Does dual oocyte retrieval with continuous FSH administration increase the number of mature oocytes in low responders? An open-label randomized controlled trial.

STUDY QUESTION: Is there an increase in the total number of metaphase II (MII) oocytes between a conventional ovarian stimulation (OS) and a double uninterrupted stimulation? SUMMARY ANSWER: There is no increase in the total number of MII oocytes when comparing one conventional OS to a continuous stimulation with double oocyte aspiration. WHAT IS KNOWN ALREADY: Based on the concept of multiple follicular waves, the combination of two stimulations in the same ovarian cycle has gained interest in patients with a low ovarian reserve. This so-called dual stimulation approach is usually characterized by a discontinuation of FSH administration for ∼5 days and appears to have a favourable impact on the number of retrieved oocytes without affecting the embryo quality or ploidy status. The outcomes of dual uninterrupted OS have not yet been studied. STUDY DESIGN, SIZE, DURATION: This was an open-label randomized controlled trial (RCT) with superiority design, performed in a single tertiary centre. Subjects were randomized with a 1:1 allocation into two groups between October 2019 and September 2021. All patients underwent a conventional stimulation with recombinant FSH. When two or more follicles of 17 mm were present, the final inclusion criterion was assessed; randomization occurred only in the presence of ≤9 follicles of ≥11 mm. In Group A, ovulation was triggered with hCG, and oocyte retrieval (OR) was performed 34-36 h later, followed by a fresh single or double embryo transfer (SET or DET) on Day 3/5. In Group B, ovulation was triggered with GnRH agonist, followed by another OS, without discontinuation of the FSH administration. In the presence of one or more follicles of ≥17 mm, the second stimulation was completed with hCG. A freeze-all strategy (Day 3/5) was applied for both retrievals, followed by transfer of one or two embryos in an artificially prepared frozen-thawed cycle. In the absence of one or more follicles of ≥17 mm after 13 additional days of stimulation, the second cycle was cancelled. All ORs were executed by a senior fertility specialist who was blinded for the first treatment, and all follicles >10 mm were aspirated, according to routine clinical practice. The primary outcome was the total number of MII oocytes. Patients were followed up until all embryos were transferred, or until live birth was achieved. Other secondary outcomes included the number of cumulus-oocyte complexes (COCs), the number of good quality embryos (Day 3/5), the ongoing pregnancy rate, and gonadotropin consumption. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients between 25 and 40 years old, with an anti-Müllerian hormone level of ≤1.5 ng/ml, antral follicle count of ≤6, or ≤5 oocytes after a previous stimulation, were included. At the start, 70 patients were eligible for participation in the trial, of whom 48 patients fulfilled the final inclusion criterium and were randomized. After drop-out of two patients, 23 patients were randomized to a single round of OS (Group A), and 23 patients were randomized to two uninterrupted rounds of OS (Group B). MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were similar between both groups. The cumulative number of COCs and MII oocytes after completion of the second OR was similar in Group A and Group B [5.3 ± 2.7 versus 5.3 ± 3.0 (P = 0.95); 4.1 ± 2.4 versus 4.3 ± 2.7 (P = 0.77)]. Likewise, a comparable number of excellent and good quality embryos was available on Day 3 (3.0 ± 2.0 versus 2.7 ± 2.0; P = 0.63). In Group B, the cancellation rate due to insufficient response to the second round of stimulation was 39.1% (9/23). When focusing on the first stimulation in both groups, there were no significant differences regarding basal FSH, gonadotropin consumption, and the number of preovulatory follicles. After the first OR, the mean number of COC and MII oocytes was significantly higher in Group A (who had hCG triggering), compared to Group B (who had GnRH agonist triggering) [5.3 ± 2.7 versus 3.3 ± 2.2; difference 95% CI (0.54 to 3.45), P = 0.004 and 4.1 ± 2.4 versus 3.0 ± 2.2; difference 95% CI (-0.15 to 2.6), P = 0.05, respectively]. Likewise, the number of excellent and good quality embryos on Day 3 was significantly higher (3.0 ± 2.0 versus 1.9 ± 1.7; P = 0.02) in Group A. LIMITATIONS, REASONS FOR CAUTION: This study was powered to demonstrate superiority for the number of MII oocytes after dual stimulation. Investigating the impact of dual stimulation on pregnancy rates would have required a larger sample size. Furthermore, the heterogeneity in embryo vitrification and transfer policies precluded a correct comparison of embryologic outcomes between both groups. WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT investigating the role of continuous stimulation with double aspiration in low responders. Our results show no statistically significant differences in the cumulative number of MII oocytes between one conventional stimulation with fresh ET and two consecutive stimulations with a freeze-only approach. Furthermore, the observed suboptimal oocyte yield after agonist ovulation triggering in low responders in the dual uninterrupted OS group is a reason for concern and further scrutiny, given that previous RCTs have shown similar outcomes in normal and high responders after hCG and GnRH agonist triggers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by a research grant from Organon. H.T. received honoraria for lectures and presentations from Abbott, Cooper Surgical, Gedeon-Richter, Cook, Goodlife, and Ferring. L.B. received fees for lectures from Merck & Organon and support for attending ESHRE 2023. M.D.V. reports fees for lectures from Ferring, Merck, Organon, IBSA, Gedeon Richter, and Cooper Surgical and support for attending ASRM 2023. S.M. received honoraria for lectures and presentations from Abbott, Cooper Surgical, Gedeon-Richter, IBSA, and Merck. C.B. was on the Advisory board and received consulting fees from Theramex and received honoraria for lectures and presentations from Abbott, Ferring, Gedeon-Richter, IBSA, and Merck. TRIAL REGISTRATION NUMBER: NCT03846544. TRIAL REGISTRATION DATE: 19 February 2019. DATE OF FIRST PATIENT'S ENROLMENT: 28 October 2019.

