPs using unbalanced embryos allowed accurate prediction of whether transferred embryos were balanced translocation carriers or truly normal in all cases completed to date (100 % concordance with conventional karyotyping of newborns). No difference in implantation potential of balanced or normal embryos was observed. CONCLUSIONS: This study demonstrates the validity of a CCS method capable of distinguishing normal from balanced translocation carrier embryos. The only prerequisite is the availability of parental DNA and an unbalanced IVF embryo, making the method applicable to the majority of carrier couples. In addition, the SNP array platform allows simultaneous CCS for aneuploidy with the same platform and from the same biopsy. Future work will involve prospective predictions to select normal embryos with subsequent karyotyping of the resulting newborns.

Is there an association between assisted reproductive technologies and time and complications of the third stage of labor?

PURPOSE: To determine if vaginal deliveries exposed to assisted reproductive technologies (ART) are associated with an increased time between delivery of the neonate and placenta and select complications. METHODS: A retrospective cohort of patients enrolled in an infertility practice who had term, singleton, vaginal deliveries at two academic hospitals from 2008 to 2013 was analyzed. Controls were patients with spontaneous conceptions after infertility consultations. The exposure groups were patients with controlled ovarian hyper-stimulation (COH) with in vivo fertilization, COH with in vitro fertilization and fresh embryo transfer (COH/IVF), and frozen embryo transfer or oocyte donation recipients without COH (non-COH ET). Multiple gestations and stillbirths were excluded. Median time of third stage was compared using the Mann-Whitney U test. Secondary outcomes of retained placenta, manual placental extraction, and post-partum hemorrhage (PPH) were compared using Chi-square or Fisher's exact analyses. RESULTS: A total of 769 patients met criteria and were analyzed. While there were no differences in time of third stage of labor, retained placenta, or PPH, manual extraction was significantly more common among non-COH ET [age-adjusted OR 5.6 (95 % CI 2.2-13.8); p < 0.001]. CONCLUSIONS: Patients who conceived after non-COH ET were at increased risk for manual placental extraction. This association was not influenced by age differences between groups. Further research must be done to determine which elements of the ART process are responsible for these differences.

Vitamin D levels do not affect IVF outcomes following the transfer of euploid blastocysts.

OBJECTIVE: We sought to characterize the relationship between serum 25-hydroxy vitamin D (25-OH D) levels and implantation and clinical pregnancy rates in women who undergo a euploid blastocyst embryo transfer. STUDY DESIGN: This retrospective cohort study, conducted in an academic setting, included 529 cycles in which comprehensive chromosome screening was performed as part of routine infertility care with an autologous transfer of 1 or 2 euploid blastocysts. After excluding repeat cycles there were 517 unique cycles representing 517 women for evaluation. Vitamin D levels from serum samples obtained on the day of ovulation trigger in the fresh in vitro fertilization cycle were analyzed. The primary outcome was ongoing pregnancy rate as defined by sonographic presence of fetal heart rate at >8 weeks' gestation. RESULTS: For the population as a whole, serum vitamin D ranges and pregnancy outcomes did not correlate. Furthermore, pregnancy rates did not differ when comparing women in different strata of vitamin D levels (<20, 20-29.9, and ≥30 ng/mL). No meaningful breakpoint for vitamin D levels and ongoing pregnancy rate was identified using receiver operating characteristic analysis with the resultant line possessing an area under the curve of 0.502. Multivariate logistic regression controlling for age, transfer order, race, season, and body mass index did not yield a different result. The study was powered to detect an 18% difference in ongoing pregnancy rates between patients grouped by the 3 vitamin D ranges. CONCLUSION: In women undergoing euploid embryo transfer, vitamin D status was unrelated to pregnancy outcomes. Measuring serum 25-OH vitamin D levels does not predict the likelihood that euploid blastocysts will implant. These results may not apply to women who do not undergo extended embryo culture, blastocyst biopsy for comprehensive chromosome screening, and euploid embryo transfer.

Endometrial disruption does not improve implantation in patients who have failed the transfer of euploid blastocysts.

PURPOSE: To assess the impact of single pass outpatient endometrial biopsy in patients at the highest risk for an endometrial cause for failed implantation; those that have failed to conceive despite the transfer of morphologically normal euploid embryos. METHODS: This is a retrospective cohort study consisting of all patients less than 42 years old who failed their first euploid blastocyst transfer and subsequently completed a second transfer cycle of euploid blastocysts. Cycles were analyzed to determine if a single pass endometrial biopsy, termed 'endometrial disruption', was performed in a cycle preceding their second embryo transfer. Transfer outcomes were analyzed and implantation rates calculated. Data analysis was performed to compare outcomes between patients who had endometrial disruption performed versus those that did not. RESULTS: Two hundred ninety patients failed their first euploid embryo transfer and subsequently completed a second euploid embryo transfer and were included. Thirty-nine patients underwent endometrial disruption and 251 did not. There were no statistical differences in clinical implantation rate or sustained implantation rate between the group with endometrial disruption and subjects without any intervention (Clinical IR, 43.6 % vs. 55.0 %, p = 0.13; 38.5 % vs. 42.6 %, p = 0.60). When controlling for transfer order there was no statistical difference noted in implantation rates. CONCLUSIONS: Single pass endometrial biopsy has no impact on endometrial receptivity in the highest risk subgroup- patient's that have failed to sustain the transfer of morphologically normal euploid embryos- as evidenced by equivalent implantation rates. It is possible that variations in technique may alter outcomes and randomized trials are needed to answer this question.

Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates.

OBJECTIVE: We sought to determine whether performing elective single embryo transfer (eSET) after trophectoderm biopsy and rapid aneuploidy screening results in improved obstetrical and neonatal outcomes compared with transferring 2 untested embryos. STUDY DESIGN: The Blastocyst Euploid Selective Transfer (BEST) Trial enrolled infertile couples with a female partner up to age 42 years who were undergoing in vitro fertilization. They were randomized to receive transfer of a single euploid embryo (eSET) or to the standard of care with transfer of 2 embryos that were not biopsied for aneuploidy screening (untested 2-embryo transfer). Gestational age at delivery, birthweight, and neonatal intensive care unit (NICU) lengths of stay were compared with Mann-Whitney U. The risk of preterm delivery, low birthweight, and NICU admission were compared with χ(2). RESULTS: Among the 175 randomized patients, the delivery rates were similar (69% after euploid eSET vs 72% after untested 2-embryo transfer; P = .6) through the fresh cycle and up to 1 frozen transfer, with a dramatic difference in multiple births (1.6% vs 47%; P < .0001). The risk of preterm delivery (P = .03), low birthweight (P = .002), and NICU admission (P = .04) were significantly higher after untested 2-embryo transfer. Babies born after untested 2-embryo transfer spent >5 times as many days in the NICU (479 vs 93 days; P = .03). CONCLUSION: By enhancing embryo selection with a validated method of aneuploidy screening, a single euploid embryo with high reproductive potential can be selected for transfer. Using this approach, eSET can be performed without compromising delivery rates and improving the chance of having a healthy, term singleton delivery after in vitro fertilization.

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

PURPOSE: To characterize each chromosome's risk for being involved in embryonic aneuploidy. METHODS: This 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. RESULTS: The 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. CONCLUSIONS: The 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.

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

OBJECTIVE: To determine if comprehensive embryology training for clinical Reproductive Endocrinology fellows could be completed to a level of proficiency equivalent to that of experienced embryologists. METHOD: Clinical 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. RESULTS: Clinical 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. CONCLUSIONS: When 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.

Lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

PURPOSE: A recent experiment indicated that a loss of function mutation in the murine Katnal1 gene resulted in male factor infertility due to premature exfoliation of spermatids. This study investigated the relevance of this gene to infertility in humans. METHODS: Multiple methods of genetic analysis were employed to investigate whether mutations in human KATNAL1 have a causative role in male infertility. This was a genetic association study, which included DNA samples from 105 men with non-obstructive azoospermia (NOA) and 242 anonymous sperm donor controls. 28 commercially available TaqMan SNP assays were used to haplotype samples from both groups and genetically tag regions of interest across the entire gene. AmpliSeq primers were then designed for identified regions so that targeted next-generation sequencing (NGS) could be used to identify causative variants. RESULTS: Four SNPs in the 3'UTR demonstrated a putative association with NOA. The AmpliSeq primers designed for the 3'UTR provided 83 % coverage of the 7,202 basepairs within the regions of interest. Variant sites were analyzed against genetic models to identify sequence polymorphisms which associated with NOA. No variants met standard criteria for significance when tested between the groups. CONCLUSIONS: This study suggests a lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

Trophectoderm DNA fingerprinting by quantitative real-time PCR successfully distinguishes sibling human embryos.

PURPOSE: To validate a novel and more practical system for trophectoderm DNA fingerprinting which reliably distinguishes sibling embryos from each other. METHODS: In this prospective and blinded study two-cell and 5-cell samples from commercially available sibling cell lines and excess DNA from trophectoderm biopsies of sibling human blastocysts were evaluated for accurate assignment of relationship using qPCR-based allelic discrimination from 40 single nucleotide polymorphisms (SNPs) with low allele frequency variation and high heterozygosity. RESULTS: Cell samples with self relationships averaged 95.1 ± 5.9 % similarity. Sibling relationships averaged 57.2 ± 5.9 % similarity for all 40 SNPs, and 40.8 ± 8.2 % similarity for the 25 informative SNPs. Assignment of relationships was accomplished with 100 % accuracy for cell lines and embryos. CONCLUSIONS: These data demonstrate the first trophectoderm qPCR-based DNA fingerprinting technology capable of unequivocal discrimination of sibling human embryos. This methodology will empower research and development of new markers of, and interventions that influence embryonic reproductive potential.

