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Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Background: Oocyte cryopreservation and gonadal suppression during chemotherapy with long-acting GnRH agonists (GnRHa) are standard fertility preservation (FP) methods in women with breast cancer. When both strategies are accepted by the patient, the first injection of GnRHa is frequently administered few days after oocyte retrieval in order to start oncological therapies as soon as possible. GnRHa suppress gonadal function after an initial stimulation phase due to a gonadotropins surge. Consequently, some cases of ovarian hyperstimulation (OHSS) have been described following GnRHa administration during the luteal phase of an oocyte cryopreservation cycle, as a consequence of the initial flare-up effect on recently stimulated ovaries. OHSS is associated with specific complications, such as an increased thromboembolic risk and a possible delay in the start of chemotherapy. These risks may discourage physicians from proposing gonadal suppression in combination with oocyte cryopreservation, denying women a FP opportunity with proven efficacy. However, since the same flare up effect of short-acting GnRHa (e.g. Triptorelin 0.2 mg) is commonly used to trigger ovulation in high responding infertile patients, we propose the initiation of long-acting GnRHa administration at triggering. Our hypothesis is that by inducing ovulation with long-acting GnRHa we meet the last requirement for oocytes maturation and create suppression by the time patients start chemotherapy, without the need of a further injection in the luteal phase. Methods From 2016 to 2020, 70 consecutive ovarian stimulation cycles for oocyte cryopreservation in oncological patients before chemotherapy were performed in a single university centre. Cycle outcomes were evaluated accordingly to the trigger method. Maturation rate was defined as number of cryopreserved mature oocytes/total number of oocytes retrieved. When the long-acting GnRHa was used for triggering, luteal phase hormones were assessed. Results After controlled ovarian stimulation (COS) with standard or random start antagonist protocol, ten women received the long-acting GnRHa trigger (Decapeptyl 3.75 mg, Ipsen. Group A) 36 hours before oocyte retrieval, 37 received highly purified Chorionic Gonadotrophin (Gonasi HP 10000 UI, IBSA. Group B) and 23 the short-acting GnRHa (Decapeptyl 0.2 mg, Ipsen. Group C). The groups were comparable in terms of demographic and clinical parameters. Median number of mature cryopreserved oocytes in group A was 14 (range 8-22) with a maturation rate of 81% (68-100), versus 9 (0-24) with a maturation rate of 78% (43-100) in group B, and 9 (3-34), 78% (38-100) in group C. There was no case of OHSS in Group A. Two patients developed OHSS after administration of long-acting GnRH in the luteal phase after COS, one in Group B and one in Group C. Five days after oocyte retrieval (7 days after trigger), serum FSH median level in group A was 1.28 mUI/ml (0.48-2.50) and LH median level was 1.04 mUI/ml (0.26-2.46). Conclusion We report for the first time the efficacy of long-acting GnRHa in obtaining mature oocytes and, at the same time, in guaranteeing complete suppression by chemotherapy initiation. We are aware that our data should be confirmed by more robust studies and higher numbers. Nevertheless, the feasibility of this strategy is an important step in reducing the risk of OHSS after ovarian stimulation while giving the opportunity to combine oocyte cryopreservation and ovarian suppression during chemotherapy, with proven efficacy as options to preserve fertility and ovarian function, respectively. Citation Format: Claudia Massarotti, Matteo Lambertini, Sara Stigliani, Fausta Sozzi, Paola Scaruffi, Paola Anserini. Long-acting GnRH agonist ovulation trigger to avoid ovarian hyperstimulation and to combine oocyte cryopreservation with ovarian suppression during chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-18-05.

Supplementary Data 1. This supplementary data comprises a step-by-step description on how our gene-expression based classification models were generated and validated

Supplementary Figure 1. Highlights the EFS and OS for the cohort of neuroblastoma patients with MYCN-amplified disease (n=114, Fig 1a) and for th subcohort of patients >18 months of age with stage 4, MYCN non-amplified disease (n=102, fig. 1b). F, favorable; UF, unfavorable.

Purpose: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression–based classification and established prognostic markers.Experimental Design: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan–Meier estimates and Cox regression analyses.Results: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients [5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001] and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 [EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20–8.02; OS: HR, 25.54; 95% CI, 8.40–77.66; both P < 0.001]. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation.Conclusions: Combination of gene expression–based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial. Clin Cancer Res; 21(8); 1904–15. ©2014 AACR.See related commentary by Attiyeh and Maris, p. 1782

Supplementary Table 1. Clinical co-variates for the 709 patients who participated in the study. This supplementary table comprises detailed information on clinical co-variates for all 709 neuroblastoma patients who participated in the study.

Supplementary Table 2. External performance validation of the top five classifiers. This supplementary table comprises the external performance metrics of the top five classifiers. Indicated are the values for classification accuracy, sensitivity , specificity and Matthew's correlation coefficient (MCC) in the prediction of 325 patients of the test set who fulfilled the criteria for classifier training (Favorable, n=187; Unfavorable, n=138).

Supplementary Table 4. Transcripts that contribute to the SVM_th10 classifier. This supplementary table highlights the transcripts that constitute the SVM_th10 classifier. Indicated for each feature are the Oligo-ID, the Gene symbols, the Entrez gene IDs, the RefSeq IDs, the Gene IDs and the Transcript IDs.

