Initial Sperm Concentration Research Articles

Circular Economy, Dairy Cow Feed Leftovers, and Withania somnifera Supplementation: Effects on Black Belly Ram's Libido, Sperm Quality, Sexual Behavior, and Hemogram Values.

Considering a circular economy perspective, this study evaluates the possible effect of targeted short-term supplementation with Withania somnifera L. (WS; Ashwagandha) on ram's seminal quality, socio-sexual behaviors, and blood constituents. Black Belly rams (n = 20) received a basal diet comprising feed-leftovers from dairy cows in the north-arid Mexico (i.e., Comarca Lagunera CL). The experimental units, with proven libido and fertility, were homogeneous in terms of age (3.41 ± 0.21 yr.), live weight (LW; 53.8 ± 3.3 kg), body condition (BC; 2.96 ± 0.01 units), initial sperm concentration (2387 ± 804 × 106), and viability (23.9 ± 15.6%). Rams were randomly assigned during the transition reproductive period (i.e., May to Jun; 25° NL) to three treatment groups: non-supplemented control group (CONT; n = 6), low WS-supplemented (LWS; i.e., 100 mg kg LW-1 d-1 × 40 d; n = 7), and high-WS-supplemented (HWS; i.e., 200 mg kg LW-1 d-1 × 40 d; n = 7). The basal leftover diet was offered twice daily (0700 and 1600 h); the experimental period (EP) lasted 47 d. No differences (p > 0.05) among treatments occurred regarding LW and BCS at the onset of the EP. Whereas the greater scrotal circumference (SCRC, cm) arose in the LWS and CONT rams, an increased ejaculated volume (VOLEJA, mL) occurred in the WS-rams. A total of 5/9 (i.e., 55%) appetitive and 3/3 (i.e., 100%) consummatory sexual behaviors favored (p < 0.05) the WS-rams, particularly the HWS rams, towards the final EP. The same was true (p < 0.05) regarding the hemogram variables white blood cell count (×109 cells L-1), hemoglobin concentration (g dL-1), and medium corpuscular volume (fL). This study, based on a rethink-reuse-reduce enquiry approach, enabled connectedness between two noteworthy animal systems in the CL: dairy cows and meat sheep schemes. Certainly, the use of dairy cow feed-leftovers aligned with the short-term supplementation with WS promoted enhanced testicular function, augmented seminal volume, and an increased sexual behavior in Black Belly rams in northern Mexico. Finally, while our research outcomes should enhance not only the resilience and sustainability of sheep production and the well-being of sheep-producers and their families, it may also embrace clinical translational applications.

(081) Fertility Trends in Men with Prior History of Anabolic Steroid Use: Results from a Standardized Treatment Regimen

Abstract Introduction The management of infertility in men who have previously used anabolic steroids poses a complicated clinical challenge. Addressing the infertility issues in this specific population requires a comprehensive approach that considers the underlying physiological alterations caused by anabolic steroid use and tailors treatment interventions accordingly to restore and optimize reproductive function. In this study, we report an analysis of sperm parameter recovery and fertility outcomes in men who had azoospermia or severe oligospermia caused by anabolic steroid use and underwent a standardized treatment regimen for recovery of spermatogenesis. Objective The study aims to assess the longitudinal trends in sperm concentrations, determine the proportion of men achieving normozoospermia, and evaluate subsequent pregnancy rates in this specific population. Additionally, the study seeks to identify potential predictors for improved semen parameters and successful pregnancy outcomes. Methods A retrospective analysis of the period spanning from 2018 to 2022 was conducted on a cohort of men with a prior history of anabolic steroid use who complained of infertility. The standardized treatment approach involved the cessation of testosterone replacement therapy (TRT) and the administration of a combination regimen comprising clomiphene citrate and human chorionic gonadotropin (hCG) for a minimum of 3 to 6 months. Following adequate treatment, men were contacted in March 2023 to determine the most up-to-date pregnancy outcomes. Statistical analysis was performed utilizing SPSS, employing univariate logistic regression analysis to establish a predictive mathematical model. The level of statistical significance was defined as p &amp;lt; 0.05. Results A total of 45 men (median age 37 years, IQR 32-45) met inclusion criteria for this analysis. Median duration of prior T use was 4 years (IQR 1.3-10), with the two most common modalities consisting of injection therapy (43.5%) and oral therapy (34.8%). Median initial sperm concentration was 0 million/cc (IQR 0-1.15), and 23 (51.1%) men initially presented with azoospermia. The median duration of combination hCG/clomid therapy was 5 months (IQR 3-12). In initially azoospermic men (N:23), 5 were lost to follow-up, 6 (33.3%) progressed to severe oligospermia (&amp;lt;5 million/cc), 6 (33.3%) to oligospermia (&amp;lt;15 million/cc), 1 (5.6%) to normozoospermia (&amp;gt;15 million/cc), and 5 (27.8%) remained azoospermic following medical treatment for 6 months. Among the 24 couples who responded to the follow-up call, a total of 9 (37.5%) achieved a successful subsequent pregnancy. Of these, 33.3% (3 couples) used assisted reproductive technology (ART), while 66.7% (6 couples) conceived naturally. On logistic regression analysis, no significant predictors for improved sperm parameters or successful pregnancy were identified. Conclusions Despite the implementation of appropriate treatment regimens, a significant proportion of men with a prior history of anabolic steroid use continue to exhibit severe oligospermia, with more than half failing to demonstrate substantial improvement in their semen parameters even after undergoing a six-month medical treatment protocol. Moreover, only a fraction of men, estimated to be 1 in 6, achieve normozoospermia following medical treatment after anabolic steroid use. Future work should continue to explore predictors for improved sperm parameters on follow-up as well as subsequent successful pregnancy in men with prior history of anabolic steroid use. Disclosure No.

