Does follicular flushing increase oocyte number in poor responders? An update of a systematic review.

RESEARCH QUESTION: Does flushing of the follicles at ovum retrieval increase the number of retrieved oocytes in poor-response IVF patients? DESIGN: An update of an electronic literature search was performed to identify randomized controlled trials (RCT) investigating follicular flushing versus no flushing in women with a poor response to IVF treatment. No exclusion criteria for type of needle, stimulation or protocol were applied. A meta-analysis was conducted using the software RevMan 5.4. RESULTS: Six RCT were identified that had the primary objective of testing for an increase in mean number of cumulus-oocyte complexes or/and metaphase II oocytes between flushing and no flushing. A double-lumen needle was used in five trials, one study investigated a pseudo-double-lumen needle, and a conventional single-lumen needle was used in all the control groups. The main risk of bias in all the included studies is a lack of blinding of the physicians performing the puncture and incomplete data in four trials. A heterogeneity of direction and size of effect of follicular flushing on mean oocyte number retrieved was detected (I2 = 80, P = 0.0001), which precludes a synthesis of the data. Two studies showed a decrease or tendency towards a decrease in oocyte numbers, one study showed similar oocyte numbers, and one study showed a strong tendency towards increased oocyte numbers with flushing. A similar picture was seen for metaphase II oocytes (I2 = 73, P = 0.002). CONCLUSIONS: It is uncertain whether follicular flushing in poor-response IVF patients affects the number of retrieved oocytes. Larger pragmatic trials are warranted to clarify the effect of flushing on oocyte numbers and clinical outcomes in poor responders and monofollicular patients.

Dydrogesterone and 20α-dihydrodydrogesterone plasma levels on day of embryo transfer and clinical outcome in an anovulatory programmed frozen-thawed embryo transfer cycle: a prospective cohort study.