Legends to Supplementaries. This documents comprises the legends to the supplementary figures and tables

Supplementary Data 2. This supplementary data comprises the r-algorithm scripts required to perform the both model selection and validation as described in the step-by-step protocol

This study aims at detecting and evaluating differences in quantitative response to controlled ovarian stimulation (COS) with high doses of gonadotropins in women with low serum anti-Müllerian hormone (AMH). About 369 first cycles in a real-life scenario in women between 21 and 43 years old and with AMH ≤0.9 ng/ml were analyzed. Older women had a significantly worse outcome with respect to young women, not only qualitatively, but also in terms of quantitative ovarian response to COS [odd ratio (OR) to obtain at least three MII oocytes with each increasing year of female age: 0.89, 95% CI: 0.85 - 0.94

In Italy during the first pandemic wave of SARS-CoV-2 the activity of fertility centers was stopped, with the exception of fertility preservation in oncological patients. We adopted telehealth and we evaluated whether it could help in the management of infertile couples at a fertility center.A longitudinal study performed at a public fertility center. Telehealth was offered to 72 couples referred to our center for a first consultation from March 17th to May 31st, 2020. Percentage of patients who performed the first assisted reproduction technology (ART) cycle or intrauterine insemination (IUI) within 6 months from the first visit and drop-out rate were analyzed during COVID-19 pandemic and compared to historical controls (couples admitted to our center in 2017-2019).Eighty-five (61/72) percent of couples accepted telehealth. Time to first treatment after online consultation in telehealth group (4.5 (1.8) months) was significantly shorter (p = 0.033) respect to time to first treatment after face-to-face visit of historical controls (7.5 (6.9) months). After telehealth consultation, we observed a significant reduction (p = 0.002) of drop-out rate from 39% in historical controls to 17% of telehealth group. Telehealth significantly diminished the drop-out rate also during the COVID-19 pandemic respect to 73% after traditional face-to-face visits (p = 0.0005), with a time to first treatment of 3.7 (2.1) months in couples who refused telehealth.Telehealth could be a useful tool to facilitate the path of patients in a fertility center.

Beneficial effects of hyaluronic acid (HA)-based selection of spermatozoa for intracytoplasmic sperm injection (ICSI) are still controversial, and further studies are needed to categorize patients that might benefit from such a method.We investigated whether HA sperm selection improved ICSI outcome of couples with previous ICSI cycle failure.In this retrospective study, we prospectively collected data of (i) Group 1: 96 couples who performed one failed ICSI cycle ("1st procedure," n = 96) followed by another ICSI cycle ("2nd procedure," n = 101); ii) Group 2: 87 couples who performed one failed ICSI cycle (n = 87) followed by an HA-ICSI cycle (n = 104). Differences between procedures and groups were measured by paired and independent statistical tests, respectively. A generalized linear mixed model analyzed the effect of procedure on the outcomes and the interaction between procedures and groups.Injection of HA-bound sperm significantly improved cleavage rate with respect to standard ICSI (p = 0.026). No evolutive pregnancies were obtained in the 1st ICSI attempts. The 2nd ICSI cycles resulted in successfully seven pregnancies. In HA-ICSI cycles, the better quality of embryos with respect to ICSI (p = 0.034) increased the choice of day 5 embryo transfer (p = 0.030), which resulted in successfully 28 pregnancies. No differences were observed in clinical outcomes of the two ICSI procedures in Group 1, while pregnancy and implantation rates were significantly higher in HA-ICSI with respect to ICSI cycles (p = 0.001, p 0.0001, respectively). No negative perinatal outcomes were recorded.In couples where previous 1st ICSI failed, selection of HA-bound spermatozoa significantly improved clinical outcomes with respect to further standard ICSI.This study identified couples with previous ICSI cycles failure as a category of infertile patients that really may benefit from HA sperm selection before ICSI.

Study question Does ejaculatory frequency during the three months preceding semen collection influence semen parameters in oligospermic men? Summary answer A frequency of 2–3 ejaculations/week during the three months preceding semen collection significantly optimizes sperm motility, without any reduction in sperm concentration. What is known already Male gametes undergo crucial physiological and biochemical changes during epididymal transit, but a longer storage is known to have negative effects on semen quality, especially on motility. Previous studies focused on abstinence prior to semen collection, while few data are available on the effect of ejaculation frequency. On one hand, a longer storage could increase exposure to reactive oxygen species and a pro-inflammatory environment, with a reduction in vitality and motility. On the other, an increased ejaculation frequency could cause a reduction in sperm volume and concentration. The effects of ejaculatory frequency are particularly understudied in men with oligospermia. Study design, size, duration This is a retrospective study performed at a tertiary level public infertility center. We included all semen samples, collected both for diagnostic purposes and ART cycles between September 2019 and September 2020, with a sperm concentration of 15 million/ml or less, and an abstinence of 3- 5 days. Exclusion criteria were surgically collected or collected for fertility preservation semen samples. Participants/materials, setting, methods Standard demographic and clinical data were recorded, as well as semen parameters. Ejaculation frequency was considered “optimal” (at least 2–3/week) or “reduced” ( Limitations, reasons for caution The study is retrospective and ejaculatory frequency was self-reported as an estimate of the mean of the number of ejaculations per week. Wider implications of the findings: Optimizing ejaculatory frequency may improve ART outcomes as well as success of spontaneous conceptions. There is no reason to limit ejaculatory frequency in oligospermic men for a hypothesized benefic in sperm concentration. Trial registration number Not applicable

