A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm.

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.

Occurrence of smooth endoplasmic reticulum aggregates in metaphase II oocytes: relationship with stimulation protocols and outcome of ICSI and IVF cycles.

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 < 0.0001) reduced in SERa+ cycles and oocytes compared to SERa- counterparts. Embryos of SERa+ cycles had a lower blastocyst formation rate compared to embryos of SERa- cycles (P = 0.059). Statistical analysis according to a generalized estimating equation model performed at patient level demonstrated that the duration of ovarian stimulation was predictive of SERa+ oocytes appearance. The clinical success of SERa+ cycles was lower than SERa- cycles, although no differences in neonatal birthweights or malformations were recorded in sibling unaffected oocytes of SERa+ cycles. 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.

Pronuclear score improves prediction of embryo implantation success in ICSI cycles.

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<0.05) were included in a multivariable analysis for calculating the adjusted odds ratio (OR) and 95% confidence interval (CI). 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.

Contraception in cancer survivors: insights from oncofertility follow-up visits.

OBJECTIVE: Current literature suggests that cancer survivors are less likely to receive adequate contraception counseling. However, limited data existed on barriers to contraception usage in this population and on the efficacy of dedicated consultations. This study aims to describe how contraception is perceived by cancer survivors after counseling and acceptance rates of highly effective contraceptives. METHODS: We retrospectively analyzed clinical records from 313 consecutive cancer survivors at their first follow-up visit at the Oncofertility Unit of a tertiary hospital, from 2014 to 2019. Contraception acceptance and choice were examined stratified for the type of malignancy (hormone-sensible or not). A multivariate logistic regression model was used to evaluate possible predictors of acceptance. RESULTS: Thity-three women were excluded from the analysis because trying to conceive or already pregnant. Out of the remaining 280, only 9 (3.2%) asked spontaneously for contraception, in all the other visits the issue was brought up by the physician. After counseling 44.3% of the women without contraindications still opted out effective methods for fear of hormones or refusal of more medications. Age < 33 years and being in a relationship were correlated with acceptance. CONCLUSIONS: Even after a complete counseling in a dedicated service, fears of hormones and refusal of more medications remain strong issues for these patients. Family planning needs to be discussed with cancer survivors, preferably in the context of a long-term healthcare relationship. The Oncofertility Unit should become a privileged place for this type of counseling.

Influence of age on response to controlled ovarian stimulation in women with low levels of serum anti-Müllerian hormone.

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; p < .001]. This study endorses that age is a significant factor when counseling patients with low AMH. AMH levels per se are not a reason to exclude patients from a COS treatment, since pregnancy and live birth can be achieved, especially in younger patients. However, with an AMH equally low, the ovarian response worsens with age, making questionable the effectiveness of a stimulation with high-dose gonadotropins in the older subgroup.

Failure modes and effects analysis for testicular sperm extraction management process.

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.

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study.

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 < 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

Beyond fertility preservation: role of the oncofertility unit in the reproductive and gynecological follow-up of young cancer patients.

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.

Impact of infertility and infertility treatments on quality of life and levels of anxiety and depression in women undergoing in vitro fertilization.

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.

A successful healthy childbirth and an ongoing evolutive pregnancy in a case of partial globozoospermia by hyaluronic acid sperm selection.

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.

Gonadotropin Releasing Hormone Agonists Have an Anti-apoptotic Effect on Cumulus Cells.

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 < 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.

Presence of aggregates of smooth endoplasmic reticulum in MII oocytes affects oocyte competence: molecular-based evidence.

STUDY QUESTION: Does the presence of aggregates of smooth endoplasmic reticulum (SERa) impact the transcriptome of human metaphase II (MII) oocytes?. SUMMARY ANSWER: The presence of SERa alters the molecular status of human metaphase II oocytes. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: This study included 28 women, aged <38 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: Raw microarray data are accessible through GEO Series accession number GSE106222 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106222). LIMITATIONS, REASONS FOR CAUTION: Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change >3 or <1/3 and FDR < 0.1). Surveys on clinical outcomes, malformation rates and follow-up of babies born after transfer of embryos from SERa+ oocytes are necessary. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(s): This study was partially supported by Ferring Pharmaceuticals. The authors have no conflicts of interest to declare.

