Superovulation with human chorionic gonadotropin (hCG) trigger and gonadotropin releasing hormone agonist (GnRHa) trigger differentially alter essential angiogenic factors in the endometrium in a mouse ART model†.

Gonadotropin-releasing hormone agonists (GnRHa) are used as an alternative to human chorionic gonadotropin (hCG) to trigger ovulation and decrease the risk of ovarian hyperstimulation syndrome. GnRHa is less potent at inducing ovarian vascular endothelial growth factor (VEGF), but may also affect endometrial angiogenesis and early placental development. In this study, we explore the effect of superovulation on endometrial angiogenesis during critical periods of gestation in a mouse model. We assigned female mice to three groups: natural mating or mating following injection with equine chorionic gonadotropin and trigger with GnRHa or hCG trigger. Females were killed prior to implantation (E3.5), post-implantation (E7.5), and at midgestation (E10.5), and maternal serum, uterus, and ovaries were collected. During peri-implantation, endometrial Vegfr1 and Vegfr2 mRNA were significantly increased in the GnRHa trigger group (P < 0.02) relative to the hCG group. Vegfr1 is highly expressed in the endometrial lining and secretory glands immediately prior to implantation. At E7.5, the ectoplacental cone expression of Vegfa and its receptor, Vegfr2, was significantly higher in the hCG trigger group compared to the GnRHa group (P < 0.05). Soluble VEGFR1 and free VEGFA were much higher in the serum of mice exposed to the hCG trigger compared to GnRHa group. At midgestation, there was significantly more local Vegfa expression in the placenta of mice triggered with hCG. GnRHa and hCG triggers differentially disrupt the endometrial expression of key angiogenic factors during critical periods of mouse gestation. These results may have significant implications for placental development and neonatal outcomes following human in vitro fertilization.

Water-fat separation and parameter mapping in cardiac MRI via deep learning with a convolutional neural network.

BACKGROUND: Water-fat separation is a postprocessing technique most commonly applied to multiple-gradient-echo magnetic resonance (MR) images to identify fat, provide images with fat suppression, and to measure fat tissue concentration. Recently, Numerous advancements have been reported. In contrast to early methods, the process of water-fat separation has become complicated due to multiparametric analytic models, optimization methods, and the absence of a unified framework for diverse source data. PURPOSE: To determine the feasibility and performance of MRI water-fat separation and parametric mapping via deep learning (DL) with a range of inputs. STUDY TYPE: Retrospective data usage. POPULATION/SUBJECTS: Ninety cardiac MR examinations from normal control, acute, subacute, and chronic myocardial infarction subjects were obtained, providing 1200 multiple gradient-echo acquisitions. FIELD STRENGTH/SEQUENCE: 1.5 T/2D multiple gradient-echo pulse sequence ASSESSMENT: Ground-truth training and validation water-fat separation were obtained using a graph cut method with R2 *, off-resonance correction, and a multipeak fat spectrum. U-Net DL training with single and multiecho, complex, and magnitude inputs were compared using quantitative and three-observer subjective analysis. STATISTICAL TESTS: DL methods' image structural similarity, and quantitative proton density fat fraction (PDFF), R2 *, and off-resonance quantitative values were statistically compared with the GraphCut reference standard using Student's t-test and Pearson's correlation. RESULTS: Myocardial fat deposition in chronic myocardial infarction and intramyocardial hemorrhage in acute myocardial infarction were well visualized in the DL results. Predicted values for R2 *, off-resonance, water, and fat signal intensities were well correlated with a conventional model-based water fat separation (R2 ≥ 0.97, P < 0.001) with appropriate inputs. DL parameter maps had a 14% higher signal-to-noise ratio (P < 0.001) when compared with a conventional method. DATA CONCLUSION: DL water-fat separation is feasible with a wide range of inputs, while R2 * and off-resonance mapping requires multiple echoes and complex images. With appropriate inputs, DL provides quantitative and subjective results comparable to conventional model-based methods. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 1 J. Magn. Reson. Imaging 2019;50:655-665.

Effects of transcytolemmal water exchange on the assessment of myocardial extracellular volume with cardiovascular MRI.

