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.

An LC-MS/MS method for simultaneous determination of LB-100 and its active metabolite, endothall, in human plasma.

We have developed and validated a novel LC-MS/MS method for the simultaneous quantification of LB-100 and its active metabolite, endothall, in human plasma following solid-phase extraction. LB-105 and endothall-D6 were used as internal standards. Chromatographic separation was achieved on a Hypercarb™ column using 5 mM (NH4)2CO3 and 30:70 (v/v) 100 mM (NH4)2CO3:acetonitrile as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 2.5-500 ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. LB-100 and endothall recoveries were 78.7 and 86.7%, respectively. The validated LC-MS/MS method enabled the accurate measurement of LB-100 and endothall in patient samples from an ongoing clinical trial (NCT04560972).

Opening the black box: why do euploid blastocysts fail to implant? A systematic review and meta-analysis.

BACKGROUND: A normal chromosomal constitution defined through PGT-A assessing all chromosomes on trophectoderm (TE) biopsies represents the strongest predictor of embryo implantation. Yet, its positive predictive value is not higher than 50-60%. This gap of knowledge on the causes of euploid blastocysts' reproductive failure is known as 'the black box of implantation'. OBJECTIVE AND RATIONALE: Several embryonic, maternal, paternal, clinical, and IVF laboratory features were scrutinized for their putative association with reproductive success or implantation failure of euploid blastocysts. SEARCH METHODS: A systematic bibliographical search was conducted without temporal limits up to August 2021. The keywords were '(blastocyst OR day5 embryo OR day6 embryo OR day7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)'. Overall, 1608 items were identified and screened. We included all prospective or retrospective clinical studies and randomized-controlled-trials (RCTs) that assessed any feature associated with live-birth rates (LBR) and/or miscarriage rates (MR) among non-mosaic euploid blastocyst transfer after TE biopsy and PGT-A. In total, 41 reviews and 372 papers were selected, clustered according to a common focus, and thoroughly reviewed. The PRISMA guideline was followed, the PICO model was adopted, and ROBINS-I and ROB 2.0 scoring were used to assess putative bias. Bias across studies regarding the LBR was also assessed using visual inspection of funnel plots and the trim and fill method. Categorical data were combined with a pooled-OR. The random-effect model was used to conduct the meta-analysis. Between-study heterogeneity was addressed using I2. Whenever not suitable for the meta-analysis, the included studies were simply described for their results. The study protocol was registered at http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329). OUTCOMES: We included 372 original papers (335 retrospective studies, 30 prospective studies and 7 RCTs) and 41 reviews. However, most of the studies were retrospective, or characterized by small sample sizes, thus prone to bias, which reduces the quality of the evidence to low or very low. Reduced inner cell mass (7 studies, OR: 0.37, 95% CI: 0.27-0.52, I2 = 53%), or TE quality (9 studies, OR: 0.53, 95% CI: 0.43-0.67, I2 = 70%), overall blastocyst quality worse than Gardner's BB-grade (8 studies, OR: 0.40, 95% CI: 0.24-0.67, I2 = 83%), developmental delay (18 studies, OR: 0.56, 95% CI: 0.49-0.63, I2 = 47%), and (by qualitative analysis) some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavage patterns, spontaneous blastocyst collapse, longer time of morula formation I, time of blastulation (tB), and duration of blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR, even in the context of PGT-A, was reported among women ≥38 years (7 studies, OR: 0.87, 95% CI: 0.75-1.00, I2 = 31%), while obesity was associated with both lower LBR (2 studies, OR: 0.66, 95% CI: 0.55-0.79, I2 = 0%) and higher MR (2 studies, OR: 1.8, 95% CI: 1.08-2.99, I2 = 52%). The experience of previous repeated implantation failures (RIF) was also associated with lower LBR (3 studies, OR: 0.72, 95% CI: 0.55-0.93, I2 = 0%). By qualitative analysis, among hormonal assessments, only abnormal progesterone levels prior to transfer were associated with LBR and MR after PGT-A. Among the clinical protocols used, vitrified-warmed embryo transfer was more effective than fresh transfer (2 studies, OR: 1.56, 95% CI: 1.05-2.33, I2 = 23%) after PGT-A. Lastly, multiple vitrification-warming cycles (2 studies, OR: 0.41, 95% CI: 0.22-0.77, I2 = 50%) or (by qualitative analysis) a high number of cells biopsied may slightly reduce the LBR, while simultaneous zona-pellucida opening and TE biopsy allowed better results than the Day 3 hatching-based protocol (3 studies, OR: 1.41, 95% CI: 1.18-1.69, I2 = 0%). WIDER IMPLICATIONS: Embryo selection aims at shortening the time-to-pregnancy, while minimizing the reproductive risks. Knowing which features are associated with the reproductive competence of euploid blastocysts is therefore critical to define, implement, and validate safer and more efficient clinical workflows. Future research should be directed towards: (i) systematic investigations of the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how lifestyle and nutrition may accelerate or exacerbate their consequences; (ii) improved evaluation of the uterine and blastocyst-endometrial dialogue, both of which represent black boxes themselves; (iii) standardization/automation of embryo assessment and IVF protocols; (iv) additional invasive or preferably non-invasive tools for embryo selection. Only by filling these gaps we may finally crack the riddle behind 'the black box of implantation'.

