CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line.

BACKGROUND: Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. METHODS: The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). RESULTS: In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. CONCLUSION: High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Safety of gender affirming treatment in assigned female at birth transgender people and association of androgen and estrogen ß receptor polymorphisms with clinical outcomes.

PURPOSE: Gender affirming hormone treatment (GAHT) with androgens in assigned female at birth (AFAB) people with Gender Incongruence (GI) can induce and maintain variable phenotypical changes, but individual response may be genetically determined. To clarify the role of AR and ERß polymorphisms we prospectively evaluated AFAB subjects undergoing virilizing GAHT. METHODS: Fifty-two AFAB people with confirmed GI were evaluated before (T0) and after 6 (T6) and 12 months (T12) of testosterone enanthate 250 mg i.m. every 28 days. Hormone profile (testosterone, estradiol), biochemical (blood count, glyco-metabolic profile) and clinical parameters (Ferriman-Gallwey score, pelvic organs) were evaluated at each time-point, as well as number of CAG and CA repeats for AR and ERß, respectively. RESULTS: All subjects have successfully achieved testosterone levels within normal male ranges and improved their degree of virilization, in absence of significant side effects. Hemoglobin, hematocrit and red blood cells were significantly increased after treatment, but within normal ranges. Ultrasound monitoring of pelvic organs showed their significant reduction already after 6 months of GATH, in absence of remarkable abnormalities. Furthermore, a lower number of CAG repeats was associated with a higher Ferriman-Gallwey score post treatment and a higher number of CA repeats was associated with uterine volume reduction. CONCLUSION: We confirmed safety and efficacy of testosterone treatment on all measured parameters. This preliminary data hints a future role of genetic polymorphisms to tailor GAHT in GI people, but evaluation on a larger cohort is necessary as the reduced sample size could limit data generalization at this stage.

Bladder Neck Obstruction: Experience and Management in a Sperm Bank.

Primary Bladder Neck Obstruction (PBNO) management provides medical and surgical treatment, such as transurethral incisions that can lead to retrograde ejaculation. The aim of this study was to investigate the maintenance of anterograde ejaculation and semen quality before and after this surgical procedure. A retrospective evaluation was carried out between 2011 and 2020. A total of 73 patients diagnosed with PBNO were recruited. Ejaculatory function, semen quality, and the fertility of recruited subjects were evaluated. Semen parameters-Baseline, 8.2% of patients were oligozoospermic and 12.3% had a semen volume below the WHO 2010 fifth percentile. Post-surgery, 20% of patients were oligozoospermic. We detected a significant decrease in total sperm number, a significant increase in the number of abnormal forms, and a reduction in the leukocyte concentration. Ejaculatory function-A total of 7.7% of patients reported anejaculation after transurethral incision of the bladder neck. Fertility-9.2% of the patients already had children before surgery; 13.8% had naturally conceived children in the years following surgery; 76.9% had no desire for paternity at the time. Our data have important implications for sperm bank management. The alterations in semen parameters and the risk of anejaculation suggest that the use of sperm cryopreservation before surgery for PBNO should be encouraged.

A decade of WHO 2010: total sperm number temporal trend and role of lifestyle factors.

