El laboratorio de fertilidad masculina, su evolución en los últimos 20 años / The male fertility laboratory, its evolution over the last 20 years / O laboratório de fertilidade masculina, sua evolução ao longo dos últimos 20 anos

Resumen La mayor accesibilidad a los tratamientos de reproducción asistida (RA) y los avances de la criobiología produjeron cambios en los laboratorios de andrología. El objetivo de este trabajo fue analizar la demanda y evolución de las variables seminales en las últimas dos décadas, caracterizar el laboratorio andrológico actual, evaluar el impacto de la incorporación del aseguramiento de la calidad y la inclusión de los sistemas computarizados (CASA). Se utilizaron datos de las medias mensuales del control de calidad interno (n=22 528) y encuestas a profesionales de laboratorios andrológicos (n=65) y a médicos especialistas en fertilidad (n=33). La demanda global se redujo significativamente con el aumento de las solicitudes de primera vez. El volumen y recuento, variables dependientes de andrógenos, disminuyeron con los años. El criterio estricto en morfología disminuyó el porcentaje de normales; la mitad de los médicos encuestados recibieron resultados entre 0 y 10% y el 40% consideró que ponía en riesgo el valor clínico de la variable. El sistema CASA permitió objetivar la cinética espermática e incrementar el porcentaje de progresivos rápidos, pero pocos laboratorios lo incorporaron. El 66% de los médicos resuelven el factor andrológico severo por tratamientos clínicos y el 95% utiliza técnicas de RA. El análisis de semen es ejecutado fundamentalmente por bioquímicos especializados, con baja adhesión a la automatización y acreditación del laboratorio, pero con participación en programas de evaluación externa de calidad. La demanda disminuyó como consecuencia del aumento del tratamiento por RA. La reducción del porcentaje de formas normales compromete su utilidad clínica.

Abstract Increasing availability to assisted reproduction (AR) treatments in Argentina and advances in cryobiology resulted in changes in andrology laboratories. The aim of this study was to evaluate the demand and evolution of seminal variables in the last two decades, characterise the current andrology laboratory, evaluate the impact of the incorporation of quality assurance and the introduction of computer assisted semen analysis (CASA). Data were taken from internal quality control (IQC) monthly means (n=22 528) and professionals in charge of laboratories (n=65) and fertility physicians' (n=33) surveys. Overall demand decreased significantly while first-time orders increased. Sperm volume and sperm count -androgen dependent parameters- decreased over the years. Strict morphology criteria reduced the percentage of normal results; half of the physicians received results between 0 and 10% and 40% considered that it compromised the clinical value of the variable. The CASA system made it possible to objectify sperm kinetic, increasing the percentage of fast progressives, but few laboratories have incorporated it. Sixty-six percent of physicians resolve severe andrological factor by clinical treatments and 95% use AR techniques in those cases. Semen analysis is mainly performed by specialised biochemists, with low adherence to laboratory automatisation and accreditation, but with participation in external quality assessment programmes. The demand decreased because of the increase in AR treatment. The lower percentage of normal forms compromises their clinical utility.

Resumo O aumento do acesso aos tratamentos de reprodução assistida (RA) e os avanços na criobiologia levaram a mudanças nos laboratórios de andrologia. O objetivo deste trabalho foi analisar a demanda e a evolução das variáveis de sêmen nas últimas duas décadas, caracterizar o laboratório de andrologia atual, avaliar o impacto da incorporação da garantia da qualidade e a inclusão dos sistemas computadorizados (CASA). Foram utilizados dados das médias mensais do controle de qualidade interno (n= 22 528) e pesquisas a profissionais de laboratórios andrológicos e a médicos especialistas em fertilidade (n=33). A demanda geral diminuiu significativamente com o aumento das solicitações de primeira vez. O volume e a contagem de esperma, parâmetros dependentes de andrógenos, diminuíram ao longo dos anos. O critério morfológico rigoroso diminuiu a porcentagem de normais; metade dos médicos entrevistados recebeu resultados entre 0 e 10% e 40% considerou que isso comprometía o valor clínico do parâmetro. O sistema CASA, permitiu objetivar a cinética espermática e aumentar o percentual de progressões rápidas, mas poucos laboratórios o incorporaram. 66% dos médicos resolvem o fator andrológico grave por tratamentos clínicos e 95% utilizam técnicas de RA nesses casos. A análise do sêmen é realizada principalmente por bioquímicos especializados, com baixa aderência à automação e acreditação laboratorial, mas com participação em programas de avalação externa de qualidade. A demanda diminuiu como consequência do aumento do tratamento por RA. A diminuição em percentagem de formas normais compromete sua utilidade clínica.

Comparison of SPAG11A gene expression in infertile men with grade 1 and 2 varicocele before and after treatment.

