Standard Stimulation Protocol Research Articles

Progestin-primed ovarian stimulation outcomes in in-vitro fertilization (IVF) – A systematic review of the literature

Background: Progestin-primed ovarian stimulation (PPOS) stimulates ovaries to block the premature surge of luteinizing hormone (LH) by using micronized progesterone or a progestin during the follicular phase instead of the conventional gonadotropin-releasing hormone (GnRH) analogues or GnRH antagonists downregulating LH to obtain multi-follicle engagement. Current work aims to assess the influence of progestogen treatment on ovarian stimulation and the ability to control LH surge, its efficacy and suitability in retrieving oocytes, without affecting the embryo quality and its benefit among infertile women long-term outcomes on children compared to standard stimulation protocols. Materials and Methods: The literature review used the randomized control trials published in the Pubmed database from January 2015 to April 2021. To generate the citation list, the following keywords were used: ‘progestin-primed ovarian stimulation’, ‘PPOS’, ‘micronized progesterone’, ‘medroxyprogesterone’, and/or ‘dydrogesterone’. The selected articles analyzed the cohort, intervention, and scheme of the progestin-primed ovarian stimulation protocol in controlled ovarian stimulation (COS) for in-vitro fertilization (IVF)/intra cytoplasmic sperm injection (ICSI) used in Assisted Reproductive Technologies (ART). Results: Overall we concluded that PPOS for IVF/ICSI in ART results in a higher number of obtained embryos, lower incidence of OHSS, equal duration of stimulation, number of retrieved oocytes, and number of MII oocytes. It is also suggested that long-term safety in children shows no significant difference between the study and control groups. Conclusions: Despite the outcomes of progestin stimulation cycles among all cohorts, we concluded that poor ovarian responders, patients with PCOS, women of advanced age and oocyte donors benefit the most from using PPOS.

Disrupting abnormal neuronal oscillations with adaptive delayed feedback control.

Closed-loop neuronal stimulation has a strong therapeutic potential for neurological disorders such as Parkinson's disease. However, at the moment, standard stimulation protocols rely on continuous open-loop stimulation and the design of adaptive controllers is an active field of research. Delayed feedback control (DFC), a popular method used to control chaotic systems, has been proposed as a closed-loop technique for desynchronisation of neuronal populations but, so far, was only tested in computational studies. We implement DFC for the first time in neuronal populations and access its efficacy in disrupting unwanted neuronal oscillations. To analyse in detail the performance of this activity control algorithm, we used specialised in vitro platforms with high spatiotemporal monitoring/stimulating capabilities. We show that the conventional DFC in fact worsens the neuronal population oscillatory behaviour, which was never reported before. Conversely, we present an improved control algorithm, adaptive DFC (aDFC), which monitors the ongoing oscillation periodicity and self-tunes accordingly. aDFC effectively disrupts collective neuronal oscillations restoring a more physiological state. Overall, these results support aDFC as a better candidate for therapeutic closed-loop brain stimulation.

Prevalence of epileptiform electroencephalographic abnormalities in people without a history of seizures: A systematic review and meta-analysis.

Abnormal patterns identified on electroencephalogram (EEG) are one of the primary diagnostic tests for epilepsy. However, epidemiological studies have established that both benign and epileptiform abnormalities (EAs) occur on the EEG of nonepileptic, seizure-free people as well. The reported rates of EAs in nonepileptic, seizure-free populations vary, and the true prevalence is unknown. The primary objective of this systematic review and meta-analysis was to estimate the overall prevalence of EAs in the EEG of people without a history of seizures. Secondary aims were to characterize (1) the cortical localization of focal abnormalities, (2) the proportion of findings that occurred during standard EEG stimulation protocols, and (3) the persistence and implications of abnormalities at follow-up. A comprehensive electronic search of six bibliographic databases was completed: Embase, MEDLINE, PsycInfo, Cumulative Index of Nursing and Allied Health Literature, Cochrane Central Register for Controlled Trials, and Web of Science. No search date restrictions were applied. Overall effect size was calculated using a generalized linear mixed-effects model. Fifty-three studies, totaling 73 990 individuals, met our inclusion criteria. The overall point prevalence of EAs was 1.74% (95% confidence interval [CI] = 1.13-2.67). Due to the risk of bias in the literature, especially from participant selection, we believe this to be an overestimate of the true prevalence. Prevalence of EAs was greater in children (2.45%, 95% CI = 1.41-4.21) and the elderly (5.96%, 95% CI = 1.39-22.13) compared with adults (.93%, 95% CI = .48-1.80). Reports of developing epilepsy after an EA-positive EEG were rare. The likelihood of subsequent positive findings on follow-up EEG may be as high as 50%. Our study has limitations in that males were overrepresented in the study samples, there is substantial heterogeneity among studies, and many studies provided insufficient detail about their exclusion criteria. Nonetheless, our estimates provide benchmark data for future studies examining EAs in clinical populations, particularly behavioral and psychiatric populations.

