In vitro manipulation of human spermatozoa during Assisted Reproductive Technology (ART) can induce several damages to sperm structure and functions. This study investigated the protective effects of Raphanus sativus seed extract and its active compounds on several sperm parameters during in vitro incubation and cryopreservation. Extracts from five seed-batches were characterized by HPLC-DAD-MS and 1H-NMR, identifying sinapine and sinipic glycosides as the main characteristic compounds. Sperm DNA fragmentation (sDF) was detected by the Sperm Chromatin Dispersion test and LiveTUNEL. Excessive reactive oxygen species (ROS) production was detected by MitoSOX Red in viable spermatozoa. Caspase activity was detected by FLICA. Cryopreservation was conducted with two alternative freezing media. In vitro incubation with the extract protected against the loss of motility and reduced the induction of sDF, sperm ROS production, and caspase activity. Similarly, during cryopreservation, it allowed much better recoveries of sperm viability, motility, and DNA integrity by decreasing sperm ROS production with both freezing media. Sinapine and sinapic acid completely mimicked the protective effects of the whole extract during both in vitro incubation and cryopreservation, suggesting that they are included among the active principles. These findings support Raphanus sativus seed extract and its active compounds as candidates for inclusion in handling and freezing media for human spermatozoa in ART.

BACKGROUND:Semen analysis remains the first-line diagnostic test for male infertility, although it is affected by operator subjectivity. Computer-assisted sperm analysis (CASA) is proposed to improve objectivity; however, its accuracy and clinical relevance remain uncertain. OBJECTIVES:We evaluated 424 samples from men attending routine semen analysis and 179 samples from male partners of ICSI couples to compare manual assessment with a CASA system (Ceros II), and to explore whether CASA-derived kinematic parameters, in particular hyperactivated motility (HA), predict ICSI outcomes. MATERIALS AND METHODS:Manual (according to WHO manual) and CASA assessments were compared using correlation analysis, Passing-Bablok regression, and Bland-Altman plots; kinematic parameters were evaluated in relation to ICSI outcomes. RESULTS:Significant correlations were found between CASA and manual assessment for rapid progressive motility and for sperm count for samples with less than 50 × 106/mL spermatozoa, whereas agreement was lost above this value, unless dilution was performed. Among CASA kinematic parameters, HA measured in whole semen was significantly higher in cycles achieving clinical pregnancy and resulted an independent pregnancy predictor also after adjustment for female age and female factor, showing good discriminative performance. DISCUSSION:These findings suggest that CASA provides objective, reproducible assessment of sperm motility, and that hyperactivated motility may serve as a clinically relevant marker to improve prediction of ICSI outcomes. CONCLUSIONS:CASA may provide useful support for routine semen analysis and identify clinically meaningful kinematic parameters. TRIAL REGISTRATION:Not applicable.

In order to acquire progressive motility, complete maturation and compaction of chromatin, regulate their volume, and acquire molecules necessary for fertilization, spermatozoa released from the testis must transit through the epididymis, a long convoluted tubule that connects the efferent ducts to the vas deferens, where they undergo several molecular modifications. Sperm modifications occurring during transit in the three segments that compose the epididymis (caput, corpus, and cauda) are accomplished by epididymal epithelium secretions, including epididymosomes (extracellular microvesicles enriched in cholesterol and proteins), miRNA, and other macromolecules. Epididymal pH and electrolytes composition of the luminal fluid are also important for a correct sperm maturation. Epididymal secretions are regulated by a variety of factors, mostly androgens and estrogens, to create a different luminal environment in each epididymal segment supporting progressive sperm maturation and allowing maintenance of sperm viability and motility during storage in the cauda. Finally, epididymal contraction allows sperm emission at ejaculation. Overall, the role of epididymis on the development of sperm functions is essential for male reproduction, and alterations in any of its functions may lead to subfertility or infertility. Due to its importance for a successful male reproductive function, the epididymis appears to be a promising target for post-testicular male contraception.