Study question Does hyaluronic acid (HA) sperm selection improve the intracytoplasmic sperm injection outcome of couples with previous ICSI cycles failure? Summary answer In couples where previous first ICSI failed, selection of HA-bound spermatozoa significantly improved clinical outcomes respect to further standard ICSI. What is known already HA is the major component of the matrix surrounding the human oocyte and in physiological fertilization it plays an important role in sperm selection since only mature spermatozoa express specific binding protein and are able to bind to HA. Although several studies demonstrated better outcomes of ICSI with selection of mature HA-bound spermatozoa, such a beneficial effect of HA-ICSI is still controversial and to date no firm clinical guidance for the routine use of HA can be drawn. Further studies are needed to categorize patients that really might benefit from HA sperm selection before ICSI. Study design, size, duration A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We selected 164 couples who performed one or more failed ICSI cycles with low fertilization rate and poor embryo quality in the period 2010–2020 (n = 164 cycles, group A), followed by other standard ICSI (n = 99, group B) and/or HA-ICSI (n = 96 group C) cycles. We included only fresh ejaculated sperm and fresh oocytes. Participants/materials, setting, methods Endpoints were fertilization, cleavage, top quality embryo, implantation (IR), clinical pregnancy (CPR), pregnancy loss (PLR), and live birth (LBR) rates. Comparisons among groups were performed using a generalized estimating equation model performed at patient level, to take into account the correlation between observations originating from the same woman. A p-value Main results and the role of chance The three groups were similar for number of retrieved, MII and injected oocytes. As regarding embryological outcomes, there was no difference in fertilization and cleavage rates between group A and C (fertilization: 47.55 + 29.88% versus 54.10 + 28.51%, p = 0.096; cleavage: 96.19 + 12.70% versus 97.52 + 10.50%, p = 0.519), nor between group B and C (fertilization: 60.30 + 30.73% versus 53.71 + 28.61%, p = 0.112; cleavage: 92.26 + 20.540% versus 97.55 + 10.44%, p = 0.106). Selection of HA-bound spermatozoa in ICSI significantly improved the embryo quality rate (63.78 + 35.55% versus 51.42 + 34.31% p = 0.024) and the blastulation rate (43.44 + 25.55% versus 17.93 + 25.52%, p = 0.001) respect to standard ICSI. Comparisons of clinical outcomes between group B and group C highlighted significant higher IR (26.16 + 40.47% versus 7.34 + 22.16%, p = 0.0001), CPR/cycle (32.29% (31/96) versus 12.12% (12/99), p = 0.0007, chi-square test), and lower PLR (12.90% (4/31) versus 41.67% (5/12), p = 0.0398, chi-square test) in HA-ICSI respect to standard ICSI cycles. The LBR/cycle in group B was 10.10% (10/99) and in group C was 32.29% (31/96) (p = 0.0029, chi-square test). No stillbirths as well as no malformations in newborns were recorded. Limitations, reasons for caution We are aware of the retrospective nature of the study performed in a single ART center. Wider implications of the findings: This study identified couples with previous ICSI cycles failure as a category of infertile patients that really may benefit from HA sperm selection before ICSI. Trial registration number Not applicable

STUDY QUESTION Are there reasons that motivate young cancer survivors to ask for follow-up visits at an oncofertility unit? SUMMARY ANSWER Cancer survivors request oncofertility follow-up visits for the management of treatment-related side effects or ovarian reserve evaluation, even if not (or not yet) wishing for a pregnancy. WHAT IS KNOWN ALREADY Personalised oncofertility counselling before gonadotoxic therapies is considered standard of care for young women with newly diagnosed cancer. However, the long-term follow-up of these patients in an oncofertility unit is not described in the literature other than for the use of cryopreserved material. STUDY DESIGN, SIZE, DURATION We retrospectively examined rates and reasons for the first follow-up visits of 154 consecutive young female cancer patients (age range: 18–40 years) who underwent a pre-treatment consultation between January 2012 and June 2017. Demographic and clinical data were collected, as well as information about the chosen fertility preservation method, if any. PARTICIPANTS/MATERIALS, SETTING, METHODS Rates and reasons for follow-up visits were collected and expressed as percentages. Different reasons were examined in the whole cohort and stratified for type of malignancy. Possible predictive factors for return to the follow-up visit (age, nulliparity, presence of a partner, neoplasm, having cryopreserved material) were investigated through logistic regression. MAIN RESULTS AND THE ROLE OF CHANCE Out of 154 patients, 74 returned to the oncofertility unit (48.1%) for a follow-up visit. The first visit was requested mostly at the end of anticancer therapies (51.3% versus 40.5% during therapies and 8.1% after cancer relapse). Among these patients, only 10.8% returned for the first time because they were actively desiring a pregnancy. For the others, the most common reasons for consultations were management of gynecological adverse effects of therapies (29.7%) and evaluation of ovarian reserve not linked to an immediate desire for a pregnancy (39.2%). Other patients asked for contraception (4.1%), menopause counselling (5.4%), or new fertility preservation counselling because of cancer relapse (10.8%). None of the examined factors were significantly predictive of return to the oncofertility unit. LIMITATIONS, REASONS FOR CAUTION These findings represent the experience of a single centre. A longer duration of follow-up would be needed to provide more precise information on this regard. WIDER IMPLICATION OF THE FINDINGS The role of an oncofertility unit should not be limited to proposing fertility preservation procedures. In the management of young adult cancer patients, the reproductive medical specialist should be considered a key figure not only before but also during and after anticancer treatments to explore salient aspects of gynecological and reproductive health. STUDY FUNDING/COMPETING INTEREST(S) This research did not receive any specific funding. M.L. served as a consultant for Teva and received honoraria from Theramex outside the submitted work. The other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N.A.

In ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P

Background In assisted reproduction technology embryo competence is routinely evaluated on morphological criteria but efficacy remains relatively low. Additional information could be obtained by evaluating pronuclear (PN) morphology. Up to now controversial results have been reported about the prognostic value of PN score. One of the main limitations of literature data is the use of different PN classification methods. In this regard, in 2011 the ESHRE and Alpha Scientists in Reproductive Medicine defined three PN categories to standardize zygote assessment. In this study we evaluated whether the consensus ESHRE-Alpha system for the pronuclear scoring could be an useful additional criterion to improve prediction of embryo implantation potential. Methods This is a retrospective, longitudinal, observational, cohort study. We included 3004 zygotes from 555 women who underwent ICSI treatment at our Center between January 2014 and June 2019. The PN were categorized as score 1: symmetrical, 2: non-symmetrical, 3: abnormal. A subset of 110 zygotes did not cleaved. On day 2–3 1163 embryos were transferred, 232 arrested, and 9 were cryopreserved. Among the 1490 embryos cultured up to day 5–7, 516 became blastocysts: 123 were transferred on day 5 and 393 were cryopreserved. Comparisons of age, cleavage and blastocyst rate, quality of embryos, implantation success among PN score groups were evaluated by chi-square test or Kruskal-Wallis test as appropriate. Potential predictors of embryo implantation were first tested in univariable analysis using generalized estimating equations taking into account correlation between embryos originated from the same patient. Then, variables potentially associated with implantation success (P Results There was no significant difference in patients’age, cleavage and blastulation rates, and embryo morphology among the three PNscore groups. The PN score 1-embryos had a greater implantation success respect to score 2-3-ones (OR 1.83; 95% CI 1.34-2.50, P=0.0001). Consistently, the pronuclear score remained predictive of implantation in top quality embryos (OR 1.68; 95%CI 1.17-2.42, P= 0.005). Conclusions The consensus pronuclear score may be routinely included among criteria for embryo evaluation to increase patients’ chance of becoming pregnant.

Some 30% to 80% of male sub-fertility may be associated with oxidative stress that damages spermatozoa and can decrease success of in vitro fertilization (IVF) techniques. This multicenter, longitudinal, prospective study aimed to investigate whether oral antioxidant supplementation improved the reproductive competence of men who had had low fertilization rates in their previous intracytoplasmic sperm injection (ICSI) cycles without azoospermia or severe oligozoospermia or any identifiable andrological disease. Seventy-seven men from couples who had an ICSI attempt with unexplained &lt, 60% fertilization rate took an antioxidant mix of myo-inositol, alpha-lipoic acid, folic acid, coenzyme Q10, zinc, selenium, and vitamins B2, B6, and B12. Semen parameters were analyzed before (T0) and after 90 days (T90) of treatment, and outcomes of the paired T0 and T90 cycles were compared. After the treatment there was an increase in sperm concentration (p = 0.027), total motile sperm count (p = 0.003), progressive motility (p &lt, 0.0001), and a decreasing trend of DNA-fragmented spermatozoa. Embryological outcomes (fertilization, embryo quality, blastocyst development) were significantly higher in T90 than T0 cycles. No T0 cycle resulted in an evolutive pregnancy. Conversely, in T90 cycles 29 singleton clinical pregnancies were obtained. No negative neonatal outcomes were recorded in newborns after antioxidant treatment. Diet supplementation of men who have had low fertilization rates in their previous ICSI cycles with a combination of myo-inositol, alpha-lipoic acid, folic acid, coenzyme Q10, zinc, selenium, betaine, and vitamins may improve semen reproductive potential and ICSI clinical outcome.

Simple Summary Sperm cryopreservation before gonadotoxic iatrogenic treatments is the only method currently available to preserve fertility in men with cancer. The aims of this study were to report our 15 years of experience, the clinical outcomes of assisted reproductive treatments as well as neonatal characteristics of babies born. We retrospectively reviewed 682 oncological patients who were referred to our center from 2004 to 2019 for fertility preservation. Data regarding cancer diagnosis, age, and the use of frozen semen were analyzed. The cumulative live-birth delivery rate per couple was 35%. No stillbirths, as well as no malformations in the babies born, were recorded. These successful findings demonstrated that pregnancy could be safely achieved using frozen-thawed sperm of cancer survivors who cryopreserved before gonadotoxic therapies. Abstract Cancer treatments frequently impair the reproductive ability of patients by damaging spermatogenesis. International guidelines recommend semen cryopreservation to preserve the fertility of oncological adult males and pubertal boys. However, due to the low usage rate of banked samples, not a lot of data on assisted reproductive treatments (ART) success rates in this population and follow-up data for children born are available in the literature. The aims of this study were to report our 15 years of experience, the clinical outcomes of ART as well as neonatal characteristics of babies born. We retrospectively reviewed 682 oncological patients who were referred to our center from 2004 to 2019 for fertility preservation. Over the years, only 26 patients (4%) returned to use their sperm by ART. They were survivors of leukemia and lymphomas (52%), testicular cancer (20%), and other malignant diseases (28%). These couples performed 45 cycles: 34 intracytoplasmic sperm injection (ICSI) plus 11 frozen embryo transfers. A total of 13 children were born, with 35% of the cumulative live-birth delivery rate per couple. No stillbirths or malformations were recorded. These successful findings demonstrated that pregnancy could be safely achieved using frozen-thawed sperm of cancer survivors who cryopreserved before gonadotoxic therapies.

