Vitrification versus slow freezing of human ovarian tissue: a systematic review and meta-analysis of histological outcomes.

A systematic review and meta-analysis of pertinent literature published from 2006 to January 2022 were conducted to study and compare vitrification and slow freezing, the two prominent methods of ovarian tissue cryopreservation. The primary outcome measures for this study were (1) proportion of intact primordial follicles, (2) proportion of intact stromal cells, (3) proportion of DNA fragmentation in primordial follicles, and (4) mean primordial follicle density. This meta-analysis of 19 studies revealed a significantly greater proportion of intact stromal cells in vitrified tissue versus slow-frozen tissue. No significant differences upon pooled analyses were observed between the two cryopreservation methods with respect to the proportion of intact primordial follicles, proportion of DNA fragmentation, or mean primordial follicle density. Due to differences seen in stromal cell viability, vitrification may be a preferred option to preserve histology of tissue. However, more work should be done to compare the two freezing techniques with less heterogeneity caused by patients, samples, and protocols.

Embryo Blastomere Exclusion Identified in a Time-Lapse Culture System Is Associated with Embryo Ploidy.

This study examined blastomere exclusion which is seen during embryo development and could represent imperfect cell division or a mechanism of aneuploidy correction. This was a retrospective cohort study which included embryos cultured in a time-lapse incubator undergoing preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm biopsy. Embryos were evaluated for blastomere exclusion early in development, late in development, both, or neither. Blastomere exclusion was compared to embryo ploidy. Embryos with no blastomere exclusion had an aneuploidy rate of 52.9%, while embryos displaying blastomere exclusion at any stage had an aneuploidy rate of 68.5% (p < .001). Early blastomere exclusion was not significantly associated with an increased aneuploidy risk (59.2% vs. 52.9% in no blastomere exclusions; p = 0.22). However, embryos with late blastomere exclusion were significantly more likely to be aneuploid, compared to embryos with no blastomere exclusions (77.5% vs. 52.9%; p < 0.001) as were embryos with both early + late blastomere exclusions (71.2% vs. 52.9%; p < 0.001). Upon restricting the analysis to aneuploid embryos, the presence of any blastomere exclusion was not significantly associated with complex aneuploidy, defined as 2 more affected chromosomes (43.9% vs. 38.7%; p = 0.28). However, the proportion with adverse embryo genetics significantly increased with the timing of blastomere exclusion (38.7%, 37%, 45.5%, and 50% for none, early, late, and early + late; p = 0.043). Late blastomere exclusion or a combination of both early + late blastomere exclusion was associated with an increased risk of aneuploid embryo genetics. Embryo selection using time-lapse culture systems should incorporate these findings when untested embryos are transferred.

Comparison of live birth rates after IVF-embryo transfer with and without preimplantation genetic testing for aneuploidies.

RESEARCH QUESTION: Does the use of preimplantation genetic testing for aneuploidies (PGT-A) result in higher live birth rates when compared with both fresh and frozen embryo transfers (FET) not utilizing PGT-A? DESIGN: Retrospective cohort study at a single tertiary centre using inverse probability of treatment weighting (IPTW) to adjust for differences in baseline characteristics between groups. RESULTS: A total of 107 FET using PGT-A from 74 patients, along with 321 fresh and 286 FET not using PGT-A from 381 patients met the inclusion criteria for this study. In the IPTW-adjusted analysis of transfer-level data, PGT-A transfers resulted in a significantly higher live birth rate when compared with both non-PGT-A fresh (49.5% versus 38.6%, P = 0.036) and FET (50.6% versus 35.8%, P = 0.016). When data were analysed per retrieval level, the live birth rate was similar and acceptably high with or without PGT-A (63.7% versus 52.3%, P = 0.09). CONCLUSION: When comparing PGT-A to non-PGT-A fresh and FET, PGT-A embryo transfers have a significantly higher live birth rate. However, this difference did not persist at a per-retrieval level. Further investigation is needed to understand in what scenarios PGT-A has clinical significance and whether differences in the number of available embryos for transfer negates the benefit of PGT-A.

Extreme laryngeal candidiasis: airway obstruction.

We describe the case of a 33-year-old female smoker who presented to the Accident and Emergency department with a 1-day history of rapidly evolving airway compromise. She had no preceding illness or other objective signs/symptoms on presentation, had a history of Chronic Obstructive Pulmonary Disease (COPD) and a previous opioid addiction. Following failed endotracheal intubation, the airway was secured with an emergency surgical tracheostomy. Subsequent direct laryngoscopy revealed a severely diseased glottis and supraglottic area, from which biopsy samples revealed a multiple drug-resistant strain of Candida albicans requiring specialist microbiology input and antifungal treatment. We describe the presentation, investigation, management and outcome of this rare case, along with a literature review of the subject.