After a huge decline in sperm concentration between 1938 and 1991 was reported, many researchers investigated the possibility of a worsening of human sperm quality. Despite massive efforts, published evidence is still controversial. Similarly, the role of lifestyle factors on semen parameters is debated. We conducted a monocentric Italian study to evaluate the total sperm number trend over the last 10 years (from 2010 to 2019). Additionally, we evaluated the association between lifestyle factors and total sperm number in order to identify possible damaging factors. We performed a retrospective study analyzing subjects aged 18-55 years who had their semen analyzed between 2010 and 2019. A total of 3329 subjects were included: 1655 subjects referred to our department (Department of Experimental Medicine, Sapienza University of Rome, Roma, Italy) for idiopathic infertility and 1674 subjects referred for preconceptional or andrological screening with no confirmed andrological diseases. Semen samples were examined according to World Health Organization (WHO) 2010 criteria by two seminologists with the same training and the same equipment. For statistical evaluations, only total sperm number (×10 6 per ejaculate) was taken into consideration. We detected no significant changes in mean total sperm number during the last decade, in either the entire population or the two subgroups (infertile group and control group). In a multivariate analysis total sperm number was significantly associated with the history of infertility, body mass index (BMI) and cigarette smoking. Our results suggest that infertile men are "vulnerable" subjects, particularly susceptible to several negative factors, many of which still remain unknown. Our study highlights the need for studies addressing men's lifestyle in order to find and reduce deleterious agents.

Sperm DNA Fragmentation and Sperm-Borne miRNAs: Molecular Biomarkers of Embryo Development?

The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile­we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.

COVID-19 and its treatments: lights and shadows on testicular function.

PURPOSE: The SARS-CoV-2 pandemic has rapidly spread worldwide and, among the others, the male gender was quickly recognized as an independent risk factor for both the disease and its consequences. Since the possibility of long-term hormonal axis changes and male gamete impairment have been hypothesized but a relatively low levels of evidence has been reached, we focused this narrative mini-review on summarizing key state-of-the-art knowledge on male reproductive effects of COVID-19 as a quick reference for reproductive health specialists. METHODS: A comprehensive Medline/PubMed and Embase search was performed selecting all relevant, peer-reviewed papers in English published from 2020. Other relevant papers were selected from the reference lists. RESULTS: Available evidence indicates that the likelihood of direct testicular damage from SARS-CoV-2 is somewhat low, but there are many indirect ways (fever, cytokine imbalance, and drugs) through which the pituitary-gonadal axis and spermatogenesis may be disrupted. These alterations are probably transient, but as available evidence is low quality, it cannot be excluded that previous pathologies or comorbidities might modulate the risk of their persistence. On the other hand, available evidence shows high safety regarding andrological health for available vaccines, although studies are mainly focused on mRNA vaccines. CONCLUSION: A careful andrological evaluation of men recovering from COVID-19 is highly recommended. Since available evidence is relatively scarce, a careful andrological follow-up and counseling of these patients are mandatory.

COVID-19 Severity and Androgen Receptor Polymorphism.

During the COVID-19 pandemic, the most severe form of the disease was most often seen in male patients. The aim of this study was to identify any male predispositions that could be used to predict the outcome of the disease and enable early intervention. We investigated CAG polymorphism in the androgen receptor gene and serum levels of testosterone and LH, which were considered as probably responsible for this predisposition. The study involved 142 patients who had recovered from COVID-19 at least three months previously and were classified according to their disease severity using the World Health Organization (WHO) classification. We observed a significant increase in the number of CAG repeats with increasing disease severity: the percentage of patients with more than 23 repeats increased two-fold from Grade I to Grade IV. Furthermore, testosterone levels were significantly lower in patients with severe disease. Reduced androgenic signaling could predispose men to a more severe form: low testosterone levels and a reduced androgen receptor activity (CAG > 23) expose the host to an excessive inflammatory response, leading downstream to the multi-organ damage seen in severe COVID-19.

Environmental disruptors and testicular cancer.