Study question Could telemedicine help in the management of the infertile couple’s path at a fertility center? Summary answer The introduction of telemedicine increased the number of cycles within 6 months from the first consultation and reduced the drop-out rate. What is known already In Italy during the complete lockdown due to the first pandemic wave of SARS-CoV–2 the activity of fertility centers were stopped, with the exception of fertility preservation procedures for oncological patients. We therefore proposed a service of telemedicine to our patients, that we called SmartPMA. Study design, size, duration A longitudinal study performed at a public infertility center. The SmartPMA service was offered to 93 couples referred to our center from March 9th to May 31st, 2020. Initially 72 couples was interested in SmartPMA. Our center gradually re-opened and the first oocytes retrieval was performed on June 9th . Participants/materials, setting, methods Sixty-one out of 72 couples (85%) performed the SmartPMA from April 7th to June 16th, 2020. After acquiring informed consent and sending two anamnestic questionnaires, we booked a video-consultation with a gynecologist and a biologist. Afterwards, we sent medical prescriptions for appropriate clinical tests. At the re-opening, we offered the chance to start the ART cycle. Time to the first treatment and drop-out rates were compared to historical controls (2017–2019) using chi-square test. Main results and the role of chance Eleven couples declined the SmartPMA and booked an appointment at the reopening. Only 2 of these couples (18%) actually performed an IVF treatment within six months. Three out the 61 couples that accepted the SmartPMA did not perform IVF treatments because the age of women exceeded the legal limit to access to a public ART center. Twelve out of 58 couples (21%) did not performed any IVF treatment for the following reasons: 4 women spontaneously got pregnant, 1 couple gave up for medical reasons, 3 were referred to ovodonation, and 4 were lost to follow-up. Thirty-eight out of 58 couples (66%) (median age of woman: 36 + 4 years, range 27–43) performed at least one ART treatment (14 IUI, 12 ICSI cycle, 12 FIVET cycle). Eight out of 58 couples (14%) needed further clinical tests and their treatments are ongoing. The mean time to first treatment in the SmartPMA couples was 4 + 1 months (range 1–6). After SmartPMA, 66% of the couples performed the first cycle within 6 months, compared to 37% of controls (333/898 couples at their first access to our center from 2017 to 2019), p Limitations, reasons for caution We cannot exclude that the couples that joined the SmartPMA service during the pandemic period were particularly motivated to perform IVF treatments. We are aware of the small sample size and that this is a monocentric study. Wider implications of the findings: Even after the pandemic, telemedicine can be an useful tool for fertility centers to reduce the discomfort of several visits in hospital, without losing patients but rather ultimately reducing the time to treatments and drop-out. Trial registration number Not applicable

Background: In assisted reproduction technology embryo competence is routinely evaluated on morphological criteria but their efficacy remains relatively low. Additional information could be obtained by evaluation of pronuclear (PN) morphology. Up to now controversial results have been reported about prognostic value of PN score. One of the main limitation of literature data is the use of different methods of PN classification. To this regard, in 2011 the ESHRE and Alpha Scientists in Reproductive Medicine defined three PN categories to standardize the zygote assessment. In this study we evaluated whether the consensus ESHRE-Alpha system for the pronuclear scoring could be an useful additional criterion to improve prediction of embryo implantation potential.Methods: This is a retrospective, longitudinal cohort study. We included 3004 zygotes from 539 women who underwent ICSI treatment at our Center between January 2014 and June 2019. The pronuclear were categorized as score 1: symmetrical, 2: non-symmetrical, 3: abnormal. A subset of 110 zygotes did not cleaved. On day 2-3 1163 embryos were transferred, 232 arrested, and 9 were cryopreserved. Among the 1490 embryos cultured up to day 5-7, 516 became blastocysts: 123 were transferred on day 5 and 393 were cryopreserved. Relationship between pronuclear score and cleavage rate, quality of embryos, blastocyst development, and implantation rate was evaluated by chi-square test. Multivariate regression analyses corrected the results for putative confounders (age of patient; infertility cause; cleavage-stage embryo morphology grade; day of transfer). Results: There was not significant difference in patients’ age, cleavage rate and embryo morphology among the three pronuclear score groups. No reduction of blastulation rates was found in score 2-3 (34%) groups respect to score 1 (35%). The pronuclear score 1-embryos had a higher implantation rate respect to score 2-3-ones (15% and 9%, respectively, P=0.0121; OR 0.46; 95% CI 0.25-0.76, P=0.004). Consistently, the pronuclear score remained predictive of implantation in top quality embryos (OR 0.51; 95% CI 0.28-0.87, P= 0.01).Conclusions: The consensus pronuclear score may be routinely included among criteria for embryo evaluation to increase patient’s chance of becoming pregnant.

STUDY QUESTION Is there any association between the appearance of smooth endoplasmic reticulum aggregates (SERa) in oocytes and ovarian stimulation, embryological, clinical and neonatal outcomes of ICSI and IVF cycles? SUMMARY ANSWER A suboptimal prolonged ovarian stimulation is detrimental to oocytes by inducing the occurrence of SERa, which reduces the reproductive potential of oocytes. WHAT IS KNOWN ALREADY Controlled ovarian stimulation recruits oocytes of different qualities. Based on current evidence, it was agreed that non-homogeneous cytoplasm may represent the normal variability among oocytes rather than a dysmorphism with developmental significance. The only exception is the appearance of SERa within the ooplasm. Owing to the lack of univocal evidence in this literature about the safety of injecting oocytes with SERa and the mechanism responsible for the occurrence of SERa, this topic is still a matter of debate. STUDY DESIGN, SIZE, DURATION A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We included 1662 cycles (180 SERa+ and 1482 SERa−) from 1129 women (age: 20–44 years) who underwent IVF/ICSI treatments in 2012–2019. The SERa+ cycles had at least one SERa+ oocyte in the oocyte cohort. The SERa− cycles had morphologically unaffected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS We collected stimulation data and embryological, clinical, neonatal outcomes of SERa− and SERa+ cycles and oocytes. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 347 out of 12 436 metaphase II oocytes (2.8%) were affected by SER. We performed only 12 transfers involving at least one SERa+ embryo. Stimulation length (P = 0.002), serum progesterone (P = 0.004) and follicle size (P = 0.046) at trigger, number of retrieved (P = 0.004) and metaphase II (P = 0.0001) oocytes were significantly higher in SERa+ than SERa− cycles. Fertilization rate was significantly (P LIMITATIONS, REASONS FOR CAUTION Given that SERa+ oocytes were discarded in our center for years and transfers of embryos originating from affected oocytes were generally avoided, clinical outcomes of SERa+ cycles are largely attributable to the transfer of embryos derived from unaffected oocytes of SERa+ cycles and we did not have data about newborns from affected oocytes, since none of the transfers involving SERa+ embryos resulted in a progressive clinical pregnancy. WIDER IMPLICATIONS OF THE FINDINGS For the first time, we speculate that the late-follicular phase elevated serum progesterone caused by a suboptimal prolonged ovarian stimulation may be detrimental to the oocytes by inducing the occurrence of SERa, resulting in negative effects on their reproductive potential. This raises the question of whether some stimulation regimens could be worse than others and a change in stimulation protocol would reduce the possibility of producing oocytes with suboptimal maturation. In particular, our data highlight the importance of correct timing of the trigger in order to maximize oocyte collection, not only in terms of numerosity but also their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

