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.

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.

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.

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.

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.

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.

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.

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.

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.

Are fear memories made and maintained by the same NMDA receptor-dependent mechanisms?

A recent finding indicates that inducible knockout of the NR1 NMDA receptor subunit promotes the loss of fear memories formed months earlier. One view is that posttraining NMDA receptor activation protects modified synapses from "synaptic drift." An alternative view is that NMDA receptors help maintain appropriate connectivity in memory-encoding networks.

Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats.

NMDA receptor antagonists block conditioned fear extinction when injected systemically and also when infused directly into the amygdala. Here we evaluate the ability of D-cycloserine (DCS), a partial agonist at the strychnine-insensitive glycine-recognition site on the NMDA receptor complex, to facilitate conditioned fear extinction after systemic administration or intra-amygdala infusions. Rats received 10 pairings of a 3.7 sec light and a 0.4 mA footshock (fear conditioning). Fear-potentiated startle (increased startle in the presence vs the absence of the light) was subsequently measured before and after 30, 60, or 90 presentations of the light without shock (extinction training). Thirty non-reinforced light presentations produced modest extinction, and 60 or 90 presentations produced nearly complete extinction (experiment 1). DCS injections (3.25, 15, or 30 mg/kg) before 30 non-reinforced light exposures dose-dependently enhanced extinction (experiment 2) but did not influence fear-potentiated startle in rats that did not receive extinction training (experiment 3). These effects were blocked by HA-966, an antagonist at the glycine-recognition site (experiment 4). Neither DCS nor HA-966 altered fear-potentiated startle when injected before testing (experiment 5). The effect of systemic administration was mimicked by intra-amygdala DCS (10 microg/side) infusions (experiment 6). These results indicate that treatments that promote NMDA receptor activity after either systemic or intra-amygdala administration promote the extinction of conditioned fear.

Insulin and messenger ribonucleic acid expression of insulin receptor isoforms in ovarian follicles from nonhirsute ovulatory women and polycystic ovary syndrome patients.

Insulin action is mediated by two insulin receptor (IR) isoforms, differing in mitogenic and metabolic function. IR isoform expression might occur in human granulosa cells and could be altered in polycystic ovary syndrome (PCOS) from hyperinsulinemia. To determine the relationship between granulosa cell IR isoform expression and follicular fluid insulin concentration in individual follicles, 18 normal women and seven PCOS patients receiving gonadotropins for in vitro fertilization were studied. Glucose tolerance testing was performed before pituitary desensitization, and fasting serum insulin was measured at oocyte retrieval. Granulosa cells and fluid aspirated from the first follicle were used to determine IR isoform mRNA expression and insulin concentration, respectively. IR isoform A mRNA expression was greater than that of IR isoform B expression in normal mural granulosa and cumulus cells, without a cell type effect. Intrafollicular insulin levels increased with adiposity and serum insulin levels at oocyte-retrieval but did not predict IR mRNA expression. Total IR mRNA expression, but not intrafollicular insulin levels, was elevated in PCOS patients, whereas intrafollicular insulin levels were increased in women with impaired glucose tolerance. Granulosa cell IR heterogeneity, together with adiposity-dependent intrafollicular insulin availability, introduces a novel mechanism by which insulin may affect granulosa cell function within the follicle.

Group II metabotropic glutamate receptors within the amygdala regulate fear as assessed with potentiated startle in rats.

The contribution to fear and fear learning of amygdala Group II metabotropic glutamate receptors was examined in rats. Pretest intra-amygdala infusions of the Group II receptor agonist LY354740 (0.3 or 1.0 microg/side) significantly disrupted fear-potentiated startle. The same rats were unimpaired when later tested without drug. The Group II receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (3.0 microg/side) mimicked the effect of LY354740, and coadministration of the Group II receptor antagonist LY341495 (0.3 microg/side) prevented it. Pretraining LY354740 (0.3 microg/side) infusions also blocked learning. The effects on learning and performance were significantly less pronounced in rats with misplaced cannulas. Thus, Group II metabotropic receptors within or very near the amygdala regulate fear and fear learning and are a potential target for anxiolytic compounds.