STUDY QUESTION: What are the plasma concentrations of dydrogesterone (DYD) and its metabolite, 20α-dihydrodydrogesterone (DHD), measured on day of embryo transfer (ET) in programmed anovulatory frozen embryo transfer (FET) cycles using 10 mg per os ter-in-die (tid) oral DYD, and what is the association of DYD and DHD levels with ongoing pregnancy rate? SUMMARY ANSWER: DYD and DHD plasma levels reach steady state by Day 3 of intake, are strongly correlated and vary considerably between and within individual subjects, women in the lowest quarter of DYD or DHD levels on day of FET have a reduced chance of an ongoing pregnancy. WHAT IS KNOWN ALREADY: DYD is an oral, systemic alternative to vaginal progesterone for luteal phase support. The DYD and DHD level necessary to sustain implantation, when no endogenous progesterone is present, remains unknown. While DYD is widely used in fresh IVF cycles, circulating concentrations of DYD and DHD and inter- and intraindividual variation of plasma levels versus successful treatment have never been explored as measurement of DYD and DHD is currently only feasible by high-sensitivity chromatographic techniques such as liquid chromatography/tandem mass spectroscopy (LC-MS/MS). STUDY DESIGN, SIZE, DURATION: Prospective, clinical cohort study (May 2018-November 2020) (NCT03507673); university IVF-center; women (n = 217) undergoing a programmed FET cycle with 2 mg oral estradiol (tid) and, for luteal support, 10 mg oral DYD (tid); main inclusion criteria: absence of ovulatory follicle and low serum progesterone on Days 12-15 of estradiol intake; serum and plasma samples were taken on day of FET and stored at -80°C for later analysis by LC-MS/MS; in 56 patients, two or more FET cycles in the same protocol were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women undergoing FET on Day 2 or Day 3 (D2, D3, cleavage) or Day 5 (D5, blastocyst) of embryonic development had blood sampling on the 3rd, 4th or 6th day of 10 mg (tid) DYD oral intake, respectively. The patient population was stratified by DYD and DHD plasma levels by percentiles (≤25th versus >25th) separately by day of ET. Ongoing pregnancy rates (a viable pregnancy at >10th gestational week) were compared between ≤25th percentile versus >25th percentile for DYD and DHD levels (adjusted for day of ET). Known predictors of outcome were screened for their effects in addition to DYD, while DYD was considered as log-concentration or dichotomized at the lower quartile. Repeated cycles were analyzed assuming some correlation between them for a given individual, namely by generalized estimating equations for prediction and generalized mixed models for an estimate of the variance component. MAIN RESULTS AND THE ROLE OF CHANCE: After exclusion of patients with 'escape ovulation' (n = 14, 6%), detected by the presence of progesterone in serum on day of ET, and patients with no results from LC-MS/MS analysis (n = 5), n = 41 observations for cleavage stage ETs and n = 157 for blastocyst transfers were analyzed. Median (quartiles) of plasma levels of DYD and DHD were 1.36 ng/ml (0.738 to 2.17 ng/ml) and 34.0 ng/ml (19.85 to 51.65 ng/ml) on Day 2 or 3 and 1.04 ng/ml (0.707 to 1.62 ng/ml) and 30.0 ng/ml (20.8 to 43.3 ng/ml) on Day 5, respectively, suggesting that steady-state is reached already on Day 3 of intake. DHD plasma levels very weakly associated with body weight and BMI (R2 < 0.05), DYD levels with body weight, but not BMI. Levels of DYD and DHD were strongly correlated (correlation coefficients 0.936 for D2/3 and 0.892 for D5, respectively). The 25th percentile of DYD and DHD levels were 0.71 ng/ml and 20.675 ng/ml on day of ET. The ongoing pregnancy rate was significantly reduced in patients in the lower quarter of DYD or DHD levels: ≤25th percentile DYD or DHD 3/49 (6%) and 4/49 (8%) versus >25th percentile DYD or DHD 42/149 (28%) and 41/149 (27%) (unadjusted difference -22% (CI: -31% to -10%) and -19% (CI: -29% to -7%), adjusted difference -22%, 95% CI: -32 to -12, P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: Some inter- and intraindividual variations in DYD levels could be attributed to differences in time between last 10 mg DYD intake and blood sampling, as well as concomitant food intake, neither of which were registered in this study. Ninety percent of subjects were European-Caucasian and DYD/DHD blood concentrations should be replicated in other and larger populations. WIDER IMPLICATIONS OF THE FINDINGS: Daily 10 mg DYD (tid) in an artificial FET cycle is potentially a suboptimal dose for a proportion of the population. Measurement of DYD or DHD levels could be used interchangeably for future studies. The pharmacokinetics of oral DYD and associated reproductive pharmacodynamics need further study. STUDY FUNDING/COMPETING INTEREST(S): The trial was financed by university funds, except for the cost for plasma and serum sample handling, storage and shipment, as well as the liquid chromatography-mass spectrometry (LC-MS/MS) analysis of DYD, DHD and progesterone, which was financially supported by Abbott Products Operations AG (Allschwil, Switzerland). Abbott Products Operations AG had no influence on the study protocol, study conduct, data analysis or data interpretation. K.N. has received honoraria and/or non-financial support (e.g. travel cost compensation) from Ferring, Gedeon-Richter, Merck and MSD. A.M. has no competing interests. R.V. has no competing interests. M.D. has received honoraria and/or non-financial support from Ferring and Merck. A.S.-M. has no competing interests. T.K.E. has received honoraria and/or non-financial support from Roche, Novartis, Pfizer, Aristo Pharma, Merck. G.G. has received honoraria and/or non-financial support (e.g. travel cost compensation) from Abbott, Ferring, Gedeon Richter, Guerbet, Merck, Organon, MSD, ObsEva, PregLem, ReprodWissen GmbH, Vifor and Cooper. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03507673.

Improving analysis of the vaginal microbiota of women undergoing assisted reproduction using nanopore sequencing.