BACKGROUND: The recent report of SARS-CoV-2 presence in semen samples of six patients, including two subjects who were recovering from the clinical disease, re-opened the discussion on possible male genital tract infection, virus shedding in semen, sexual transmission and safety of fertility treatments during the pandemic period. OBJECTIVES: To explore current data and hypothesis on the possible sites of SARS-CoV-2 infection in the male reproduction system. MATERIALS AND METHODS: We reviewed the current literature to describe: a) the evidences on angiotensin-converting enzyme 2 (AC2E) and transmembrane serine protease 2 (TMPRSS2) expression in the testes, accessory glands (including prostate) and the urinary tract; b) other coronaviruses' (SARS and MERS) ability to infect these sites. RESULTS: The co-expression of both ACE2 and TMPRSS2 genes was reported in spermatogonial stem cells, elongated spermatids, in at least a small percentage of prostate hillock cells and in renal tubular cells. Testicular damage was described in autopsies of SARS patients, without evidence of the virus in the specimens. Prostate is a known infection site for MERS-CoV. SARS-CoV-2 was detected in urines. DISCUSSION: There are still al lot of open questions on the effects of SARS-CoV-2 infection on the male reproductive tract. The presence of receptors is not a proof that the testis provides a site for viral infection and it is still unknown if SARS-CoV-2 is capable to pass the blood-testis barrier. The possibility of a prostate involvement has not been investigated yet: we have no data, but theoretically it cannot be excluded. Moreover, the RNA detected in semen could have been just a residual of urinary shedding. CONCLUSION: Opening our prospective beyond the testis could be the key to better understand the possibility of a semen-related viral transmission as well as COVID19 short and long-term effects on male reproductive function.

Failure modes and effects analysis (FMEA) is a proactive risk evaluation to identify and reduce potential failures that may occur during a procedure within a quality management programme. One of the procedures performed in assisted reproduction technology centres is testicular sperm extraction (TESE) as treatment of azoospermic patients. To examine the risks associated with the 'TESE management' process, we applied the FMEA method, before and after implementation of corrective measures defined in a standard operative procedure (SOP). A multidisciplinary team was formed. Possible causes of failures and their potential effects were identified, and risk priority number (RPN) for each failure was calculated. The FMEA team identified 4 process activities, 19 process steps and 19 potential failure modes. The re-evaluation after the corrective measures disclosed a reduction in the number of phases with high/moderate risk (pre-SOP: n = 13; post-SOP: n = 3). Improvements in the traceability system removed 11 out of 13 (85%) steps with a low risk of occurrence. In our experience, FMEA is efficient in helping multidisciplinary groups to strengthen knowledge and awareness on routine processes, identifying critical steps and planning practical improvements for a better compliance with criteria of traceability and conformity of biological samples and patients.

During the last decades, important advances in therapeutic options have led to increased survival rates in cancer patients; however, cancer treatments are associated with several potential adverse effects including infertility in those diagnosed during their reproductive years. A proper discussion about fertility preservation options before the use of therapies with potential gonadotoxicity (i.e. oncofertility counseling) is standard of care and should be offered to all patients of childbearing age. Temporary ovarian suppression with LH-RH analogs, oocyte and embryo cryopreservation are standard strategies for fertility preservation in female cancer patients. Oocyte cryopreservation should be preferred to embryo cryopreservation when this latter is prohibited by law, avoided for ethical or religious issues and in single women refusing sperm donation. Despite the increasing use of this strategy, data are still lacking about the efficacy and safety of the procedure in female cancer patients, with most of the evidence on this regard deriving from infertile non-oncologic women. This article aims at critically review the available evidence about the success of oocyte cryopreservation in female cancer patients with the final goal to further improve the oncofertility counseling of these women.

// Fabio Morandi 1 , Sebastiano Barco 2 , Sara Stigliani 3 , Michela Croce 4 , Luca Persico 5 , Corrado Lagazio 5 , Francesca Scuderi 6 , Maria Luisa Belli 6 , Mariapina Montera 7 , Giuliana Cangemi 2 , Sarah Pozzi 8 , Valentina Rigo 4 , Paola Scaruffi 3 , Loredana Amoroso 6 , Giovanni Erminio 9 , Vito Pistoia 10 , Silvano Ferrini 4 and Maria Valeria Corrias 1 1 UOC Laboratory of Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy 2 UOC Clinical Pathology Laboratory, IRCCS Istituto Giannina Gaslini, Genoa, Italy 3 UOS Physiopathology of Human Reproduction, IRCCS AOU SanMartino-IST, Genoa, Italy 4 UOC Biotherapy, IRCCS AOU SanMartino-IST, Genoa, Italy 5 Department of Economy, University of Genoa, Genoa, Italy 6 UOC Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy 7 UOC Immune-Hematology and Transfusion Medicine, IRCCS Istituto Giannina Gaslini, Genoa, Italy 8 UOC Immune-Hematology and Transfusion Medicine, IRCCS AOU SanMartino-IST, Genoa, Italy 9 UOS Epidemiology, Biostatistics and Committees, IRCCS Istituto Giannina Gaslini, Genoa, Italy 10 Immunology Area, IRCCS Bambino Gesu, Rome, Italy Correspondence to: Fabio Morandi, email: fabiomorandi@gaslini.org Keywords: neuroblastoma, erythrocytes, bone marrow, survival, microenvironment Received: February 14, 2017 Accepted: May 09, 2017 Published: May 30, 2017 ABSTRACT Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages’ maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

