Conventional Sperm Parameters Research Articles

7394 Testicular Function in Young Adults with Growth Hormone (GH) Deficiency Previously Treated with GH: Comparison with Normal Controls

Abstract Disclosure: R. Cannarella: None. A. Crafa: None. S. Sapienza: None. M.M. Caruso: None. A.E. Calogero: None. Background: We have previously reported that treatment with recombinant human growth hormone (rhGH) can influence testicular growth and puberty onset in children with GH deficiency (GHD), and suggested GH as a determinant of testicular growth in childhood (doi: 10.3389/fendo.2021.619895). Limited evidence is available on testicular function in post-pubertal GHD patients. Objective: This prospective controlled study was undertaken to evaluate testicular function in young GHD adults previously undergone to GH treatment. Patients and Methods: Post-pubertal patients with non-syndromic GHD (>18 years), in whom GH therapy was discontinued after reaching the therapeutic goal, were required to have a complete evaluation of testicular function. Those who agreed were enrolled in our study. Age-matched healthy men older than 18 years served as controls. Exclusion criteria were: the presence of varicocele, urogenital infections, cryptorchidism, hypospadias, hypogonadism, testicular trauma or torsion, and other congenital or acquired disorders capable of interfering with testicular function. The study outcomes were: serum gonadotropins and total testosterone (TT), conventional sperm parameters, and testicular volume (TV) measured by ultrasound examination. Between-group analysis was performed using the Student’s t-test for independent samples or the Mann-Whitney test, for normally or non-normally distributed variables, respectively. Data were reported as mean±standard deviation for non-skewed data and median (interquartile range) for skewed data. Results: 26 patients with GHD and 25 age-matched controls (18.8±3.3 years vs. 20.1±3.6 years; p=0.18) were enrolled. Patients and controls did not differ for serum luteinizing hormone [2.6 (1.5-4.9) IU/L vs. 3.5 (2.8-4.8) IU/L); p=0.2], follicle-stimulating hormone [3.4 (2.2-4.3) IU/L vs. 3.1 (2.3-4.3) IU/L); p=1.0], and TT [5.7 (4.7-6.8) ng/dL vs. 6.0 (5.1-7.4) ng/dL); p=0.4] levels. At semen analysis, GHD patients had lower semen volume (2.1±1.4 mL vs. 3.5±1.5 mL; p=0.002), total sperm count [135.0 miL/ejaculate (54.3-229.0) vs. 231.3 miL/ejaculate (124.8-285.0); p=0.03], progressive motility [25.5% (15.0-34.0%) vs. 32.0% (30.0-36.5); p=0.02], and total motility [56.0% (49.0-62.0) vs. 66.5% (62.5-70.0); p=0.0001] compared to controls. No difference was found in sperm concentration [73.0 miL/mL (45.0-92.0) vs. 68.0 miL/mL (39.0-90.0); p=1.0] and normal morphology [8.5% (7.0-12.0) % vs. 10.0% (7.5-14.0); p=0.3]. Importantly, GHD patients had lower right (13.3±3.4 mL vs. 16.9±4.1 mL; p=0.001) and left (12.5±3.4 mL vs. 16.3±3.8 mL; p=0.0005) TV. Conclusion: This study provides the first evidence of lower TV and slightly worse semen parameters in patients with GHD compared to healthy controls. Serum GH levels during growth may be important for achieving normal testicular volume and spermatogenesis in adulthood. Presentation: 6/2/2024

9277 Testicular Function in Young Adults with Growth Hormone (GH) Deficiency Previously Treated with GH: Comparison with Normal Controls