ANTI-MÜLLERIAN HORMONE (AMH) CAN BE A PREDICTABLE PARAMETER OF CLINICAL OUTCOMES AFTER SINGLE VITRIFIED BLASTOCYST TRANSFER CYCLES (SVBT)

Anti-Müllerian hormone (AMH) serum level significantly correlates with an ovarian reserve and useful quantitative marker in in-vitro fertilization (IVF) cycle. Recent studies reported AMH serum level positively correlated with clinical outcomes, which used standard ovarian stimulation protocol often. Clomiphene-citrate (CC) based minimal stimulation protocol is one of the mild stimulations, which uses low dose or no gonadotropin. This study aims to investigate the correlation between clinical outcomes and AMH serum level after CC based minimal stimulation.

To curette or not to curette; efficiency of oocyte retrieval technique.

Follicular curetting is the gentle and rapidly rotating movement of the aspiration needle in a clockwise and counterclockwise fashion. The aim of our study is to assess this retrieval technique comparing cycle and pregnancy outcomes between curetting and non-curetting. This was a retrospective review of 817 patients that underwent oocyte retrieval at our fertility center from January 1st, 2016 until August 31st, 2019. All patients enrolled in the study underwent standard ovarian stimulation protocols. Three physicians routinely utilized follicle curetting during oocyte retrieval, while a fourth physician did not curette. Retrievals and embryo transfers were performed based on a rotating physician schedule. Retrievals that were performed using a curetting technique had a significantly higher number of total oocytes retrieved 12.4 ± 8.1 vs 10.7 ± 7.5 (p = 0.01), and number of M2 oocytes retrieved 8.1 ± 6.4 vs 6.9 ± 6.0 (p = 0.03), resulting in an increased M2 oocyte yield 63.7% ± 41.3 vs 50.9% ± 30.6 (p = 0.0001). These patients also had a significant increase in clinical pregnancy rate per retrieval 55 vs 41% (p = 0.0016), live birth rate per retrieval 42 vs 34% (p = 0.04) and with no significant difference in multiple gestations or OHSS. A significant improvement in clinical pregnancy rates and live birth per retrieval was detected in patients undergoing curetting. These patients had more embryos frozen, leading to more transfers and more successful pregnancies.

Fertility preservation for women with breast cancer: a multicentre randomized controlled trial on various ovarian stimulation protocols.

Does ovarian stimulation with the addition of tamoxifen or letrozole affect the number of cumulus-oocyte complexes (COCs) retrieved compared to standard ovarian stimulation in women with breast cancer who undergo fertility preservation? Alternative ovarian stimulation protocols with tamoxifen or letrozole did not affect the number of COCs retrieved at follicle aspiration in women with breast cancer. Alternative ovarian stimulation protocols have been introduced for women with breast cancer who opt for fertility preservation by means of banking of oocytes or embryos. How these ovarian stimulation protocols compare to standard ovarian stimulation in terms of COC yield is unknown. This multicentre, open-label randomized controlled superiority trial was carried out in 10 hospitals in the Netherlands and 1 hospital in Belgium between January 2014 and December 2018. We randomly assigned women with breast cancer, aged 18-43 years, who opted for banking of oocytes or embryos to one of three study arms; ovarian stimulation plus tamoxifen, ovarian stimulation plus letrozole or standard ovarian stimulation. Standard ovarian stimulation included GnRH antagonist, recombinant FSH and GnRH agonist trigger. Randomization was performed with a web-based system in a 1:1:1 ratio, stratified for oral contraception usage at start of ovarian stimulation, positive estrogen receptor (ER) status and positive lymph nodes. Patients and caregivers were not blinded to the assigned treatment. The primary outcome was number of COCs retrieved at follicle aspiration. During the study period, 162 women were randomly assigned to one of three interventions. Fifty-four underwent ovarian stimulation plus tamoxifen, 53 ovarian stimulation plus letrozole and 55 standard ovarian stimulation. Analysis was according to intention-to-treat principle. No differences among groups were observed in the mean (±SD) number of COCs retrieved: 12.5 (10.4) after ovarian stimulation plus tamoxifen, 14.2 (9.4) after ovarian stimulation plus letrozole and 13.6 (11.6) after standard ovarian stimulation (mean difference -1.13, 95% CI -5.70 to 3.43 for tamoxifen versus standard ovarian stimulation and 0.58, 95% CI -4.03 to 5.20 for letrozole versus standard ovarian stimulation). After adjusting for oral contraception usage at the start of ovarian stimulation, positive ER status and positive lymph nodes, the mean difference was -1.11 (95% CI -5.58 to 3.35) after ovarian stimulation plus tamoxifen versus standard ovarian stimulation and 0.30 (95% CI -4.19 to 4.78) after ovarian stimulation plus letrozole versus standard ovarian stimulation. There were also no differences in the number of oocytes or embryos banked. There was one serious adverse event after standard ovarian stimulation: one woman was admitted to the hospital because of ovarian hyperstimulation syndrome. The available literature on which we based our hypothesis, power analysis and sample size calculation was scarce and studies were of low quality. Our study did not have sufficient power to perform subgroup analysis on follicular, luteal or random start of ovarian stimulation. Our study showed that adding tamoxifen or letrozole to a standard ovarian stimulation protocol in women with breast cancer does not impact the effectiveness of fertility preservation and paves the way for high-quality long-term follow-up on breast cancer treatment outcomes and women's future pregnancy outcomes. Our study also highlights the need for high-quality studies for all women opting for fertility preservation, as alternative ovarian stimulation protocols have been introduced to clinical practice without proper evidence. The study was supported by a grant (2011.WO23.C129) of 'Stichting Pink Ribbon', a breast cancer fundraising charity organization in the Netherlands. M.G., C.B.L. and R.S. declared that the Center for Reproductive Medicine, Amsterdam UMC (location VUMC) has received unconditional research and educational grants from Guerbet, Merck and Ferring, not related to the presented work. C.B.L. declared a speakers fee for Inmed and Yingming. S.C.L. reports grants and non-financial support from Agendia, grants, non-financial support and other from AstraZeneca, grants from Eurocept-pharmaceuticals, grants and non-financial support from Genentech/Roche and Novartis, grants from Pfizer, grants and non-financial support from Tesaro and Immunomedics, other from Cergentis, IBM, Bayer, and Daiichi-Sankyo, outside the submitted work; In addition, S.C.L. has a patent UN23A01/P-EP pending that is unrelated to the present work. J.M.J.S. reported payments and travel grants from Merck and Ferring. C.C.M.B. reports her role as unpaid president of the National guideline committee on Fertility Preservation in women with cancer. K.F. received unrestricted grants from Merck Serono, Good Life and Ferring not related to present work. K.F. declared paid lectures for Ferring. D.S. declared former employment from Merck Sharp & Dohme (MSD). K.F. declared paid lectures for Ferring. D.S. reports grants from MSD, Gedeon Richter and Ferring paid to his institution; consulting fee payments from MSD and Merck Serono paid to his institution; speaker honoraria from MSD, Gedeon Richter, Ferring Pharmaceuticals and Merck Serono paid to his institution. D.S. has also received travel and meeting support from MSD, Gedeon Richter, Ferring Pharmaceuticals and Merck Serono. No payments are related to present work. NTR4108. 6 August 2013. 30 January 2014.