Quantitative analysis of the myocardial interstitial space is gaining increased interest as a biomarker in the MRI and clinical cardiovascular communities. To investigate the effect of water exchange on the calculation of myocardial extracellular volume (ECV), we employed two tissue models: the standard ECV two-point model (SM) and the shutter speed model (SSM). Twenty individuals (18 men and two women; age 61.9 ± 10.3 years) underwent MRI at 1.5 T with pre-contrast and post-contrast dynamic T1 quantification. Means, standard deviations and ranges for SM and SSM model parameters were calculated. Infarct and viable myocardial model parameters as well as apparent ECV values calculated with the SM and SSM were statistically compared. Viable ECV(SM) remained temporally constant (27.3-28.0%: P = 0.5) and infarcted myocardial ECV(SM) changed significantly (49.3-58.8%; P < 0.001), reaching a steady-state value after 15 min. The intracellular lifetime of water was three times greater in infarcted myocardium when compared with viable myocardium (τi: 66.6 ± 115 versus 208.7 ± 72.7 ms) and accompanied a twofold increase in ECV (ECV(SSM) : 30.3 ± 11.1 versus 71.0 ± 13.1%; P < 0.001). There was a consistent significant difference in ECV values of infarcted myocardium at different timepoints between the SM and SSM, but not viable myocardium, presumably due to slower water exchange. In summary, we found a significant change in apparent ECV and water exchange in infarcted myocardium when compared with viable myocardium. This was visualized by changes in dynamic contrast enhanced curve shapes and quantified using the SSM as not only an increase in apparent ECV but also a decrease in water exchange.

Water-fat separation imaging of the heart with standard magnetic resonance bSSFP CINE imaging.

PURPOSE: To study balanced steady-state free precession CINE phase-sensitive water-fat separation imaging in four cardiac imaging planes to determine the necessary phase correction and image artifacts particular to this technique. METHODS: Ten healthy volunteers and two subjects with known heart pathologies were studied with standard balanced steady-state free precession CINE imaging. Water-only and fat-only images were calculated using sign detection of the real part of the complex image after phase correction with constant and linear terms. Phase correction values were determined using both manual and automated methods. Differences in phase correction values between imaging planes, cardiac phases, coil elements, automated image reconstruction parameters as well as artifact scores between the automated and manual methods were studied with statistical tests. RESULTS: Water-fat separation performed well in the heart after constant and linear phase correction. Both constant (p = 0.8) and linear x (p = 1) and y (p = 1) phase correction values did not vary significantly across cardiac phases, but varied significantly among the coils (p < 0.001) and imaging planes (p < 0.001). False water-fat separation artifacts were most frequent in the chest/back and also were present at the mitral and aortic valves. CONCLUSION: Constant and linear phase correction is necessary to provide consistent results in standard imaging planes using a balanced steady-state free precession water-fat separation postprocessing algorithm applied to standard cardiac CINE imaging.

Magnetic resonance susceptibility weighted phase imaging for the assessment of reperfusion intramyocardial hemorrhage.

PURPOSE: To quantitatively investigate myocardial phase in multiple-gradient-echo images to determine normal phase angle ranges as a function of echo time and anatomical position and then compare phase values from patients with myocardial infarction with those normal ranges to determine the feasibility of intramyocardial hemorrhage detection. METHODS: Fifteen normal control and 11 patients with reperfused myocardial infarction participated in this prospective study. A 1.5 T magnetic resonance system was used to perform volumetric CINE, high-pass filtered (HPF) phase, T2-weighted, T2*-weighted and late gadolinium-enhanced infarct imaging at four times points after myocardial infarction. HPF-phase analyzed using a 16-segment model was compared with late gadolinium-enhanced infarct imaging and T2* measurements. RESULTS: Myocardial HPF-phase angle in the normal control group was small (-0.008 ± 0.027 radians). There was a difference between anatomical segments, with less variation in septal segments compared with cyclic variations in non-septal segments. Abnormal phase was only shown in myocardial segments with transmural late gadolinium-enhanced and microvascular obstruction consistent with intramyocardial hemorrhage. There were six studies from three patients (seven segments at 3 days, five segments at follow-up) with HPF-phase outside of normal range indicative of intramyocardial hemorrhage. CONCLUSION: Myocardial HPF-phase angle is normally small and varies by anatomical myocardial segment. intramyocardial hemorrhage causes a phase decrease beyond normal variations.

Left atrial late gadolinium enhancement with water-fat separation: the importance of phase-encoding order.