Abstract Disclosure: R. Cannarella: None. A. Crafa: None. S. Sapienza: None. M.M. Caruso: None. A.E. Calogero: None. Background: We have previously reported that treatment with recombinant human growth hormone (rhGH) can influence testicular growth and puberty onset in children with GH deficiency (GHD), and suggested GH as a determinant of testicular growth in childhood (doi: 10.3389/fendo.2021.619895). Limited evidence is available on testicular function in post-pubertal GHD patients. Objective: This prospective controlled study was undertaken to evaluate testicular function in young GHD adults previously undergone to GH treatment. Patients and Methods: Post-pubertal patients with non-syndromic GHD (>18 years), in whom GH therapy was discontinued after reaching the therapeutic goal, were required to have a complete evaluation of testicular function. Those who agreed were enrolled in our study. Age-matched healthy men older than 18 years served as controls. Exclusion criteria were: the presence of varicocele, urogenital infections, cryptorchidism, hypospadias, hypogonadism, testicular trauma or torsion, and other congenital or acquired disorders capable of interfering with testicular function. The study outcomes were: serum gonadotropins and total testosterone (TT), conventional sperm parameters, and testicular volume (TV) measured by ultrasound examination. Between-group analysis was performed using the Student’s t-test for independent samples or the Mann-Whitney test, for normally or non-normally distributed variables, respectively. Data were reported as mean±standard deviation for non-skewed data and median (interquartile range) for skewed data. Results: 26 patients with GHD and 25 age-matched controls (18.8±3.3 years vs. 20.1±3.6 years; p=0.18) were enrolled. Patients and controls did not differ for serum luteinizing hormone [2.6 (1.5-4.9) IU/L vs. 3.5 (2.8-4.8) IU/L); p=0.2], follicle-stimulating hormone [3.4 (2.2-4.3) IU/L vs. 3.1 (2.3-4.3) IU/L); p=1.0], and TT [5.7 (4.7-6.8) ng/dL vs. 6.0 (5.1-7.4) ng/dL); p=0.4] levels. At semen analysis, GHD patients had lower semen volume (2.1±1.4 mL vs. 3.5±1.5 mL; p=0.002), total sperm count [135.0 miL/ejaculate (54.3-229.0) vs. 231.3 miL/ejaculate (124.8-285.0); p=0.03], progressive motility [25.5% (15.0-34.0%) vs. 32.0% (30.0-36.5); p=0.02], and total motility [56.0% (49.0-62.0) vs. 66.5% (62.5-70.0); p=0.0001] compared to controls. No difference was found in sperm concentration [73.0 miL/mL (45.0-92.0) vs. 68.0 miL/mL (39.0-90.0); p=1.0] and normal morphology [8.5% (7.0-12.0) % vs. 10.0% (7.5-14.0); p=0.3]. Importantly, GHD patients had lower right (13.3±3.4 mL vs. 16.9±4.1 mL; p=0.001) and left (12.5±3.4 mL vs. 16.3±3.8 mL; p=0.0005) TV. Conclusion: This study provides the first evidence of lower TV and slightly worse semen parameters in patients with GHD compared to healthy controls. Serum GH levels during growth may be important for achieving normal testicular volume and spermatogenesis in adulthood. Presentation: 6/2/2024

Effects of Switching FSH Preparations on Sperm Parameters and Pregnancy: A Prospective Controlled Study

Objective: To study the effect of switching to a follicle-stimulating hormone (FSH) preparation other than that to which infertile male patients have not had an effective response. Patients and methods: Seventy-four normogonadotropinemic, non-obstructive, oligozoospermic patients who were poor responders to the administration of highly purified FSH (hpFSH) (Group 1 (n = 22) and Group 3 (n = 15)) or to recombinant human FSH (rhFSH) (Group 2 (n = 22) and Group 4 (n = 15)) were selected for this prospective study. After 3 months of washout from treatment with the first FSH preparation of choice, rhFSH was administered to patients in Groups 1 and 4 and hpFSH to those in Groups 2 and 3. Serum luteinizing hormone, FSH, total testosterone levels, conventional sperm parameters, testicular volume, and the number of pregnancies were evaluated at study entry and after the first and second treatment cycles. Results: Comparing treatment groups, the greatest improvement in sperm parameters was recorded in the groups of patients prescribed the switch in FSH preparation. Group 1 had the greatest benefit from therapy, with the highest pregnancy rate after the second treatment cycle. Indeed, eight couples achieved pregnancy (36.4%), compared to Groups 2 (n = 4; 18.2%), 3 (n = 1; 6.7%), and 4 (n = 2; 13.3%) (p = 0.04). Conclusions: The results of this study suggest that a therapeutic scheme involving the “switching” of the FSH preparation yields better results than a protocol using the same FSH preparation for six months. These findings, if confirmed by further studies, will help us better design a treatment strategy with FSH for infertile patients with oligozoospermia.

Hyperglycemia adversely affects critical physiological events related to rat sperm capacitation

Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (EmR, DiSC3(5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na+ ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while EmR was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies.

Correlation between standard sperm parameters and sperm DNA fragmentation from 11,339 samples

Conventional semen parameters have long been considered fundamental in male fertility analyses. However, doubts have been raised regarding the clinical utility of the assessment of spermatozoa (sperm) DNA damage. In this retrospective study, we investigated the potential correlation between conventional semen parameters and semen DNA fragmentation (SDF) assessed as sperm DNA damage, in 11,339 semen samples collected between January 2019 and June 2022. We observed significant negative correlations between the DNA fragmentation index (DFI) and sperm viability (correlation coefficient [r] = −0.514) as well as progressive sperm motility (r = −0.512, p < 0.05). Samples were categorized into three groups according to DFI levels (Groups A, B, and C: ≤15%, 15 < DFI ≤30%, and >30%, respectively). Furthermore, the percentage of semen samples with normal sperm conventional parameters in Groups A, B, and C was 76.7% (4369/5697), 61.4% (2351/3827), and 39.7% (721/1815), respectively. Moreover, according to the reference values of conventional sperm parameters, the samples were divided into Groups F, G, and H with all normal, only one abnormal, and > two abnormal parameters, respectively. In addition, the proportions of samples with abnormal DFI values (>30) in Groups F, G, and H were 9.7% (721/7441), 23.1% (618/2676), and 39.0% (476/1222), respectively. Multivariate logistic regression models demonstrated that sperm vitality, progressive sperm motility, normal sperm form, total sperm count, semen volume, age, and some sperm kinematics collectively improved the area under the receiver operating characteristic curve (AUROC) to 0.861, surpassing the predictive value of a single predictor of pathologically damaged sperm DNA. Our study suggests that samples with abnormal sperm parameters may have a higher likelihood of high DNA fragmentation. Furthermore, certain semen parameters could be potential indicators of sperm DNA fragmentation, aiding sperm selection in assisted reproductive procedures.