Consult and procedure incidence outcomes following establishment of a fertility preservation program for children with cancer.

PURPOSE: Fertility is a quality of life outcome adversely affected by cancer therapy. Many childhood cancer patients, however, are not offered options to preserve their fertility. Providers acknowledge difficulty discussing impaired fertility to patients due to lack of knowledge of available options. Our objective was to review the impact of a pediatric multidisciplinary fertility preservation program on providers' fertility preservation counseling and discussion of options. METHODS: A retrospective medical chart review was conducted for pediatric cancer patients prior to and following program establishment. Fertility preservation discussions, consults, and incidence were noted. Following filtering and stratification, 198 and 237 patients were seen prior to and following program establishment, respectively. RESULTS: Following program establishment, provider-patient discussions of impaired fertility (p = 0.007), fertility preservation consults (p = 0.01), and incidence of fertility preservation procedures (p < 0.001) increased among patients. Furthermore, the number of patients who received fertility preservation consults after receiving gonadotoxic treatment decreased (p < 0.001). This trend was particularly noted in pre-pubertal and female patients, for whom fertility preservation options are limited without an established program. CONCLUSION: The establishment of a formal program greatly improved access to fertility preservation consults and procedures in children with cancer.

Mechanisms underlying long-term fear memory formation from a metaplastic neuronal state.

We previously showed that a single weak fear conditioning trial, that does not produce a long-term fear memory (LTM), appeared to prime memory formation such that when a second trial followed within a circumscribed time window a robust and long-lasting fear memory was formed. We also showed that this priming effect could be blocked if we interfered with protein kinase A (PKA) signaling in the amygdala during the first conditioning trial. The goals of the current study were to determine if LTM formation after the second trial depends on PKA signaling in the amygdala and to characterize the underlying memory processes engaged during the second trial that allows for LTM formation. Our interpretation of the original findings is that the second conditioning trial triggers LTM from a metaplastic state that is engaged by the first conditioning trial. However, it is also possible that the second conditioning trial acts as a reminder of the first and engages a reconsolidation-like process. Several experiments were conducted to distinguish between these two possibilities. We show that interfering with PKA signaling during the second conditioning trial disrupts memory formation. However, if a third trial follows the second or if the second trial was presented without shock, the PKA inhibitor was no longer effective. Our findings demonstrate that the induction of fear memory from a metaplastic state involves new learning that is distinct from retrieval-dependent updating of memories.

Role of bed nucleus of the stria terminalis and amygdala AMPA receptors in the development and expression of context conditioning and sensitization of startle by prior shock.

A core symptom of post-traumatic stress disorder is hyper-arousal-manifest in part by increases in the amplitude of the acoustic startle reflex. Gewirtz et al. (Prog Neuropsychopharmacol Biol Psychiatry 22:625-648, 1998) found that, in rats, persistent shock-induced startle increases were prevented by pre-test electrolytic lesions of the bed nucleus of the stria terminalis (BNST). We used reversible inactivation to determine if similar effects reflect actions on (a) BNST neurons themselves versus fibers-of-passage, (b) the development versus expression of such increases, and (c) associative fear versus non-associative sensitization. Twenty-four hours after the last of three shock sessions, startle was markedly enhanced when rats were tested in a non-shock context. These increases decayed over the course of several days. Decay was unaffected by context exposure, and elevated startle was restored when rats were tested for the first time in the original shock context. Thus, both associative and non-associative components could be measured under different conditions. Pre-test intra-BNST infusions of the AMPA receptor antagonist NBQX (3 µg/side) blocked the non-associative (as did infusions into the basolateral amygdala) but not the associative component, whereas pre-shock infusions disrupted both. NBQX did not affect baseline startle or shock reactivity. These results indicate that AMPA receptors in or very near to the BNST are critical for the expression and development of non-associative shock-induced startle sensitization, and also for context fear conditioning, but not context fear expression. More generally, they suggest that treatments targeting the BNST may be clinically useful for treating trauma-related hyper-arousal and perhaps for retarding its development.

CGRP antagonist infused into the bed nucleus of the stria terminalis impairs the acquisition and expression of context but not discretely cued fear.