Sperm preparation is a critical step in assisted reproduction, aiming to isolate spermatozoa with optimal characteristics and high fertilizing potential. Traditional sperm selection methods involve centrifugation, which may cause sperm damage. Microfluidic sperm sorting (MSS) offers an alternative approach, mimicking the female reproductive tract environment, avoiding centrifugation, and reducing manipulation and processing time. This study aims to compare the performance of MSS and Swim-up (SU) in 26 normozoospermic, 31 hyperviscous normozoospermic, 15 oligozoospermic, and 9 asthenozoospermic subjects. Semen samples were collected from male subjects undergoing routine semen analysis at Careggi University Hospital, Florence. Sperm selection was carried out using both SU and MSS. The parameters assessed included sperm motility, viability, concentration, kinematics, DNA fragmentation (sDF), chromatin compaction, and oxidative status. Both SU and MSS improved sperm characteristics compared to unselected samples. MSS isolated high-quality spermatozoa with lower sDF and higher chromatin compaction than SU, not only in normozoospermic samples but also in samples with semen defects like hyperviscosity, low concentration and/or motility, and high sDF. In conclusion, the use of microfluidics may enhance the chances of successful fertilization and improve reproductive outcomes, especially for individuals with compromised semen quality where conventional methods may fail.

Does the microfluidic device select high-quality spermatozoa from samples with compromised semen quality? Microfluidics selects high-quality spermatozoa compared to Swim-up technique, even in samples with compromised semen parameters. Sperm preparation is essential for isolating high-quality spermatozoa to be used in Assisted Reproductive Technology (ART). Traditional methods, such as Density Gradient Centrifugation (DGC) and Swim-up, can lead to oxidative stress and DNA damage, particularly in compromised semen samples. Microfluidic devices, which mimic the female reproductive tract, offer a promising alternative by reducing processing time, avoiding centrifugation, and preserving sperm quality. While studies have shown microfluidic sorting improves sperm quality compared to conventional methods in normozoospermic semen samples, its effectiveness in compromised semen samples requires further investigation. A comparative study was conducted on 81 semen samples from male subjects undergoing routine semen analysis for couple infertility at the Andrology Laboratory of Careggi University Hospital of Florence, from April to December 2024. The study compared two different methods for sperm selection: pellet Swim-up technique (SU) and microfluidic devices (MSS). The study included 26 normozoospermic, 31 hyperviscous normozoospermic, 15 oligozoospermic and 9 asthenozoospermic samples. In these samples, MSS and conventional SU were compared by evaluating sperm motility, concentration and viability, kinematic parameters and hyperactivated motility (by Computer-Assisted Sperm Analysis, CASA), sperm DNA fragmentation (sDF, by TUNEL/PI method), oxidative status (by CellROX® Orange probe) and chromatin compaction (by Chromomycin A3 staining, CMA3), before and after selection procedures. As expected, compared to unselected samples, a significant increase in all analysed sperm parameters was observed after selection with both MSS and SU in all groups of samples. Notably, MSS selection statistically improved all parameters compared to SU in both non-viscous and hyperviscous samples. In 10 out of 34 normozoospermic samples, SU selection did not improve, and in some cases even increased, sDF, whereas MSS reduced it in all samples. In oligozoospermic samples, characterized by a sperm concentration of less than 10x106/mL, MSS significantly increased the percentage of total motile and viable spermatozoa compared to SU. However, no significant difference was observed in the percentage of progressive motile spermatozoa, kinematic parameters, or hyperactivated motility. MSS also showed a notable improvement in chromatin integrity, with a significant reduction in CMA3 positivity compared to SU. Similar results were obtained also in asthenozoospermic samples (n = 9), characterized by progressive motility of less than 32%. Our study demonstrated that microfluidic sorting is an effective method for selecting the optimal sperm fraction, not only in normozoospermic samples but also in semen samples exhibiting defects such as hyperviscosity, low concentration, reduced motility, and high sDF levels. The number of semen samples included in the study is not very elevated. All results should be confirmed in a greater number of samples. Our data suggest that MSS selects a sperm fraction with better characteristics and chromatin compaction, especially in compromised semen samples. This method is faster than traditional techniques and reduces operator variability. Despite its high cost, microfluidic sorting could enhance fertilization success and reproductive outcomes in challenging cases. No

