Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

STUDY QUESTION: Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY: FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION: This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE: No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION: The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S): This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. TRIAL REGISTRATION NUMBER: The study was approved by WCG Connexus IRB (Study Number 1298156).

Utilization of Cryopreserved Oocytes in Patients With Poor Ovarian Response After Planned Oocyte Cryopreservation.

Importance: Poor ovarian response (POR) to stimulation may impact patients' desire or need to utilize cryopreserved oocytes for family building in the future. These findings, captured by Society for Assisted Reproductive Technology (SART) national data, underscore the need for tailored counseling and further research into the decision-making processes influencing oocyte utilization. Objective: To examine the association of ovarian response to stimulation and the number of vitrified oocytes with the likelihood and timing of patients returning for oocyte utilization following planned oocyte cryopreservation (OC). Design, Setting, and Participants: This cohort study used data in the SART Clinical Outcome Reporting System for patients in US fertility clinics and data was used for eligible patients who underwent planned OC from January 2014 through December 2020. Data were analyzed from November 2022 to June 2023. Main outcomes and measures: The association between number of oocytes cryopreserved on return rate to utilize cryopreserved oocytes and the time from vitrification to warming. Results: A total of 67 893 autologous oocyte freezing cycles were performed in the US between 2014 and 2020, among 47 363 patients (mean [SD] age, 34.5 [4.7] years). Of these, 6421 (13.5%) were classified as patients with POR, with fewer than 5 oocytes vitrified across all ovarian stimulation cycles. A total of 1203 patients (2.5%) returned for oocyte warming and utilization. The rate of return was significantly higher in the POR group, with 260 (4.0%) returning compared with 943 (2.3%) in the normal responder group (P < .001). This trend was most notable in the age 30 to 34 years (warm cycle, 46 of 275 [16.7%] vs no warm cycle, 982 of 11 743 [8.4%]; P < .001) and age 35 to 39 years groups (warm cycle, 124 of 587 [21.1%] vs no warm cycle, 3433 of 23 012 [14.9%]; P < .001). The time elapsed from vitrification to warming was comparable between patients with POR (mean [SD], 716.1 [156.1] days) and normal responders (803.8 [160.7] days). A multivariate analysis adjusted for age, clinic region in the US, body mass index, and history of endometriosis was conducted to identify factors associated with the utilization of oocytes. The analysis revealed that having fewer than 5 oocytes vitrified was associated with higher odds of utilizing oocytes (OR, 1.52; 95% CI, 1.32-1.76). Conclusions and Relevance: This cohort study reveals a distinct pattern in the utilization of cryopreserved oocytes among patients undergoing planned OC in the US. Despite the increase in number of patients pursuing OC, there is a notably low rate of return to utilize previously vitrified oocytes; notably, patients with POR are more likely to return, although the time to return is similar to those with normal ovarian response.

Routine ketorolac at oocyte retrieval decreases postoperative narcotic use by more than 50.

OBJECTIVE: To study the impact of routine ketorolac administration during oocyte retrieval on the proportion of patients who require postoperative narcotics for analgesia. DESIGN: Retrospective cohort study. SETTING: Single, university-affiliated infertility clinic. PATIENTS: All women undergoing oocyte retrieval between July and November 2016 (non-ketorolac group [NKG]; n = 826) and April-August 2017 (ketorolac group, KG; n = 1780). INTERVENTIONS: A single 30 mg intravenous dose of ketorolac was administered after the oocyte retrieval procedure. MAIN OUTCOME MEASURES: The number of patients who required postoperative narcotic analgesia, postoperative complication rate, and fresh embryo transfer pregnancy outcomes were examined. RESULTS: In the KG, we found a significant decrease in the patients who required narcotics after oocyte retrieval compared with the NKG (12% KG vs. 25.5% NKG). We found no significant change in the clinical pregnancy rate (CPR) resulting from fresh embryo transfer after our intervention (NKG CPR 32.6%, KG CPR 32.4%). Furthermore, there was no increase in postoperative bleeding complications in the KG. CONCLUSIONS: Routine use of ketorolac at the time of oocyte retrieval may decrease the rate of postoperative opioid use without adversely impacting pregnancy and complication rates.

Blasts from the past: is morphology useful in PGT-A tested and untested frozen embryo transfers?