PURPOSE: Testicular cancer (TC) is the most common malignancy among young adult males. The etiology is multifactorial, and both environmental and genetic factors play an essential role in the origin and development of this tumor. In particular, exposure to environmental endocrine disruptors (EEDs), resulting from industrialization and urbanization, seems crucial both in pre-and postnatal life. However, the lack of long-term studies on a wide caseload and the difficulty in evaluating their toxic effects in vivo make it challenging to establish a causal link. This review aims to discuss the main human epidemiological studies currently available in the literature to define a possible association between these chemicals and TC. METHODS: A comprehensive Medline/PubMed and Embase search was performed, selecting all relevant, peer-reviewed papers in English published from 2002 to January 2022. Other relevant papers were selected from the reference lists. RESULTS: To date, literature evidence is limited due to the scarcity and heterogeneity of human studies and shows controversial data, highlighting the complexity of the topic. However, most human epidemiological studies seem to point toward a correlation between EEDs exposure and TC. CONCLUSION: Although the molecular mechanisms are not yet fully understood, the role of EEDs in TC onset is plausible, but several factors, such as the individual genetic background, the exposure time, and the complex mechanism of action of these chemicals, do not allow defining the causal link with certainty and make further studies necessary to investigate this complex topic.

Comment on Boitrelle et al. The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis. Life 2021, 11, 1368.

We wish to congratulate Boitrelle and colleagues for their comprehensive critical review on the Sixth Edition of the WHO Laboratory Manual for Human semen examination [...].

4BNT162b2 mRNA COVID-19 vaccine and semen: What do we know?

BACKGROUND: The effects of an mRNA COVID-19 vaccine on spermatozoa parameters are not known. The aim of this study was to evaluate the effect of the BNT162b2 mRNA COVID-19 vaccine on human semen, comparing spermatozoa parameters before and after vaccine inoculation. MATERIALS AND METHODS: In this single-center prospective study, voluntary subjects who received mRNA vaccines from February to July 2021 were enrolled. The study population included male subjects aged between 18 and 45 years who completed the BNT162b2 mRNA COVID-19 vaccine cycle. All subjects were evaluated before the first dose of vaccine (T0) and after 3 months (T1) with semen analysis and further analysis of seminal plasma, including colorimetric determination of reactive oxygen metabolites (d-ROM test), electrolytes, and interleukin 6 (IL-6) assessment by enzyme-linked immunosorbent assay technology. RESULTS: The experimental sample included 47 subjects (age: 29.3 ± 6.0 years, range 24-32; body mass index: 23.15 ± 2.5 kg/m2 , range 19.2-28.0). All the subjects reported no systemic side effects. No significant differences were observed in any spermatozoa parameter between T0 and T1. A subanalysis was performed in oligoazoospermic and asthenozoospermic subjects, confirming the same results. Electrolyte analysis also showed no significant differences before and after vaccine inoculation. Finally, no significant differences were observed in T0, compared to T1 for the d-ROM test and IL-6. DISCUSSION AND CONCLUSION: In this study, no significant differences in spermatozoa parameters before and after vaccine inoculations were found. Furthermore, oxidative stress analysis,, the activity of the cell membrane, and IL-6, as a marker of inflammation, was not affected by the mRNA COVID-19 vaccine. These results suggest that this vaccine is safe for male semen quality.

Epigenetic Effects of Gender-Affirming Hormone Treatment: A Pilot Study of the ESR2 Promoter's Methylation in AFAB People.