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.

Genetics in reproductive endocrinology and infertility.

Tremendous advances in genetics have transformed the field of reproductive endocrinology and infertility over the last few decades. One of the most prominent advances is preimplantation genetic testing (PGT), which allows for the screening of embryos obtained during in vitro fertilization before transfer. Moreover, PGT can be performed for aneuploidy screening, detection of monogenic disorders, or exclusion of structural rearrangements. Refinement of biopsy techniques, such as obtaining samples at the blastocyst rather than the cleavage stage, has helped optimize results from PGT, and technological advances, including next-generation sequencing, have made PGT more efficient and accurate. The continued evolution of the approach to PGT has the potential to further enhance the accuracy of results, expand the application to other conditions, and increase access by reducing cost and improving efficiency.

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.

Evaluating the utility of a global webinar for mentoring medical students and OBGYN residents in REI.

BACKGROUND: In medical school and residency, clinical experiences influence trainee's decisions on what medical specialty they choose. Most trainees have limited access to opportunities to engage in the field of reproductive endocrinology and infertility (REI). Due to the COVID-19 pandemic and the shutdown of away electives, exposure to REI was especially limited. This study aims to evaluate the effectiveness of a live Q and A webinar on improving trainees' access to mentorship and knowledge of the path to becoming a reproductive endocrinology and infertility (REI) physician. MATERIALS AND METHODS: This study is a prospective paired cohort study. Medical students and OBGYN residents participated in a global Q and A webinar featuring REI physicians and fellows. 70 pre- and post-webinar surveys were included in the analysis. Paired nonparametric tests (Wilcoxon signed-rank test) were performed to assess whether post-webinar knowledge was significantly different from pre-webinar knowledge. RESULTS: Of the 268 registrants, 162 (60%) attended the live webinar. A majority of the respondents who completed both surveys were female (90%) and allopathic medical students (80%). Seventy-seven percent reported receiving only minimal advice about an REI career from their medical school or residency program, while 22% reported receiving some advice, and 1% extensive advice. Thirty-four percent had previously shadowed an REI physician and 23% had rotated in an REI office. Post-webinar significantly more trainees had a better understanding of the REI field, the path required to become an REI physician, opportunities to find mentors in the field, opportunities that are conducive to learning more about REI, and applying for rotations in the REI field (p = <.00001). Eighty-two percent agreed that their interest in REI increased due to this webinar. CONCLUSIONS: A webinar featuring REI physicians and fellows was effective in providing mentorship and career advisement for prospective REI trainees who otherwise expressed having limited access to the field.

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.

A review of the pathophysiology of recurrent implantation failure.

Implantation is a critical step in human reproduction. The success of this step is dependent on a competent blastocyst, receptive endometrium, and successful cross talk between the embryonic and maternal interfaces. Recurrent implantation failure is the lack of implantation after the transfer of several embryo transfers. As the success of in vitro fertilization has increased and failures have become more unacceptable for patients and providers, the literature on recurrent implantation failure has increased. While this clinical phenomenon is often encountered, there is not a universally agreed-on definition-something addressed in an earlier portion of this Views and Reviews. Implantation failure can result from several different factors. In this review, we discuss factors including the maternal immune system, genetics of the embryo and parents, anatomic factors, hematologic factors, reproductive tract microbiome, and endocrine milieu, which factors into embryo and endometrial synchrony. These potential causes are at various stages of research and not all have clear implications or immediately apparent treatment.

Considerations on the restriction of Assisted Reproductive Technology (ART) due to COVID-19.