Fertility outcomes in men with prior history of anabolic steroid use

To study sperm parameters recovery and fertility outcomes in men with azoospermia or severe oligospermia caused by anabolic steroid use who underwent a standardized treatment regimen for spermatogenesis recovery. A retrospective analysis of a cohort of men with a prior history of anabolic steroid use and infertility complaints (between 2018 and 2022) was conducted. The standardized treatment approach involved discontinuing testosterone replacement therapy and administering a combination regimen of clomiphene citrate and human chorionic gonadotropin for a minimum of 3 to 6 months. The main outcome measures included changes in sperm parameters, predominantly sperm concentration, and subsequent pregnancy outcomes. A total of 45 men (median age 37 years, IQR 32-45) met the inclusion criteria for this analysis. Median duration of prior T use was 4 years (IQR 1.3-10), with the 2 most common modalities consisting of injection therapy (43.5%) and oral therapy (34.8%). The median initial sperm concentration was 0 million/cc (IQR 0-1.15), and 23 (51.1%) men initially presented with azoospermia. The median duration of combination human chorionic gonadotropin/clomid therapy was 5 months (IQR 3-12). In initially azoospermic men (N: 23), 5 were lost to follow-up, 6 (33.3%) progressed to severe oligospermia (<5 million/cc), 6 (33.3%) to oligospermia (<15 million/cc), 1 (5.6%) to normozoospermia (>15 million/cc), and 5 (27.8%) remained azoospermic after medical treatment for 6 months. Among the 24 couples who responded to the follow-up call, a total of 9 (37.5%) achieved a successful subsequent pregnancy. Of these, 33.3% (3 couples) used assisted reproductive technology, whereas 66.7% (6 couples) conceived naturally. On logistic regression analysis, no significant predictors for improved sperm parameters or successful pregnancy were identified. Despite appropriate treatment regimens, a significant proportion of men with a prior history of anabolic steroid use continue to exhibit severe oligospermia, with more than half showing limited improvement in semen parameters after 6 months of treatment. Only a fraction of men achieves normozoospermia after treatment. Further research is needed to explore predictors for improved sperm parameters and successful pregnancy outcomes in men with a history of anabolic steroid use.

PD18-05 IDIOPATHIC SECONDARY AZOOSPERMIA OCCURRENCE IN INFERTILE MEN OVER TIME

You have accessJournal of UrologyCME1 Apr 2023PD18-05 IDIOPATHIC SECONDARY AZOOSPERMIA OCCURRENCE IN INFERTILE MEN OVER TIME Gilad Karavani, Kirk C. Lo, Ethan D. Grober, Mohamed S. Kattan, Susan Lau, and Keith Jarvi Gilad KaravaniGilad Karavani More articles by this author , Kirk C. LoKirk C. Lo More articles by this author , Ethan D. GroberEthan D. Grober More articles by this author , Mohamed S. KattanMohamed S. Kattan More articles by this author , Susan LauSusan Lau More articles by this author , and Keith JarviKeith Jarvi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003273.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: While the occurrence of azoospermia in the male population and its risk factors are well studied, only scarce data exists on the unique group of patients without known risk factors for sperm quality deterioration who eventually develop idiopathic secondary azoospermia (ISA). Early identification of such cases will allow interventions to preserve fertility by sperm banking. This study aimed to investigate the occurrence of ISA with time in men with initial oligospermia to identify those with higher risk of becoming azoospermic. METHODS: This was a retrospective cohort study from a single university-affiliated male infertility clinic. Patients that had at least two semen analyses (SA) 3 months or more apart between 2005 and 2021 were included. Patients with normal sperm concentration (above 15 million/ml), those with risk factors for deterioration in semen quality (chemo-radiotherapy exposure, Kleinfelter syndrome, etc.) and those with an initial semen analyses showing azoospermia, were excluded. Patients were allocated to three groups according to their initial sperm concentration – ≤1, 1-5 and 5-15 million/ml. Basic characteristics as well as the first and last SA and hormonal levels were compared between the groups. The primary outcome was the occurrence of ISA in each group with time. RESULTS: Overall, 1044 patients with two SA and no other known risk factors were included in the analysis. Thirty-one patients (3.0%) eventually became azoospermic with time. The ≤1 million/ml group (280 patients) had significantly higher rates of ISA in each time period compared to the 1-5 million/ml (312 patients) and 5-15 million/ml (452 patients) group (p<0.005 for all comparisons). CONCLUSIONS: The group of men with ≤1 million/mL have a dramatically increased chances of becoming azoospermic with time, reaching a peak of over 25% after 3 years. Therefore, sperm banking should be recommended, as early as possible, in case of extremely severe oligospermia. Men with sperm counts > 1million/mL could be counseled that their chances of ISA are low, even after 3 or more years. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e504 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gilad Karavani More articles by this author Kirk C. Lo More articles by this author Ethan D. Grober More articles by this author Mohamed S. Kattan More articles by this author Susan Lau More articles by this author Keith Jarvi More articles by this author Expand All Advertisement PDF downloadLoading ...