PURPOSE: Subclinical alterations of the vaginal microbiome have been described to be associated with female infertility and may serve as predictors for failure of in vitro fertilization treatment. While large prospective studies to delineate the role of microbial composition are warranted, integrating microbiome information into clinical management depends on economical and practical feasibility, specifically on a short duration from sampling to final results. The currently most used method for microbiota analysis is either metagenomics sequencing or amplicon-based microbiota analysis using second-generation methods such as sequencing-by-synthesis approaches (Illumina), which is both expensive and time-consuming. Thus, additional approaches are warranted to accelerate the usability of the microbiome as a marker in clinical praxis. METHODS: Herein, we used a set of ten selected vaginal swabs from women undergoing assisted reproduction, comparing and performing critical optimization of nanopore-based microbiota analysis with the results from MiSeq-based data as a quality reference. RESULTS: The analyzed samples carried varying community compositions, as shown by amplicon-based analysis of the V3V4 region of the bacterial 16S rRNA gene by MiSeq sequencing. Using a stepwise procedure to optimize adaptation, we show that a close approximation of the microbial composition can be achieved within a reduced time frame and at a minimum of costs using nanopore sequencing. CONCLUSIONS: Our work highlights the potential of a nanopore-based methodical setup to support the feasibility of interventional studies and contribute to the development of microbiome-based clinical decision-making in assisted reproduction.

A randomised, multi-center, open trial comparing a semi-automated closed vitrification system with a manual open system in women undergoing IVF.

STUDY QUESTION: What are outcome and procedural differences when using the semi-automated closed Gavi® device versus the manual open Cryotop® method for vitrification of pronuclear (2PN) stage oocytes within an IVF program? SUMMARY ANSWER: A semi-automated closed vitrification method gives similar clinical results as compared to an exclusively manual, open system but higher procedure duration and less staff convenience. WHAT IS KNOWN ALREADY: A semi-automated closed vitrification device has been introduced to the market, however, little evaluation of its performance in a clinical setting has been conducted so far. STUDY DESIGN, SIZE, DURATION: This prospective, randomised, open non-inferiority trial was conducted at three German IVF centers (10/2017-12/2018). Randomization was performed on day of fertilization check, stratified by center and by indication for vitrification (surplus 2PN oocytes in the context of a fresh embryo transfer (ET) cycle or 'freeze-all' of 2PN oocytes). PARTICIPANT/MATERIAL, SETTING, METHODS: The study population included subfertile women, aged 18-40 years, undergoing IVF or ICSI treatment after ovarian stimulation, with 2PN oocytes available for vitrification. The primary outcome was survival rate of 2PN oocytes at first warming procedure in a subsequent cycle and non-inferiority of 2PN survival was to be declared if the lower bound 95% CI of the mean difference in survival rate excluded a difference larger than 9.5%; secondary, descriptive outcomes included embryo development, pregnancy and live birth rate, procedure time and staff convenience. MAIN RESULTS AND THE ROLE OF CHANCE: The randomised patient population consisted of 149 patients, and the per-protocol population (patients with warming of 2PN oocytes for culture and planned ET) was 118 patients. The survival rate was 94.0% (±13.5) and 96.7% (±9.7) in the Gavi® and the Cryotop® group (weighted mean difference -1.6%, 95% CI -4.7 to 1.4, P = 0.28), respectively, indicating non-inferiority of the Gavi® vitrification/warming method for the primary outcome. Embryo development and the proportion of top-quality embryos was similar in the two groups, as were the pregnancy and live birth rate. Mean total procedure duration (vitrification and warming) was higher in the Gavi® group (81 ± 39 min vs 47 ± 15 min, mean difference 34 min, 95% CI 19 to 48). Staff convenience assessed by eight operators in a questionnaire was lower for the Gavi® system. The majority of respondents preferred the Cryotop® method because of practicality issues. LIMITATIONS, REASON FOR CAUTION: The study was performed in centers with long experience of manual vitrification, and the relative performance of the Gavi® system as well as the staff convenience may be higher in settings with less experience in the manual procedure. Financial costs of the two procedures were not measured along the trial. WIDER IMPLICATIONS OF THE FINDINGS: With increasing requirements for standardization of procedures and tissue safety, a semi-automated closed vitrification method may constitute a suitable alternative technology to the established manual open vitrification method given the equivalent clinical outcomes demonstrated herein. STUDY FUNDING/COMPETING INTERESTS: The trial received no direct financial funding. The Gavi® instrument, Gavi® consumables and staff training were provided for free by the distributor (Merck, Darmstadt, Germany) during the study period. The manufacturer of the Gavi® instrument had no influence on study protocol, study conduct, data analysis, data interpretation or manuscript writing. J.H. has received honoraria and/or non-financial support from Ferring, Merck and Origio. G.G. has received honoraria and/or non-financial support from Abbott, Ferring, Finox, Gedeon Richter, Guerbet, Merck, MSD, ObsEva, PregLem, ReprodWissen GmbH and Theramex. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03287479. TRIAL REGISTRATION DATE: 19 September 2017. DATE OF FIRST PATIENT'S ENROLMENT: 10 October 2017.