Calcitonin gene-related peptide (CGRP) infusions into the bed nucleus of the stria terminalis (BNST) evoke increases in startle amplitude and increases in anxiety-like behavior in the plus maze. Conversely, intra-BNST infusions of the CGRP antagonist CGRP8₋37 block unconditioned startle increases produced by fox odor. Here we evaluate the contribution of CGRP signaling in the BNST to the development and expression of learned fear. Rats received five pairings of a 3.7-sec light and footshock and were tested for fear-potentiated startle one or more days later. Neither pre-training (Experiment 1) nor pre-test (Experiment 2) infusions of the CGRP antagonist CGRP8₋37 (800 ng/BNST) disrupted fear-potentiated startle to the 3.7-sec visual cue. However, in both experiments, CGRP8₋37 infusions disrupted baseline startle increases that occurred when rats were tested in the same context as that in which they previously received footshock (Experiment 3). Intra-BNST CGRP8₋37 infusions did not disrupt shock-evoked corticosterone release (Experiment 4). These data confirm previous findings implicating BNST CGRP receptors in fear and anxiety. They extend those results by showing an important contribution to learned fear and, specifically, to fear evoked by a shock-associated context rather than a discrete cue. This pattern is consistent with previous models of BNST function that have posited a preferential role in sustained anxiety as opposed to phasic fear responses. More generally, the results add to a growing body of evidence indicating behaviorally, possibly clinically, relevant modulation of BNST function by neuroactive peptides.

Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid.

OBJECTIVE: To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. DESIGN: Experimental laboratory study. SETTING: University-based laboratory. ANIMAL(S): FVB and CF1 mice crossed to create embryos used in experiments. INTERVENTION(S): Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). MAIN OUTCOME MEASURE(S): Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). RESULT(S): The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were <50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C. CONCLUSION(S): The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.

Mouse strain and quality control testing: improved sensitivity of the mouse embryo assay with embryos from outbred mice.

OBJECTIVE: To determine the relative sensitivities of embryos from different strains of mice to in vitro stress. DESIGN: Laboratory experiment with embryos from different mouse strains. SETTING: University hospital-based fertility clinic. ANIMAL(S): Mice. INTERVENTION(S): Fresh one-cell embryos from outbred (CF1), inbred (FVB), F1 hybrid (B6/CBA), and cryopreserved F2 hybrid embryos (bcl/B6 × B6/bcl) compared in a mouse embryo assay (MEA) using six doses of each of three in vitro stressors: cumene hydroperoxide in mineral oil, Triton X-100 (TX-100) in media, and hyperosmolality. MAIN OUTCOME MEASURE(S): Blastocyst rate at 96 hours. RESULT(S): All studies were conducted in triplicate; data were analyzed with chi-square analysis based on fitting a logistic regression model. Both cumene hydroperoxide and Triton X-100 affected blastocyst formation in the outbred strain at concentrations that were less than half of the concentration that affected the other strains. The total number of cells was affected by the treatments in all strains. CONCLUSION(S): Outbred CF1 embryos are genetically diverse and more sensitive to toxins than either inbred or hybrid mouse embryos. Outbred embryos provide an additional tool for effective quality-control testing.

Potential of inner cell mass outgrowth and amino acid turnover as markers of quality in the in vitro fertilization laboratory.

OBJECTIVE: To compare sensitivity of inner cell mass (ICM) outgrowth assay and analysis of culture media amino acid turnover with the sensitivity of the human sperm motility assay (HSMA) and murine embryo assay (MEA) for detection of formaldehyde toxicity. DESIGN: Prospective in vitro study. SETTING: University hospital-based infertility center. ANIMAL(S): Murine embryos. INTERVENTION(S): The HSMA, MEA, and ICM outgrowth assays were performed with media containing 0-64-µM concentrations of formaldehyde. These assays were compared with dynamics of amino acid turnover in culture media. MAIN OUTCOME MEASURE(S): The lowest concentration of formaldehyde in culture media detected by each quality control assay. RESULT(S): Sperm forward progression, but not motility, detected formaldehyde at a concentration of 32 µM. Sperm motility index identified formaldehyde toxicity at 64 µM, whereas blastocyst rates in the MEA were affected at 32 µM formaldehyde. Evaluation of ICM using outgrowth and grade detected 16 µM formaldehyde. Leucine turnover in culture media detected 64 µM formaldehyde in the amino acid assay. CONCLUSION(S): Inner cell mass outgrowth is a more sensitive bioassay than MEA and HSMA for the detection of formaldehyde in culture media. Amino acid metabolism may also provide a sensitive quality control measure for detection of formaldehyde.