Virilization of gender-incongruent subjects to whom were assigned the female gender at birth (AFAB) is achieved through testosterone administration. Inter-individual differences in the timing and acquisition of phenotypic characteristics, even if the same hormone preparations and regimens are used, are frequently observed. Polymorphisms of sex hormone receptors and methylation of their gene promoters, as well of several imprinted genes as H19, may underlie the differential response to treatment. Thus, the aim of this study was to examine the possible relationship between the CpG methylation profile of the estrogen receptor 2 gene (ESR2) and H19 promoters and their influence on phenotype modifications in a cohort of AFAB people at baseline (T0) and after 6 mo (T6) and 12 mo (T12) of testosterone therapy (testosterone enanthate, 250 mg i.m. every 28 d). A total of 13 AFAB subjects (mean age 29.3 ± 12.6) were recruited. The percentage of methylation of the ESR2 promoter significantly increased at T6 (adj. p = 0.001) and T12 (adj. p = 0.05), while no difference was detected for H19 (p = 0.237). Methylation levels were not associated with androgen receptor (AR)/estrogen receptor beta (ERß) polymorphisms nor hormone levels at baseline and after six months of treatment. On the other hand, total testosterone level and patient age resulted in being significantly associated with ESR2 methylation after twelve months of treatment. Finally, the difference in ESR2 promoter methylation between T6 and baseline was significantly associated with the number of CA repeats of the ERß receptor, adjusted vs. all considered variables (R2 = 0.62, adj. R2 = 0.35). No associations were found with CAG repeats of the AR, age, and estradiol and testosterone levels. Despite the small sample size, we can hypothesize that treatment with exogenous testosterone can modify the ESR2 methylation pattern. Our data also indicated that epigenetic changes may be regulated, suggesting that the modulation of estrogen signaling is relevant shortly after the beginning of the treatment up to T6, with no further significant modification at T12. Furthermore, estrogen receptor methylation appears to be associated with the age of the subjects and exogenous testosterone administration, representing a marker of androgenic treatment. Nonetheless, it will be necessary to increase the number of subjects to evaluate how epigenetic regulation might play a relevant role in the modulation of phenotypical changes after testosterone treatment.

Molecular Analysis of DPY19L2, PICK1 and SPATA16 in Italian Unrelated Globozoospermic Men.

This study aims to evaluate genetic contribution and sperm DNA fragmentation (SDF) in a cohort of 18 unrelated globozoospermic Italian men (Group G). Semen samples were assessed according to the WHO 2010 Laboratory Manual and compared with 31 fertile controls. We focused our genetic analysis on the exons of the main globozoospermia-associated genes, performing qualitative PCR to assess deletion of DPY19L2 and sequencing to detect mutations of SPATA16 and PICK1. SDF was evaluated using the TUNEL assay. In Group G, 10 patients had a complete form of globozoospermia, whereas 8 patients had a partial form. Molecular analysis revealed deletion of DPY19L2 in six of the patients, all of them with complete globozoospermia, while no mutations were found in the examined exons of PICK1 and SPATA16. TUNEL analysis showed a higher SDF% in Group G. Our findings confirm DPY19L2 defects as the most frequent genetic alteration in Italian patients contributing to globozoospermic phenotypes. Furthermore, spermatozoa with acrosomal defects could also display high levels of SDF as a possible consequence of abnormally remodeled chromatin. The possible effect on offspring of chromatin structure abnormalities and altered DNA integrity should be carefully evaluated by clinicians, especially regarding the feasibility and safety of artificial reproductive techniques, which represent the only treatment that allows these patients to conceive.

Sperm DNA damage and cytokines in varicocele: A case-control study.

Varicocele is a vascular disease characterised by the abnormal enlargement of the pampiniform plexus veins and a well-known cause of male infertility. The aim of this study was to investigate the relationship between sperm DNA fragmentation (SDF) and inflammation in the pathogenesis of varicocele. We included 84 varicocele patients and 85 normozoospermic healthy controls, further analysed according to the body mass index, the smoking habit (smokers/non-smokers) and the varicocele severity (low/high grade). Semen parameters, SDF (by TUNEL) and inflammatory cytokines (by Luminex xMAP analysis) were evaluated. Varicocele patients showed significantly reduced semen parameters (volume, total sperm number, progressive motility, normal morphology) and increased SDF. Moreover, we observed a significant reduction of IFN-γ, IL-6, TNF-α and an increase of IL-10. No difference was reported according to the smoking habit, body mass index and varicocele severity. The observed cytokines pathway suggests the establishment of a chronic inflammatory condition, which may contribute to the alteration of semen quality. A thorough knowledge of the cytokine network might contribute to better understanding the link between inflammation and semen quality in varicocele and its impact on reproductive health.

Effects of endocrine disruptors on fetal testis development, male puberty, and transition age.