To date, the Gardner Grading System remains the gold standard for the conventional evaluation of blastocysts. However, the use of Time-lapse technology in Assisted Reproductive Technologies (ART) cycles provides a dynamic, morphokinetic assessment that complements traditional morphological evaluation, as vitrification and subsequent warming process may induce morphological and biochemical variations in blastocysts. After warming, 203 blastocysts, obtained from homologous ART cycles, were cultured in EmbryoScope to evaluate their morphokinetic modifications using Time-Lapse Technology (TLT). (I) The degree of trophectoderm expansion at insertion into the incubator (t0 exp), (II) the first signal of trophectoderm re-expansion (t first exp), and (III) the degree of expansion and morphology at two hours (t2h exp, t2h grad) were recorded. Statistical analysis was performed by SPSS version 29.0 and relations between I, II, III parameters and beta-human Chorionic Gonadotropin (beta-hCG) outcome, heartbeat, and live birth were assessed. RESULTS: Showed that t first exp was significantly associated with beta-hCG levels, heartbeat, and live birth outcomes. Additionally, blastocysts achieving complete re-expansion within two hours and presented optimal morphology exhibited a higher likelihood of successful implantation. Early biomarkers were identified through morphokinetic parameters derived from Time-Lapse technology, and pregnancy outcome prediction was improved by the combination of morphological evaluation with post-warming kinetic assessments.

BACKGROUND AND AIMS:It is not known whether the gut microbiota (GM) may vary according to the endoscopic severity of diverticular disease (DD). We aimed to profile the GM in DD patients according to the severity of the diverticular inflammation and complication assessment (DICA) classification (DICA 1 vs. DICA 2 vs. DICA 3). METHODS:We retrospectively assessed the GM in a population of patients with DD. We analyzed stool samples collected by fecal swab for microbiological studies. Among them, we identified DD patients in whom DD was scored according to DICA classification. The severity of the abdominal pain was measured using a 10-point visual analogue scale (VAS). RESULTS:The GM of 71 DD patients [49 (69.0%) were scored as DICA1, 18 (25.4%) as DICA2, and 4 (5.6%) as DICA3 was analysed. The three groups did not differ in alpha diversity, but significantly separated in the PCoA of beta diversity (p=0.018). Taxonomically, DICA1 group was characterized by higher relative abundances of the phylum Actinobacteriota, the families Erysipelatoclostridiaceae and Bacteroidaceae, and the genera Lachnospiraceae ND3007 group and Bacteroides (p≤0.1); DICA2 group was mainly discriminated by higher proportions of Streptococcaceae (p=0.018); DICA3 group was mainly discriminated by the phylum Bacteroidota, the families Prevotellaceae and Succinivibrionaceae, and the genera Prevotella, Alloprevotella and Dialister (p≤0.045). Stratifiyng patients by abdominal pain severity, only for the DICA2 group the PCoA of beta diversity showed a significant separation between the moderate and severe groups (p=0.024), with the latter also showing higher alpha diversity (p=0.05). Taxonomically, the severe group was enriched in the families Enterobacteriaceae and Erysipelotrichaceae, and the genera Megasphaera and Veillonella, while depleted in Sutterellaceae and Blautia compared to the moderate group (p≤0.08). CONCLUSIONS:GM in DD may vary according to endoscopic disease severity and clinical characteristics. Such associations may improve patient stratification and clinical management.

The role of gut microbiota (GM) in the pathogenesis of Symptomatic Uncomplicated Diverticular Disease (SUDD) remains controversial. Here, we assessed the efficacy of a butyrate formulation in modulating GM and abdominal pain in patients with SUDD. A retrospective study was conducted in patients with SUDD who were treated with a delayed- and colonic-release formulation of butyrate (two capsules bid, for a total dose of 400 mg butyrate) for 3 months. GM was profiled before (T0) and after 90 days of treatment (T2) using 16S rRNA amplicon sequencing. The primary endpoint was change in GM at T2; secondary endpoints were reduction in abdominal pain severity according to VAS (Visual Analog Scale, 0: absence; 10: maximum severity) at T1 (45 days) and T2, stool characteristics according to the Bristol stool form scale at T0, T1 and T2, and safety of treatment. Fifty-nine patients with SUDD (59.3% male; median age 65.5 years, interquartile range 55-71 years) completed treatment. The butyrate formulation increased GM diversity and resulted in several compositional changes that were closely related to baseline abdominal pain severity. Regarding secondary endpoints, abdominal pain decreased significantly over time, while the Bristol stool form scale did not. Mild adverse events were recorded in 3 (5.08%) patients. This study showed that a microencapsulated and colonic-release formulation of butyrate favorably modulates GM and reduces abdominal pain in patients with SUDD.