This study aims to evaluate levels of anxiety and depression in women, correlated with infertility per se and with infertility treatments, highlighting predictors of higher levels of distress. Two validated standardized questionnaires, the Hospital Anxiety and Depression Scale (HADS) and the Fertility Quality of Life (FertiQoL), were administered to 89 women both before their first cycle of infertility treatment and again at the end of the ovarian stimulation for in vitro fertilization (IVF). Women's levels of anxiety were significantly higher before the treatment than during the treatment itself. Stratifying the women in three groups based on principal cause of infertility (male infertility, female infertility, or both male and female), we found significantly higher levels of anxiety and general distress in patients under treatment for female infertility. Higher anxiety levels in our sample before the treatment are probably an effect of not knowing what they are expected to do to solve their problem. Moreover, when the cause of infertility is exclusively female, women experience higher levels of anxiety and general distress both before and during the treatment, probably correlated to a sense of guilt. These data help the treating physician to better counsel patients and to provide a more focused psychological support.

We here report a successful healthy childbirth and an ongoing evolutive pregnancy in a case of partial globozoospermia after selection of mature spermatozoa bound to hyaluronic acid (HA). The couple underwent two in vitro fertilisation (IVF) cycles. In the first attempt, 14 MII oocytes were retrieved. Randomly, seven oocytes were injected by conventional PVP-ICSI and seven by HA-ICSI. Fertilised oocytes were 2/7 and 4/7 after PVP-ICSI and HA-ICSI respectively. Transfer of two grade A embryos from HA-ICSI lead to birth of a healthy baby. The surplus embryo of the HA-ICSI group was vitrified at blastocyst stage. The two embryos from PVP-ICSI arrested their development. Two years after the childbirth, the vitrified blastocyst was transferred into the uterus, but implant failed. In the second cycle, 14 MII oocytes were retrieved and they were injected by HA-ICSI. Fertilised oocytes were 10 out of 14 injected oocytes. On day 5, two blastocysts were transferred into uterus and a single evolutive pregnancy is ongoing. On day 6, one blastocyst was vitrified. The remaining surplus embryos arrested their development. A healthy childbirth and an ongoing evolutive pregnancy in two consecutive ICSI attempts of the same couple suggest that HA sperm selection might assist in cases with partial globozoospermia.

// Sara Stigliani 1, * , Fabio Morandi 2, 5, * , Luca Persico 3 , Corrado Lagazio 3 , Giovanni Erminio 4 , Paola Scaruffi 1 and Maria Valeria Corrias 2 1 Physiopathology of Human Reproduction, Ospedale Policlinico San Martino, Genoa, Italy 2 Experimental Therapy in Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy 3 Department of Economy, University of Genoa, Genoa, Italy 4 Epidemiology, Biostatistics and Committees, IRCCS Istituto Giannina Gaslini, Genoa, Italy 5 Present address: Stem Cell Laboratory and Cell Therapy Center, IRCCS Istituto Giannina Gaslini, Genoa, Italy * These authors contributed equally to this work Correspondence to: Maria Valeria Corrias, email: mariavaleriacorrias@gaslini.org Keywords: miRNA; neuroblastoma; bone marrow; mitochondria; gene expression Received: August 29, 2017 Accepted: March 02, 2018 Published: April 10, 2018 ABSTRACT The miRNA expression profiles of bone marrow resident cells from children with neuroblastoma were compared to that of healthy children. No significant difference was found between localized and metastatic neuroblastoma, or between children with neuroblastoma and healthy children. By considering the fold change we identified six miRNAs over-expressed by more than 150 fold in neuroblastoma. Validation confirmed miR-221 over-expression in BM resident cells from children with neuroblastoma, regardless of localized or metastatic disease. MiR-221 over-expression was unlikely derived from neuroblastoma primary tumors or from bone marrow-infiltrating metastatic cells, since neuroblastoma cells expressed lower or similar amount of miR-221 than BM cells, respectively. To get insight on the genes potentially regulated by miR-221 we merged the list of miR-221 potential targets with the genes under-expressed by BM resident cells from children with neuroblastoma, as compared with healthy children. In silico analysis demonstrated that none of the miR-221 target genes belonged to heme biosynthetic processes found altered in children with neuroblastoma, whereas two genes associated with mitochondria. However, the encoded proteins were not under-expressed in children with neuroblastoma, making unlikely that altered erythrocyte maturation in children with neuroblastoma was mediated by miR-221. In conclusion, miRNA expression profiles of BM resident cells from children with localized and metastatic neuroblastoma were similar to that of BM resident cells from healthy children. Moreover, miRNAs expressed by neuroblastoma primary tumors or by BM-infiltrating NB cells do not appear to be involved in mediating the functional defect of erythrocyte maturation recently observed in children with neuroblastoma.