PURPOSE: Endocrine disruptors (EDs) are exogenous substances able to impair endocrine system; consequently, they may cause numerous adverse effects. Over the last years, particular focus has been given to their harmful effects on reproductive system, but very little is known, especially in males. The aim of this review is to discuss the detrimental effects of EDs exposure on fetal testis development, male puberty, and transition age. METHODS: A search for the existing literature focusing on the impact of EDs on fetal testis development, male puberty, andrological parameters (anogenital distance, penile length, and testicular volume), and testicular cancer with particular regard to pubertal age provided the most current information available for this review. Human evidence-based reports were given priority over animal and in vitro experimental results. Given the paucity of available articles on this subject, all resources were given careful consideration. RESULTS: Information about the consequences associated with EDs exposure in the current literature is limited and often conflicting, due to the scarcity of human studies and their heterogeneity. CONCLUSIONS: We conclude that current evidence does not clarify the impact of EDs on human male reproductive health, although severe harmful effects had been reported in animals. Despite controversial results, overall conclusion points toward a positive association between exposure to EDs and reproductive system damage. Further long-term studies performed on wide number of subjects are necessary in order to identify damaging compounds and remove them from the environment.

SARS-CoV-2 presence in seminal fluid: Myth or reality.

Great concerns have been raised on SARS-CoV-2 impact on men's andrological well-being, and one of the critically unanswered questions is whether it is present or not in the seminal fluid of infected subjects. The expression of ACE2 and TMPRSS2 in the testis and in the male genital tract allows speculations about a possible testicular involvement during the infection, possibly mediated by local and/or systemic inflammation that might allow a high viral load to overcome the hemato-testicular barrier. To date, few investigations have been carried out to ascertain the presence of SARS-CoV-2 in the seminal fluid with contrasting results. Furthermore, the cumulative number of subjects is far too low to answer the question unambiguously. Therefore, great caution is still needed when evaluating this data; otherwise, we risk unleashing unmotivated concerns in the scientific world with troublesome consequences in reproductive medicine.

Cryopreserved Gamete and Embryo Transport: Proposed Protocol and Form Templates-SIERR (Italian Society of Embryology, Reproduction, and Research).

Introduction: In Italy, the transport of cryopreserved biological material is controlled by several Decrees (Legislative Decree No. 191/2007 and No. 16/2010 and Health Ministry's Decree of October 10, 2012). Given the nature of their applications, the transport of reproductive cells has peculiar quality and safety requirements that must be applied universally, minimizing the chance of error. To standardize the cross-border shipping procedure to meet the quality, traceability, and safety criteria for cells and tissues, it is appropriate to establish a unified process using the same tools, forms, and communication channels. Methods: A working group has been created by SIERR. This "FOCUS Group" was constituted by representatives from Italian-assisted reproductive technology centers and sperm banks who worked together to define joint procedural steps and create specific forms to support the movement of cryopreserved samples. Results: The FOCUS Group identified the critical steps in the communication procedures between Italian centers and created the related forms: patient authorization, request from the recipient center, critical checks carried out by both sending and recipient centers, start of samples transfer, collection, transport and taking responsibility of the biological material, acknowledgment of samples arrival, and acknowledgement of any adverse event that occurred. Discussion: Indications on shipping between tissue institutions and legal responsibilities are important points and a working protocol with shared transport forms has been defined. Standard Operating Procedures are necessary in light of the increasingly widespread movement of biological samples between the various countries, and represent a valid means of support for the patients who could have a higher awareness of safety and traceability during each stage of gamete transport.

Semen quality in non-Hodgkin lymphoma survivors: a monocentric retrospective study.