Involvement of CATSPER 2 mutation in a familial context of unexplained infertility and fertilization failure associated with hearing loss: a case report

To explore the functional implications of a homozygous CATSPER 2 (cation channel for sperm) deletion within the acrosome reaction pathway during fertilization in 2 brothers, who have unexplained infertility and hearing loss. Case report. Two twin brothers aged 30 years with hearing loss and unexplained infertility. Molecular genetic diagnosis of deafness. Evaluation of the acrosome reaction and calcium mobilization assays after induction by progesterone and ionomycin on spermatozoa of the CATSPER 2-mutated patient and on fertile controls. Fertilization rate during conventional in vitro fertilization. Molecular genetic test. Percentage of acrosome-reacted spermatozoa with peanut agglutinin lectin staining. Recording of progesterone and ionomycin-induced intracellular calcium signals with a fluorescent probe. Mr. S and his brother have normal, conventional sperm parameters. Both brothers have had repeated intrauterine insemination failures and one fertilization failure after conventional in vitro fertilization. Mr. S obtained 2 healthy babies after intracytoplasmic sperm injection. Genetic analysis found a homozygote deletion of the STRC (stereocilin) gene (NM 153700: c.1-? 5328+?del) that removes the CATSPER 2 gene. Mutation of the STRC gene is known to be associated with hearing loss. Sperm functional tests revealed an inability of progesterone to activate intracellular calcium signaling and to induce acrosome reaction. We demonstrate the absence of a calcium signal and acrosome reaction after progesterone in our patient with a CATSPER 2 mutation. We emphasize the importance of the male medical interview and of the genetic investigation of hearing loss. We show that in vitro fertilization-intracytoplasmic sperm injection is necessary, even where normal sperm parameters are present.

Sperm chromatin structure assay (SCSA®) and flow cytometry-assisted TUNEL assay provide a concordant assessment of sperm DNA fragmentation as a function of age in a large cohort of approximately 10,000 patients

BackgroundSperm DNA integrity is increasingly seen as a critical characteristic determining reproductive success, both in natural reproduction and in assisted reproductive technologies (ART). Despite this awareness, sperm DNA and nuclear integrity tests are still not part of routine examinations for either infertile men or fertile men wishing to assess their reproductive capacity. This is not due to the unavailability of DNA and sperm nuclear integrity tests. On the contrary, several relevant but distinct tests are available and have been used in many clinical trials, which has led to conflicting results and confusion. The reasons for this are mainly the lack of standardization between different clinics and between the tests themselves. In addition, the small number of samples analyzed in these trials has often weakened the value of the analyses performed. In the present work, we used a large cohort of semen samples, covering a wide age range, which were simultaneously evaluated for sperm DNA fragmentation (SDF) using two of the most frequently used SDF assays, namely the TUNEL assay and the sperm chromatin structure assay (SCSA®). At the same time, as standard seminal parameters (sperm motility, sperm morphology, sperm count) were available for these samples, correlations between age, SDF and conventional seminal parameters were analyzed.ResultsWe show that the SCSA® and TUNEL assessments of SDF produce concordant data. However, the SDF assessed by TUNEL is systematically lower than that assessed by SCSA®. Regardless of the test used, the SDF increases steadily during aging, while the HDS parameter (High DNA stainability assessed via SCSA®) remains unchanged. In the cohort analyzed, conventional sperm parameters do not seem to discriminate with aging. Only sperm volume and motility were significantly lower in the oldest age group analyzed [50–59 years of age].ConclusionsIn the large cohort analyzed, SDF is an age-dependent parameter, increasing linearly with aging. The SCSA® assessment of SDF and the flow cytometry-assisted TUNEL assessment are well correlated, although TUNEL is less sensitive than SCSA®. This difference in sensitivity should be taken into account in the final assessment of the true level of fragmentation of the sperm nucleus of a given sample. The classical sperm parameters (motility, morphology, sperm count) do not change dramatically with age, making them inadequate to assess the fertility potential of an individual.