RESEARCH QUESTION:What effect does the use of a rapid warming method of vitrified embryos have on survival and pregnancy? DESIGN:A comparison of two different warming embryos protocols: long protocol (group 1, n = 486) and a new fast protocol (group 2, n = 413) for blastocyst and cleavage stage frozen embryo transfers at two IVF centres: Assisted Reproductive Technology Centre, Careggi University Hospital Firenze and IVF Unit, 'Cervesi' Hospital Cattolica. Total pregnancy rate was considered primary outcome, and embryo survival rate, clinical pregnancy rate, miscarriage rate, ongoing pregnancy rate and mean time required for warming procedure were considered secondary outcomes. RESULTS:The same embryo survival rate was observed in the two groups. Total and clinical pregnancy rates seemed to be higher, but not statistically different, in group 2 compared with group 1 (29.5% versus 26.7% and 27.6% versus 22.6%, respectively). Conversely, a statistically significant reduction in miscarriage rate was found in group 2 compared with group 1 (18.0% versus 32.3%, P = 0.009). Similarly, a significant increase in pregnancy rate at 26 gestational weeks or over was observed in group 2 compared with group 1 (66.9% versus 80.3%, P = 0.016). Finally, a difference in time required for warming procedures was found in favour of group 2. CONCLUSIONS:The use of the fast warming protocol is a valid alternative to the classic warming protocol, and allows a reduction in the time needed to carry out the procedure and the workload of embryologists in an IVF laboratory.

Abstract Study question What is the best technique to select spermatozoa in hyper viscous semen samples? Summary answer Microfluidics is the best technique that allows to select spermatozoa with better seminal characteristics and hyperactivated motility, and less sperm DNA fragmentation. What is known already Density gradient centrifugation and Swim-up (SU) are the two traditional selection methods used during assisted reproduction techniques (ART) to collect the highly motile sperm fraction. Many studies reported that centrifugation increase sperm DNA fragmentation and oxidative status which could affect reproductive outcomes. To overcome the limitations of centrifugation, an alternative approach by using microfluidic-based devices was recently designed. In microfluidic protocol, sample is applied to the device’s inlet port, connected to a lower chamber. This chamber consists of a microporous filter, where the most motile spermatozoa migrate upward through the filter. Study design, size, duration An observational study was conducted on 29 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from January 2023 to January 2024. In these samples we compare two different methods for sperm selection: indirect SU technique and microfluidic devices (MSS). Participants/materials, setting, methods We evaluated seminal parameters (sperm progressive and total motility, viability, and concentration), sperm kinematic parameters by Computer-Assisted Sperm Analysis (C.A.S.A. system), sperm DNA fragmentation by TUNEL/PI assay and sperm oxidative status by CellROX® Orange (which we have recently shown to identify a fraction of viable oxidized spermatozoa with better characteristics (Traini G., et al. 2022)) in 29 normozoospermic and 18 hyper viscous semen samples selected by SU and MSS. Main results and the role of chance In normozoospermic samples, sperm selection with both indirect SU technique and microfluidic devices, ameliorated sperm characteristics compared to not selected samples. Spermatozoa selected by MSS showed statistically significant higher progressive and total motility, viability and concentration compared to those selected by indirect SU. Moreover, an increase in most of kinematic semen parameters (like VAP, VCL and VSL) and hyperactivated motility was observed in MSS selected samples. Also, sperm DNA fragmentation was significantly reduced after MSS selection compared to indirect SU. We evaluated the percentage of viable oxidized spermatozoa by using CellROX® Orange. A significant increase of the percentage of CellROX® Orange positive spermatozoa was observed in most of MSS selected samples. In all 18 hyper viscous samples, the results obtained in normozoospermic samples were confirmed. Indeed, an overall improvement of sperm progressive and total motility, viability and concentration together with an increase in most of kinematic semen parameters and in hyperactivated motility were evidenced in MSS selected spermatozoa respect to those selected by indirect SU. Again, a significant decrease of sperm DNA fragmentation and an increase of the percentage of CellROX® Orange positive spermatozoa, were observed after MSS selection. Limitations, reasons for caution The number of semen samples included in the study is not very elevated. Results obtained in hyper viscous semen samples should be confirmed in a greater number of samples. Wider implications of the findings Our data indicate that MSS selects the sperm fraction with better characteristics and less DNA fragmentation. These results suggest that MSS could improve ART outcomes, particularly for specific categories of patients, including hyper viscous semen samples, for which the selection by SU does not always guarantee good yields. Trial registration number not applicable

High levels of sperm DNA fragmentation(SDF)are associated with reduced assisted reproductive technology(ART)outcomes.