DNA extraction methods of canine sperm cells to sexing by quantitative PCR

Despite similarity to other cell types, the sperm has a compacted chromatin which makes DNA extraction more difficult. Based on this, this study compared different sperm concentrations and three DNA extraction techniques to use in qPCR. Four protocols were tested: a non-commercial using phenol:chloroform (M1) alone or associated with a preparatory kit (Differex-System-Kit-M2), and two commercial with mini-columns extraction: M3 (Illustra-Blood-Genomic-Prep-Mini-Spin) and M4 (Wizard®-Genomic-DNA-Purification). Four sperm concentrations were used (1, 10, 30 and 50x106) from ejaculates of 3 dogs. After extraction, the DNA was used for qPCR with primers directed against X and Y chromosomes. M1 and M2 protocols recovered high DNA concentration independently of initial sperm concentration, with a satisfactory ratio of 260/280 absorbances (mean 1.80 and 2.10, M1 and M2 respectively). In contrast, other methods recovered low DNA concentration and with poor quality in M3 (mean 1.60 in M3 and 1.85 in M4). The DNA extraction of canine sperm cells using protocols with phenol:chloroform (M1 and M2) was efficient. The M1 protocol notably allowed the quantification of X and Y chromosomes in qPCR using low number of sperm cells (1x106). This study provides the first comparison of DNA extraction techniques of canine sperm and it is concluded that independent of sperm concentration used, including use of only 1x106 sperm, DNA extraction from these cells using phenol:chloroform has satisfactory results regarding the quantity and quality of the extracted sample, and enabled quantitation of target gene sequences in qPCR, allowing subsequent use in sperm sexing and other biotechnologies.

Inhibin-B and FSH Are Good Indicators of Spermatogenesis but Not the Best Indicators of Fertility.

Biochemical markers of spermatogenesis and fertility assessment are important in the practical management of infertile males and the determination of an individual’s prognosis. We performed an analysis on 100 males with a male infertility factor. The following study inclusion parameters were analyzed: seminogram, FSH, LH, testosterone, estradiol, prolactin, TSH, and inhibin B concentrations. The patients were subsequently treated by reproductive endocrinologists in accordance with AUA/ASRM and EAU guidelines. The reproductive status was evaluated over a period of 3 years. We found a strong correlation of sperm count with inhibin B (r = 0.74, p < 0.001) and FSH concentration levels (r = −0.46, p < 0.001). Among 95 patients at follow-up, pregnancies occurred for 59 of their partners (48 spontaneous, 5 after IVF–ET, and 6 after IUI). Thirty-six patients remained childless despite the therapy. Sperm count and inhibin B level were the best predictors of natural fertilization (ROC AUC: 0.86 and 0.84; cut-off: 2.7 mln/mL and 45 pg/mL). Although inhibin B and FSH can be used to evaluate spermatogenesis and fertility, the initial sperm concentration appeared to be the best predictor of success. Pregnancy was achieved in a surprisingly large proportion of patients with a very low concentration of inhibin B and a low initial sperm count. It is noteworthy that 81% of the pregnancies were achieved without medically assisted reproduction.

Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10–14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0–d5), T2 (d6–d10), and T3 (d11–d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

EFFICACY OF CLOMIPHENE CITRATE IN SUBFERTILE MEN STRATIFIED BY PRE-TREATMENT SEX HORMONE LEVELS AND SPERM CONCENTRATION

Selective estrogen receptor modulators (SERMs) are known to improve sperm count. Men with low testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) are thought to benefit most from SERMs like clomiphene citrate (CC), though predictors of efficacy have not been robustly characterized. We sought to identify changes in sperm counts with CC, stratifying by pre-treatment sex hormones and semen parameters. With IRB approval, we retrospectively reviewed sex hormone levels and semen parameters in subfertile men before and after 3 months of CC use (25 mg qd or 50 mg qod) during 10/2015-6/2020. Men were stratified by initial hormone levels (T≥300 vs T<300, LH≥7 vs LH<7, FSH≥7 vs FSH<7) and separately by initial sperm concentration (azoospermia, oligozoospermia, and normospermia). Men who previously underwent hormone therapy, genital surgery, or had previously conceived were excluded. Men with or without varicoceles were included. Student t-test was conducted with alpha=0.05. Out of 137 patients, 34% improved, 13% worsened, and 53% had no change in sperm concentration with CC use. 77% of azoospermic men had no change in sperm concentration. Table 1 summarizes changes in T and sperm concentration stratified by initial parameters. No significant difference was found in magnitude of changes in semen parameters stratified by testosterone. Men with lower LH had greater improvements in total sperm count (TSC) and total motile sperm count. Men with lower FSH had greater improvements in T, sperm concentration, and TSC. Azoospermic men showed least improvement across all semen parameters. No significant differences in magnitude of improvement were observed between oligozoospermia sub-categorizations (mild, moderate, severe) and normospermia. Not all patients experience the same benefit from CC. Our data suggest that men with nonobstructive azoospermia do not significantly benefit from CC use. Degree of oligospermia did not affect magnitude of improvement. Men with lower gonadotropin levels may expect greater degree of improvement in both hormone and semen parameters.

MP31-17 PRE-TREATMENT SEX HORMONE LEVELS AND SPERM CONCENTRATION INFLUENCE MAGNITUDE OF RESPONSE TO CLOMIPHENE IN SUBFERTILE MEN

You have accessJournal of UrologyInfertility: Therapy (MP31)1 Sep 2021MP31-17 PRE-TREATMENT SEX HORMONE LEVELS AND SPERM CONCENTRATION INFLUENCE MAGNITUDE OF RESPONSE TO CLOMIPHENE IN SUBFERTILE MEN Tommy Jiang, Alvaro Santamaria, Neilufar Modiri, Michael Zheng, Vadim Osadchiy, John Sigalos, Jesse Mills, and Sriram Eleswarapu Tommy JiangTommy Jiang More articles by this author , Alvaro SantamariaAlvaro Santamaria More articles by this author , Neilufar ModiriNeilufar Modiri More articles by this author , Michael ZhengMichael Zheng More articles by this author , Vadim OsadchiyVadim Osadchiy More articles by this author , John SigalosJohn Sigalos More articles by this author , Jesse MillsJesse Mills More articles by this author , and Sriram EleswarapuSriram Eleswarapu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002035.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Selective estrogen receptor modulators (SERMs) are known to improve sperm counts. Men with low testosterone and gonadotropins are thought to benefit most from SERMs like clomiphene, though predictors of efficacy have not been robustly characterized. We sought to identify changes in sperm counts with clomiphene, stratifying by pre-treatment sex hormones and sperm parameters. METHODS: With IRB approval, we retrospectively analyzed sex hormone levels and sperm parameters in subfertile men before and after 3 months of clomiphene (25 mg qd or 50 mg qod) during 10/2015-6/2020. Men were stratified by initial hormone levels (T≥300 vs T<300, LH≥7 vs LH<7, FSH≥7 vs FSH<7) and separately by initial sperm concentration (azoospermia, oligozoospermia, and normospermia). Men who previously underwent hormone therapy, genital surgery, or conceived a pregnancy were excluded. Student t-test was conducted with α=0.05. RESULTS: Out of 137 patients, 34% improved, 13% worsened, and 53% had no change in sperm concentration categorization (Fig. 1). 77% of azoospermic men had no change in sperm concentration. Table 1 summarizes changes in T and semen analyses stratified by initial parameters. No significant difference was found in magnitude of changes in semen parameters stratified by testosterone. Men with lower LH had greater improvements in total sperm count (TSC) and total motile sperm count (TMSC). Men with lower FSH had greater improvements in T, sperm concentration, and TSC. Azoospermic men showed least improvement across all semen parameters. No significant differences in magnitude of improvement were observed between oligozoospermia sub-categorizations (mild, moderate, severe) and normospermia. CONCLUSIONS: Not all patients experience the same benefit from clomiphene. Our data suggest that men with azoospermia do not significantly benefit. Degree of non-azoospermia did not affect magnitude of improvement. Men with lower gonadotropin levels may expect greater degree of improvement in both hormone and semen parameters. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e561-e562 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tommy Jiang More articles by this author Alvaro Santamaria More articles by this author Neilufar Modiri More articles by this author Michael Zheng More articles by this author Vadim Osadchiy More articles by this author John Sigalos More articles by this author Jesse Mills More articles by this author Sriram Eleswarapu More articles by this author Expand All Advertisement Loading ...