Development and validation of an automated robotic system for preparation of embryo culture dishes.

OBJECTIVE: To study the development and clinical validation of the ART Pipetting Robot for the IVF Laboratory (APRIL), a liquid-handling robot customized for the precise preparation of microdroplet culture dishes in the field of in vitro fertilization (IVF). DESIGN: A prospective randomized study conducted at an academic IVF center comparing mouse and human embryo outcomes and quantitative measures of accuracy in embryo dishes prepared using APRIL compared with standard manual preparation. SETTING: Academic IVF center. SUBJECTS: The study involved the assessment of the automated culture dish preparation system, APRIL, compared with manual preparation methods in the context of IVF treatment. INTERVENTION: ART Pipetting Robot for the IVF Laboratory is an enclosed liquid-handling robot equipped with custom three-dimensional-printed adapters and designed to dispense embryo culture media and mineral oil into microdroplet culture dishes. MAIN OUTCOME MEASURES: The study evaluated the precision and consistency of APRIL in culture dish preparation by looking at droplet mass, pH of prepared media droplets, and mouse and human embryo development rates. Clinical implementation was assessed by comparing embryo development and outcomes in dishes prepared by APRIL and human embryologists. RESULTS: Compared with embryo culture dishes prepared using standard manual procedures, embryo culture dishes prepared using APRIL demonstrated a greater than 10-fold improvement in consistency (coefficient of variation, 0.46% vs. 6%-7%), maintained optimal pH levels (pH range, 7.281-7.33 vs. 7.275-7.311), and had a greater mouse embryo blastocyst rate (100% vs. 90%-91%). Human embryos cultured in dishes prepared by APRIL had a higher rate of development on days 3 (92.4% vs. 82.6%) and 5 (19.75% vs. 15.57%), and a total number of usable embryos (50.3% vs. 46.1%) compared with manually prepared dishes, although the last two outcomes did not reach statistical significance. CONCLUSION: The results suggest that the use of an automated robotic system for preparation of embryo culture dishes may improve accuracy and outcome measures while reducing the need for trained laboratory personnel to prepare the dishes manually.

Incidental identification of balanced translocation carrier patients through comprehensive chromosome screening of IVF-derived blastocysts.

PURPOSE: To demonstrate that translocation carrier patients can be identified by analysis of chromosomes in preimplantation human embryos. METHODS: A report of 3 cases in which multiple embryos were found to possess consistent segmental imbalances by CCS. The parents then had a conventional karyotype performed. RESULTS: In each case, parental karyotyping revealed the presence of an otherwise unknown balanced translocation. Original blastocyst CCS results were then reinterpreted to consider the presence of unbalanced derivative chromosomes and to modify the diagnosis of embryos eligible for transfer. CONCLUSIONS: It is possible to identify patients that are carriers of balanced translocations through the analysis of chromosomes in their IVF-derived embryos. Given that translocation carrier screening is not routinely performed, the growing use of CCS may facilitate discovery and provide both an etiology of reproductive failure and an improved more focused treatment strategy going forward. Future work will involve a large retrospective study to define the sensitivity and frequency of detection using this methodology.

Transfer of the fittest: using preimplantation genetic testing for aneuploidy to select embryo(s) most likely to lead to live birth.

Preimplantation genetic testing for aneuploidy (PGT-A) was developed to identify euploid embryos from a cohort of embryos with unknown ploidy produced during an in vitro fertilization (IVF) cycle. In recent years, the ability of PGT-A to improve IVF outcomes has come into question. The goal of this review was to summarize the major randomized controlled trials (RCTs) and nonselection studies evaluating the benefit of PGT-A to improve the live birth rates (LBRs). We argue that the LBR per transfer is more relevant to the individual patient than the cumulative LBR as a means to minimize the burden of IVF by reducing futile transfers, pregnancy losses, and ongoing aneuploidy. The early RCTs demonstrate improved implantation rates and LBRs with PGT-A for embryo selection vs. traditional morphology. However, these studies are limited by the small sample size and a bias toward good-prognosis patients. Further studies using next-generation sequencing found more variable results but did confirm an improvement in the LBRs per transfer in an older population with a higher baseline risk of aneuploidy. The largest RCT to date showed similar cumulative LBRs in the PGT-A and control groups after biopsy and sequential transfer of up to 3 blastocysts with a significant reduction in the cumulative clinical pregnancy loss rate in the PGT-A group. Nonselection studies evaluating pregnancy outcomes after transfer of euploid vs. aneuploid embryos demonstrate near-perfect negative predictive value for an aneuploid result to predict live birth. Putative mosaic embryos had similar LBRs compared with euploid embryos. The available RCTs and nonselection studies support the practice of using PGT-A to identify euploid embryos for transfer, especially in an older population, while simultaneously selecting against aneuploid embryos, without negative impact on the total reproductive potential of the cycle.