RESEARCH QUESTION: Day of cryopreservation, inner cell mass (ICM) grade, trophectoderm grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. This study sought to examine the likelihood of live birth and whether the morphological grade of the blastocyst is more or equally useful in FET cycles among preimplantation genetic testing for aneuploidies (PGT-A) tested and untested blastocysts. DESIGN: This was a retrospective cohort study of 6271 vitrified-warmed, autologous, single-embryo transfer cycles among patients undergoing IVF from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equations. RESULTS: Among PGT-A tested embryos, inferior ICM grade was associated with a lower chance of live birth (ICM grade B versus A: adjusted risk ratio [aRR] 0.91, 95% confidence interval [CI] 0.84-0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved on day 6 versus day 5 (aRR 0.87, 95% CI 0.78-0.96), and those with inferior pre-vitrification trophectoderm grade (trophectoderm grade B versus A: aRR 0.86, 95% CI 0.79-0.94). Blastocysts with a higher pre-vitrification expansion grade (pre-vitrification expansion grade 5 versus 4: aRR 1.1, 95% CI 1.01-1.2) were associated, but ICM grade was not associated (ICM grade B versus A: aRR 0.93, 95% CI 0.86-1.02), with chance of live birth. CONCLUSIONS: Among PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, trophectoderm grade and expansion grade may help to identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphological grade and day of cryopreservation less important.

Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia.

PURPOSE: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. METHODS: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. RESULTS: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. CONCLUSIONS: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Double trouble? Clinic-specific risk factors for monozygotic twinning.

OBJECTIVE: To investigate clinic-specific risk factors for monozygotic twinning (MZT) using a large, electronic database. DESIGN: Retrospective case-control study. SETTING: Infertility clinics. PATIENT(S): Using an electronic medical record system, viable clinical pregnancy (confirmation of a gestational sac(s) and presence of at least one fetal pole with a heartbeat on first trimester ultrasound), data were obtained from homologous in vitro fertilization (IVF) cycles after single ET from June 1, 2004, to December 31, 2016. Monozygotic twinning was defined as a pregnancy with two fetal heartbeats on ultrasound with sex concordance at birth. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Risk factors for MZT including cycle type, method of insemination, and method of cryopreservation. RESULT(S): Of the 28,265 IVF cycles that met inclusion criteria over the study period, 8,749 (31.0%) resulted in a viable intrauterine clinical pregnancy. There were 102 (2.7%) MZT in the fresh cycle cohort and 133 (2.7%) in the frozen cycle cohort. Neither cryopreservation nor the method of cryopreservation was a significant risk factor for MZT. However, the use of sequential media was an independent risk factor for MZT in fresh, but not frozen, ETs (odds ratio = 1.72, 95% confidence interval, 1.10-2.68). Significant differences were seen in the incidence of MZT between clinics, and this difference persisted after controlling for known risk factors (clinic 0, reference; clinic 2, odds ratio = 2.22; 95% confidence interval, 1.48-3.32; clinic 3, odds ratio = 1.93; 95% confidence interval, 1.30-2.87). CONCLUSION(S): Differences in MZT rates exist between individual IVF clinics, suggesting that variations in practice patterns may contribute to this event. The present study noted the use of sequential media was an independent risk factor for fresh but not frozen cycles.

Choice of statistical model in observational studies of ART.

Analyzing data on ART presents unique and sometimes complicated challenges related to choosing the unit(s) of analysis and the statistical model. In this commentary, we provide examples of how these challenges arise and guidance for overcoming them. We discuss the implications of different ways to count treatment cycles, considering the perspectives of research questions, data management and analysis and patient counseling. We present the advantages and disadvantages of different statistical models, and finally, we discuss the definition and calculation of the cumulative incidence of live birth, which is a key outcome of research on ART.

To test or not to test? A framework for counselling patients on preimplantation genetic testing for aneuploidy (PGT-A).

STUDY QUESTION: What is the treatment path and cumulative live birth (CLB) rate from a single oocyte retrieval of patients who intend to pursue PGT-A at the start of an IVF cycle compared to matched controls? SUMMARY ANSWER: The choice of PGT-A at the start of the first IVF cycle decreases the CLB per oocyte retrieval for patients <38 years of age, however patients ≥38 years of age benefit significantly per embryo transfer (ET) when live birth (LB) is evaluated. WHAT IS KNOWN ALREADY: PGT-A has been shown to reduce the practice of transferring multiple embryos and to confer a higher live birth rate per transfer. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study from December 2014 to September 2016, involving 600 patients: those intending PGT-A for their first IVF cycle (N = 300) and their matched controls. Post-hoc power calculations (alpha of 0.05, power of 0.80) indicated that our study was powered adequately to demonstrate significant differences in CLB per retrieval and LB per transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was performed at a large academically affiliated infertility practice where approximately 80% of patients have insurance coverage for fertility care. Patients were identified through electronic medical records, and those who intended to pursue PGT-A at the start of stimulation were assessed. Patients were matched by age, time of oocyte retrieval and oocyte yield to the same number of controls. CLB outcomes per single retrieval, including the fresh and frozen transfers arising from the initial stimulation cycle, were calculated. MAIN RESULTS AND THE ROLE OF CHANCE: PGT-A was not beneficial when CLB rate was assessed per retrieval, however its benefits were significant when LB rate was assessed per transfer. First cycle, <38 year-old patients who intended to have PGT-A had a significantly (P < 0.001) lower CLB rate per oocyte retrieval compared to controls (49.4% vs. 69.1%). Conversely, patients ≥ 38 years in the PGT-A group had similar CLB rates compared to controls per oocyte retrieval, while LB rates per transfer were doubled compared to controls (62.1% vs. 31.7%; P < 0.001). Of the first-cycle PGT-A and control patients, 25.3% and 2.3% failed to achieve a transfer, respectively. LIMITATIONS, REASONS FOR CAUTION: This is not a true intention-to-treat study, due to its retrospective nature. Additionally, the number of patients with two or more previous miscarriages was significantly greater in the PGT-A group as compared to controls, however a sub-analysis showed that this failed to impact outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The findings indicate that PGT-A may be detrimental for those <38 years old undergoing their first IVF cycle. PGT-A has the greatest clinical impact when a transfer is achieved in the ≥38 years old population. This study evaluates the typical treatment path following a patient's choice to pursue PGT-A at the cycle start, and can be used as a guide for counselling patients in relation to age and cycle number. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Will noninvasive methods surpass invasive for assessing gametes and embryos?