The WOEST 2 registry : A prospective registry on antithrombotic therapy in atrial fibrillation patients undergoing percutaneous coronary intervention.

BACKGROUND: Patients on oral anticoagulants (OACs) undergoing percutaneous coronary intervention (PCI) also require aspirin and a P2Y12 inhibitor (triple therapy). However, triple therapy increases bleeding. The use of non-vitamin K antagonist oral anticoagulants (NOACs) and stronger P2Y12 inhibitors has increased. The aim of our study was to gain insight into antithrombotic management over time. METHODS: A prospective cohort study of patients on OACs for atrial fibrillation or a mechanical heart valve undergoing PCI was performed. Thrombotic outcomes were myocardial infarction, stroke, target-vessel revascularisation and all-cause mortality. Bleeding outcome was any bleeding. We report the 30-day outcome. RESULTS: The mean age of the 758 patients was 73.5 ± 8.2 years. The CHA2DS2-VASc score was ≥ 3 in 82% and the HAS-BLED score ≥ 3 in 44%. At discharge, 47% were on vitamin K antagonists (VKAs), 52% on NOACs, 43% on triple therapy and 54% on dual therapy. Treatment with a NOAC plus clopidogrel increased from 14% in 2014 to 67% in 2019. The rate of thrombotic (4.5% vs 2.0%, p = 0.06) and bleeding (17% vs. 14%, p = 0.42) events was not significantly different in patients on VKAs versus NOACs. Also, the rate of thrombotic (2.9% vs 3.4%, p = 0.83) and bleeding (18% vs 14%, p = 0.26) events did not differ significantly between patients on triple versus dual therapy. CONCLUSIONS: Patients on combined oral anticoagulation and antiplatelet therapy undergoing PCI are elderly and have both a high bleeding and ischaemic risk. Over time, a NOAC plus clopidogrel became the preferred treatment. The rate of thrombotic and bleeding events was not significantly different between patients on triple or dual therapy or between those on VKAs versus NOACs.