Is oxytocin receptor antagonist administration around embryo transfer associated with IVF treatment success? A systematic review and meta-analysis.

A systematic literature review and meta-analysis was conducted to evaluate whether the administration of an oxytocin receptor antagonist (OTR-a) around embryo transfer is associated with live birth and pregnancy achievement in IVF treatment. Multiple databases were searched for randomized controlled trials (RCT) comparing the outcome of IVF treatment with administration of an OTR-a before, during or after embryo transfer versus administration of placebo/nil. The literature search identified 11 eligible RCT. The active compound was intravenous atosiban (n = 7), subcutaneous barusiban (n = 1) and oral nolasiban (n = 3). Clinical pregnancy rate was significantly higher in women receiving an OTR-a around embryo transfer (relative risk [RR] 1.31, 95% confidence interval [CI] 1.13-1.51, P = 0.0002, I2 = 61%, n = 11 studies, n = 3611); however, live birth rate was not statistically significantly affected (RR 1.09, 95% CI 0.98-1.20, P = 0.11, I2 = 25%, n = 5 studies, n = 2765). A sensitivity analysis on low risk of bias studies likewise indicates a higher clinical pregnancy chance (RR 1.11, 95% CI 1.01-1.22, P = 0.03, I2 = 5%, n = 5 RCT, n = 2765). OTR-a administration in IVF treatment has the potential to increase IVF efficacy, although the treatment effects observed so far are small and have not been sufficiently corroborated.

Characterization of early pregnancy placental progesterone production by use of dydrogesterone in programmed frozen-thawed embryo transfer cycles.

RESEARCH QUESTION: When and how does the gradual transition of the endocrine control of early pregnancy from the corpus luteum to the placenta, termed luteoplacental shift, take place? DESIGN: Prospective analysis of serum progesterone levels in pregnancies (n = 88) resulting from programmed frozen-thawed embryo transfer cycles in which ovulation was suppressed and no corpus luteum was present. Dydrogesterone, which does not cross-react with progesterone in immunoassay or spectrometric assay, was used for luteal phase and early pregnancy support. Progesterone, oestradiol and hCG were measured at regular intervals from before pregnancy achievement until +65 to 71 days after embryo transfer by Roche Elecsys electrochemiluminescence immunoassay (Elecsys ECLIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Serum progesterone remained at baseline levels on first blood analysis +9 to 15 days after embryo transfer and increased only marginally independently from the type of pregnancy up to +16 to 22 days after embryo transfer. From +23 to 29 days after embryo transfer, progesterone increased non-linearly above 1.0 ng/ml and increased further throughout the first trimester with elevated levels in multiples. Oestradiol levels increased in parallel with progesterone; hCG plateaued around +37 to 43 days. Progesterone levels were significant predictors for pregnancy viability from +23 to 29 days after embryo transfer onwards with best accuracy +37 to 43 days after embryo transfer (receiver operator characteristic analysis area under the curve 0.98; 95% CI 0.94 to 1; P = 0.0009). CONCLUSIONS: The onset of substantial progesterone production is the 7th gestational week. Progesterone increase is non-linear, depends on chorionicity and zygosity, and may have predictive potential on the outcome of pregnancies originating from frozen embryo transfer cycles.

Follicular flushing in patients with poor ovarian response: a systematic review and meta-analysis.