Storage time does not modify the gene expression profile of cryopreserved human metaphase II oocytes.

STUDY QUESTION: Does storage time have any impact on the transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? SUMMARY ANSWER: The length of cryostorage has no effect on the gene expression profile of human MII oocytes. WHAT IS KNOWN ALREADY: Oocyte cryopreservation is a widely used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e. women undergoing gonadotoxic therapies) and oocyte donation programs. Although cryopreservation has negative impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. STUDY DESIGN, SIZE, DURATION: This study included 27 women, ≤38 years aged, without any ovarian pathology, undergoing IVF treatment. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pools of ≈10 oocytes for each 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. MAIN RESULTS AND THE ROLE OF CHANCE: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle, cellular response to DNA damage and to stress, DNA repair, calcium ion binding, malate dehydrogenase activity and mitochondrial activity. Among the probes significantly up-regulated in cryopreserved oocytes, two corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs. LIMITATIONS, REASONS FOR CAUTION: Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change >3 or <1/3 and FDR < 0.1). Further research should be undertaken to verify experimentally that the length of cryostorage has no effect on gene expression profile of vitrified/warmed MII oocytes, as well as to include in analyses 'older' frozen oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Confirmation that the length of storage does not alter the gene expression profile of frozen oocytes is noteworthy for the safety issue of long-term oocyte banking, i.e. fertility preservation, gamete donation. STUDY FUNDING/COMPETING INTEREST: This study was supported by a grant of the Italian Ministry of Health (CCM 2012) and by Ferring Pharmaceutical company. The authors have no conflicts of interest to declare.

Segmental chromosome aberrations converge on overexpression of mitotic spindle regulatory genes in high-risk neuroblastoma.

Integration of genome-wide profiles of DNA copy number alterations (CNAs) and gene expression variations (GEVs) could provide combined power to the identification of driver genes and gene networks in tumors. Here we merge matched genome and transcriptome microarray analyses from neuroblastoma samples to derive correlation patterns of CNAs and GEVs, irrespective of their genomic location. Neuroblastoma correlation patterns are strongly asymmetrical, being on average 10 CNAs linked to 1 GEV, and show the widespread prevalence of long range covariance. Functional enrichment and network analysis of the genes covarying with CNAs consistently point to a major cell function, the regulation of mitotic spindle assembly. Moreover, elevated expression of 14 key genes promoting this function is strongly associated to high-risk neuroblastomas with 1p loss and MYCN amplification in a set of 410 tumor samples (P < 0.00001). Independent CNA/GEV profiling on neuroblastoma cell lines shows that increased levels of expression of these genes are linked to 1p loss. By this approach, we reveal a convergence of clustered neuroblastoma CNAs toward increased expression of a group of prognostic and functionally cooperating genes. We therefore propose gain of function of the spindle assembly machinery as a lesion potentially offering new targets for therapy of high-risk neuroblastoma.

Telehealth for infertile patients during SARS-CoV-2 pandemic: far, and yet close.

BACKGROUND: 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. METHODS: 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). RESULTS: Eighty-five (61/72) percent of the 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. CONCLUSIONS: Telehealth could be a useful tool to facilitate the path of patients in a fertility center.

Collecting semen samples at home for fertility assessment: time for a new standard?

BACKGROUND: In fertility clinics the standard approach to semen collection involves a private room close to the laboratory to avoid fluctuations in temperature and to control the time between collection and processing. There are still no firm conclusions whether collecting semen at home has any influence on sperm quality and reproductive competence. The purpose of this study was to assess whether the site of semen collection affects semen parameters. METHODS: This retrospective cohort study performed at a tertiary level public fertility center included 8634 semen samples from 5880 men undergoing fertility assessment from 2015 to 2021. The impact of sample collection site was evaluated using a generalized linear mixed model. A subgroup analysis comparing clinic to home collection within the same patient was performed on 1260 samples from 428 men by paired t-test or Wilcoxon Signed Rank Test. RESULTS: Samples collected at home (N.=3240) had significantly higher semen volume, sperm concentration and total sperm count respect to samples collected at clinic (N.=5530) (median (range): 2.9 (0.0-13.9) mL versus 2.9 (0.0-11.5) mL, P=0.016; 24.0 (0.0-252.0) million/mL versus 18.0 (0.0-390.0), P<0.0001; 64.6 (0.0-946.0) million versus 49.3 (0.0-1045.0), P<0.0001, respectively). There was no difference in abstinence period and sperm motility. Paired comparisons of semen characteristics in 428 patients with home-collected (N.=583) and clinic-collected (N.=677) samples confirmed a no negative effect on volume and total sperm count. CONCLUSIONS: Our data provide evidence for a not disadvantage with collection at home.