P-681 Balance of Estrogen:Androgen ratio (Estradiol E2: Dehydroepiandrosterone-sulphate DHEAS) and its symbiotic correlationship with Vascular-Endothelial Growth-Factor (VEGF) critically foments implantation and pregnancy outcomes in IVF cycles

Abstract Study question Is the estrogen/androgen balance vital for implantation and does this E2:DHEAS ratio coordinate with neoangiogenesis/neovascularisation (VEGF) to facilitate establishment of viable pregnancy in IVF cycles? Summary answer The Estradiol E2:DHEAS ratio independently as well as in symbiotic correlationship with VEGF is strongly predictive of invasive implantation and pregnancy outcomes in IVF cycles What is known already Decidualization of endometrial stroma and vascularization at feto-maternal interface is necessary for implantation. Estrogen Estradiol E2 is among the factors widely known to regulate these processes. Androgen Dehydroepiandrosterone (DHEA) is the designated primary precursor for natural estrogens. DHEA-induced PCOS mice have demonstrated adverse impacts on Embryo implantation and decidualization. However, role of DHEA/S in human implantation has not yet been explored. Vascular-Endothelial growth-factor (VEGF) is known to influence vascularization, angiogenesis, endometrial receptivity. Higher VEGF expression in endometrium is associated with pregnancy. However endometrial-biopsy for VEGF estimation is an invasive method for implantation studies and cannot be applied to ongoing cycles. Study design, size, duration Prospective study of n = 235 (Power of study>85%) non-PCOS, normal-responder infertile females (mean age 31.77±2.42 years, BMI 24.4±4.1, W/H ratio 0.82±0.07) undergoing fresh IVF treatment cycles using standard antagonist stimulation protocol at our fertility clinic during January 2019-December 2020. Elderly women (age>35 years), women with polycystic-ovaries, endometriosis and frozen-thawed cycles were excluded.7 cycles were cancelled owing to no embryo transfer (ET). Day3-cleavage stage or day5-blastocyst stage embryos were transferred (n = 228). Luteal-phase was supported with Micronized progesterone. Participants/materials, setting, methods Serum levels of Estradiol E2 and DHEAS were measured by Radio-Immunoassay and VEGF estimation by ELISA on day ET, day7, day14 post ET. Cycles were divided into Pregnant (P:n=73) and Non-Pregnant (NP=n=155) groups. Main outcome measures: Clinical Pregnancy (CPR:n=70=30.70%), Live-Birth (LBR:28.94%) rates. Secondary outcome measures: Biochemical-pregnancy (BCP:n=3=1.31%), Early-Miscarriage (EM:n=4=1.75%) rates. Pregnant=Serum βhCG>50 on d14ET. BCP=No exponential rise/tapering βhCG levels post d14ET, CP=positive cardiac activity at 6-8 weeks gestation, EM=non-viable intra-uterine pregnancy upto 12 weeks gestation. Main results and the role of chance dET: E2 did not differ significantly (780±50 vs. 750±30 pg/ml;p=0.61) whereas DHEAS = (212±11 vs. 170±6 ng/dL;p=0.0005), E2:DHEAS = (3.72±0.13 vs. 4.65±0.17;p=0.0011), VEGF = (658±20 vs. 495±16;p<0.0001) differed significantly between P vs. NP groups; indicating importance of E2/DHEAS ratio. D7ET: E2 = (790±57 vs. 222±19;p<0.0001), DHEAS = (381±28 vs. 1262±70;p<0.0001), E2:DHEAS = (2.37±0.25 vs. 0.30±0.029;p<0.0001), VEGF = (636±23.61 vs. 480±10.13;p<0.0001) displayed statistically robust differences between P vs. NP groups. d14ET: E2 = (855±73.45 vs. 193.2±13.31;p<0.0001), VEGF = (544.2±15.80 vs. 452.3±16.77;p=0.0011) displayed statistically robust differences between P vs. NP groups. Interestingly, although DHEAS = (685±41.05 vs. 806±55.37;p=0.18) did not differ significantly, E2:DHEAS = (1.643±0.26 vs. 0.45±0.048;p<0.0001) showed highly significant difference; again highlighting importance of E2/DHEAS balance. Ratio E2:DHEAS on d7ET correlated strongly with d7VEGF (Pearson r = 0.31,p<0.0001). ROC curve for d7 E2:DHEAS had AUC 78%, p < 0.0001 Hormone values and their rising trends (d/ET