A systematic literature review and meta-analysis was conducted to evaluate the effect of follicular flushing on clinical outcomes (primary outcome: mean number of cumulus-oocyte-complexes [COC]) in poor-response IVF patients). The bibliographic databases OvidMedline (includes Pubmed), Cochrane Library and Web of Science were searched electronically for randomized controlled trials (RCT) comparing follicular flushing with no flushing. Three RCT with a total of 210 patients could be included. The mean number of COC did not increase with flushing (weighted mean difference: -0.45 COC, 95% CI -1.14 to 0.25, I2 = 70%; P = 0.21; three RCT, n = 210). Mean number of metaphase II oocytes and the proportion of randomized patients having at least one COC retrieved were no different between groups. No difference was observed between groups for mean number of embryos, the proportion of randomized patients achieving embryo transfer, clinical pregnancy and live birth rates. Procedure duration was significantly increased with flushing (P = 0.0006). A positive effect of flushing on any of the investigated outcomes could not be observed in the existing literature in patients with poor ovarian response. Flushing is unlikely to significantly increase the number of oocytes, and the routine use of follicular flushing should, therefore, be scrutinized.

The use of intracytoplasmic sperm injection is associated with a shift in the secondary sex ratio.

RESEARCH QUESTION: What is the association between assisted reproductive technologies and human sex ratio as a proportion of male offspring at birth. DESIGN: A total of 59,628 singleton deliveries resulting from IVF, intracytoplasmic sperm injection (ICSI) and intrauterine insemination (IUI) or ovulation induction from 101 IVF clinics in Germany, that had been documented in a national German IVF registry, were analysed. Sex ratio after assisted reproductive technology was also compared with the sex ratio reported in the birth records of the German Federal Statistical Office. RESULTS: The sex ratio was 50.0% (95% CI 49.5% to 50.5%) for ICSI, 52.2% (95% CI 51.5% to 52.9%) for IVF, 52.2% (95% CI 50.9% to 53.5%) for IUI or ovulation induction and 51.3% in the national birth records, respectively. Significant differences existed across the three treatment groups (P = 6.86 × 10-7) as well as in pairwise comparisons between ICSI versus IVF (P = 6.88 × 10-7) and ICSI versus IUI or ovulation induction (P = 0.003). No difference existed between the groups IUI or ovulation induction versus IVF. Same results were also present after stratification by maternal age: IVF versus ICSI (P = 6.433 × 10-7), ICSI versus IUI or ovulation induction (P = 0.003), and IVF versus IUI or ovulation induction (non-significant). Compared with the national birth records, ICSI is associated with a lower sex ratio compared with the reference group (P < 0.001), whereas IVF is associated with a higher sex ratio (P = 0.015). CONCLUSIONS: The use of ICSI is associated with an equal proportion of sexes at birth, which is not the case for IVF, IUI or ovulation induction, or natural conception. This phenomenon is not influenced by maternal age.

Is surgical plume developing during routine LEEPs contaminated with high-risk HPV? A pilot series of experiments.

INTRODUCTION: Growing evidence shows a causal role of high-risk humane papillomavirus (HPV) infections in the development of head and neck cancer. A recent case report shows two patients suffering from tonsillar cancer without any risk factors apart from their work as gynecologists doing laser ablations and loop electrosurgical excision procedures (LEEP). The aim of the present investigation is to evaluate whether surgical plume resulting from routine LEEPs of HSIL of the cervix uteri might be contaminated with the DNA of high-risk HPV. MATERIALS AND METHODS: The prospective pilot study is done at the Department of Gynecology and Obstetrics of the University of Lübeck, Germany. The primary outcome was defined as HPV subtype in resected cone and in surgical plume resulting from LEEPs of HSIL of the cervix uteri. Plume resulting from LEEPs was analyzed using a Whatman FTA Elute Indicating Card which was placed in the tube of an exhaust suction device used to remove the resulting aerosols. For detection of HPV and analysis of its subtype, the novel EUROArray HPV test was performed. Resected cones of LEEPs were evaluated separately for HPV subtypes. RESULTS: Four samples of surgical plume resulting from routine LEEPs indicated contamination with high-risk HPV and showed the same HPV subtype as identified in the resected cones. CONCLUSION: Surgical plume resulting from routine LEEPs for HSIL of the cervix uteri has the risk of contamination with high-risk HPV. Further investigations of infectiousness of surgical plume are necessary for evaluation of potential hazards to involved healthcare professionals.