PURPOSE: To compare two late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) methods: a Dixon LGE sequence with sequential phase-encoding order, reconstructed using water-fat separation, and standard fat-saturated LGE. MATERIALS AND METHODS: We implemented a dual-echo Dixon LGE method for reconstructing water-only images and compared it to fat-saturated LGE in 12 patients prior to their first pulmonary vein isolation (PVI) procedure. Images were analyzed for quality and fat-suppression. Regions of the left atrium were evaluated by a blinded observer (1 = prominent enhancement, 0 = mild or absent enhancement) on two sets of images (fat-saturated and water-only LGE) and agreement was assessed. RESULTS: Water-only LGE showed a trend toward better fat-suppression (P = 0.06), with a significantly more homogeneous blood pool signal and reduced inflow artifacts (both P < 0.01). Agreement between fat-saturated LGE and water-only methods was found in 84% of regions, significantly correlated by chi-squared test (P < 0.001). The kappa value was 0.52 (moderate). The average number of enhancing segments was higher for fat-saturated LGE than water-only LGE (4.2 ± 2.7 vs. 3.2 ± 2.9, P = 0.03). CONCLUSION: The two-point Dixon LGE technique reduces artifacts due to a centric k-space order. A similar enhancement pattern was observed irrespective of the LGE technique, with more enhancement detected by fat-saturated LGE.

Does embryo biopsy, independent of vitrification, impact perinatal outcomes? An analysis of perinatal outcomes following preimplantation genetic testing biopsy in fresh and frozen embryo transfer cycles.

Objective: To compare neonatal outcomes in pregnancies resulting from embryos that have undergone preimplantation genetic testing (PGT) biopsy compared with no biopsy in both fresh and frozen embryo transfers (ETs) and determine whether findings are mediated by multiple births. Design: Retrospective cohort study. Setting: Society of Assisted Reproductive Technologies-Clinical Outcomes Reporting System data, 2014-2015. Patients: Autologous in vitro fertilization treatment cycles using fresh or frozen blastocyst ET, with or without PGT biopsy. Interventions: Not applicable. Main Outcome Measures: Large for gestational age (LGA), small for gestational age, and preterm delivery. Secondary outcomes included high birthweight, low birthweight, and clinical pregnancy measures. Outcomes were evaluated using log-binomial regression models with repeated measures. Models were used to estimate the controlled direct effects of biopsy on birth outcomes that were not mediated by multiple gestations. Results: In fresh ET, biopsy was associated with an increase in LGA (relative risk [RR] 1.45, confidence interval [CI] 1.04-2.02) that persisted in the model mediated for multiple gestation (RR 1.36, 95% CI 1.01-1.83) but was not present in an analysis restricted to elective single ET (RR 0.99, 95% CI 0.91-1.09). In frozen ET, there were no differences in any of the primary outcomes after accounting for multiple gestations. Conclusions: In a large multicenter database, there were no differences in neonatal outcomes after PGT biopsy in frozen ET cycles, and an increase in LGA was noted in fresh transfers that persisted even after accounting for multiple gestations but was not present in analysis restricted to elective single ET.

Trends in magnetic resonance and computed tomography angiography utilization among Medicare beneficiaries between 2013 and 2020.

PURPOSE: To evaluate relative and absolute utilization trends and practice patterns in the United States for MRA and CTA. METHODS: Using Medicare Part B physician payment databases (2013-2020), MRA and CTA interpreting physicians and exams were identified using the unique MRA and CTA Healthcare Common Procedure Coding System codes. The number of exams, physicians, demographics, use of contrast, and payments were summarized annually and analyzed to evaluate trends before and during the first year of the COVID-19 pandemic. RESULTS: From 2013 to 2019, the annual number of MRA exams performed decreased by 17.9 %, while the number of CTA exams increased by 90.3 %. The number of physicians interpreting MRA decreased in both hospital (-17.2 %) and outpatient (-7.5 %) environments. The number of physicians interpreting CTA increased in both hospital (+29.4 %) and outpatient (+54.3 %) environments. During the first year of the COVID-19 pandemic, MRA utilization decreased across all imaging environments by 25.0 % whereas CTA only decreased by 5.5 %. Intracranial MRA studies were most often performed without contrast, while contrast use for neck MRA was performed at similar rates as non-contrast exams. CONCLUSION: The overall utilization of MRA and the number of interpreting physicians are decreasing. On the other hand, CTA use and its number of interpreting physicians are increasing. During the first year of the COVID-19 pandemic, use of both MRA and CTA decreased, but the utilization of MRA decreased at five times the rate of CTA.

A machine learning-derived risk score to predict left ventricular diastolic dysfunction from clinical cardiovascular magnetic resonance imaging.