Effect of Hydroxyurea Exposure before Puberty on Sperm Parameters in Males with Sickle Cell Disease

Background: Sperm parameters alteration is documented in untreated men with sickle cell disease (SCD). An aggravating effect of hydroxyurea (HU) on sperm parameters is also well established: HU, at current doses, causes significant, rapid, and unpredictable impairment of spermatogenesis. Reversal of its effect when treatment is stopped has also been documented, albeit in patients given very low doses of treatment (10 mg/kg/d). In France, sperm banking is free of charge and recommended whenever possible before initiation of HU treatment. While recent guidelines have broadened the indication of HU for asymptomatic infantssuch banking is impossible in younger boys before puberty. In addition, little is known on the effect of HU on sperm parameters when given at this specific period. Objectives and Methods: The main objective of this study was to compare sperm parameters after treatment resolution in young males treated by HU prior to puberty with sperm parameters of untreated males. Secondary objective was to analyze longitudinally sperm parameters during HU washout in those treated prior to puberty.Data regarding indication of HU, dosage, date of initiation and stop, clinical profile including vaso occlusive events (VOE) transfusion episodes and date of puberty was collected. Alternative treatment before or during semen analysis was also documented. A period of 3 months of HU wash out was required prior to semen analysis in treated patients. Results: A total of 26 patients (43 semen samples) were studied, with 16 patients treated with HU prior to puberty (HU-PP) and 10 untreated (HU-naive). Characteristics of patients are presented in Table 1. Indication of HU was cerebral vasculopathy (n=3), VOE (n=5), severe anemia (n=1) or combined (n= 7). The median stopping of the HU before CECOS is 4.5 years [0.5-11.0]. An alternative treatment based on a transfusion program was initiated in 14 patients (87.5%) at the time of sperm analysis in the HU-PP group versus 5 patients (50%) in the HU-naive group. Duration of transfusion program was 126 months [3-188] in HU-PP versus 13 [7-100] in the HU naive group. We compared the fraction of abnormal values in semen samples in both groups (25 samples in the HU-PP group and 18 in the HU-naive). No significant difference was observed regarding volume of ejaculate, spermatozoid concentration, total sperm count, spermatozoid motility, morphology and vitality, and sexual abstinence before sampling in both groups (Table 1). In the HU-PP group, there was a trend in improvement of sperm parameters with the duration of transfusion program. In addition, a kinetic analysis of sperm parameters during the period of HU wash out was performed in 3 patients exposed to HU prior to puberty, demonstrating reversibility of HU toxicity in all. All 26 patients were offered semen cryopreservation. For 22 of them, semen parameters after thawing indicated a possible use for assisted reproductive technologies, mostly in vitro fertilization with intracytoplasmic sperm injection. In the remaining 4 (3 in HU-PP group and 1 in HU naïve), there was serious concern about the possible use because of a very low initial sperm concentration and the absence of motile spermatozoids after thawing. Discussion/Conclusion: Toxicity of HU upon spermatogenesis has been well documented in animal studies and in adult males. In many settings, such issues may be a drawback for parents and/or caregivers to treat young boys before semen banking can be performed, given the lack of robust information on reversibility after treatment resolution. Abnormalities of sperm count are also common in non-treated males so that demonstrating the specific effect of HU may be complex. Oseggbe et al. and Berthaud et al. showed that 91% of untreated SCD males have at least one abnormal sperm parameter. Here, we show that after treatment resolution, there are no differences in sperm parameters in patients exposed to HU before puberty compared to untreated males. Because a majority of patients benefitted from transfusion therapy at the time of analysis, we were however unable to demonstrate whether reversal of toxicity occurs spontaneously or requires transfusion. Notwithstanding possible limitations due to small sample size, this study shows that in boys with severe disease requiring HU treatment before puberty, toxicity of HU on sperm parameters should not be a major drawback. Disclosures Bernaudin: AddMedica: Honoraria, Other: Help for travel; GBT: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Consultancy. De Montalembert:Novartis: Consultancy, Honoraria; Addmedica: Consultancy, Honoraria; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Brousse:bluebird bio: Consultancy; Add medica: Consultancy.