BackgroundSperm cryopreservation is a consolidate option for long-term male fertility preservation. The freezing/thawing procedure causes detrimental effects to spermatozoa, including damage to viability, motility, membrane composition, and DNA, whereas the effect on sperm chromatin compaction is less studied.ObjectivesThe primary aim of this study was to investigate the impact of cryopreservation on sperm chromatin compaction. Furthermore, the effect of cryopreservation on sperm parameters (motility, viability, chromatin compaction, and DNA fragmentation) was also assessed in relation to the storage time in liquid nitrogen.Materials and MethodsSemen samples, collected from 126 (92 normozoospermic and 34 oligozoospermic) patients undergoing routine semen analysis in the Andrology Laboratory of Careggi University Hospital of Florence, were frozen by conventional fast vapor freezing method. Sperm motility, viability, kinematic parameters (by computer-aided sperm analysis [CASA]), chromatin compaction (by staining with both aniline blue [AB] and Chromomycin A3 [CMA3]), and sperm DNA fragmentation (sDF, by TUNEL/Propidium Iodide [PI]) were evaluated before freezing and after thawing at different timepoints.ResultsAfter 7 days of storage, a significant decline in sperm motility, viability, and kinematics parameters, as well as a significant increase in the percentage of sperm positivity to CMA3, AB, and sDF, were observed. It is noteworthy that while motility and viability decreased in almost all subjects, the increase in CMA3 and AB positivity was observed in 68.0% and 79.2% of samples, respectively. A progressive deterioration of sperm motility and viability, less evident for chromatin structure, was observed at longer times of storage (28 and 180 days).DiscussionOur results indicate that freezing/thawing procedures can alter chromatin structure. A reduction in protamine content and/or a modification in chromatin assembly can be hypothesized. Furthermore, the length of storage in liquid nitrogen appears to progressively affect sperm parameters, although it should be confirmed in larger cohort of subjects.ConclusionCurrent sperm cryopreservation protocols need to be improved with new strategies and personalized procedures aimed to minimize the damage.

Background Hyperglycemia can promote the development of prostate cancer (PCa). Differential expression levels of miRNAs between PCa patients and controls were also reported. Therefore, we examined the relationship between hyperglycemia and miRNA levels in PCa. Methods Relative expression of urinary miR-574-3p, miR-375, miR-205-5p, miR-200b-3p, miR-187-3p, miR-182-5p, and miR-100-5p were investigated in 105 PCa patients and 138 noncancer controls by Real-Time quantitative PCR. Fasting plasma glucose measurements were retrieved from clinical records. The differential miRNA expressions among groups were compared using non-parametric tests. Correlations with glucose and prostate-specific antigen (PSA) were tested using Pearson correlation coefficient. Results When we analyzed miRNA expression according to glycemic state, significant down-regulations were found for miR-200b-3p, miR-187-3p, miR-182-5p, and miR-100-5p in noncancer controls with high glucose. The lowest down-regulations were observed for miR-187-3p, miR-182-5p, and miR-100-5p. Subsequently, when hyperglycemia was considered in PCa, significant dysregulations of selected miRNAs were found in hyperglycemic PCa patients than in controls with high glucose. In particular, miR-375 and miR-182-5p showed a 3-FC in hyperglycemic PCa patients than controls who left hyperglycemia untreated. Conversely, only a down-regulation of miR-574-3p was observed in PCa patients regardless of glycemic status and only modest down-regulation of miR-574-3p, miR-200b-3p, miR-187-3p and miR-182-5p were found in normoglycemic PCa patients. Next, significant correlations between miRNAs and glucose (miR-200b-3p, miR-100-5p) and PSA (miR-205-5p and miR-187-3p) were detected in controls. Similarly, miR-205-5p and miR-187-3p were correlated with glucose in PCa patients, while miR-574-3p and miR-375 showed inverse relationships. Conclusions miRNA dysregulations can occur in hyperglycemic PCa patients as compared to noncancer controls who left hyperglycemia untreated. Hyperglycemia can consistently promote the expression of miR-375 and miR-182-5p. Uncontrolled hyperglycemic state could contribute to the creation of a suitable microenvironment for later PCa development by promoting gene expression.