Light-enhanced startle: further pharmacological and behavioral characterization.

RATIONALE: Previous findings indicate that the acoustic startle response is elevated when rats are tested in bright light. The phenomenon is disrupted by the 5HT1A partial agonist and the D2 receptor antagonist buspirone, a compound that also blocks the effect on startle of conditioned fear, and it was suggested that light-enhanced startle reflects an anxious state produced by bright light. It was also suggested that pre-test handling may be necessary for light-enhanced startle. To characterize this phenomenon further, we evaluate here the sensitivity of light-enhanced startle to the anxiolytic compound chlordiazepoxide, to the noradrenergic beta-receptor antagonist propranolol, and to pre-test handling. METHODS: Startle was measured for 20 min in the dark (phase I), followed 5 min later by a second test (phase II) either in the dark or the light. Immediately prior to testing, rats received IP injections of chlordiazepoxide (5, 10, or 20 mg/kg; experiment 1), propranolol (10 or 20 mg/kg; experiment 2) or saline. Using the minimally effective doses from the light-enhanced startle experiments, conditioned fear to a shock-paired cue was also evaluated. In a third experiment, rats were (a) removed from the test cages and briefly handled between phases I and II, (b) were not handled during this interval, or (c) were tested without the interposed delay. RESULTS: Propranolol (10 and 20 mg/kg) and chlordiazepoxide (10 mg/kg) disrupted light-enhanced startle at doses comparable to those required to disrupt fear-potentiated startle to a shock-paired cue. There was no effect of handling. CONCLUSION: These results further characterize the pharmacology of light-enhanced startle, provide additional support for the view that the effects of light on startle reflect an influence of anxiety, and offer additional information concerning the procedural variables that influence this behavior.

Quantifying fear potentiated startle using absolute versus proportional increase scoring methods: implications for the neurocircuitry of fear and anxiety.

RATIONALE: The fear-potentiated startle paradigm [increased startle in the presence of a conditioned fear stimulus (CS)] has become increasingly popular as a tool for evaluating the potential efficacy of putative anxiolytic compounds. However, when the tested compounds also influence baseline startle, it is unclear how comparisons with control groups can best be made. OBJECTIVE: To evaluate the validity of absolute difference (startle amplitude on CS minus non-CS test trials) vs. proportional increase (the absolute difference score divided by startle amplitude on non-CS test trials) scoring methods. METHODS: The effect on proportional increase and absolute difference scores of baseline shifts that occur with or without concomitant increases in fear was evaluated in rats. A reliable measure should yield similar scores across shifting baselines, provided that fear levels remain constant. RESULTS: Preexisting baseline differences, and those brought about by different startle-eliciting noise burst intensities, by strychnine injections, or by CRH infusions, each increased absolute difference scores without markedly influencing proportional change scores. These baseline differences were not associated with different fear levels. Increases in baseline startle brought about by unsignaled footshocks or by a second CS - increases which are associated with increased fear - partially occluded additional CS-induced increases using either measure. CONCLUSIONS: Across different baselines, CS-elicited fear is most accurately reflected in proportional change scores. Under certain conditions saturation effects may interfere with an accurate assessment using either measure. However, these same saturation effects may provide opportunities to explore the neural circuitry of fear and anxiety in novel ways.

Role of the amygdala in fear extinction measured with potentiated startle.

Although much is now known about the neural basis of excitatory fear conditioning, much less is known about the neural basis of inhibitory conditioning. One type of inhibitory conditioning is extinction, a process in which stimuli that elicit fear by virtue of previous associations with aversive stimuli such as shock (excitatory fear conditioning) are now presented in the absence of the aversive stimuli (extinction training). As a result, the ability of the conditioned stimulus to elicit fear gradually diminishes. Extinction is different from forgetting and does not reflect an erasure of the original fear memory. Instead, extinction is an active form of inhibitory learning that competes with excitatory fear conditioning. Infusions into the amygdala (a brain area essential for excitatory fear conditioning) of either NMDA receptor antagonists or inhibitors of the NMDA-receptor-linked mitogen-activated protein kinase cascade block extinction learning. Conversely, the NMDA receptor agonist D-cycloserine facilitates extinction after either systemic administration or intra-amygdala infusion. Because therapeutic interventions based on extinction procedures are commonly used to treat fear disorders, and because D-cycloserine is a widely available and safe compound, D-cycloserine or similar agents might be usefully combined with traditional extinction-based approaches in the treatment of clinical fear.

Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety.

The bed nucleus of the stria terminalis is a limbic forebrain structure that receives heavy projections from, among other areas, the basolateral amygdala, and projects in turn to hypothalamic and brainstem target areas that mediate many of the autonomic and behavioral responses to aversive or threatening stimuli. Despite its strategic anatomical position, initial attempts to implicate the bed nucleus of the stria terminalis in conditioned fear were largely unsuccessful. Recent studies have shown, however, that the bed nucleus of the stria terminalis does participate in certain types of anxiety and stress responses. In this work, we review these findings and suggest from the emerging pattern of evidence that, although the bed nucleus of the stria terminalis may not be necessary for rapid-onset, short-duration behaviors which occur in response to specific threats, the bed nucleus of the stria terminalis may mediate slower-onset, longer-lasting responses that frequently accompany sustained threats, and that may persist even after threat termination.

Equivalent blastocyst rates after freezing murine embryos in Cryo Bio System high security or standard instruments-medicine-veterinarian straws.

OBJECTIVE: To validate the Cryo Bio System (CBS) straw in our current cryopreservation system before using it in clinical practice. DESIGN: A prospective comparison of blastocyst development rates in 278 murine embryos after refreezing and thawing at the two-cell stage against the standard Instruments-Medicine-Veterinarian (IMV) straw used in our cryopreservation program. SETTING: Private IVF laboratory. PATIENT(S): No human subjects or material was used in this study. INTERVENTION(S): Frozen two-cell murine embryos were thawed and randomized into three treatments [1] refreezing in the CBS straws, [2] refreezing in IMV 0.25-mL straws, and [3] control embryos remaining in culture without refreezing. Embryos were refrozen using identical cryoprotectants and identical programmed controlled-rate freezers. After cryopreservation, straws were held in liquid nitrogen for a brief period before thawing and continued culture. MAIN OUTCOME MEASURE(S): Postthaw murine blastocyst development rate. RESULT(S): When the manufacturer's filling and loading protocol was used for the CBS straw there was no significant difference in the blastocyst development rate between CBS (75.0%) and IMV (76.4%) straws. CONCLUSION(S): The CBS straw may be a viable and potentially safer alternative for cryopreservation of human embryos, particularly for patients with known infections.

The role of amygdala glutamate receptors in fear learning, fear-potentiated startle, and extinction.

Using a paradigm known as fear-potentiated startle, we have examined the neurobiological substrates of Pavlovian fear conditioning. In these experiments, rats are trained to fear an initially neutral stimulus by pairing that stimulus with shock. The amount of fear elicited by the stimulus [i.e., now a conditioned stimulus (CS)] is later assessed by presenting startle-eliciting noise bursts both in the presence and also the absence of the CS. After training, startle responses are typically greater in the presence of the CS. Findings reviewed here suggest that amygdala N-methyl-D-aspartate (NMDA) receptors play a key role in triggering the neural changes that support fear learning and also the loss of fear that accompanies extinction training. Amygdala (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors also participate in fear learning. However, unlike NMDA receptor antagonists, AMPA receptor antagonists also block fear-potentiated startle when infused prior to testing. Very recent data indicate that glutamate metabotropic Group II receptor agonists also block fear learning when infused into the amygdala prior to training, and block fear-potentiated startle when infused prior to testing. A fuller understanding of the role of amygdala glutamate systems in fear and fear learning may suggest novel pharmacological approaches to the treatment of clinical anxiety disorders.