Cumulus-corona gene expression analysis combined with morphological embryo scoring in single embryo transfer cycles increases live birth after fresh transfer and decreases time to pregnancy.

PURPOSE: Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies. METHODS: A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and ß2M). Patients (n = 62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes. RESULTS: Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p < 0.001) and day 5 (43% and 39%: p = 0.022 and p = 0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3. CONCLUSION: Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy. TRIAL REGISTRATION NUMBER: This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).

A contemporary look at pericardiocentesis.

Percutaneous drainage is the default strategy for evacuating a pericardial effusion. A pericardiocentesis can be necessary or required in a wide variety of clinical settings ranging from urgent tamponade to relieve in iatrogenic hemorrhagic effusions in the electrophysiology or catheterization room, to planned diagnostic procedures in patients with suspected or known malignancy or infections. With the help of several procedural improvements over the past decades, echocardiography and fluoroscopy-guided percutaneous pericardiocentesis has become the standard intervention for evacuating pericardial effusions, as well as an essential tool in the diagnostic work-up of an unexplained pericardial effusion. When performed by skilled physicians assisted by appropriate imaging it is a very safe procedure, and provided that an indwelling catheter is placed, it is also very effective with an acceptably low risk of recurrences. In this review, the indications and standard techniques for pericardiocentesis are discussed, as well as their consequences for patients with iatrogenic and malignant effusions.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

STUDY QUESTION: What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? SUMMARY ANSWER: Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. WHAT IS KNOWN ALREADY: Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. STUDY DESIGN, SIZE, DURATION: Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. STUDY FUNDING AND COMPETING INTEREST(S): A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

5-Year clinical follow-up of the COBRA (complex coronary bifurcation lesions: Randomized comparison of a strategy using a dedicated self-expanding biolimus A9-eluting stent vs. a culotte strategy using everolimus-eluting stents) study.

OBJECTIVES: We evaluated healing responses with optical coherence tomography, and long-term clinical outcomes after treatment with a dedicated stent versus a conventional culotte technique. BACKGROUND: Dedicated bifurcation stents have been proposed as an alternative treatment for coronary bifurcation lesions. The long-term performance of dedicated stents versus conventional dual-stent techniques for the treatment of complex coronary bifurcation lesions is unknown. METHODS: Forty patients with true coronary bifurcation lesions were randomized to treatment with a dedicated Axxess bifurcation stent in the proximal main vessel and additional Biomatrix stents in branches versus culotte stenting using Xience stents. RESULTS: The percentage of uncovered struts in each bifurcation segment at 9 months (primary endpoint) was similar between groups. Five-year clinical follow-up was available for all patients and included major adverse cardiac events [MACE; a composite of cardiac death, myocardial infarction (MI) and ischemia-driven target lesion revascularization (TLR)], target-vessel (TVR) and non-target-vessel revascularization (non-TVR), non-TLR and stent thrombosis. At 5 years, in the culotte group, one patient had undergone TLR and another suffered a clinical MI, resulting in 10% MACE versus none in the Axxess group. TVR (5% vs. 10%, P = 0.54) and non-TVR (5% vs. 20%, P = 0.39) rates were similar between the Axxess and culotte groups, respectively. There was no stent thrombosis. CONCLUSION: Compared with culotte stenting with Xience, complex coronary bifurcation stenting using a dedicated strategy combining the Axxess and Biomatrix stents results in similar stent strut coverage at 9 months, and excellent clinical outcomes at 5 years.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

STUDY QUESTION: Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? SUMMARY ANSWER: The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. WHAT IS KNOWN ALREADY: ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. STUDY DESIGN, SIZE, DURATION: This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte. WIDER IMPLICATIONS OF THE FINDINGS: Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Complex bifurcation lesions: Randomized comparison of a fully bioresorbable modified t stenting strategy versus bifurcation reconstruction with a dedicated self-expanding stent in combination with bioresorbable scaffolds, an OCT study: Rationale and design of the COBRA II trial.