OBJECTIVE: Sperm Associated Antigen 11A (SPAG11A) protein is a family of the epididymis-specific secretory proteins implicated in sperm maturation and function. Varicocele might cause pathophysiological difficulties in the testis and epididymis, with a harmful effect on the environment for spermatogenesis and sperm maturation. The aim of this study was to evaluate the expression level of the SPAG11A gene and sperm parameters in infertile men with grade 1 and 2 varicocele before and after treatment. METHODS: Semen specimens were collected from 20 infertile men with varicocele pre-and post-treatment and 10 healthy volunteers. Semen analysis was conducted according to world health organization guidelines. Real time PCR (qRT-PCR) reaction was applied for determination of SPAG11A mRNA expression. RESULTS: The results showed that there was a significant difference between the concentration and normal morphology between pre- and post-treatment groups and the controls. There were significant differences between pre-treatment and control groups in terms of progressive and non-progressive mobility. SPAG11A mRNA levels were significantly lower in the pre-treatment group than in healthy control subjects (p=0.007). There was no statistically significant difference in the expression of SPAG11A as well as semen parameters in the post-treatment group compared to the pre-treatment group. CONCLUSIONS: SPAG11A gene expression and semen parameters may be affected by varicocele. Whether varicocele treatment is an effective approach to reduce the adverse effect of this disease on SPAG11A expression and semen parameters needs further investigation.

Early Pharmacologic Approaches to Avert Anabolic Steroid-induced Male Infertility: A Narrative Review.

PURPOSE: To review the impact of testosterone and other androgenic-anabolic steroids (AASs) on male fertility, exploring potential drugs that can be used to preserve or restore male fertility upon AAS use or prior contact. METHODS: A review was performed to provide a unifying clinical link between drugs used to preserve or restore male fertility (ie, clomiphene citrate, human chorionic gonadotropin, selective estrogen receptor modulators, recombinant luteinizing and follicle-stimulating hormones, and human menopausal gonadotrophin) in the context of AAS-induced infertility and related aspects. FINDINGS: Human chorionic gonadotropin (125-500 IU every other day), clomiphene citrate (12.5-50 mg/d), recombinant luteinizing hormone (125-500 IU every other day), recombinant follicle-stimulating hormone (75-150 IU 1-3×/wk), and human menopausal gonadotrophin (75-150 IU 1-3×/wk) are promising early pharmacologic approaches to avert AAS-induced male infertility. Additionally, a full partner assessment is crucial to the success of a couple planning to have children. The partner's age and gynecopathies must be considered. Egg or sperm cryopreservation can also be alternatives for future fertility. Reinforcing AAS cessation is imperative to achieving better success in misusers. IMPLICATIONS: The exponential increase in AAS misuse raises concerns about the impact on male fertility. This review suggests that gonadotropin analogs and selective androgen receptor modulators (clomiphene citrate) are viable approaches to early preserve or restore fertility in men on AAS use or with previous contact. However, proper standardization of doses and combinations is required and hence physicians should also be aware of patients' and partners' fertility.

Surgically retrieved spermatozoa for ICSI cycles in non-azoospermic males with high sperm DNA fragmentation in semen.

Intracytoplasmic sperm injection (ICSI) using surgically retrieved spermatozoa outside the classic context of azoospermia has been increasingly used to overcome infertility. The primary indications include high levels of sperm DNA damage in ejaculated spermatozoa and severe oligozoospermia or cryptozoospermia, particularly in couples with ICSI failure for no apparent reason. Current evidence suggests that surgically retrieved spermatozoa for ICSI in the above context improves  outcomes, mainly concerning pregnancy and miscarriage rates. The reasons are not fully understood but may be related to the lower levels of DNA damage in spermatozoa retrieved from the testis compared with ejaculated counterparts. These findings are consistent with the notion that excessive sperm DNA damage  can be a limiting factor responsible for the failure to conceive. Using testicular in preference of low-quality ejaculated spermatozoa bypasses post-testicular sperm DNA damage caused primarily by oxidative stress, thus increasing the likelihood of oocyte fertilization by genomically intact spermatozoa. Despite the overall favorable results, data remain limited, and mainly concern males with confirmed sperm DNA damage in the ejaculate. Additionally, information regarding the health of ICSI offspring resulting from the use of surgically retrieved spermatoa of non-azoospermic males is still lacking. Efforts should be made to improve the male partner's reproductive health for safer ICSI utilization. A comprehensive andrological evaluation aiming to identify and treat the underlying male infertility factor contributing to sperm DNA damage is essential for achieving this goal.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp) kept in captivity.