Does SARS-CoV-2 Affect the Prostate? A Molecular Analysis from a Case Series of COVID-19 Patients.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) causes COVID-19 that has been spreading worldwide since December 2019. Viral entry into cells requires expression of both angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) on the surface of the host cell. The male reproductive system, including the prostate, was supposed to be a potential target for SARS-CoV-2 since the presence of ACE and TMPRS2 receptors. This paper investigated for the first time the presence of SARS-CoV-2 mRNA in the prostatic tissue of a patient with active infection. In addition, we searched for the virus in the prostate of five patients after their recovery from COVID-19. The SARS-CoV-2 RNA was not detected in any of the prostate tissues tested even during the acute phase of infection. As case series have limitations, causality cannot be excluded and sporadic evidence of prostatic tissue invasion by SARS-CoV-2 may be detectable.

The Role of Cyclin-Dependent Kinases (CDK) 4/6 in the Ovarian Tissue and the Possible Effects of Their Exogenous Inhibition.

The combination of cyclin-dependent kinase (CDK) 4/6 inhibitors with endocrine therapy is the standard treatment for patients with HR+/HER2- advanced breast cancer. Recently, this combination has also entered the early setting as an adjuvant treatment in patients with HR+/HER2- disease at a high risk of disease recurrence following (neo)adjuvant chemotherapy. Despite their current use in clinical practice, limited data on the potential gonadotoxicity of CDK4/6 inhibitors are available. Hence, fully informed treatment decision making by premenopausal patients concerned about the potential development of premature ovarian insufficiency and infertility with the proposed therapy remains difficult. The cell cycle progression of granulosa and cumulus cells is a critical process for ovarian function, especially for ensuring proper follicular growth and acquiring competence. Due to the pharmacological properties of CDK4/6 inhibitors, there could be a potentially negative impact on ovarian function and fertility in women of reproductive age. This review aims to summarize the role of the cyclin D-CDK4 and CDK6 complexes in the ovary and the potential impact of CDK4/6 inhibition on its physiological processes.

Age-dependent accumulation of genomic aberrations and deregulation of cell cycle and telomerase genes in metastatic neuroblastoma.

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.

Bone marrow of neuroblastoma patients shows downregulation of CXCL12 expression and presence of IFN signature.

BACKGROUND: At diagnosis, children with neuroblastoma (NB) present with either localized or metastatic disease. Since the mechanisms responsible for BM invasion are not well known, we investigated the transcriptome of resident BM cells from NB patients as compared to healthy children. PROCEDURE: Ninety-two and 88 children with localized and metastatic NB, respectively, and 15 healthy children were included in the study. BM resident cells recovered from BM aspirates by immunomagnetic bead manipulation were subjected to genome-wide microarray analysis. After validation in an independent set of samples, the genes significantly modulated in resident BM cells from NB patients were tested for their diagnostic/prognostic values. RESULTS: BM resident cells, irrespective of neoplastic cell invasion, significantly overexpressed genes involved in innate immune responses, and interferon (IFN) and IFN-DRS signatures were enriched. Genes coding for metallothioneins and zinc finger proteins, and involved in histone and nucleosome/chromatin organization were also overexpressed. Resident BM cells from NB patients significantly downregulated genes involved in cell adhesion, and in erythrocyte, myeloid, and platelet differentiation pathways. Among downregulated genes, CXCL12 expression reached near complete silencing in patients with metastatic disease. The downregulation of CXCL12 expression was independent of contact between NB cell and resident BM cell. CONCLUSIONS: We demonstrated that NB tumor growth at the primary site can alter the BM microenvironment, and the presence of BM-infiltrating NB cells makes the alterations more pronounced. Therefore, the restoration of a BM physiological state by means of IFN-α monoclonal antibody, Sifalimumab, and selective noradrenaline receptor blockers should be further studied to ameliorate patients' clinical management.