to d7/ET to d14/ET) varied with pregnancy outcome status E2= LB:(d/ET:780±50 to d7/ET:790±57 to d14/ET:855±73.45) vs. BCP:(d/ET:723.3±157.6 to d7/ET:743.3±159 to d14/ET:770±153) vs. EM:(d/ET:906.3±104 to d7/ET:1150±113 to d14/ET:1758±127) DHEAS= LB:(d/ET:212±11 to d7/ET:381±28 to d14/ET:685±41.05) vs. BCP:(d/ET:248.3±55 to d7/ET:266.7±71.2 to d14/ET:400±120.3) vs. EM:(d/ET:243.8±27 to d7/ET:303.8±11.43 to d14/ET:391.3±49) E2:DHEAS= LB:(d/ET:3.72±0.13 to d7/ET:2.37±0.25 to d14/ET:1.643±0.26) vs. BCP:(d/ET:2.93±0.15 to d7/ET:2.97±0.4 to d14/ET:2.12±0.32) vs. EM:(d/ET:3.78±0.40 to d7/ET:3.77±0.26 to d14/ET:4.68±0.59) Decreasing E2:DHEAS ratio (d/ET-d7/ET-d14/ET) is vital for successful implantation/Live-birth VEGF= LB:(d/ET:658±20 to d7/ET:636±23.61 to d14/ET:544.2±15.80) vs. BCP:(d/ET:513.3±37.56 to d7/ET:628.3±40.45 to d14/ET:468.3±35.63) vs. EM:(d/ET:647.5±78.8 to d7/ET:887.5±60 to d14/ET:1138±94.4) Limitations, reasons for caution This study is limited by small sample size and is not a randomized controlled trial. We have taken only representative molecules into consideration. There is a plethora of factors involved during the implantation process. Multi-centric systematic studies are needed to corroborate these findings and account for the probable confounding factors. Wider implications of the findings This novel study explores and highlights significance of maintenance of a critical estrogen/androgen ratio (E2:DHEAS), rather than individual estrogen or androgen markers, in the implantation process. We have also underscored how this ratio coordinates symbiotically with VEGF as a critical non-invasive determinant of implantation and pregnancy-outcomes in ongoing IVF cycles. Trial registration number Not Applicable

Proteomic Analysis of Human Follicular Fluid Reveals the Pharmacological Mechanisms of the Chinese Patent Drug Kunling Pill for Improving Diminished Ovarian Reserve

Objective To explore the pharmacological mechanism of a Chinese patent drug (Kunling Pill (KLP)) on improving diminished ovarian reserve based on proteomic analysis. Methods A total of 18 patients divided into three groups (the normal ovary reserve (NOR), diminished ovary reserve (DOR), and KLP groups) undergoing assisted reproductive technology by standard ovarian stimulation protocols were recruited to collect follicular fluid. Data-independent acquisition mass spectrometry was used to identify differentially expressed proteins by nano-LC-MS/MS. Bioinformatic analysis was conducted to predict the functions and pathways of the identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in the three groups. Results A total of 144 differentially expressed proteins were screened out, including 56 proteins that were downregulated and 88 proteins that were upregulated in the DOR group compared with the NOR group, while 27 proteins were shared in the KLP-treated group. Among them, 10 proteins were upregulated and 17 proteins were downregulated in the KLP-treated group compared with the DOR group. The most enriched biological processes accounted for 28 GO terms, including cellular process, biological regulation, metabolic process, and regulation of biological process. Significant pathways were associated with fatty acid elongation, fatty acid degradation, fatty acid metabolism, nicotinate and nicotinamide metabolism, and valine, leucine, and isoleucine degradation. Conclusion Our study provides the proteome profiles of human follicular fluid from DOR patients treated by KLP. Functional analyses of proteome datasets revealed that core proteins (SAA1, MIF, and PRDX5) and related pathways (fatty acid metabolism, nicotinate and nicotinamide metabolism, and tyrosine and purine metabolism) are possible pharmacological mechanisms through which KLP improves DOR. Therefore, these findings may help better understand the complex mechanisms through which DOR is treated by the Chinese patent drug KLP.