Calcitonin gene-related peptide in the bed nucleus of the stria terminalis produces an anxiety-like pattern of behavior and increases neural activation in anxiety-related structures.

Calcitonin gene-related peptide (CGRP) evokes anxiety-like responses when infused into the lateral ventricle of rats. Because the bed nucleus of the stria terminalis (BNST) lies immediately adjacent to the lateral ventricle, is rich in CGRP receptors, and has itself been implicated in anxiety, we evaluated the hypothesis that these effects are attributable to stimulation of CGRP receptors within the BNST itself. Bilateral intra-BNST, but not dorsal, CGRP infusions (0, 200, 400, 800 ng/side) enhanced startle amplitude in a dose-dependent manner, and produced an anxiety-like response on the elevated plus maze. Intra-BNST infusion of the CGRP antagonist, αCGRP(8-37), blocked the effect of CGRP on startle, and also blocked startle potentiation produced by exposure to trimethylthiazoline (a component of fox feces that induces anxiety-like behavior in rats). Intra-BNST, but not dorsal, CGRP infusions also increased c-Fos immunoreactivity in a number of anxiety-related brain areas (central nucleus of the amygdala, locus ceruleus, ventrolateral septal nucleus, paraventricular hypothalamic nucleus, lateral hypothalamus, lateral parabrachial nucleus, dorsal raphe nucleus, and nucleus accumbens shell), all of which receive direct projections from the BNST. Together, the results indicate that the activation of BNST CGRP receptors is both necessary and sufficient for some anxiety responses and that these effects may be mediated by activation of a wider network of BNST efferent structures. If so, inhibition of CGRP receptors may be a clinically useful strategy for anxiety reduction.

Washing mineral oil reduces contaminants and embryotoxicity.

OBJECTIVE: To determine if washing improves the quality of mineral oil used for embryo culture. DESIGN: A 2 × 3 factorial experimental study. SETTING: University hospital-based infertility center. ANIMAL(S): Mice. INTERVENTION(S): The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined. MAIN OUTCOME MEASURE(S): The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media. RESULT(S): Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%. CONCLUSION(S): Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil.

Peroxides in mineral oil used for in vitro fertilization: defining limits of standard quality control assays.

PURPOSE: To determine the relative sensitivities of the 1 and 2-cell mouse embryo assays (MEA) and the human sperm motility assay (HSMA) for peroxides in mineral oil. The effect of peroxide on blastocyst cell number and apoptosis was also studied. METHODS: One and two-cell MEA and HSMA were performed using mineral oil containing cumene hydroperoxide (CH). RESULTS: The 1-cell MEA was twice as sensitive as the 2-cell MEA and 20-times more sensitive than the HSMA for CH in mineral oil. The sensitivity of the 1-cell MEA doubled when embryos were cultured individually versus group culture. CH decreased blastocyst cell number in a dose dependent manner. CONCLUSIONS: Individually cultured 1-cell embryos had the highest sensitivity for peroxides in mineral oil. Current quality control assays, including group cultured murine embryos and human sperm motility, have limited sensitivity for peroxides in mineral oil and may not detect levels of peroxides that cause sub-lethal cellular damage.

Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety.

Data will be reviewed using the acoustic startle reflex in rats and humans based on our attempts to operationally define fear vs anxiety. Although the symptoms of fear and anxiety are very similar, they also differ. Fear is a generally adaptive state of apprehension that begins rapidly and dissipates quickly once the threat is removed (phasic fear). Anxiety is elicited by less specific and less predictable threats, or by those that are physically or psychologically more distant. Thus, anxiety is a more long-lasting state of apprehension (sustained fear). Rodent studies suggest that phasic fear is mediated by the amygdala, which sends outputs to the hypothalamus and brainstem to produce symptoms of fear. Sustained fear is also mediated by the amygdala, which releases corticotropin-releasing factor, a stress hormone that acts on receptors in the bed nucleus of the stria terminalis (BNST), a part of the so-called 'extended amygdala.' The amygdala and BNST send outputs to the same hypothalamic and brainstem targets to produce phasic and sustained fear, respectively. In rats, sustained fear is more sensitive to anxiolytic drugs. In humans, symptoms of clinical anxiety are better detected in sustained rather than phasic fear paradigms.

Parthenogenic activation of surplus in vitro-matured human oocytes: a tool for validation of oocyte cryopreservation.

Controlled-rate frozen and vitrified mature human oocytes were thawed or warmed and parthenogenically activated using ionomycin and 6-dimethylaminopurine to assess pronuclear development compared with fresh controls. This chemical activation model provides a useful tool for laboratories to assess proficiency before offering oocyte cryopreservation to their patients.