Effect of Micronutrients and L-Carnitine as Antioxidant on Sperm Parameters, Genome Integrity, and ICSI Outcomes: Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

The evaluation of sperm DNA integrity is recommended in the sixth edition of the 2021 World Health Organization guidelines. Oxidative stress has been identified as a crucial factor leading to genome decay, lipid peroxidation, and nucleoprotein oxidation. This double-blind, placebo-controlled clinical trial aimed to assess the effect of oral antioxidant treatment (Fertilis), which contains L-carnitine and some micronutrients, in the improvement of conventional sperm parameters, sperm DNA integrity and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. A total of 263 participants were enrolled and randomly divided into two groups: 131 participants received the antioxidant treatment, while 132 participants received a placebo. The male partners in both groups underwent the antioxidant treatment or the placebo for a duration of three months. For each participant, we performed a hormonal test, an infectious test, a spermogram, a TUNEL assay for sperm DNA fragmentation, a toluidine blue staining for sperm DNA decondensation, and an IVF/ICSI procedure. Sperm characteristics analysis (volume, count, motility, and vitality), sperm DNA fragmentation, and sperm DNA decondensation were assessed and compared to the results preceding the antioxidant treatment. The study outcome revealed a significant decrease in the DNA fragmentation index and a significant increase in sperm motility after 3 months of treatment (p = 0.01 and p = 0.02, respectively). Additionally, a significant improvement in clinical pregnancy rate (p = 0.01) and life birth rate (p = 0.031) was observed. No significant changes were observed in conventional sperm parameters (volume, count, and vitality) or sperm DNA decondensation (SDI). Antioxidant therapy has a beneficial impact on achieving pregnancy, whether through spontaneous conception or assisted reproductive procedures (ART).

OR25-02 Hunting The Magic Bullet In Treating Male Secondary Hypogonadism Associated With Metabolic Syndrome: Results Of a Prospective Study Comparing Liraglutide Vs. Gonadotropins

Abstract Disclosure: S. La Vignera: None. R.A. Condorelli: None. A.E. Calogero: None. R. Cannarella: None. E. Greco: None. A. Aversa: None. Background: Previously, we hypothesized a role for glucagon-like peptide-1 (GLP1) analogs in the treatment of male infertility (DOI: 10.1111/andr.13015), although no studies have so far investigated this issue. Purpose: To prospectively investigate the effects of treatment with liraglutide, a GLP1 analog, on reproductive and sexual function in men with functional hypogonadism of childbearing age. Patients and Methods: 110 obese men of childbearing age (18-35 years) with functional hypogonadism were enrolled and divided into three groups based on their desire to have children. Group A consisted of men actively seeking paternity, Group B of men who did not seek paternity, and Group C of men who had already fathered a child. Group A was treated with gonadotropins [urofollitropin 150 IU three times a week and human chorionic gonadotropin (hCG) 2000 IU twice a week], Group B with liraglutide (3 mg daily), and Group C with transdermal testosterone (60 mg daily). The following endpoints were measured at enrolment and after 4 months: anthropometric and metabolic parameters, gonadotropins, total testosterone, conventional sperm parameters (Group A and B only), the score of the International Index of Erectile Function 5 Items (IIEF5), and frequency of phosphodiesterase type 5 inhibitors (PDE5i) use. Results: The groups did not differ on any of the baseline endpoints. Weight, body mass index, and waist circumference significantly decreased after treatment in Group B, but not in the other Groups. Liraglutide significantly increased LH (3.2±0.2 IU/L vs. 2.0±0.3 IU/L; p&amp;lt;0.01), FSH (2.6±0.2 IU/L vs. 1.6±0.3 IU/L; p&amp;lt;0.01), total testosterone (4.1±0.5 ng/mL vs. 1.4±0.6 ng/mL; p&amp;lt;0.05), and sex hormone-binding globulin (36.0±4.0 nmol/L vs. 14.0±3.0 nmol/L) levels from baseline, while no improvement was observed in the other Groups. Liraglutide increased sperm concentration (16±4 mil/ml vs. 6±2 mil/ml; p&amp;lt;0.05), progressive motility (34±4 % vs. 14±2 %; p&amp;lt;0.01), and morphology (10±2 % vs. 4±2 %; p&amp;lt;0.05), while treatment with gonadotropins only increased sperm progressive motility (19±2 % vs. 12±3 %; p&amp;lt;0.05) compared to baseline. The IIEF-5 score improved after liraglutide treatment (4±2 vs. 21±4; p&amp;lt;0.05), while no significant changes were observed in the other Groups. Similarly, PDE5i use decreased significantly in Group B (31%), compared with Groups A (48%) and C (45%). Conclusions: Liraglutide is an effective weight loss drug, which also improves serum gonadotropin and total testosterone levels, sperm conventional parameters, and sexual function in obese patients with functional hypogonadism seeking parenthood. This is the first study documenting the efficacy of liraglutide on the testicular function of these patients. Presentation Date: Saturday, June 17, 2023

Predictive role of 17α-hydroxy-progesterone serum levels of response to follicle-stimulating hormone in patients with abnormal sperm parameters