Auricular Transcutaneous Vagus Nerve Stimulation Acutely Modulates Brain Connectivity in Mice.

Brain electrical stimulation techniques take advantage of the intrinsic plasticity of the nervous system, opening a wide range of therapeutic applications. Vagus nerve stimulation (VNS) is an approved adjuvant for drug-resistant epilepsy and depression. Its non-invasive form, auricular transcutaneous VNS (atVNS), is under investigation for applications, including cognitive improvement. We aimed to study the effects of atVNS on brain connectivity, under conditions that improved memory persistence in CD-1 male mice. Acute atVNS in the cymba conchae of the left ear was performed using a standard stimulation protocol under light isoflurane anesthesia, immediately or 3 h after the training/familiarization phase of the novel object-recognition memory test (NORT). Another cohort of mice was used for bilateral c-Fos analysis after atVNS administration. Spearman correlation of c-Fos density between each pair of the thirty brain regions analyzed allowed obtaining the network of significant functional connections in stimulated and non-stimulated control brains. NORT performance was enhanced when atVNS was delivered just after, but not 3 h after, the familiarization phase of the task. No alterations in c-Fos density were associated with electrostimulation, but a significant effect of atVNS was observed on c-Fos-based functional connectivity. atVNS induced a clear reorganization of the network, increasing the inter-hemisphere connections and the connectivity of locus coeruleus. Our results provide new insights into the effects of atVNS on memory performance and brain connectivity extending our knowledge of the biological mechanisms of bioelectronics in medicine.

Cell-Extrinsic Priming Increases Permissiveness of CD4+ T Cells to Human Immunodeficiency Virus Infection by Increasing C-C Chemokine Receptor Type 5 Co-receptor Expression and Cellular Activation Status.

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28 in vitro stimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5+ T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increase ex vivo infection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12.

P–446 Controlled ovarian stimulation protocols for oocyte vitrification induce differential gene expression profiles in primary tumours of breast cancer

Abstract Study question Could controlled ovarian stimulation (COS) protocols used in fertility preservation (FP) impact on malignant cell proliferation and tumour molecular profiling of breast cancer (BC) patients? Summary answer Letrozole supplementation during ovarian stimulation for oocyte vitrification could be considered as a safe procedure in estrogen-dependent BC patients undergoing FP. What is known already High estradiol levels associated to COS could promote changes in gene expression in estrogen-positive BC tumors. Estradiol levels reached during the ovarian stimulation could aggressively promote malignant cell proliferation and cell migration to adjacent organs. Aromatase inhibitors such as letrozole, are added to standard stimulation protocols to avoid this undesirable potential side effect. Despite the reassuring clinical results achieved by using letrozole for FP in BC patients, there is still a lack of evidence regarding its impact on malignant cell behaviour. For this reason, specific molecular studies to properly evaluate safety of letrozole in this specific population are still required. Study design, size, duration Experimental in vivo study. Thirty 5-week-old Nude-nu female mice were divided into three different groups: BC (n = 10), BC and FSH stimulation (BC-FSH, n = 10), or BC and letrozole stimulation (BC-LTZ, n = 10). BC was considered the control group, whereas BC-FSH and BC-LTZ represented distinct COS protocols. Hormone-dependent BC was induced in all mice. Animals were followed-up for 5 months and then euthanized to collect kidney, ovary, spleen, and liver tissues for gene expression and immunohistochemistry (IHC) analysis. Participants/materials, setting, methods One million of human MCF–7 BC cells were injected into the mouse left kidney capsule. Two days after xenograft, COS was induced by 10IU FSH or 1mg/ml letrozole + 10IU FSH, followed by ovarian triggering with 10IU hCG at 48h. Human BC RT2 Profiler PCR Arrays were performed to evaluate the impact of COS on tumour behaviour. BC biomarkers (Ki67, Erα, PR and HER–2) were also analyzed by IHC to validate gene expression results. Main results and the role of chance The differential gene expression was firstly assessed in kidney samples, as they represent the xenograft site, and different expression profiles were obtained depending on the COS protocol used. The BC-FSH group showed a global over-expression pattern of all genes of the array when compared to BC and BC-LTZ. Further gene ontology analysis revealed that cellular process, biological regulation, metabolic process, and proteases were the most over-represented biological terms, with a 20.5-fold over-expression for MMP2 compared to the other groups. On the other hand, BC-LTZ mice presented gene expression profiles similar to that of controls. When other tissues were analysed to detect malignant cell presence, our results revealed a significant up-regulation of matrix-proteases, cell cycle and proliferation related-genes, in liver samples from the BC-FSH group, but no amplification of any of the studied genes was detected in ovarian tissue or spleen. IHC findings confirmed the presence of human BC cells in 100% of samples from kidney tissue and in 30% of samples from liver tissue in the BC-FSH group. No human cells were detected by IHC in the BC and BC-LTZ groups. Limitations, reasons for caution Since this is an animal model of estrogen-dependent BC induced through a cell line, further validation with human tumour breast cancer samples would be required. Wider implications of the findings: Adjuvant letrozole in COS protocols prevents BC cell migration. The present study suggests that this protective effect could be mediated by interfering ER-pathway downstream genes involved in cell proliferation and matrix digestion. Altogether, letrozole could safely be used as a supplement during COS procedures for oocyte vitrification in BC women. Trial registration number Not applicable