The rapid rise of novel coronavirus disease 2019 (COVID-19) cases led the American Society for Reproductive Medicine (ASRM) to recommend immediate cessation of all new fertility treatment cycles on March 17, 2020. Controversial from the start, providers and patients expressed their opposition through online petitions, surveys, and other forums. While the impact of a delay in access to reproductive care is unknown, previous studies are reassuring that a delay in the timespan of months may not affect clinical outcomes. However, dropout from care during this pandemic remains a serious concern. Effective therapies against the virus and a vaccine are not on the immediate horizon. Accepting COVID-19 will likely be a part of our lives for the near future necessitates the modification of fertility protocols to keep patients, providers, and staff as safe as possible. We believe fertility treatment is an urgent, essential service that can be performed safely and responsibly during this pandemic.

Embryonic aneuploidy rates are equivalent in natural cycles and gonadotropin-stimulated cycles.

OBJECTIVE: To determine if natural selection and follicular stimulation produces a lower risk for embryonic aneuploidy than that attained following superovulation with exogenous gonadotropins. DESIGN: Prospective observational with historical control group. SETTING: Large academically affiliated private practice. PATIENT(S): All patients presenting for their evaluation for infertility were offered participation in the study. INTERVENTION(S): All participants in the natural cycle group underwent an unstimulated in vitro fertilization (IVF) cycle. A subsequent frozen embryo transfer was performed if a euploid blastocyst was attained. MAIN OUTCOME MEASURE(S): Rates of embryonic aneuploidy attained in unstimulated IVF cycles were compared to those observed in age-controlled historical cohort undergoing conventional stimulated IVF cycles with exogenous gonadotropins. RESULT(S): Aneuploidy rates were equivalent in unstimulated and stimulated IVF cycles. The prevalence of aneuploidy in natural cycles increased with the age of the female partner in a manner identical to that seen in stimulated IVF cycles. Finally, sustained implantation rates of euploid blastocysts were equivalent in natural and stimulated IVF cycles. CONCLUSION(S): Rates of embryonic aneuploidy are not impacted by follicular stimulation with exogenous gonadotropins. Prior concerns of inducing a higher risk of embryonic aneuploidy are not supported by this data. CLINICAL TRIAL REGISTRATION NUMBER: NCT01866618.

Use of fertility preservation services in female reproductive-aged cancer patients.

OBJECTIVE: The objective of the study was to determine the rates and predictors of fertility preservation services among reproductive-aged women with common cancers in the United States. STUDY DESIGN: We used the MarketScan database to identify women 18-45 years of age with lung, breast, colorectal, or cervical cancer who underwent surgery and chemotherapy from 2009 through 2016. Services from 3 months before to 3 months after chemotherapy for evaluation for fertility preservation, laboratory testing for fertility evaluation, and fertility-preserving procedures were captured. Multivariable models were used to assess the factors associated with the use of fertility-preservation services. RESULTS: A total of 18,781 women, including 386 cervical, 1372 colorectal, 246 lung, and 16,777 with breast cancer, were identified. In women 18-35 years old, 11.7% underwent evaluation for fertility preservation, 13.7% underwent laboratory testing, and 6.3% pursued fertility-preserving procedures. The rates of office evaluation, laboratory testing, and performance of procedure were 3.3%, 7.5%, and 1.9 % in women aged 36-40 years and 0.5%, 7.2%, and 0.3% in those aged 41-45 years, respectively. The rate of fertility preservation evaluation rose from 1.0% in 2009 to 5.5% in 2016 (risk ratio, 4.66, 95% confidence interval, 2.38-9.11) while use of fertility-preserving procedures increased from 1.0% to 4.6% (risk ratio, 3.84, 95% confidence interval, 1.94-7.59) during the same time period. In a multivariable model, use of any fertility-preserving interventions were more common in patients with breast cancer (adjusted risk ratio, 2.30, 95% confidence interval, 1.30-4.06), those in the Northeast (adjusted risk ratio, 1.24, 95% confidence interval, 1.10-1.40), and in younger women (18-35 years) (adjusted risk ratio, 2.59, 95% confidence interval, 2.32-2.89). CONCLUSION: Although limited by lack of information regarding cancer stage and desire for future fertility, only a small fraction of reproductive-aged female cancer patients receiving chemotherapy are evaluated in a nationwide sample for fertility preservation or undergo fertility-preserving procedures.

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.

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.