To study the possible role of serum 17α-hydroxy-progesterone (17αOH-P) levels in predicting favorable responses to follicle-stimulating hormone (FSH) administration in patients with normal serum FSH levels and idiopathic abnormal sperm parameters. Prospective cohort study. University-affiliated fertility center. Fifty patients with oligozoospermia, asthenozoospermia, and/or teratozoospermia and normal serum levels of gonadotropins and total testosterone (TT). Treatment with exogenous FSH is administered subcutaneously at a dose of 150 IU 3 times a week for 3 consecutive months. Luteinizing hormone levels, FSH levels, TT levels, 17αOH-P levels, testicular volume, conventional sperm parameters, and seminal spermatid concentration were evaluated before and after therapy. To evaluate the predictive role of pretreatment serum 17αOH-P levels on FSH responsiveness, the doubling of sperm concentration at the end of the FSH administration was considered a positive outcome. After therapy, patients showed a significant increase in sperm concentration, total sperm count (TSC), progressive motility, percentage of normal forms, FSH levels, TT levels, and testicular volume. There was a negative correlation between pretreatment 17αOH-P levels and the posttreatment increase in sperm concentration, TSC, progressive motility, and normal morphology, and a positive correlation with the posttreatment increase in spermatids. Predictive analysis showed that 17αOH-P levels (<1.18 ng/mL) foretold a doubling of sperm concentration with a sensitivity of 90.0% and a specificity of 73.3%, and of TSC with a sensitivity of 91.3% and a specificity of 81.48%. The results of this study suggest that pretreatment serum levels of 17αOH-P, a marker of steroidogenic function, appear to be able to predict the success of subcutaneous administration of exogenous FSH in terms of spermatogenesis improvement. Receiver operating characteristic curves indicated that 17αOH-P levels (<1.18 ng/mL) predict a doubling of sperm concentration and TSC after exogenous FSH administration to patients with idiopathic abnormal sperm parameters and normal gonadotropin levels.

The role of total antioxidant capacity and malondialdehyde of seminal plasma in the association between air pollution and sperm quality

Accumulating evidence has suggested that men exposed to air pollution are associated with decreased sperm quality, and seminal plasma plays a pivotal role in maintaining sperm viability. However, the role of seminal plasma in air pollution related sperm quality decline remain unestablished. In current study, we recruited 524 participants from couples who underwent in vitro fertilization treatment due to female factors at a fertility clinic in China from March to August 2020. Conventional sperm parameters, total antioxidant capacity (T-AOC), malondialdehyde (MDA) and testosterone were measured using semen samples. The six main air pollutants (PM2.5, PM10, NO2, SO2, CO, O3) during four key periods of sperm development (meiotic stage, spermiogenesis stage, epididymal stage and total sperm cycle period) were estimated using inverse distance weighting method. Multiple linear regression models were employed to investigate the exposure-outcome relationships. And we found that PM10 exposures were negatively related to sperm total motility and the exposures of PM2.5 and PM10 were inversely associated with sperm progressive motility during epididymal stage. Furthermore, PM2.5 and PM10 exposures were positively associated with seminal plasma MDA and PM10 was negatively related to seminal plasma T-AOC during epididymal stage. PM2.5, PM10 and CO exposures during total sperm cycle period might relate to increased seminal plasma testosterone. Mediation analysis indicated seminal plasma MDA and T-AOC partially mediated PM10 associated reduction of sperm motility during epididymal stage. Our study suggested MDA and T-AOC of seminal plasma played a role in air pollution associated decline of sperm motility.

P-059 Sperm DNA fragmentation (SDF) after cryopreservation and sperm selection: implications for clinical pregnancies and live births after intrauterine insemination with donor sperm