OBJECTIVE:Patients with symptomatic uncomplicated diverticular disease (SUDD) may have a disrupted gut microbiota. However, current data are from small sample studies, and reported associations vary widely across studies. We aimed to profile the fecal microbiota in SUDD patients enrolled in primary care. METHODS:A retrospective study was conducted in SUDD ( N  = 72) and asymptomatic diverticulosis (AD) ( N  = 30), the latter serving as a control group. RESULTS:No significant differences in alpha and beta diversity were found between SUDD and AD, but SUDD was discriminated by a higher relative abundance of the family Streptococcaceae and the genera Alistipes , Agathobacter , and Butyricimonas . Interestingly, the gut microbiota of SUDD patients stratified by the severity of abdominal pain [according to the visual analog scale (VAS)]. In particular, higher diversity and health-associated taxa (such as Bifidobacterium , Eubacterium coprostanoligenes group, and Dorea ) characterized mild (VAS score 1-3) SUDD, Proteobacteria , Veillonellaceae and Blautia moderate (VAS score 4-7) SUDD, and Prevotellaceae and Megasphaera severe (VAS score 8-10) SUDD. CONCLUSION:Our analysis suggests that specific taxa may be related to SUDD, but the associations vary depending on the severity of abdominal pain. In addition to advancing our ecological understanding of this complex disease, our findings may pave the way for the incorporation of gut microbiota profiling into clinical practice to aid patient management, including stratification and treatment.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 106/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure.

Abstract Study question Can blastocyst first expansion time after thawing, complete expansion, and morphology evaluated by time-lapse technology predict implantation? Summary answer Lower blastocyst expansion time after thawing and higher blastocyst quality assessed at the time of transfer are associated with a higher probability of clinical pregnancy What is known already Conflicting results have been published concerning the time of first and complete re-expansion of blastocyst after thawing or its quality and the attainment of pregnancy. Some studies reported no differences in pregnancy rate (PR) with different re-expansion duration, whereas other retrospective studies identified the degree of re-expansion, assessed within 6 hours after thawing, as the best predictor. Similarly, both studies reporting associations and non-associations between blastocyst morphology and PR have been published. Study design, size, duration We prospectively evaluated the time of first and complete expansion and quality grade of 141 homologous single blastocysts after thawing and relate the results to PR. The enrolled patients had a mean age of 38.7 ± 5.4 years. Morphokinetic parameters were evaluated using the Embryoscope time-lapse system. The study is currently ongoing. Participants/materials, setting, methods The study was conducted on 141 patients iundergoing homologous PMA cicles. Time lapse Embryoscope was used to assess post-thawing morphokinetic parameters as possible early markers of successful implantation. Time to first trophectoderm expansion, time and morphology two hours after thawing, and time and morphology of the blastocyst at the time of transfer were evaluated and recorded. Statistical analysis was performed using SPSS statistical package 28.0. Main results and the role of chance Both time of first (TfE) and maximum (TmE) blastocyst expansion were associated with a higher probability of pregnancy. Median TfE in women achieving pregnancy was 0.97 hours (range: 0.29-2.2) vs 1,4 (range 0.2-3.3) in those not achieving pregnancy (p < 0.001). Similarly, TmE was lower in pregnant women (p < 0.05). When blastocyst expansion occurred within 2 h, a PR of 71% was obtained vs 16% in women where expansion was post-poned (p < 0.001). ROC analysis showed that a TfE of 1,19h predicted attainment of pregnancy with an accuracy of 75%, a sensitivity of 75% and a specificity of 70% (p < 0.001). Blastocyst quality was found as another independent predictor of attainment of pregnancy. PR resulted of 21, 31,5 and 63,3% respectively with 0, 1 and 2 grade of blastocyst quality (p < 0.001, OR = 7.7). Limitations, reasons for caution Our study is preliminary and to draw firm conclusions, a higher number of blastocysts should be evaluated. The study is ongoing. Statistical correction for partners semen quality should be also performed. Wider implications of the findings Our preliminary results showed that TfE, TmE after thawing, and blastocyst quality are predictors of pregnancy achievement. Our study suggests that time lapse technology is useful for predicting pregnancy with good accuracy when transfer is performed after blastocyst freezing. Trial registration number not applicable