Introduction: The evaluation of left ventricular diastolic dysfunction (LVDD) by clinical cardiac magnetic resonance (CMR) remains a challenge. We aimed to train and evaluate a machine-learning (ML) algorithm for the assessment of LVDD by clinical CMR variables and to investigate its prognostic value for predicting hospitalized heart failure and all-cause mortality. Methods: LVDD was characterized by echocardiography following the ASE guidelines. Eight demographic and nineteen common clinical CMR variables including delayed enhancement were used to train Random Forest models with a Bayesian optimizer. The model was evaluated using bootstrap and five-fold cross-validation. Area under the ROC curve (AUC) was utilized to evaluate the model performance. An ML risk score was used to stratify the risk of heart failure hospitalization and all-cause mortality. Results: A total of 606 consecutive patients underwent CMR and echocardiography within 7 days for cardiovascular disease evaluation. LVDD was present in 303 subjects by echocardiography. The performance of the ML algorithm was good using the CMR variables alone with an AUC of 0.868 (95% CI: 0.811-0.917), which was improved by combining with demographic data yielding an AUC 0.895 (95% CI: 0.845-0.939). The algorithm performed well in an independent validation cohort with AUC 0.810 (0.731-0.874). Subjects with higher ML scores (>0.4121) were associated with increased adjusted hazard ratio for a composite outcome than subjects with lower ML scores (1.72, 95% confidence interval 1.09-2.71). Discussion: An ML algorithm using variables derived from clinical CMR is effective in identifying patients with LVDD and providing prognostication for adverse clinical outcomes.

Severe teratozoospermia and its influence on pronuclear morphology, embryonic cleavage and compaction.

BACKGROUND: Fertilization, cell division and embryo development depend on genomic contributions from male and female gametes. We hypothesize that teratozoospermic sperm influences early embryo development and embryo compaction. METHODS: We conducted a retrospective analysis of embryos derived from intracytoplasmic sperm injection (ICSI) cycles. Two hundred thirty-five consecutive ICSI cycles were included in the study; all treatment was provided at the Cleveland Clinic Fertility Center. Patient cycles were divided by sperm morphology based on Kruger's strict criteria: Group A, embryos where teratozoospermic sperm (0-2% normal) were used for ICSI and Group B, embryos where dysmorphic sperm (5-13% normal) were used for ICSI. All cycles analyzed were of patients doing day 3 embryo transfers. Outcome measures assessed included pronuclear (PN) pattern, syngamy, early cleavage, cell number, rate of compaction and blastulation of embryos left in culture and not transferred on day 3. RESULTS: A total of 1762 embryos were analyzed. PN patterns were similar in Group A and Group B embryos. No differences were noted in syngamy, cleavage, cell number or blastulation rate. Studying the development of embryos in culture after day 3 transfer revealed a difference in the timeline for compaction. By day 4, 25% of Group A embryos had compacted compared to 36% in Group B (P = 0.0007). There was no difference found between Group A and Group B embryos in regards to blastulation. CONCLUSIONS: We did not find an association between sperm morphology and clinical outcomes. The impact of teratozoospermia may be masked in ICSI cycles where fertilization, implantation rate and clinical pregnancy rate are the primary outcome measures. However, by examining the timeline of development, we were better able to discern a potential paternal effect at critical transition points from fertilization through activation.

Cyclic CINE-balanced steady-state free precession image intensity variations: implications for the detection of myocardial edema.

PURPOSE: To evaluate the dependence of CINE-balanced steady-state free precession (bSSFP) image intensities on spatial location, cardiac phase, and disease state. MATERIALS AND METHODS: Eight subjects with recent myocardial infarctions and eight age- and sex-matched normal volunteers were studied using CINE-bSSFP imaging to describe cyclic image intensity variations as a function of the cardiac cycle and to optimize and assess the ability of CINE-bSSFP imaging to depict myocardial edema. Signal intensities of the left ventricular (LV) bloodpool and myocardium were measured using region-of-interest analysis across the cardiac cycle. The magnitude and time course of the cyclic variations were evaluated. Mixed-model analysis of variance was used to examine the influence of physical location, cardiac phase, and presence of myocardial infarction. RESULTS: The LV bloodpool and myocardial CINE-bSSFP signal intensities varied significantly with spatial location, cardiac phase, and disease (P < 0.001). Cardiac phase had a significant effect on the signal intensities after adjustments for spatial location. The LV bloodpool signal decreased slowly during systole and rose sharply during LV filling. There were two distinct myocardial intensity peaks, one occurring at peak systole and the other at the end of the LV rapid inflow phase. Myocardial edema was seen as a hyperintense region. Image contrast with adjacent myocardium was the greatest at the end of systole. CONCLUSION: Detection of myocardial edema using the conventional CINE-bSSFP technique is feasible, but is complicated by normal cyclic changes in myocardial image intensities during the cardiac cycle.