Can a quality-of-life assessment assist in identifying women at risk of prematurely discontinuing IVF treatment? A prospective cohort study utilizing the FertiQoL questionnaire.

PURPOSE: This study aimed at assessing quality of life (QoL) by means of a validated measurement tool (FertiQoL) in German infertile patients before a first IVF/ICSI cycle with ancillary assessment of changes in FertiQoL scores after a failed first cycle and the predictive capacity of FertiQoL scores for treatment discontinuation. METHODS: The validated FertiQoL tool consisting of 24 questions regarding fertility-specific aspects of QoL was used for this prospective cohort study conducted at a university affiliated IVF center in Germany. Female patients (n = 119) filled out the FertiQoL form and questionnaire on sociodemographic variables on initiation of a first- and second-cycle IVF/ICSI treatment, respectively. RESULTS: On initiation of a first IVF/ICSI cycle, the mean scores (± standard deviation) for subscales emotional, mind-body, relational, and social items were 62 (± 19), 75 (± 17), 82 (± 13), and 78 (± 14), respectively; the total FertiQoL score was 73 (± 12). The mean total FertiQoL score at initiation of a first treatment cycle did not differ between patients who continued vs. discontinued treatment in case of no pregnancy achievement in the first cycle (73) (± 10) vs. 74 (± 14), p = 0.46). Furthermore, the mean total FertiQoL score did not change after an unsuccessful first IVF cycle (74 vs. 76, p = 0.46). CONCLUSIONS: There was no statistical difference in a small sample size for FertiQoL scores between all groups. In this study, FertiQoL scores were, therefore, not usable to predict withdrawal from infertility treatment.

What is the net effect of introducing vitrification for cryopreservation of surplus 2PN oocytes in an IVF program?

PURPOSE: The aim of this study was to accurately describe outcome differences (cryo-survival, pregnancy rate and live birth rate, both per ET and cumulatively), between the vitrification method and slow-freezing method of surplus 2PN oocytes in an IVF program. METHODS: In 2004, the freezing method for 2PN oocytes was changed from slow-cooling to vitrification. The data of 711 patients (timespan: 1/1999-7/2011; 410 vitrification and 301 slow-cooling events) undergoing a first IVF/ICSI cycles with freezing of 2PN oocytes were retrospectively analyzed. The outcome of one, the first, IVF cycle per patient was explored. The data were analyzed per freezing-thawing attempt as well as cumulatively per one complete IVF cycle, taking pregnancy occurrence after a fresh embryo transfer preceding the cryo-cycle(s) and other confounders (such as female age, elective vs. surplus 2PN cryopreservation) into account by means of exploratory regression analyses. RESULTS: In the vitrification and slow-cooling group, 756 and 376, respectively, attempts of thawing 2PN oocytes were recorded. Each attempt of thawing 2PN oocytes showed statistically significantly higher mean cryo-survival rates after vitrification (effect size approximately 30-40%, with vitrification cryo-survival consistently above 90% in all thawing attempts). Furthermore, the incidence of "zero survival" was lower after vitrification (0.5 vs. 7.3%, p < 0.01). It is estimated that the odds of achieving a live birth per one IVF cycle (fresh and frozen transfers combined) with vitrification of 2PN oocytes is increased approximately 1.4-fold (OR of 1.405, 95% CI 0.968-2.038; p = 0.07); however, statistical significance was not achieved due to sample size. Female age and elective cryopreservation of all 2PN oocytes without a fresh transfer (e.g., hyperresponders) were found to be negatively and positively, respectively, associated with the chance of achieving a live birth. CONCLUSIONS: The introduction of vitrification has a measurable impact on the efficacy of an IVF program. However, this effect is not large despite the impressively higher cryo-survival rates with vitrification. The "true" net efficacy effect of introducing 2PN vitrification in an IVF program will, in real life, be lower due to patients not having surplus 2PN oocytes available for freezing and later transfer.

Do ART patients face higher C-section rates during their stage of delivery? A German monocenter experience.