OBJECTIVE: There is an ongoing controversy regarding the efficacy and safety of different percutaneous stenting techniques for coronary bifurcation lesions needing >1 stent. The promise of safe vessel restoration with bioresorbable scaffolds (BRS) may not be transferable to complex double BRS bifurcation techniques, and permanent metallic scaffolding of the bifurcation core may be needed. We identified modified-T stenting as the most promising fully bioresorbable 2-stent strategy in a preclinical setting. The objective of this study is to assess acute performance and compare long-term vessel healing with this strategy, versus an approach combining BRS with a dedicated metallic drug-eluting bifurcation stent. STUDY DESIGN: In a single center, 60 consecutive patients with true and complex coronary bifurcation lesions will be randomly assigned to treatment with the dedicated self-expanding Axxess™ biolimus-eluting bifurcation stent in the proximal main vessel and additional Absorb™ everolimus-eluting BRS in the branches versus a modified T technique using Absorb™ only. Angiography and optical coherence tomography (OCT) will be performed immediately after implantation and at 30 months, and clinical follow-up is foreseen up to 5 years after implantation. The primary endpoint is the change in minimal luminal area assessed with OCT from baseline to 30 months in pre-specified bifurcation segments. CONCLUSION: To date the use of Absorb™ BRS in complex coronary bifurcations has not been evaluated in a randomized clinical trial setting. The COBRA II study will examine the role and safety of a double BRS strategy in coronary bifurcations, alone or in combination with a metallic dedicated bifurcation device. © 2016 Wiley Periodicals, Inc.

T-Wave Alternans Is Linked to Microvascular Obstruction and to Recurrent Coronary Ischemia After Myocardial Infarction.

The purpose of this study is to investigate the relationship between T-wave alternans (TWA), infarct size and microvascular obstruction (MVO) and recurrent cardiac morbidity after ST elevation myocardial infarction (STEMI). One hundred six patients underwent TWA testing 1-12 months and 57 patients underwent cardiac magnetic resonance imaging (MRI) in the first 2-4 days after STEMI. During follow-up (3.5 ± 0.5 years), death (n = 2), ventricular tachycardia (n = 3), supraventricular tachycardia (n = 4), heart failure (n = 3) and recurrent coronary ischemia (n = 25) were observed. After multivariate analysis, positive TWA (HR2.59, CI1.10-6.11, p0.024) and larger MVO (HR1.08, CI1.01-1.16, p0.034) were associated with recurrent angina or ACS. Presence of MVO was correlated with TWA (Spearman rho 0.404, p0.002) and the impairment of LVEF (-0.524, p < 0.001). Patients after STEMI remain at a high risk of symptoms of coronary ischemia. The presence of MVO and TWA 1-12 months after STEMI is related to each other and to recurrent angina or ACS.

Human antral follicles <6 mm: a comparison between in vivo maturation and in vitro maturation in non-hCG primed cycles using cumulus cell gene expression.