Effects of letrozole or tamoxifen coadministered with a standard stimulation protocol on fertility preservation among breast cancer patients.

To assess the effects of letrozole or tamoxifen coadministration on fertility preservation treatment outcomes. Retrospective cohort study of 118 breast cancer patients undergoing fertility preservation treatment between 2008 and 2018. Patients who received letrozole (n = 36) or tamoxifen (n = 30) were compared to controls (n = 52) who underwent standard ovarian stimulation protocols. The primary outcome measures included the number of retrieved oocytes, mature oocytes (MII), fertilization, and top-quality embryo rates. The secondary outcome measures included duration of stimulation, gonadotropin dose and peak estradiol level. The number of oocytes retrieved, MII oocytes, fertilization rate, duration of stimulation, or gonadotropin dose were similar inthe letrozole and tamoxifen groups, compared to controls. Top-quality embryo rate was lower in the tamoxifen group compared to controls (25% vs 39.4%, respectively, P = 0.034). The abnormal fertilization rate was higher in the letrozole group compared to controls (7.8% vs 3.60%, respectively, P = 0.015). A stepwise logistic regression analysis revealed that letrozole and peak estradiol were significantly associated with abnormal fertilization (OR 11.94; 95% CI 2.35-60.4, P = 0.003 for letrozole and OR 1.075; 95% CI 1.024-1.12, P = 0.004 per 100 unit change in estradiol). There may be a negative effect of letrozole or tamoxifen on fertilization and embryo quality, in fertility preservation cycles. Further studies are needed to confirm these findings.

Induction of long-term potentiation-like plasticity in the primary motor cortex with repeated anodal transcranial direct current stimulation – Better effects with intensified protocols?

BackgroundA single session of anodal tDCS induces LTP-like plasticity which lasts for about 1 h, while repetition of stimulation within a time interval of 30 min results in late-phase effects lasting for at least 24 h with standard stimulation protocols. ObjectiveIn this pilot study, we explored if the after-effects of a recently developed intensified single session stimulation protocol are relevantly prolonged in the motor cortex by repetition of this intervention. Methods16 healthy right-handed subjects participated in this study. The effects of an intensified (3 mA-20min) and a standard anodal tDCS protocol (1 mA-15min) with short (20 min) and long (3 h) repetition intervals were compared with the effects of respective single session tDCS protocols (3 mA-20min, 1 mA-15min, and Sham). Cortical excitability alterations were monitored by single-pulse TMS-elicited MEPs. ResultsCompared to sham, both single session tDCS protocols (1 mA-15min, and 3 mA-20min) resulted in cortical excitability enhancements lasting for about 30 min after stimulation. The short repetition interval (20 min) resulted in a prolongation of after-effects for the standard protocol, which lasted for more than 24 h after stimulation. For the intensified protocol, the prolongation of after-effects was limited to 120 min after stimulation. The long repetition interval (3 h) resulted in no excitability-enhancing after-effects for the intensified, and only minor excitability enhancement within the first 30 min after the intervention for the standard protocol. ConclusionThese results suggest a non-linearity of late-phase LTP-like plasticity induction, which was dependent not only on the interval of intervention repetition, but also on other protocol characteristics, including intensity, and duration of tDCS. Further studies in larger samples are needed to confirm these results.

The relevance of the individual screening for genetic variants in predicting ovarian response.

To investigate if polymorphisms of some genes involved in folliculogenesis predict ovarian response. This prospective randomized study includes 124 egg donors genotyped for six SNPs ESR1 (rs2234693), AMHR2 (rs2002555), GDF-9 (rs10491279 and rs254286), AMH (rs10407022) and LHCBR (rs229327) genes and four STRs in ESR1 rs3138774), SHBG (rs6761), CYP19A1 (rs60271534) and AR genes (CAG repeats in exon 1). All donors followed standard ovarian stimulation protocol using a daily dose of 225 UI. The genotypes obtained were compared with the ovarian stimulation outcome. Regarding the number of retrieved oocytes, we found statistical differences for the ESR1 SNP and STR (19.3 ± 8.9 for TT vs 15.3 ± 6.2 for CC/CT, P = 0.027; 19.1 ± 8.3 for <17repeats vs 14.7 ± 6.2 for >17repeats, P = 0.020). Moreover, women carrying TT in the ESR1 at position c.-397T>C with ESR1 (TA)n=17 retrieved the highest number of oocytes (20.4 ± 9.3) (P = 0.001). Concerning AMHR2, we observed an association with the length of stimulation (9.1 ± 1.4 d for AA vs 9.7 ± 1.3 d for AG/GG, P = 0.021) and gonadotropin received (2050 ± 319 for AA vs 2188 ± 299 for AG/GG, P = 0.017). No significant differences were observed for the other polymorphisms (P > 0.05). The polymorphisms in ESR1 and AMHR2 genes showed a clear association with the number of retrieved oocytes and the stimulation data, respectively. Our results suggest that polymorphisms in the genes for key reproductive hormones receptors could be used to predict the ovarian response and to personalize the stimulation prior the treatment.