The need to identify the most viable embryo following in vitro fertilization (IVF) was established early in the history of human IVF. The stalwart of identifying the best embryos has been morphology. Other techniques have however seen wide acceptance, including the use of preimplantation genetic screening, even though concerns exist over the invasive nature of the technique. Alternatively, noninvasive assessment technologies have tried to determine an embryo's viability through measurements of factors in the media or by imaging of the embryo. We present data showing that the metabolic blueprint of an embryo is linked to viability, and argue that analysis of metabolic function, using either spent medium or by novel microscopies, could provide the basis for selecting the embryo with the highest viability. This review therefore asks, "Will noninvasive methods surpass invasive for assessing gametes and embryos?" We examine the current state of research on noninvasive technologies, including novel optical methods, and conclude noninvasive embryo viability assessment will assist in embryo selection for transfer.

Metabolic Determinants of Mitochondrial Function in Oocytes.

Mitochondrial production of cellular energy is essential to oocyte function, zygote development and successful continuation of pregnancy. This review focuses on several key functions of healthy oocyte mitochondria and the effect of pathologic states such as aging, oxidative stress and apoptosis on these functions. The effect of these abnormal conditions is presented in terms of clinical presentations, specifically maternal obesity, diminished ovarian reserve and assisted reproductive technologies.

Effectiveness of Novel Borane-Phosphine Complexes In Inhibiting Cell Death Depends on the Source of Superoxide Production Induced by Blockade of Mitochondrial Electron Transport.

Central neurons undergo cell death after axotomy. One of the signaling pathways for this process is oxidative modification of one or more critical sulfhydryls in association with superoxide generation within mitochondria. Agents that reduce oxidized sulfhydryls are neuroprotective of axotomized retinal ganglion cells, and we hypothesized that this occurs via reversal of the effects of mitochondrial-produced superoxide. To study this, we measured the ability of the novel borane-phosphine complex drugs bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester)diphenylphosphine borane complex (PB2) to inhibit the death of neuron-like RGC-5 cells induced by perturbation of the mitochondrial electron transport chain. We found that borane-phosphine complexes prevent neuronal cell death from superoxide produced by the redox-cycling agent menadione and the complex III inhibitor antimycin A, which produce superoxide towards the cytoplasm and matrix, but not the complex I inhibitor rotenone, which produces superoxide in the matrix alone. The ability of these disulfide reductants to prevent cell death may be predicted by the topology of superoxide production with respect to the mitochondrial matrix and extramitochondrial space.

Cell-autonomous generation of mitochondrial superoxide is a signal for cell death in differentiated neuronal precursor cells.

The vast majority of optic neuropathies result from retinal ganglion cell (RGC) axonal injury. This induces cell death and is associated with a burst of mitochondria-generated superoxide within the soma. It is unclear whether there is a clear causal relationship between superoxide generation and cell death. To determine whether mitochondrial-generated superoxide can cause cell-autonomous death signaling, we knocked down SOD2 in a pure population of RGC-5 cells, a neuronal precursor cell line that can be differentiated to resemble retinal ganglion cells. RGC-5 cells were differentiated and transfected with siRNA for SOD2 or a scramble control. Viability, superoxide production, cytotoxic RNA transfection efficiency, and measurement of SOD2 protein levels by immunoblotting were assayed at varying times after transfection. SOD2 knockdown increased intracellular superoxide levels and cell death was presumed triggered from knockdown. This was amplified when extramitochondrial superoxide was elevated with the redox cycling agent menadione. Dysregulation of mitochondrial superoxide in differentiated RGC-5 cells is likely a potent signal for cell death, consistent with a role of this reactive oxygen species in apoptosis signaling after axonal injury.