Within the context of an oocyte in vitro maturation (IVM) program for reproductive treatment, oocyte cumulus complexes (COCs) derived from follicles <6 mm in patients with PCOS were matured in vitro. Key transcripts related to meiotic maturation (FSHR, LHCGR, EGFR, PGR) and oocyte competence (AREG, ADAMTS, HAS2, PTGS2) were quantified in cumulus cells (CCs) before and after maturation. Control CC samples were collected from PCOS and normo-ovulatory patients who had undergone conventional gonadotrophin stimulation for IVF/ICSI. Additional control samples from a non-stimulated condition were obtained ex vivo from patients undergoing ovariectomy for fertility preservation. Expression data from CCs from follicles with a diameter of <6 mm before (IVM-CCs) and after in vitro maturation (IVM-CCs) were obtained after pooling CCs into four groups in relation to the percentage of matured (MII) oocytes obtained after 40 h of IVM (0; 40-60; 61-80; 100% MII) and values were compared with in vivo matured controls (IVO-CCs). Genes encoding key receptors mediating meiotic resumption are expressed in human antral follicles of <6 mm before and after IVM. The expression levels of FSHR, EGFR and PGR in CCs were significantly down-regulated in the IVO-CCs groups and in the 100% MII IVM group compared with the BM groups; all the receptors studied in the 100% MII IVM group reached an expression profile similar to that of IVO-CCs. However, after maturation in a conventional IVF/ICSI cycle, IVO-CCs from large follicles contained significantly increased levels of ADAMTS1, AREG, HAS2 and PTGS2 compared with IVM-CCs and IVM-CCs; the expression patterns for these genes in all IVM-CCs were unchanged compared with IVM-CCs. In conclusion, genes encoding receptors involved in oocyte meiotic resumption appeared to be expressed in CCs of small human antral follicles. Expression levels of genes-encoding factors reflecting oocyte competence were significantly altered in IVM-CCs compared with in vivo matured oocytes from large follicles. Observed differences might be explained by the different stimulation protocols, doses of gonadotrophin or by the intrinsic differences between in vivo and in vitro maturation.

Automatic segmentation of in-vivo intra-coronary optical coherence tomography images to assess stent strut apposition and coverage.

The implantation of intracoronary stents is currently the standard approach for the treatment of coronary atherosclerotic disease. The widespread adoption of this technology has boosted an intensive research activity in this domain, with continuous improvements in the design of these devices, aiming at reducing problems of restenosis (re-narrowing of the stented segment) and thrombosis (sudden occlusion due to thrombus formation). Recently, a new, light-based intracoronary imaging modality, optical coherence tomography (OCT), was developed and introduced into clinical practice. Due to its very high axial resolution (10-15 µm), it allows for in vivo evaluation of both stent strut apposition and neointima coverage (a marker of healing of the treated segment). As such, it provides valuable information on proper stent deployment, on the behaviour of different stent types in-vivo and on the effect of new types of stents (e.g. drug-eluting stents) on vessel wall healing. However, the major drawback of the current OCT methodology is that analysis of these images requires a tremendous amount of-currently manual-post-processing. In this manuscript, an algorithm is presented that allows for fully automated analysis of stent strut apposition and coverage in coronary arteries. The vessel lumen and stent struts are automatically detected and segmented through analysis of the intensity profiles of the A-lines. From these data, apposition and coverage can then be measured automatically. The algorithm was validated using manual assessments by two experienced operators as a reference. High Pearson's correlation coefficients were found (R = 0.96-0.97) between the automated and manual measurements while Bland-Altman analysis showed no significant bias with good limits of agreement. As such, it was shown that the presented algorithm provides a robust and fast tool to automatically estimate apposition and coverage of stent struts in in-vivo OCT pullbacks. This will be important for the integration of this technology in clinical routine and for the analysis of datasets of larger clinical trials.

Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients.

BACKGROUND: Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model. METHODS: From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed. RESULTS: Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%. CONCLUSIONS: Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics.

BACKGROUND: Gene expression of cumulus cells (CC) could predict oocyte developmental quality. Knowledge of the genes involved in determining oocyte quality is scanty. The aim was to correlate clinical and biological characteristics during ovarian stimulation with the expression of 10 selected genes in CC. METHODS: Sixty-three ICSI patients were stimulated with GnRH-agonist plus highly purified hMG (n = 35) or recombinant FSH (n = 28). Thirteen variables were analyzed: Age, BMI, duration of stimulation, serum concentrations of progesterone, 17beta-estradiol, FSH and LH on day of hCG, Ovarian Response, Oocyte Maturity, 2 pronuclei and three embryo morphology related variables: > or =7 cells, Low Fragmentation, Good Quality Embryos score. Expression of HAS2, VCAN, SDC4, ALCAM, GREM1, PTGS1, PTGS2, DUSP16, SPROUTY4 and RPS6KA2 was analyzed in pooled CC using quantitative PCR, and the relationship to the 13 variables was evaluated by multivariable analysis. RESULTS: All 10 genes are expressed at oocyte retrieval, with PTGS1, SPROUTY4, DUSP16 and RPS6KA2 described in human ovary for the first time. The three variables that correlated most often with differential expression were Age, BMI and serum FSH level. Significant correlation was found with Oocyte Maturity (VCAN, P < 0.005), Low Fragmentation (RPS6KA2, P < 0.05), Embryos with > or =7 cells (ALCAM and GREM1, P < 0.05). The expression of the other genes was also correlated to oocyte developmental quality but to a less extent. SDC4, VCAN, GREM1, SPROUTY4 and RPS6KA2 showed gonadotrophin preparation-dependent expression and/or interactions (all P < 0.05). CONCLUSION: The expression of ovulation related genes in CC is associated with patient and treatment characteristics, oocyte developmental potential and differs with the type of gonadotrophin used.