Abstract Study question Can SDF assessment pre-cryopreservation, post-thaw and after post-thaw sperm selection predict clinical outcome in a heterologous intra-uterine donor insemination program (IUI)? Summary answer SDF increased post-cryopreservation in donor sperm and after post-thaw density gradient without affecting clinical pregnancy, live birth and miscarriage rates. What is known already Sperm cryopreservation is effective for preservation of male fertility and facilitates the storage of donor semen, while infectious disease screening can be completed and confirmed negative. Sperm viability and motility are the most vulnerable parameters during the freeze-thaw process. Inherent reactive oxygen species may induce SDF via oxidative stress. Controversial results are reported regarding induction of SDF after cryopreservation due to differences in cryo tolerance, methods of cryopreservation and methods used to evaluate SDF. Sperm selection post-thaw is indispensable to select a normal motile fraction for insemination. Again, results are controversial regarding influence on SDF levels after sperm selection post-thaw. Study design, size, duration This was a prospective, observational study. Between March 2015 and March 2019, eighteen potential sperm donors were screened via a three step plan – semen assessment, medical assessment and serological blood tests. In accordance with the Belgian legislation (2007), a sperm donor was matched to obtain a maximum of 6 pregnancies in acceptor women/couples, allowing for more than one offspring/woman or couple. A total of 106 acceptors were matched for heterologous intra-uterine insemination. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and standard semen parameters analyzed within 60 mins after ejaculation. Within the same time frame SDF test was performed using TUNEL assay both before and after cryopreservation and after a two-step discontinuous density gradient centrifugation post-thaw. Samples were cryopreserved by the conventional slow freezing method using sperm freeze solution. A yield of ≥ 2 M progressive spermatozoa post-thaw after selection was sufficient to contemplate IUI. Main results and the role of chance Mean female age at first cycle was 33.9 ± 4.1 years and donor age 28.5 ± 5.6 years. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. Excluding three ongoing pregnancies, we counted 75 live births (17.6% of cycles or 77.3% of pregnancies), while 21 pregnancies ended in miscarriage (4.9% of cycles or 21.6% of pregnancies) and 1 resulted in stillbirth. Progressive motility (59.3 ± 12.5%) decreased significantly after cryopreservation (30.0 ± 13.2%; p &amp;lt; 0.001), but increased post-thaw after density gradient centrifugation (61.6 ± 16.0%; p &amp;lt; 0.001). On the contrary, SDF (12.0 ± 5.9%) increased post-cryopreservation (26.3 ± 14.5%; p &amp;lt; 0.001) and further increased after sperm selection (34.9 ± 22.1%; p = 0.04). Using multilevel mixed-effects logistic regression (Odds ratio [95%CI]), we found that female age significantly influenced clinical pregnancy (0.911 [0.847-0.981]; p = 0.01), live birth (0.894 [0.834-0.959]; p = 0.002) and miscarriage rates (1.180 [1.033-1.347]; p = 0.015). There was no significant age-adjusted effect of SDF post-thaw after density gradient selection on clinical pregnancy (1.007 [0.994-1.021]; p = 0.30), live birth (1.001 [0.988-1.014]; p = 0.84) or miscarriage (1.021 [0.997-1.046]; p = 0.08). Median time-to-live birth was 4 cycles. Analyses of time-to-live birth with mixed-effects Cox models revealed no significant association with progressive motility; SDF before or after cryopreservation and after sperm selection post-thaw. Limitations, reasons for caution Results concern a small donor population, with high normal semen parameters and should therefore be extrapolated cautiously to subfertile men with normal and subnormal semen parameters. Wider implications of the findings In a donor IUI program with strict selection on conventional sperm parameters, differences in SDF before and after cryopreservation and gradient centrifugation do not change IUI outcomes, although cryopreservation and centrifugation increase SDF. Findings could be useful for the optimization of sperm freezing and selection in different assisted reproductive procedures. Trial registration number not applicable

P-088 The Paternal Clock: Shedding Light on the Relationship Between Paternal Age and Sperm DNA Fragmentation

Abstract Study question What is the impact of advanced paternal age (APA) on sperm DNA fragmentation index (DFI)? Summary answer Sperm DFI levels remain relatively stable until the age of 35 and increase progressively beyond that age. What is known already APA can have a negative impact on male fertility and the health of offspring. Previous studies have shown that APA is linked to poor conventional sperm parameters, including decreased semen volume, sperm count, motility, morphology, as well as poor sperm DNA integrity. Additionally, APA has been associated with reduced natural or assisted reproduction and perinatal outcomes and a higher risk of genetic and chromosomal abnormalities in the offspring. Study design, size, duration A retrospective cohort study of 4250 consecutive semen samples from men undergoing infertility evaluation at the OVO clinic, in Montreal, Canada, between April 2016 and December 2022. Participants were stratified into seven age groups: &amp;lt;26 (n = 36; 0,8%), 26-30 (n = 500; 11,8%), 31-35 (n = 1269; 29,9%), 36-40 (n = 1268; 29,8%), 41-45 (n = 732; 17,2%), 46-50 (n = 304; 7,2%), &amp;gt;50 years (n = 141; 3,3%). The mean age was 37.4 ± 6.4 years (range 18-71 years). Participants/materials, setting, methods The study was population-based and included male patients from all ages, ethnicities, and medical histories. Semen samples were collected after 2-3 days of abstinence. For patients who underwent more than one DFI testing, only the first sample was included, and any duplicates were excluded. DFI was evaluated by flow-cytometry based TUNEL assay using the APO-Direct Kit. Data were analyzed using one-way ANOVA and T3 Dunnett post-hoc multiple comparison test, as well as Pearson’s correlation coefficient. Main results and the role of chance The results show a significant positive correlation between %DFI and age (r = 0.19, p &amp;lt; 0.001). Mean %DFI levels were relatively stable in men aged &amp;lt;26 to 35 years (17.9%, 18.1% and 18.1% in men aged &amp;lt;26, 26-30 and 31-35, respectively), with %DFI increasing progressively beyond age 35 (20.7%, 22.5%, 25.7% and 27.9% in men aged 36-40, 41-45, 46-50 and &amp;gt;50, respectively). The mean %DFI in the 36-40 age group was significantly higher than in the 31-35 age group (p &amp;lt; 0,001). Our study has uncovered that %DFI follows an exponential curve starting at age 35, indicating that the %DFI accelerates significantly as men age beyond their mid-30s. Limitations, reasons for caution This retrospective analysis has inherent limitations that may introduce confounding variables. The patient clinical background, such as medical history and lifestyle factors was not assessed. Also, the studied cohort consisted of a population under investigation for infertility and may not be representative of the general male population. Wider implications of the findings The study demonstrates the age-related increase in sperm %DFI and suggests that there may be an age cut-off below which sperm %DFI is stable and beyond which sperm %DFI increases. This information may be useful to medical specialists that offer sperm DNA testing to infertile couples. Trial registration number not applicable