PURPOSE: The mode of delivery depends on multiple parameters. After assisted reproductive technology (ART), previous studies have shown elevated C-section rates but few studies differentiated between elective and emergency operations and different protocols of cryopreservation. Because these studies did not use multiparity as exclusion criteria which reduces confounding with previous pregnancies, aim of this study is to compare mode of delivery of different techniques of ART using data of primiparae only [1, 2]. METHODS: Retrospective analysis of patient data treated at the university hospital of Luebeck in a period of 12 years. Patients were divided in different groups according to their way of conception: spontaneous conception and conception after ART. The group of ART was further divided into: (a) a group of fresh transferred embryos (IVF/ICSI), (b) vitrification and (c) slow freezing. Exclusion criteria were defined as: multiparity, delivery <24. + 0 p.m., incomplete data and treatment outside university of Luebeck. Main parameter of this study was mode of delivery which was divided into spontaneous delivery or C-section. C-sections were further differentiated into elective or emergency C-sections. RESULTS: The group of fresh transferred embryos and slow freezing showed higher risks for elective and emergency C-sections (elective C-sections odds ratio 2.0, CI 95% 1.6-2.6, emergency C-sections odds ratio 1.4, CI 95% 1.1-1.9). Moreover, all groups of ART show enhanced risk of significant perinatal bleeding. CONCLUSION: Patients after ART treatment suffer from higher C-section rates during their stage of delivery.

Introducing a new scoring system for pap smear in the detection of high-grade squamous intraepithelial lesions in pregnancy (The Luebeck Score).

PURPOSE: To construct a scoring system for pap smears to objectify cytological appraisal and to enhance the accuracy and comparability of pap smear interpretation in pregnancy. MATERIALS AND METHODS: For development of a scoring system for cell appraisal of pap smears the style of the Modified Masood's Scoring Index for appraisal of cells from fine needle aspirations of breast lesions was used. Cohort analysis of n = 54 dysplastic pap smears for polymorphology of cells, anisonucleosis, structure of the nucleus, signs of tissue destruction, nucleus/plasma relation and signs of tumordiathesis. Each criteria was classified into three stages: The first with little evidence for dysplasia (one point), second stage with sporadic evidence (two points) or third stage with frequent evidence (three points). To further evaluate if pregnancy associated cells changes interfere with this scoring system we compared the results of pregnant and non-pregnant women. Histological result was used as an indicator of correctness of the score. RESULTS: Statistical analysis showed a good correlation of the scoring system with histological results. Especially in pregnancy statistical analysis shows promising results (sensitivity 86.67 %, Specificity 100 %, receiver operating characteristic analysis p ≤ 0.05). CONCLUSION: The Luebeck Score seems to be a useful approach for appraisal of pap smears in pregnancy. Further studies containing high numbers of cases are needed for further evaluation of potential benefits of the scoring system compared to conventional evaluation of pap smears.

Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit calcineurin-induced myocardial hypertrophy.

BACKGROUND: Ca(2+)-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. METHODS AND RESULTS: We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca(2+) handling nor functional and structural changes. On pathological pressure overload and ß-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. CONCLUSIONS: We established a mouse model in which CaMKII's activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure.

Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII.

Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes' Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes' systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction.

CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation.