STUDY QUESTION: How is semen quality affected by treatment in survivors of non-Hodgkin lymphoma (NHL)? SUMMARY ANSWER: Before cancer treatment, most NHL subjects were normozoospermic and, while standard first-line treatments seemed compatible with post-treatment recovery after 18 months, salvage therapy followed by haematopoietic stem cell transplant caused permanent damage to spermatogenesis in many cases, with 66% azoospermic subjects in the long term. WHAT IS KNOWN ALREADY: Testicular function has been widely investigated in relation to the most common malignancies in men of reproductive age, such as testicular cancer and Hodgkin lymphoma, but NHL has been somewhat under-investigated. The available reports generally show a post-treatment worsening of semen parameters in NHL survivors, but they involved small caseloads or a subgroup of broader caseloads, and their results are not comparable. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective analysis of 222 subjects who attended our University Hospital Sperm Bank between 2002 and 2017 for sperm cryopreservation after a diagnosis of NHL. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 222 patients with NHL who underwent sperm cryopreservation before any antineoplastic treatment. Subjects with any comorbidity and/or other conditions interfering with sperm parameters were excluded. All patients underwent a careful medical history and physical examination at the time of sperm cryopreservation (T0) and had at least one follow-up visit at 6 (T6), 12 (T12), 18 (T18) and/or 24 months (T24) or more than 24 months (T > 24), with a median follow-up of 47.5 months (range 28-140 months). Fertility information was collected through the administration of a questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-treatment, more than 80% of NHL patients were normozoospermic and in 15.9% of cases had already fathered a child. Aggressive lymphomas were associated with worse baseline semen volume and total sperm number compared to indolent subtypes (P < 0.05). Post-treatment analyses showed that standard first-line treatments alone had a more favourable outcome than intensified regimens for semen parameters, with total sperm number returning to near-baseline values at 18 months (T0: 195.0 ± 189.8 versus T18: 113.4 ± 103.1, P = 0.278), and a 7.7% prevalence of azoospermia at 2 years. In this subgroup receiving standard first-line treatments, radiotherapy of the pelvis versus other 'high' sites (mediastinum, latero-cervical and axillary lymph nodes, etc.) was associated with an increased risk of developing post-treatment azoospermia (odds ratio 4.29, 95% CI 1.81-10.14; P = 0.001). Two-thirds of subjects who had relapsed or had disease progression after first-line treatment and then underwent salvage treatment ± haematopoietic stem cell transplant became azoospermic. Fertility data were available for 176 patients: 15.9% already had at least one child prior to the NHL diagnosis and 12.5% (22 patients) desired children after treatment. Fourteen patients achieved fatherhood: 12 through natural conception and two following ART. LIMITATIONS, REASONS FOR CAUTION: The main limitations of the study are the lack of data on blood hormones for evaluation of testicular function as a whole and the non-compliance of several patients in attending follow-up visits at all time points, resulting in a reduced sample size for the treatment subgroup analyses. Furthermore, despite a good fertility questionnaire response rate (>80%), the low number of NHL survivors actively seeking fatherhood limits the generalization of results. WIDER IMPLICATIONS OF THE FINDINGS: The increased survival of NHL patients of reproductive age makes it essential to focus on the testicular toxicity of the treatment. Sperm cryopreservation must be suggested before any treatment. Two years after first-line treatments, sperm number showed signs of recovery: this finding is of the utmost importance for oncofertility counselling, as it indicates that only a standard first-line chemotherapy in many patients may be compatible with at least a partial spermatogenesis recovery in the long term. Nonetheless, it is expected that up to 30% of subjects will require treatment intensification, which could result in permanent testicular damage; in such cases the use of banked semen might represent the patient's best chance for future fertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Italian Ministry of Education and Research (MIUR-PRIN 2015-2015XSNA83-002) and the 'Sapienza' University of Rome, Faculty of Medicine. The authors report no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Phthalates and Bisphenol A: Presence in Blood Serum and Follicular Fluid of Italian Women Undergoing Assisted Reproduction Techniques.

BACKGROUND: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. METHODS: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. RESULTS: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan-Turin, Area 2: Rome-Naples; Area 3: Catania-Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. CONCLUSIONS: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).

Correction: gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.