Effect of pesticide exposure on human sperm characteristics, genome integrity, and methylation profile analysis.

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group.

How much obesity and diabetes do impair male fertility?

BackgroundSubfertility in obese and diabetic men during the reproductive age is evident, but the mechanisms by which obesity and diabetes mellitus cause male infertility are not entirely understood. The current study aimed to evaluate the effects and potential mechanisms of obesity and diabetes on male fertility.MethodsWe enrolled control = 40, obese = 40, Lean-DM = 35, and Obese-DM = 35 individuals. The obesity-associated markers, diabetic markers, hormonal and lipid profile, inflammatory indices, and semen analysis were assessed in four experimental groups.ResultsOur finding showed that diabetic markers were significantly increased in two diabetic groups, while obesity indices were markedly increased in two obese groups. Conventional sperm parameters were significantly lower in three groups compared with the control. Serum levels of total testosterone and sex hormone-binding globulin were significantly lower in men with obesity and DM compared with the control. There was a significant difference in the concentration of high-sensitivity C-reactive protein among four experimental groups. Moreover, serum leptin was significantly increased in obese DM, lean DM, and obese groups. Serum insulin levels had a positive correlation with metabolic-associated indices and high-sensitivity C-reactive protein levels, whereas it had a negative correlation with count, motility, and morphology.ConclusionsOur findings showed the metabolic changes, hormonal dysfunction and inflammatory disturbance might be suspected mechanisms of subfertility in obese and diabetic subfertile men.

H19 Sperm Methylation in Male Infertility: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis summarize the difference in the methylation of the H19 gene in patients with abnormal versus normal conventional sperm parameters. It also evaluates the effects of age and sperm concentration on H19 methylation in spermatozoa using meta-regression analysis. It was performed according to the MOOSE guidelines for meta-analyses and Systematic Reviews of Observational Studies and the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P). The quality of the evidence reported in the studies included was assessed using the Cambridge Quality Checklists. A total of 11 articles met our inclusion criteria. Quantitative analysis showed that H19 methylation levels were significantly lower in the group of infertile patients than in fertile controls. The reduction in methylation was much more pronounced in patients with oligozoospermia (alone or associated with other sperm parameter abnormalities) and in those with recurrent pregnancy loss. Meta-regression analysis showed the results to be independent of both patient age and sperm concentration. Therefore, the H19 methylation pattern should be evaluated among couples accessing assisted reproductive techniques (ART), in order to gain prognostic information on ART outcome and offspring health.

Sexual and Reproductive Outcomes in Obese Fertile Men with Functional Hypogonadism after Treatment with Liraglutide: Preliminary Results.

Purpose: To prospectively investigate the effects of treatment with liraglutide, a glucagon-like peptide 1 (GLP1) analog, on reproductive and sexual function in men with metabolic hypogonadism who are of childbearing age. Materials and Methods: To accomplish this purpose, 110 men of childbearing age (18-35 years) with metabolic hypogonadism were enrolled and divided into three groups, according to their desire to have children. Group A was made up of men actively seeking fatherhood, Group B, of men who did not seek fatherhood, and Group C, of men who had already fathered a child. Group A patients were treated with gonadotropins (urofollitropin at 150 IU, three times a week, and human chorionic gonadotropin at 2000 IU, twice a week), Group B patients with liraglutide (3 mg daily), and Group C patients with transdermal testosterone (60 mg per day). All patients were treated for 4 months. Results: Patients treated with liraglutide (Group B) showed significant improvement in conventional sperm parameters, compared to baseline and Group A patients, and in the quality of erectile function compared to baseline and patients of Groups A and C. In addition, they had significantly higher levels of total testosterone and sex hormone-binding globulin serum levels after 4 months of treatment with liraglutide than those achieved by patients in the other two groups at the end of the respective treatments. Finally, Group B patients also showed significantly higher serum gonadotropin levels than the other groups. Conclusions: The results of this study showed, for the first time, the efficacy of liraglutide, a GLP1 analog, for the pharmacological treatment of male patients with metabolic hypogonadism. Liraglutide has also shown advantages over traditional treatments on both reproductive and sexual function and appears to offer greater benefits in terms of metabolic protection. These findings suggest that liraglutide is a useful drug for the treatment of obese males with metabolic hypogonadism.