RATIONALE: Although research suggests that diastolic Ca(2+) levels might be increased in atrial fibrillation (AF), this hypothesis has never been tested. Diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR) might increase diastolic Ca(2+) levels and play a role in triggering or maintaining AF by transient inward currents through Na(+)/Ca(2+) exchange. In ventricular myocardium, ryanodine receptor type 2 (RyR2) phosphorylation by Ca(2+)/calmodulin-dependent protein kinase (CaMK)II is emerging as an important mechanism for SR Ca(2+) leak. OBJECTIVE: We tested the hypothesis that CaMKII-dependent diastolic SR Ca(2+) leak and elevated diastolic Ca(2+) levels occurs in atrial myocardium of patients with AF. METHODS AND RESULTS: We used isolated human right atrial myocytes from patients with AF versus sinus rhythm and found CaMKII expression to be increased by 40+/-14% (P<0.05), as well as CaMKII phosphorylation by 33+/-12% (P<0.05). This was accompanied by a significantly increased RyR2 phosphorylation at the CaMKII site (Ser2814) by 110+/-53%. Furthermore, cytosolic Ca(2+) levels were elevated during diastole (229+/-20 versus 164+/-8 nmol/L, P<0.05). Most likely, this resulted from an increased SR Ca(2+) leak in AF (P<0.05), which was not attributable to higher SR Ca(2+) load. Tetracaine experiments confirmed that SR Ca(2+) leak through RyR2 leads to the elevated diastolic Ca(2+) level. CaMKII inhibition normalized SR Ca(2+) leak and cytosolic Ca(2+) levels without changes in L-type Ca(2+) current. CONCLUSION: Increased CaMKII-dependent phosphorylation of RyR2 leads to increased SR Ca(2+) leak in human AF, causing elevated cytosolic Ca(2+) levels, thereby providing a potential arrhythmogenic substrate that could trigger or maintain AF.

Mercury exposure and neurochemical impacts in bald eagles across several Great Lakes states.

In this study, we assessed mercury (Hg) exposure in several tissues (brain, liver, and breast and primary feathers) in bald eagles (Haliaeetus leucocephalus) collected from across five Great Lakes states (Iowa, Michigan, Minnesota, Ohio, and Wisconsin) between 2002-2010, and assessed relationships between brain Hg and neurochemical receptors (NMDA and GABA(A)) and enzymes (glutamine synthetase (GS) and glutamic acid decarboxylase (GAD)). Brain total Hg (THg) levels (dry weight basis) averaged 2.80 µg/g (range: 0.2-34.01), and levels were highest in Michigan birds. THg levels in liver (r(p) = 0.805) and breast feathers (r(p) = 0.611) significantly correlated with those in brain. Brain Hg was not associated with binding to the GABA(A) receptor. Brain THg and inorganic Hg (IHg) were significantly positively correlated with GS activity (THg r(p) = 0.190; IHg r(p) = 0.188) and negatively correlated with NMDA receptor levels (THg r(p) = -0245; IHg r(p) = -0.282), and IHg was negatively correlated with GAD activity (r(s) = -0.196). We also report upon Hg demethylation and relationships between Hg and Se in brain and liver. These results suggest that bald eagles in the Great Lakes region are exposed to Hg at levels capable of causing subclinical neurological damage, and that when tissue burdens are related to proposed avian thresholds approximately 14-27% of eagles studied here may be at risk.

An Economic Analysis of Aneuploidy Screening of Oocytes in Assisted Reproduction in Germany.

Background The randomized ESTEEM trial reported that preimplantation genetic aneuploidy testing of oocytes by polar body biopsy (PGT-A) with array comparative genomic hybridization (aCGH) in women aged 36 - 40 years undergoing assisted reproduction treatment reduces the number of embryo transfers and the risk of miscarriage while not impacting the live birth rate. Method A decision tree model based on data from the ESTEEM trial was created and analyzed, using three cost scenarios for assisted reproduction treatment in Germany (statutory health insurance [GKV] = the deductible is 50% of the standard medical costs; private medical insurance [PKV] = invoicing is based on the German medical fee schedule [GOÄ]; private medical insurance with a simple GOÄ factor [simple GOÄ factor] = invoicing is based on the standard medical fees multiplied by a linear GOÄ factor). The scenarios were compared for cost-effectiveness (cost per live birth), cost per prevented miscarriage and the threshold values for cost and effectiveness. Results PGT-A increased the costs per live birth in all scenarios (GKV: + 208%; PKV: + 49%; simple GOÄ factor: + 89%). A threshold analysis showed a substantial cost discrepancy between the actual cost of the intervention based on GOÄ (€ 5801) vs. the theoretically tolerable PGT-A cost (GKV: € 561, PKV: € 1037, single GOÄ-factor: € 743). The incremental cost per one prevented miscarriage was approximately € 70 000 - 75 000 for all cost scenarios. Conclusion The use of PGT-A with aCGH in assisted reproduction cannot be recommended from a cost-effectiveness perspective.