Prolonged semen cryopreservation decreases motile concentration

There is a paucity of data regarding the viability of cryopreserved sperm. Good laboratory practice involving cryopreservation include QA/QC, equipment maintenance and stable temperatures of -196°C. These ensure tissues remain viable for later use. However, cryopreservation has been shown to alter the structure and function of stored spermatozoa. This study aims to determine if sperm viability is affected by long-term storage. Before-After Study. Patients sperm samples that were abandoned over the years of 1995-2014 (average 14.5 years) were thawed and analyzed before discard. Samples were considered abandoned if patients couldn’t be contacted within the past 5 years to continue storage. Prior to initial freeze, semen analyses were performed. Semen samples were stored in a 1:1 mixture of test yolk buffer, aliquoted into a 1.5 ml cryovial and suspended in nitrogen vapor for 30 minutes prior to being plunged into liquid nitrogen. Vials were thawed by placing cryovials into 37°C heat blocks for 20 minutes. Post-thaw survival of sperm was determined by calculating sperm concentration, motility and progression. Pre and post analyses were analyzed using regression analyses with a cluster analysis to account for pairwise comparisons. 131 patient semen samples were grouped into four categories listed in Table 1. Age, initial sperm concentration and motility were not different between groups. Overall there is a significant decline in sperm motility with years of storage as vials stored for 20 years and longer show a 70% decrease compared to 10 years and less (p=0.0012). The patient age or initial sperm concentration at the time of freeze has no impact on sperm survival. Despite appropriate measures to maintain specimen in storage, it appears that prolonged storage in liquid nitrogen may impact sperm survival. This result warrants further study as viability of tissues may be affected over time and reduce the success of fertility outcomes.Table 1Summary of difference between initial and post-thaw motile concentrationsYears StorednPt AgeInitial Motile Concentration (M/ml)Post-Thaw Motile Concentration (M/ml)Δ<103038.8±4.937.6±33.519.6±29.80.26±0.2610-143637.7±7.528.2±29.316.6±220.28±0.1815-193638±7.768.8±3610.1±21.90.18±0.21>202934.7±8.745±58.211.7±24.10.09±0.13**p<0.05 Open table in a new tab

Seasonal effect on seminal constituents of swamp buffalo (Bubalus carabanesis) in the foot hill of Eastern Himalaya

ABSTRACT A total of 150 ejaculates, 10 from each of 15 swamp buffalo bulls comprising each group 5 animals were used in the study to observe the seasonal effect in sperm biochemical and semen quality. Methods: Clinical and andrological examinations, and monitoring of semen production and quality were carried out on fifteen mature, healthy swamp buffalo bulls in North East India. Seminal characteristics were compared between three seasons of the year (Summer, March-June; Monsoon, i.e. July–September; Autumn, i.e. October–November and winter, i.e. November–February) with distinct ambient temperature and humidity. Results: All bulls were diagnosed as clinically healthy and with good libido throughout the period of study. Ejaculate volume, mass activity, HOST and acrosomal integrity differ significantly (P < 0.05–0.001) between all seasons, whereas initial sperm motility, sperm concentration and abnormalities did not differ significantly. The total protein, total cholesterol and total lipid of seminal plasma of swamp buffalo semen differ significantly between all the seasons. In conclusion, climatic changes did not seem to largely affect semen sperm output or viability, although the proportions of some semen characteristics and biochemical constituents altered season wise. Conclusion: Seasonal changes moderately appear to cause deleterious changes in sperm quality in swamp buffalo.

Effect of bariatric surgery on semen parameters and sex hormone concentrations: a prospective study

Obesity has become a major health concern, with a prevalence rate approaching epidemic states. An inverse relationship between men′s body weight and semen parameters has been observed, suggesting a favourable role for weight loss in improving fertility. This prospective study included 46 patients undergoing sleeve gastrectomy, who were investigated with semen analysis and serum hormone tests before and 12 months after surgery. Patients were divided into three groups according to their initial sperm concentration; median loss of body mass index was used as a cut-off to further classify patients according to extent of weight loss. Patients' preoperative seminal investigations revealed azoospermia in 13 (28.3%), oligospermia in 19 (41.3%) and normal sperm concentration in 14 (30.4%). Overall, only serum testosterone significantly increased after surgery (P < 0.001). Between study groups, the increase in sperm concentration was statistically significant in men with azoospermia and oligospermia (both P < 0.05), whereas serum testosterone was statistically significant in all groups (P < 0.001). Changes in semen and hormone tests were not affected by the extent of weight loss experienced by patients. Weight loss from bariatric surgery had a favourable effect on serum testosterone levels and semen parameters of patients with pre-existing azoospermia and oligospermia.

Semen Discarded During Different Stages of Cryopreservation in Ongole (Bos indicus) Bulls

Indian subcontinent is a treasure house of Bos indicus cattle, one of the most popular breed amongst them is “Ongole” which by inheritance is a dual purpose breed. During the period under report 549 ejaculates were collected from eight breeding bulls, of which 192 ejaculates (34.97%) were discarded during various stages of semen processing. Out of the total ejaculates, 126 (22.95%), 40 (7.29%) and 26 ejaculates (4.74%) were discarded after collection, equilibration and freezing, respectively while most of the discards were second ejaculates. From the present study, it was concluded that the ejaculate discard in Ongole bulls was mainly based on low volume, initial progressive motility, sperm concentration and post-thaw motility. However, it was suggested that certain reproductive technologies like Sephadex or Glass wool filtration or Percoll gradients could be used to enhance the quality of these low grade ejaculates from genetically superior bulls.