Abstract Study question Is oxidative stress (OS) evaluated in human spermatozoa a predictive marker of better semen quality? Summary answer CellROX®Orange is a new fluorescent probe able to detect Reactive Oxygen Species (ROS) identifying a viable oxidized sperm fraction related to a better sperm performance. What is known already OS, defined as an unbalance between ROS production and antioxidant defences, is considered one of the causes of male infertility. OS evaluation in spermatozoa represents an important goal of research in this field because this parameter could be helpful to predict sperm fertilization ability to improve Assisted Reproductive Technology outcomes. By using different probes and methods for OS evaluation in spermatozoa or in semen, several studies were performed demonstrating a negative role of ROS on sperm functions. Such studies were not conclusive for the small number of included subjects, the high variability in the cohorts and the lack of validation. Study design, size, duration An observational study was conducted on 121 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from September 2021 to March 2022. Washed and Swim-up selected spermatozoa were incubated with two fluorescent probes, CellROX® Orange and Dihydroethidium (DHE), and revealed by flow cytometry. Participants/materials, setting, methods After routine semen analysis, the percentage of oxidized spermatozoa was evaluated with CellROX® Orange and DHE, and then correlated with standard semen parameters and sperm DNA fragmentation (sDF, evaluated by TUNEL/PI). Sperm kinematic parameters and hyperactivated motility were also assessed by C.A.S.A. system. Furthermore, CellROX® Orange positivity and phosphatidylserine membrane exposure (determined by Annexin V staining) or Caspase 3 and 7 activities (measured by FLICA™) were concomitantly evaluated. Main results and the role of chance We demonstrated that CellROX® Orange is able to detect hydrogen peroxide only in viable spermatozoa and the percentage of CellROX® Orange positive spermatozoa were positively associated with semen quality and negatively with sDF. To confirm these results, we performed the same experiments by using DHE, another probe which reveals hydrogen peroxide and superoxide anion in both viable and unviable cells. Similarly to CellROX® Orange, a positive correlation with semen quality and a negative one with sDF were found in viable spermatozoa. Conversely, in unviable spermatozoa, opposite associations were observed. These results indicate that oxidative status revealed by the two probes is related to a better sperm quality. To further investigate this possibility, we double labelled spermatozoa with CellROX® Orange and Annexin V (a marker of early signs of apoptosis) as well as with CellROX® Orange and FLICA™ (that detects caspase activity, a late sign of apoptosis). We found that CellROX® Orange mostly identifies spermatozoa without apoptotic features. Furthermore, we evaluated CellROX® Orange positivity in Swim-up selected spermatozoa, finding significantly higher levels in these samples respect to unselected samples, demonstrating once again that this probe identifies spermatozoa with better quality. Limitations, reasons for caution Our results indicate that within the viable oxidized spermatozoa there are cells with physiological and non-physiological intracellular ROS levels, however, they do not explain at what levels ROS cease to be functional and become deleterious for spermatozoa. Wider implications of the findings ROS evaluation in viable spermatozoa with the commercially available probes CellROX® Orange and DHE, allows the identification of the oxidized sperm fraction related to a sperm better performance. Therefore, the two probes may be useful to determine such fraction, likely improving the diagnostic process of male infertility. Trial registration number not applicable

Abstract Objectives The short- and long-term andrological effects of coronavirus disease 2019 (COVID-19) have not been clarifed. Our aim is to evaluate the available evidence regarding possible andrological consequences of COVID-19 either on seminal or hormonal parameters. The safety of the COVID-19 vaccines in terms of sperm quality was also investigated. Methods All prospective and retrospective observational studies reporting information on severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) mRNA semen and male genitalia tract detection (n=19), as well as those reporting data on semen analysis (n=5) and hormonal parameters (n=11) in infected/recovered patients without any arbitrary restriction were included. Results Out of 204 retrieved articles, 35 were considered, including 2092 patients and 1138 controls with a mean age of 44.1±12.6 years, and mean follow-up 24.3±18.9 days. SARS-CoV-2 mRNA can be localized in male genitalia tracts during the acute phase of the disease. COVID-19 can result in short-term impaired sperm and T production. Available data cannot clarify long-term andrological efects. Low T observed in the acute phase of the disease is associated with an increased risk of being admitted to the Intensive Care Unit or death. The two available studies showed that the use of mRNA COVID-19 vaccines does not affect sperm quality. Conclusions The results of our analysis clearly suggest that each patient recovering from COVID-19 should be monitored to rule out sperm and T abnormalities. The specifc contribution of reduced T levels during the acute phase of the infection needs to be better clarifed. Conflicts of Interest The authors declare that they have no confict of interest.