Mouse ovarian follicle cryopreservation using vitrification or slow programmed cooling: assessment of in vitro development, maturation, ultra-structure and meiotic spindle organization.

AIM: To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and to evaluate different cryo-devices for vitrification of isolated follicles. METHODS: Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slow freezing. A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolated follicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n=100), nylon mesh (n=96), electron microscopy grid (n=102), and micro-capillary tips (n=116). Subsequently, a total of 979 follicles were randomly assigned to three different groups: CT fresh (n=256), vitrification (n=399) and slow freezing (n=324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily for development. Final maturation was triggered with human chorionic gonadotrophin and rates of oocyte maturation were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electron microscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosight imaging system. RESULTS: Micro-capillary tips resulted in poor immediate post-warming survival but no differences were observed in the subsequent in vitro development characteristics between different cryo-devices. Nylon mesh proved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared to vitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the culture period (P<0.0001). However all other outcome measures were comparable between both techniques. CONCLUSIONS: Isolated follicles were more vulnerable to cryodamage after slow freezing as compared to vitrification.

Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential?

PURPOSE: Vitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable ESC line. This study tests the feasibility of cryopreserving ICM cells using vitrification. MATERIALS AND METHODS: ICMs from mouse embryos were isolated and vitrified in HSV straws or on cryoloops. Upon warming, the vitrified ICMs were cultured and observed for attachment and morphology. Colonies were passaged every 3-6 days. ICMs and ICM-derived ESC colonies were tested for expression of stem cell specific markers. RESULTS: ICMs vitrified on both the cryoloop and the HSV straw had high survival rates. ICM derived ESCs remained undifferentiated for several passages and demonstrated expression of typical stem cell markers; SSEA-1, Sox-2, Oct 4 and alkaline phosphatase. CONCLUSION: This is the first report on successful vitrification of isolated ICMs and the subsequent derivation of ESC colonies. Vitrification of isolated ICMs is a novel approach for preservation of the "stem cell source" material.

Trends in Cardiovascular MRI and CT in the U.S. Medicare Population from 2012 to 2017.

PURPOSE: To assess the characteristics and trends of cardiovascular MRI and CT practitioners and practice in the United States. MATERIALS AND METHODS: A retrospective cross-sectional analysis of 2012-2017 Medicare Part B physician payments from the Provider Utilization and Payment Data Physician and Other Supplier Public Use Files (POSPUF) was performed. Characteristics of cardiovascular MRI and CT, including the number of providers and examinations, provider sex and location, and physician reimbursement were analyzed. Variable means, standard deviations, and changes per year were reported and compared. RESULTS: In 2017, 582 physicians provided cardiovascular MRI services in 45 states, a 16.6% increase from 2016 and an 84.8% increase from 2012. A total of 1645 physicians provided cardiovascular CT services in 49 states, a 14.2% increase from 2016 and a 77.3% increase from 2012. Of the providers, 18.0% and 13.3% of cardiovascular MRI and CT providers were women, respectively, similar to providers' respective medical specialties. Only 1.0% of radiologists and 0.2% of cardiologists provided cardiovascular MRI services. A total of 3.2% of radiologists and 0.5% of cardiologists provided cardiovascular CT services. Both cardiovascular MRI use (+75.5%) and cardiovascular CT use (+97.4%) increased markedly over the 6-year study period. CONCLUSION: Although the availability of cardiovascular MRI and CT is increasing, both are used less frequently in comparison with other cardiovascular imaging modalities.See also the commentary by Bierhals in this issue.Supplemental material is available for this article.© RSNA, 2021.

Magnetic resonance separation imaging using a divided inversion recovery technique (DIRT).