Effect of Management Systems on Seminal Characteristics of Jakhrana Bucks

A study was carried out with an objective to determine effect of management systems on seminal characteristics of Jakhrana bucks. Twenty-four Jakhrana bucks (one year-old with mean body weight of 30.0±2.0 kg) were randomly allotted into two groups as Gr-I (intensive system, n=12) and Gr-ll (semi-intensive system, n=12). A total of 336 ejaculates (168 from each group) were collected (twice weekly) using artificial vagina. Each ejaculate was evaluated for volume, mass motility, pH, progressive motility, sperm concentration, viability, hypo-osmotic swelling (HOS) response and acrosomal integrity. The mean semen volume was significantly (P<0.05) higher in Gr-I, but the sperm mass motility, initial progressive motility, sperm concentration, viability and acrosomal integrity were significantly (P<0.05) higher in Gr-ll. However, non-significant difference was observed in mean pH and HOS responsive spermatozoa. The study indicated that semi-intensive system of management is better for producing quality semen from Jakhrana bucks.

Spontaneous pregnancy rates in Chinese men undergoing microsurgical subinguinal varicocelectomy and possible preoperative factors affecting the outcomes

To investigate pregnancy rates after microsurgical varicocelectomy in Chinese infertile men with clinical varicoceles and to identify preoperative factors affecting the outcomes. Retrospective data analysis. University infertility clinic. One hundred seventy-six consecutive Chinese men with infertility and clinical varicoceles who underwent varicocelectomy from January 2010 to December 2011. Semen data were available for 145 patients, and pregnancy data were available for 66. Varicocelectomy was performed on all patients. Spontaneous pregnancy rates and improvement in semen parameters after varicocelectomy. We followed 145 men (82.4%) for a median of 21 months (range 12-36 months) after varicocelectomy. For 109 patients (75.2%), both sperm concentration and forward motility improved. Overall, 45.5% (66/145) of female partners achieved spontaneous pregnancy at a mean follow-up of 11.7 ± 6.2 months. On univariate analysis, preoperative high sperm concentration (≥20 million/mL) and high sperm motility (≥25%) were positively associated with spontaneous pregnancy. On multivariate analysis, only preoperative sperm concentration was an independent predictor. Microsurgical varicocelectomy could improve semen quality and spontaneous pregnancy rates. Only preoperative sperm concentration was an independent predictor of spontaneous pregnancy. Patients with high initial sperm concentration may benefit from varicocelectomy for spontaneous pregnancy.

Should ART Patients with Severe Oligozoospermia Undergo Sperm Cryopreservation at Time of Initial Diagnosis?

Every experienced IVF clinician has been confronted by the scenario of a couple about to undergo ICSI for a diagnosis of severe oligozoospermia, only to find there is azoospermia on the day of oocyte collection. To establish the incidence of this disastrous event, a retrospective study of all men with an initial sperm concentration of <5 million/ml, who subsequently had at least 2 further samples collected was conducted. An analysis of the change in sperm parameters over time was undertaken. Of the 35 men identified, 31.4% (11/35) did not have any change in their sperm concentration, 20.0% (7/35) of men had an increase, and 34.3% (12/35) declined. Seven men (20.0%) became azoospermic. These findings suggest that all men with an initial sperm conc

Endogenous prostaglandin F2α concentrations in bovine whole semen, seminal plasma, and extended semen

A series of experiments were conducted to quantify PGF2α in bovine semen, seminal plasma, and extended semen, and to determine if PGF2α was synthesized or released during extension of bovine semen. Concentrations of PGF2α were measured in paired samples of whole and extended semen from beef and dairy bulls. Concentrations of PGF2α did not differ between beef and dairy (mean ± SEM, 273.8 ± 42.8 vs. 210.3 ± 18.5 pg/mL, respectively; P = 0.12), but tended (P = 0.08) to be greater for whole compared with extended semen (255.5 ± 29.8 vs. 194.5 ± 17.0 pg/mL). Whole semen was extended at eight dilution rates (regardless of initial sperm concentration), using a diluent consisting of two fractions. Samples collected after each dilution step resulted in four subsamples. Concentrations of PGF2α in subsamples decreased (P < 0.001) at higher dilution rates and later steps of extension. Subsequently, whole semen and seminal plasma were extended at three dilution rates. Initial PGF2α concentration was greater (P < 0.001) for whole semen compared with seminal plasma. During extension, PGF2α synthesis or release resulted in less disparity, but the amount synthesized or released was greater (P = 0.03) for semen compared with seminal plasma. We concluded that synthesis or release of PGF2α during extension resulted in concentrations similar to whole semen.