Stimulation of the ovaries in women with breast cancer undergoing fertility preservation: alternative versus standard stimulation protocols

to evaluate the effectiveness of ovarian stimulation with tamoxifen or letrozole compared to standard ovarian stimulation on the number of oocytes retrieved in women with breast cancer in the course of fertility preservation. Multi-center randomized open-label trial in the Netherlands and Belgium. Women between 18 and 43 years with breast cancer who opted for banking of oocytes or embryos in the course of fertility preservation were included. We randomly assigned them to one of the three study groups; group 1 ovarian stimulation plus tamoxifen (60 mg per day), group 2 ovarian stimulation plus letrozole (5 mg per day) or group 3 standard ovarian stimulation without additional medication. Primary outcome was the number of oocytes retrieved at follicle aspiration. Secondary outcomes were number of mature oocytes retrieved, number of oocytes or embryos banked and peak E2 levels during ovarian stimulation. Between January 2014 and December 2018, we randomised 162 women with breast cancer. We analysed the primary outcome for 148 (91%) women of which 142 women (88%) underwent ovum pick up. Mean age of the women was 32 years.51 women underwent ovarian stimulation plus tamoxifen, 51 plus letrozole and 46 standard ovarian stimulation without additional medication. The mean number of oocytes retrieved at follicle aspiration was 12.6 (group 1), versus 14.2 (group 2) versus 13.3 (group 3) (mean difference in number of oocytes: group 1 vs. group 3 -0.675 ;95% CI, -5.6 to 4.3; group 2 vs. group 3 0.871; 95% CI, -4.1 to 5.9). The mean number of oocytes banked was 10.4 (group 1) versus 10.5 (group 2) versus 10.2 (group 3) (mean difference in number of oocytes banked: group 1 vs. group 3 0.243; 95% CI,-4.2 to 4.7; group 2 vs. group 3 0.297; 95% CI, -4.2 to 4.8). Mean number of embryos banked was 5.9 (group 1) versus 4.9 (group 2) versus 5.0 (group 3) (mean difference in number of embryos banked: group 1 vs. group 3 0.857; 95% CI, -3.0 to 4.7; group 2 vs. group 3 -1.33; 95% CI, -4.0 to 3.7). These results show that the addition of tamoxifen or letrozole to standard ovarian stimulation did not effect the number of oocytes or embryos banked in the course of fertility preservation for women with breast cancer. Whether the addition of tamoxifen or letrozole to standard ovarian stimulation affects the long-term follow up in terms of safety in women with breast cancer, remains to be seen.

An Evaluation of Ovarian Response and Pregnancy Rates with the Use of Growth Hormone as an Adjunct to IVF in Women who are Poor Responders to Standard IVF Stimulation Protocols

Background: Women who fail to respond adequately to standard ovarian stimulation protocols pose a significant treatment challenge. Research has been conducted in order to identify risk factors and causes of poor ovarian response and has attempted to identify new strategies which may improve the response to ovarian stimulation with standard IVF protocols. Growth hormone (GH) supplementation is one of these strategies proposed as a management option for poor IVF responders. GH is needed for growth and development but is also involved in the modulation of both male and female fertility through both gonadotrophin-dependent and gonadotrophin-independent actions. Some studies have shown that the supplementation of GH as an adjunct to ovarian stimulation for poor ovarian responders improves oocyte quality and increases pregnancy rates; while other studies has shown it to have no significant effect.

Effects of antiarrhythmics on the electrical restitution in perfused guinea-pig heart are critically determined by the applied cardiac pacing protocol.