The prognosis of children with metastatic neuroblastoma (NB) > 18 months at diagnosis is dismal. Since the immune status of the tumor microenvironment could play a role in the history of disease, we evaluated the expression ofCD45,CD14,ARG1,CD163,CD4,FOXP3,Perforin-1(PRF1),Granzyme B (GRMB), andIL-10mRNAs in primary tumors at diagnosis from children with metastatic NB and tested whether the transcript levels are significantly associated to event-free and overall survival (EFS and OS, resp.). Children with high expression ofCD14,ARG1andFOXP3mRNA in their primary tumors had significantly better EFS. Elevated expression ofCD14, andFOXP3mRNA was significantly associated to better OS.CD14mRNA expression levels significantly correlated to all markers, with the exception ofCD4. Strong positive correlations were found betweenPRF1andCD163, as well as betweenPFR1andFOXP3. It is worth noting that the combination of high levels ofCD14,FOXP3, andARG1mRNAs identified a small group of patients with excellent EFS and OS, whereas low levels ofCD14were sufficient to identify patients with dismal survival. Thus, the immune status of the primary tumors of high-risk NB patients may influence the natural history of this pediatric cancer.

Does the presence of aggregates of smooth endoplasmic reticulum (SERa) impact the transcriptome of human metaphase II (MII) oocytes?.The presence of SERa alters the molecular status of human metaphase II oocytes.Oocytes presenting SERa are considered dysmorphic. Oocytes with SERa (SERa+) have been associated with reduced embryological outcome and increased risk of congenital anomalies, although some authors have reported that SERa+ oocytes can lead to healthy newborns. The question of whether or not SERa+ oocytes should be discarded is still open for debate, and no experimental information about the effect of the presence of SERa on the oocyte molecular status is available.This study included 28 women, aged38 years, without any ovarian pathology, and undergoing IVF treatment. Supernumerary MII oocytes with no sign of morphological alterations as well as SERa+ oocytes were donated after written informed consent. A total of 31 oocytes without SERa (SERa-) and 24 SERa+ oocytes were analyzed.Pools of 8-10 oocytes for both group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package.The expression profiles of SERa+ oocytes significantly differed from those of SERa- oocytes in 488 probe sets corresponding to 102 down-regulated and 283 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics disclosed that genes involved in three main biological processes were significantly down-regulated in SERa+ oocytes respective to SERa- oocytes: (i) cell and mitotic/meiotic nuclear division, spindle assembly, chromosome partition and G2/M transition of mitotic cell cycle; (ii) organization of cytoskeleton and microtubules; and (iii) mitochondrial structure and activity. Among the transcripts up-regulated in SERa+ oocytes, the most significantly (P = 0.002) enriched GO term was 'GoLoco motif', including the RAP1GAP, GPSM3 and GPSM1 genes.Raw microarray data are accessible through GEO Series accession number GSE106222 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106222).Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change3 or1/3 and FDR0.1). Surveys on clinical outcomes, malformation rates and follow-up of babies born after transfer of embryos from SERa+ oocytes are necessary.We provide information on the molecular status of SERa+ oocytes, highlighting possible associations between presence of SERa, altered oocyte physiology and reduced developmental competence. Our study may offer further information that can assist embryologists to make decisions on whether, and with what possible implications, SERa+ oocytes should be used. We believe that the presence of SERa should be still a 'red flag' in IVF practices and that the decision to inseminate SERa+ oocytes should be discussed on a case-by-case basis.This study was partially supported by Ferring Pharmaceuticals. The authors have no conflicts of interest to declare.

Background: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. Methods: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women &lt, 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: a) control, culture with the addition of b) GnRHa, c) cyclophosphamide, d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. Results: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. Conclusion: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.

Neuroblastoma is the most common pediatric cancer, arising from the neural crest cells of the sympathetic nervous system. Its most aggressive subtype, characterized by the amplification of the MYCN oncogene, has a dismal prognosis and no effective treatment is available. Understanding the alterations induced by the tumor on the various layers of gene expression is therefore important for a complete characterization of this neuroblastoma subtype and for the discovery of new therapeutic opportunities. Here we describe the profiling of 13 MYCN-amplified neuroblastoma cell lines at the genome (copy number), transcriptome, translatome and miRome levels (GEO series GSE56654, GSE56552 and GSE56655). We provide detailed experimental and data analysis procedures by means of which we derived the results described in [1].

International audience; About 50% of children with neuroblastoma (NB) show a metastatic disease and have a poor prognosis. However, disease progression is greatly variable and depends on patients' age and MYCN oncogene amplification. To investigate the role of patients' age in tumor aggressiveness, we performed array-CGH and gene expression profiles of three groups (G) of metastatic NBs: G1, stage 4S patients and MYCN single copy (MYCN−) tumors; G2, stage 4 patients, ≤18 months of age, MYCN− tumors and favorable outcome and G3, Stage 4 patients, ≥19 months with unfavorable outcome. G1 was characterized by numerical aberrations prevalently; on the contrary, all G3 tumors had structural rearrangements, whereas G2 showed an intermediate pattern. The average of numerical alterations decreased significantly from G1 to G2 to G3 (p < 0.01). Contrarily, the number of structural aberrations increased from G1 to G2 to G3 (p < 2.35 E−05). Noteworthy, G3/MYCN− NBs were characterized by several complex intrachromosome rearrangements. Expression analysis of the three groups showed significant differences in genes of Rho and Ras signaling pathways, development and adhesion, cell cycle regulation and telomerase activity. Accumulation of structural alterations increased with patients' age and was associated with a more aggressive disease. Abnormal expression of genes involved in cell cycle and telomerase in G3 may be responsible for the genomic instability in this cohort of patients. The higher DNA instability observed in G3/MYCN− NBs than in MYCN-amplified G3 may also explain why patients ≥19 months have a poor outcome independently by MYCN status.