The divided inversion recovery technique is an MRI separation method based on tissue T(1) relaxation differences. When tissue T(1) relaxation times are longer than the time between inversion pulses in a segmented inversion recovery pulse sequence, longitudinal magnetization does not pass through the null point. Prior to additional inversion pulses, longitudinal magnetization may have an opposite polarity. Spatial displacement of tissues in inversion recovery balanced steady-state free-precession imaging has been shown to be due to this magnetization phase change resulting from incomplete magnetization recovery. In this paper, it is shown how this phase change can be used to provide image separation. A pulse sequence parameter, the time between inversion pulses (T180), can be adjusted to provide water-fat or fluid separation. Example water-fat and fluid separation images of the head, heart, and abdomen are presented. The water-fat separation performance was investigated by comparing image intensities in short-axis divided inversion recovery technique images of the heart. Fat, blood, and fluid signal was suppressed to the background noise level. Additionally, the separation performance was not affected by main magnetic field inhomogeneities.

Three-dimensional in vitro follicle growth: overview of culture models, biomaterials, design parameters and future directions.

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.

Clinical pregnancy and live births after transfer of embryos vitrified on day 3.

Human embryo vitrification is a promising new technology but clinical outcome data is needed to gauge its effectiveness and safety. While pregnancy and live-birth data is available for blastocyst vitrification, such information is lacking for human embryo vitrification at the 6- to 8-cell stage. The current work presents clinical and obstetric outcomes from the transfer of embryos vitrified on day 3 at the cleavage stage. A total of 270 transfers were performed. The clinical pregnancy and implantation rates for patients under 38 years of age (n=200) were 45% and 24%, respectively. Corresponding rates in patients 38-42 years old declined to 29% and 13% (n=70). Embryonic compaction and/or blastulation by the time of transfer were excellent prognostic indicators of a successful pregnancy outcome. Of the 66 deliveries, 12 (18.2%) were twin pregnancies and nine were preterm (13.6%). The mean birthweight for singletons was 3281+/-644 g, compared with 2506+/-549 g in the twin pregnancies. A total of 78 infants have been born with no major congenital malformations. These data attest to the efficacy and safety of the vitrification technique for cryopreservation of human embryos at the 6- to 8-cell stage.

Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis.

Embryo cryopreservation is an important aspect of assisted reproduction. Many methods have been described, but they have been poorly investigated in randomized trials, highlighting the need for a systematic review of the literature. Meticulous electronic/hand searches were performed to locate randomized trials (RCT) comparing embryo cryopreservation methods. Primary outcomes were clinical pregnancy rate (CPR) and incidence of congenital abnormalities. Secondary outcomes included live-birth (LBR), ongoing pregnancy (OPR), implantation (IR), and miscarriage (MR) rates. Data were extracted to allow for an intention-to-treat analysis and analysed using a random-effects model. Literature search revealed 11 RCT, of which five were excluded. The quality of the included studies was variable, but generally poor. There was a significantly higher CPR, OPR and IR with vitrification compared with slow freezing (odds ratio (OR)=1.55, 95% confidence interval (CI)=1.03-2.32, OR=1.82, 95% CI=1.04-3.20 and OR=1.49, 95% CI=1.03-2.15, respectively). In addition, there was a significantly lower CPR and OPR with embryo ultra-rapid freezing compared with slow freezing (OR=0.35, 95% CI=0.16-0.76 and OR=0.37, 95% CI=0.17-0.81, respectively). Vitrification is superior to slow freezing, which in turn is superior to ultra-rapid freezing. However, more well-designed and powered studies are needed to further corroborate these findings.

A simple and efficient method for preparation of isolated ovarian follicles for transmission electron microscopy.

OBJECTIVE: A simple method for preparation of isolated ovarian follicles for transmission electron microscopy (TEM) using transwell inserts is described. MATERIALS AND METHODS: Pre-antral follicles were enzymatically isolated from mouse ovaries and cultured overnight on transwell insert polyester membranes. The following day, isolated ovarian follicles were processed for TEM by moving the transwell insert through successive wells containing the fixation and embedding reagents. After polymerization of the resin, the polyester membrane with the follicles embedded in the resin was disengaged from the transwell unit. The resin was sectioned. Semi-thin sections were stained with toluidine blue while ultra-thin sections were stained by uranyl acetate and examined by light microscopy and TEM, respectively. RESULTS: Isolated ovarian follicles were easily processed in groups for TEM. Follicles were well embedded and there appeared to be no loss of tissue during processing. The ultra-structure of processed isolated ovarian follicles was well preserved with little evidence of processing artifacts. CONCLUSIONS: In situ processing and preparation of isolated ovarian follicles by first allowing attachment on transwell insert membranes was shown to be a simple, rapid and effective method for TEM.