What is the central question of this study? Are modifications in the restitution of ventricular action potential duration induced by antiarrhythmic drugs the same when assessed with premature extrastimulus application at variable coupling intervals (the standard stimulation protocol) and with steady state pacing at variable rates (the dynamic stimulation protocol)? What is the main finding and its importance? With class I and class III antiarrhythmics, the effects on electrical restitution determined with the standard stimulation protocol dissociate from those obtained during dynamic pacing. These findings indicate a limited value of the electrical restitution assessments based on extrasystolic stimulations alone, as performed in the clinical studies, in estimating the outcomes of antiarrhythmic drug therapies. A steep slope of the ventricular action potential duration (APD) to diastolic interval (DI) relationships (the electrical restitution) can precipitate tachyarrhythmia, whereas a flattened slope is antiarrhythmic. The derangements in APD restitution responsible for transition of tachycardia to ventricular fibrillation can be assessed with cardiac pacing at progressively increasing rates (the dynamic stimulation protocol). Nevertheless, this method is not used clinically owing to the risk of inducing myocardial ischaemia. Instead, the restitution kinetics is determined with a premature extrastimulus application at variable coupling intervals (the standard stimulation protocol). Whether the two protocols are equivalent in estimating antiarrhythmic drug effects is uncertain. In this study, dofetilide and quinidine, the agents blocking repolarizing K+ currents, increased epicardial APD in perfused guinea-pig hearts, with effects being greater at long vs. short DIs. These changes were more pronounced during dynamic pacing compared to premature extrastimulations. Accordingly, although both agents markedly steepened the dynamic restitution, there was only a marginal increase in the standard restitution slope with dofetilide, and no effect with quinidine. Lidocaine and mexiletine, selective Na+ channel blockers, prolonged the effective refractory period without changing APD, and increased the minimum DI that enabled ventricular capture during extrastimulations. No change in the minimum DI was noted during dynamic pacing. Consequently, although lidocaine and mexiletine reduced the standard restitution slope, they failed to flatten the dynamic restitution. Overall, these findings imply a limited value of the electrical restitution assessments with premature extrastimulations alone in discriminating arrhythmic vs. antiarrhythmic changes during drug therapies.

Gene expression of follicle-stimulating hormone receptors in granulosa cells in poor ovarian responders

Women with poor ovarian response do not produce adequate follicles and express low estradiol levels after standard ovarian stimulation protocols; therefore, their chances of becoming pregnant are reduced. The follicle-stimulating hormone (FSH) binds to specific follicle-stimulating hormone receptors (FSHRs) in the ovary, thereby activating adenylyl cyclase/cyclic adenosine monophosphate, a gene expression regulator. The present study aimed to evaluate whether the FSHR levels in poor ovarian responders differed from those in normal ovarian responders. This cross-sectional study was conducted in the Cipto Mangunkusomo’s Hospital. The study cohort included women with poor ovarian responses (n = 17) and control women with normal ovarian responses (n = 17) who underwent IVF procedures. The FSHR levels in ovarian tissues were measured using qPCR and then compared between the two groups. No statistically significant differences were found in the FSHR levels between the groups. A polymorphism mutation may be present in the FSHR gene of poor ovarian responders.

Progestin-primed milder stimulation with clomiphene citrate yields fewer oocytes and suboptimal pregnancy outcomes compared with the standard progestin-primed ovarian stimulation in infertile women with polycystic ovarian syndrome

BackgroundOral progestin has recently been used to prevent premature LH surges in ovarian stimulation, and this progestin-primed ovarian stimulation (PPOS) is effective and safe in patients with different ovarian reserves. The current data are lacking regarding how to individualize the gonadotropin dose and regimen for women with polycystic ovarian syndrome (PCOS). A retrospective cohort trial was performed to evaluate the efficacy of progestin-primed milder stimulation with clomiphene citrate (CC) compared to the standard progestin-primed ovarian stimulation (PPOS) protocol for infertile women with PCOS.MethodsA total of 220 PCOS women were collected and classified into the study group (HMG 150 IU/d + CC 50 mg/d + MPA 10 mg/d) and control group (HMG 225 IU/d + MPA 10 mg/d). Ovulation was triggered by GnRH agonist 0.1 mg and hCG 1000 IU when dominant follicles matured. Viable embryos were cryopreserved for later transfer. The primary endpoint was the ongoing pregnancy rate. Secondary outcomes included the cycle characteristics and the live birth rate.Result(s)The study group consumed less HMG (1470.0 ± 360.1 IU vs 1943.8 ± 372.0 IU, P < 0.001) and harvested fewer oocytes than the control group (12.2 ± 7.4 vs 18.2 ± 9.7, P < 0.001). The study group showed a higher mid-follicular LH concentration (4.49 ± 2.49 mIU/ml vs 2.52 ± 2.09 mIU/ml, P < 0.05) but no endogenous LH surge. No between-group difference was found in the incidence of ovarian hyperstimulation syndrome (OHSS) (0.91% vs 0.91%, P > 0.05). The cumulative ongoing pregnancy rate and live birth rate per patient were lower but did not reach significance compared with the control group (71.8% vs 81.8 and 64.5% vs 75.5%, respectively, both P > 0.05).Conclusion(s)The milder PPOS with CC in PCOS women led to lower oocyte yields and suboptimal pregnancy outcomes compared to the standard PPOS treatment. The two regimens both achieved a low incidence of OHSS. The results from the CC combination regimen provide a new insight for developing a more patient-friendly protocol for PCOS women.

Case report: dTMS and improvement of kidney functions in a CKD patient

This is to report a case-study of a patient with stage 3b Chronic Kidney Disease (CKD) as well as Major Depressive Disorder (MDD) to examine the relationship between the brain and the renal system. This case of significant kidney function improvement following the standard Transcranial Magnetic Stimulation (TMS) protocol used for the treatment of MDD may suggest a role of TMS in the functional improvement of CKD and requires our attention to other therapeutic effects of TMS on the human body.