201 EFFECTS OF REMOVING SEMINAL PLASMA DURING 8-h STORAGE BEFORE SEX-SORTING BOVINE SPERM

We evaluated sex-sorting parameters and post-thaw motility for sperm stored with or without seminal plasma for 8 h before sorting. One first ejaculate was collected from each of 6 bulls routinely collected via artificial vagina; ejaculates contained at least 70% motile and 75% morphologically normal sperm and sperm concentrations ranged from 0.75 to 2.21 × 109 sperm mL–1. Ejaculates were divided into 2 samples and centrifuged at 1000 × g for 15 min. Seminal plasma from 1 sample was replaced with TALP (pH 7.4) to a sperm concentration of 1.4 × 109 sperm mL–1. The seminal plasma/sperm admixture of the other sample (control) was suspended to initial ejaculate sperm concentration. Both samples were stored for 8 h at 16°C before being subjected to standard sex-sorting procedures. Sperm were analyzed and bulk sorted on a MoFlo SX (XY Inc., Navasota, TX, USA) flow cytometer/cell sorter for percentage of live-oriented cells, percentage of membrane-impaired sperm (cell membranes permeable to red food colouring, which were discarded during sorting) and resolution between X- and Y-bearing sperm populations (peak to valley ratio). Sorted sperm were frozen according to standard procedures and post-thaw motility was determined immediately after thawing using computer-assisted sperm analysis. Treatments were compared using a paired t-test. Control sperm stored with seminal plasma resulted in a higher percentage of live-oriented cells (55%) versus those stored without seminal plasma (51%; P = 0.02). The percentage of membrane-impaired sperm was lower for control sperm (19%) than that of samples without seminal plasma (28%; P &lt; 0.001). Resolution was greater for sperm stored without seminal plasma (34%) than for control sperm (10%; P = 0.04). Post-thaw, both total and progressively motile sperm were higher for samples without seminal plasma (63 and 53%, respectively) compared with those of the control samples (52 and 45%, respectively; P &lt; 0.04). In conclusion, sperm stored for 8 h without seminal plasma had greater resolution between X- and Y-bearing populations and higher post-thaw motility than control sperm. However, these samples had a higher percentage of membrane-impaired sperm that were removed during sorting. Long-term storage of sperm in their seminal plasma before sex-sorting appears to be detrimental to post-sorting, post-thaw sperm motility.

277 EFFECTS OF SEMINAL PLASMA ON SEX-SORTING BOVINE SPERM

This experiment evaluated how characteristics of bovine ejaculates affect the sortability of X- and Y-bearing spermatozoa. Ejaculates were collected by artificial vagina, 2 each from 10 bulls with an average of 1 h between collections. Only ejaculates with at least 60% motile and 70% normal sperm were used. Semen was centrifuged at 1 000 × g for 15 min to separate sperm from seminal plasma; seminal plasma was clarified by 10 min of additional centrifugation at 2 000 × g. Sperm were rediluted to 160 million sperm/mL with TALP (pH 7.4) and 0, 5, 10, or 20% seminal plasma, from the same ejaculate or reciprocally from first/second ejaculates. Following incubation with Hoechst 33342 for 45 min, an equal volume of TALP (pH 5.5) containing red food dye was added, and sperm were analysed on a MoFlo (Dako, Glostrup, Denmark) flow cytometer for percentage live-oriented cells, X sort rate, coincidence rate, percentage dead or dying (sperm membrane permeable to red dye), and splitability (peaks to valley ratio). The percentage live-oriented sperm was higher for treatments with 0% seminal plasma (64.4%) than for 5 (59.6%), 10 (59.0%), and 20% (57.8%) seminal plasma (P &lt; 0.01). The percentage live-oriented sperm was higher for second (63.0%) than for first ejaculates (56.2%). Sort rate was higher for 0% seminal plasma and second ejaculates (P &lt; 0.05). Dead/dying rates were lower for 0% (16.5%) than for 5 (21.9%), 10 (23.6%), or 20% (23.4%) seminal plasma (P &lt; 0.003); and for first ejaculates (25.9%) compared with second ejaculates (18.2%). Seminal plasma percentage and ejaculate had no effect on splitability, and there was no difference in sorting parameters whether the seminal plasma was from the first or second ejaculate. The initial sperm concentration of the ejaculate (range, 1.3 to 3.4 billion sperm/mL) did not affect any sperm sorting parameter. Thus, effects of sperm concentration on sortability need to be reconsidered, as current practice is to select ejaculates to sort based on initial sperm concentration. The initial pH, percentage morphological abnormalities, initial seminal plasma pH, and age of bull did not affect sorting parameters. In conclusion, the presence of seminal plasma during staining and sorting may decrease sort rate and percentage of live-oriented cells, as well as increase death rate. In addition, sorting second ejaculates may be more advantageous than sorting first ejaculates. Future studies are needed to determine if the results hold true if sperm are stored for several hours and how the various factors affect sperm post-thaw viability.