Preimplantation genetic screening in a case of recurrent trisomy 21 offspring.

OBJECTIVE: To describe a unique case of recurrent aneuploidy and the use of preimplantation genetic screening (PGS). DESIGN: Case report. SETTING: Midwest academic medical center. PATIENT(S): A 36-year-old woman with two trisomy 21 offspring. INTERVENTION(S): Preimplantation genetic screening. MAIN OUTCOME MEASURE(S): Karyotype of embryos, liveborn eukaryotic infant. RESULT(S): Preimplantation genetic screening was performed on three cryopreserved embryos, followed by a two-embryo transfer yielding a eukaryotic infant. CONCLUSION(S): Preimplantation genetic screening may prove to be useful as a diagnostic tool to help ensure a euploid pregnancy when termination is not a viable option for a couple.

Effects of substance P in the amygdala, ventromedial hypothalamus, and periaqueductal gray on fear-potentiated startle.

The neural pathways through which substance P (SP) influences fear and anxiety are poorly understood. However, the amygdala, a brain area repeatedly implicated in fear and anxiety processes, is known to contain large numbers of SP-containing neurons and SP receptors. Several studies have implicated SP neurotransmission within the amygdala in anxiety processes. In the present study, we evaluated the effects of site-specific infusions of an SP receptor antagonist, GR 82334, on conditioned fear responses using the fear-potentiated startle paradigm. GR 82334 infusion into the basolateral (BLA) or the medial (MeA) nuclei of the amygdala, but not into the central nucleus of the amygdala (CeA), dose dependently reduced fear-potentiated startle. Similar effects were obtained with GR 82334 infusion into the ventromedial nucleus of the hypothalamus (VMH), to which the MeA projects, and into the rostral dorsolateral periaqueductal gray (PAG), to which the VMH projects, but not into the deep layers of the superior colliculus/deep mesencephalic nucleus (dSC/DpMe), an output of the CeA previously shown to be important for fear-potentiated startle. Consistent with previous findings, infusion of the AMPA receptor antagonist, NBQX, into the dSC/DpMe, but not into the PAG, did disrupt fear-potentiated startle. These findings suggest that multiple outputs from the amygdala play a critical role in fear-potentiated startle and that SP plays a critical, probably modulatory role, in the MeA to VMH to PAG to the startle pathway based on these and data from others.

Role of the extended amygdala in short-duration versus sustained fear: a tribute to Dr. Lennart Heimer.

The concept of the "extended amygdala", developed and explored by Lennart Heimer, Jose de Olmos, George Alheid, and their collaborators, has had an enormous impact on the field of neuroscience and on our own work. Measuring fear-potentiated startle test using conditioned stimuli that vary in length we suggest that the central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST(L)) are involved in short-term versus long-term fear responses we call phasic versus sustained fear, respectively. Outputs from the basolateral amygdala (BLA) activate the medial division of the CeA (CeA(M)) to very rapidly elicit phasic fear responses via CeA(M) projections to the hypothalamus and brainstem. The BLA also projects to the BNST(L), which together with other BNST(L) inputs from the lateral CeA (CeA(L)) initiate a slower developing, but sustained fear response, akin to anxiety. We hypothesize this occurs because the CeA(L) releases the peptide corticotropin releasing hormone (CRF) into the BNST(L) which facilitates the release of glutamate from BLA terminals. This activates the BNST(L) which projects to hypothalamic and brainstem areas similar to those innervated by the CeA(M) that mediate the specific signs of fear and anxiety. The generality of this idea is illustrated by selective studies looking at context conditioning, social defeat, drug withdrawal and stress induced reinstatement.

Amygdala infusions of an NR2B-selective or an NR2A-preferring NMDA receptor antagonist differentially influence fear conditioning and expression in the fear-potentiated startle test.

Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the NR1/NR2B receptor antagonist CP101,606 (0.5, 1.5, or 4.5 microg/amygdala) or the NR1/NR2A-preferring antagonist NVP-AAM077 (0.075, 0.25, 0.75, or 2.5 microg/amygdala) into the amygdala prior to either fear conditioning (i.e., light-shock pairings) or fear-potentiated startle testing. CP101,606 nonmonotonically disrupted fear conditioning but did not disrupt fear expression. NVP-AAM077 dose-dependently disrupted fear conditioning as well as fear expression. The results suggest that amygdala NR1/NR2B receptors play a special role in fear memory formation, whereas NR1/NR2A receptors participate more generally in synaptic transmission.