Expression of growth differentiation factor 9, bone morphogenetic protein 15, and anti-Müllerian hormone in cultured mouse primary follicles.

Growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and anti-Müllerian hormone (AMH) play an important role in the primary to secondary follicle transition and follicle activation in vivo. In organ culture of neonatal mouse ovaries, it was observed that significantly fewer primary follicles develop to the secondary stage. The objectives of this study were: (1) to compare ovarian follicular populations between organ-cultured neonatal mouse ovaries and freshly isolated age-matched control ovaries; (2) to quantify RNA levels of Gdf9, Bmp15, and Amh in cultured primary follicles; and (3) to immunolocalize GDF9 and AMH in cultured ovaries. Ovaries from 3-day-old (PND 3) mice were cultured for 7 or 10 days in the absence or presence of FSH. Follicular populations were counted in freshly isolated 13-day-old (PND 13) ovaries and organ-cultured ovaries. Transcripts were quantified in isolated primary follicles using real-time RT-PCR, and protein expressions were localized using immunohistochemistry. The number of secondary follicles in organ-cultured ovaries was significantly lower than in vivo controls. Gdf9 and Bmp15 mRNA expression levels were similar as in controls. Amh mRNA levels were significantly (P<0.05) lower after day 10 of culture in the absence of FSH. GDF9 and AMH proteins were respectively detected in the oocytes and the granulosa cells (GC) beginning at the primary and primordial stages onward. GDF9 and BMP15 production in cultured primary follicles are not different from in vivo controls; hence abnormal early follicular growth was not related to a deficient transcription of these factors.

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

The midgestational rabbit as a model for the creation of membrane defects after needle fetoscopy.

OBJECTIVES: The aim of the study was to determine whether the pregnant rabbit at mid gestation could be used as a suitable in vivo model for the study of membrane defects after invasive procedures. STUDY DESIGN: Pregnant rabbits at gestational ages of 22 and 18 days (term is 32 days' gestation) underwent needle insertion with different instrument diameters (1.1 mm, 1.35 mm, 2.0 mm, and 2.7 mm). Two different insertion techniques were evaluated, blind amniotic puncture and puncture through surgically exposed amnion. Membrane integrity, presence of amniotic fluid, and fetal lung/body weight ratio were evaluated at 31 days' gestation. RESULTS: Among rabbits operated on at 22 days' gestation the amniotic integrity restoration at 31 days' gestation ranged from 46% to 76% in the different diameter and access technique groups, as compared with 98% in untreated sacs (P <.05 for all groups). Fetuses from sacs with persisting membrane defects had oligohydramnios and significantly lower fetal lung/body weight ratios. Survival rates among fetuses operated on at 18 days' gestation were so poor that appropriate statistical analysis was not possible in this group. CONCLUSIONS: The rabbit at mid gestation can be used as a model to reproduce permanent membrane defects after fetoscopy, reproducing oligohydramnios and pulmonary hypoplasia. This may provide a suitable in vivo model for the study of iatrogenic membrane defects.