Alpha-Lipoic Acid Supplementation for Male Partner of Couples with Recurrent Pregnancy Loss: A Post hoc analysis in Clinical Trial.

Increased sperm DNA damage is known as one of the causes of recurrent pregnancy loss (RPL) which can be due to increased levels of oxidative stress. Therefore, the aim of this study was to assess the effect of alpha-lipoic acid (ALA) on sperm parameters and sperm functions in couples with a history of RPL. In this post hoc analysis in clinical trial study, a total of 37 couples with RPL (n=12 and n=25 for placebo and ALA groups, respectively) were considered. Men were treated with ALA (600 mg/day) or placebo for 80 days. Semen samples were acquired from the participants before initiation and after completion of the medication course and assessed regarding conventional sperm parameters, chromatin damage/integrity, intracellular oxidative stress, lipid peroxidation, and seminal antioxidant characteristics. Individuals were further followed up for twelve months for pregnancy occurrence and outcomes. Finally, after excluding patients with no history of RPL, the data was analyzed. No significant differences were observed between the baseline measures of the aforementioned parameters except for seminal volume. After the intervention, the mean sperm DNA damage, protamine deficiency, and persisted histones were significantly lower in the ALA group than in placebo receivers (P<0.05). A decrease in the mean of seminal total antioxidant capacity (P=0.03), malondialdehyde (P=0.02), and sperm DNA damage (P=0.004) as well as an increase in sperm total motility (P=0.04) after treatment with ALA was noticed. In addition, the mean of protamine deficiency and persisted histones were declined post-ALA therapy (P=0.003 and 0.002, respectively). The percentage of spontaneous pregnancy in the ALA group (4 of 25 cases; 16%) was higher than in the placebo group (1 of 12, 8.3%). ALA-therapy attenuates sperm DNA damage and lipid peroxidation while enhancing sperm total motility and chromatin compaction in the male partner of couples with PRL (registration number: IRCT20190406043177N1).

The Impact of a Very Short Abstinence Period on Conventional Sperm Parameters and Sperm DNA Fragmentation: A Systematic Review and Meta-Analysis.

In recent years, a growing number of studies have supported the beneficial effects of a very short abstinence period on sperm parameters, especially in patients with oligoasthenozoospermia. However, the results are controversial and no consensus exists regarding whether to request a second semen collection in clinical practice. Therefore, this systematic review and meta-analysis aimed to evaluate the influence of a very short abstinence period (within 4 h) on conventional sperm parameters and sperm DNA fragmentation (SDF) rate. The literature search was performed using Scopus and PubMed databases. The meta-analysis was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocol (PRISMA-P) guidelines. All eligible studies were selected according to the Population, Intervention, Comparison/Comparator, Outcomes, and Study design (PICOS) model. The quality of evidence of the included studies was analyzed through the Cambridge Quality Checklists. The standardized mean difference (SMD) was used to analyze the outcomes. Cochran-Q and I2 statistics were used to evaluate statistical heterogeneity. We assessed for eligibility 1334 abstracts, and 19 studies were finally included. All 19 articles evaluated the effects of a very short abstinence period on sperm parameters and, among these, 5 articles also evaluated the effects on SDF rate. The quantitative analysis showed a significant reduction in semen volume after a very short abstinence period in both normozoospermic men and patients with oligozoospermia, asthenozoospermia, and/or teratozoospermia (OAT) patients. We found a statistically significant increase in sperm concentration and total and progressive motility in the second ejaculation of patients with OAT. In contrast, the SDF rate decreased significantly in the second ejaculate of OAT patients. This is the first systematic review and meta-analysis investigating the impact of a very short abstinence period on sperm parameters and SDF rate. The results suggest that collecting a second consecutive ejaculation after a very short time from the first could represent a simple and useful strategy for obtaining better-quality spermatozoa, especially in patients with abnormal sperm parameters.

THE IMPACT OF VARICOCELE REPAIR ON CONVENTIONAL SEMEN PARAMETERS IN INFERTILE MEN: A META-ANALYSIS USING PRE- AND POST-OPERATIVE DATA

Varicocele is a widespread disorder affecting about 15% of the normal male population, 25% of men with abnormal semen analysis, and 35-40% of primary infertile men. There are few systematic reviews and meta-analyses (SRMAs) employing the “before-after” varicocele repair (VR) to assess the effect of VR on conventional sperm parameters. Indeed, the pregnancy rate is the most common outcome chosen by SRMAs on VR. The present SRMA aims to evaluate the impact of varicocele repair on conventional semen parameters in infertile patients with clinical varicocele using a before-after analysis.