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Testicular and epididymal dysfunctions: searching a new index for the differential diagnosis / Disfunciones testiculares y epididimarias: búsqueda de un nuevo índice para el diagnóstico diferencial

Objective: This proof-of-principle aims to develop an index to aid the differential diagnosis of disorders affecting testicular and/or epididymis. A total of 202 subject data were evaluated in two groups: fertile men with children naturally conceived within 1 year of unprotected intercourse (n = 36) and infertile men (n = 166) who had attempted a pregnancy more than 1 year with unprotected intercourse. Materials and methods: Semen parameters (sperm count, vitality, motility, morphology, and hypoosmotic swelling test [HOST]) were evaluated. The index was calculated by dividing the percentage HOST by the percentage of sperm progressive motility in the fertile group (n = 36). Results: A normal range from 1.23 to 1.53 was determined. Using this index, the outcomes of semen analysis from infertile men were grouped in three study groups: below 1.23 (n = 24), normal (n = 44), and higher than 1.53 (n = 98). These parameters were significantly decreased in semen with normal range (p < 0.01) and in indexes higher than 1.53 (p < 0.0001). Receiver operating characteristic curves compared progressive motility and morphology in infertile men with indexes higher than 1.53 shows that semen samples with normal sperm progressive motility and morphology did not suggest dysfunctions in testis and epididymis. Semen samples with asthenozoospermia suggested epididymal dysfunction (area under the curve [AUC] 0.889, confidence interval [CI] 0.783-1), whereas semen samples with teratoasthenozoospermia suggested dysfunction in both testicles and epididymis (AUC 0.891, CI 0.77-1). Conclusions: The current index proof-of-principle of the success of such a strategy provides valuable information about whether a disorder individually affects testicular and/or epididymal function.

Objetivo: Esta prueba de principio tiene como objetivo desarrollar un índice que ayude al diagnóstico diferencial de los trastornos testiculares y/o epidídimales. Métodos: Se evaluaron 202 individuos divididos en dos grupos: hombres fértiles con hijos concebidos de forma natural en el plazo no mayor a un año (n = 36) y hombres infértiles (n = 166), los cuales habían intentado un embarazo por más de un año. Se evaluaron los parámetros seminales (concentración, viabilidad, movilidad, morfología y prueba de hinchazón hipoosmótica [HOST]). El índice se calculó dividiendo el porcentaje de HOST por el porcentaje de movilidad espermática progresiva en el grupo fértil (n = 36). Resultados: Se determinó un rango normal de 1,23 a 1,53. Utilizando este índice, los resultados del análisis del semen de los hombres infértiles se agruparon en tres grupos de estudio: por debajo de 1,23 (n = 24), normal (n = 44) y superior a 1,53 (n = 98). En contraste, estos parámetros disminuyeron significativamente en el semen de rango normal (p < 0,01) y en los índices superiores a 1,53 (p < 0,0001). Las curvas ROC comparadas con la movilidad espermática progresiva y la morfología en los hombres infértiles con índices superiores a 1,53 muestran que las muestras de semen con movilidad progresiva y morfología normales no sugieren disfunciones en los testículos y epidídimos. Las muestras de semen con astenozoospermia sugerirían una disfunción del epidídimo (AUC 0,889, IC 0,783-1), mientras que las muestra de semen que presentaban teratoastenozoospermia sugerirían una disfunción tanto en los testículos como en el epidídimo (AUC 0,891, IC 0,77-1).

Insights on sperm assays and cryopreservation in six Neotropical pit vipers.

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Evaluation of the effect of conventional and natural antifungals on motility and kinetics of cooled stallion semen.

Microbial growth in semen may cause a decline of sperm quality and fertility; however, the addition of antifungals to semen extender has been shown to impair the overall fertility of the sperm. The aim of this study was to evaluate the antifungal activity of conventional and natural compounds, and their effect on the motility and kinetics of cooled stallion semen. A total of 15 ejaculates from five stallions were collected using the artificial vagina. Each ejaculate was supplemented with: fluconazole at 12.5 (F1), 25 (F2) and 50 (F3) mg/ml; amphotericin-B at 6.5 (A1), 12.5 (A2) and 25 (A3) mg/ml (A3); clotrimazole at 12.5 (C1), 25 (C2) and 50 (C3) mg/ml; isoespintanol at 50 (I1), 100 (I2) and 150 (I3) µM; thymol at 50 (T1), 100 (T2) and 150 (T3) µM; and a control without supplementation. Motility and kinetics of semen at 0, 24 and 48 hr of cooling at 15°C were assessed using computer-assisted sperm analysis (CASA). At hour 48 of cooling, the antifungal effect of the treatments was evaluated. At hour 0 of cooling, amphotericin-B and I3 showed a reduction in most of the motility and kinetic parameters evaluated (p < .05). These treatments, and also C2 and C3, showed similar results at 24 and 48 hr of cooling. Thymol maintained motility and kinetics of the spermatozoa at all evaluated refrigeration times. Besides, I2 showed a decrease (p < .05) in the colony-forming unit compared to that in the control. It is concluded that thymol and isospintanol could be added as natural antifungals in extenders for stallion semen refrigeration.

Comparison of spermiograms of infertile men before and during the COVID-19 pandemic

SUMMARY OBJECTIVE: Since the start of the COVID-19 pandemic, there has been interest in the impact of both SARS-CoV-2 infection and pandemic-induced social restrictions on male reproductive health. This study aimed to evaluate the spermiogram values of men who presented for infertility during the pandemic compared with the previous 2 years. METHODS: Patients who presented to a urology outpatient clinic for the first time due to infertility were included. The patients' age, semen volume, and spermiogram results were recorded. Based on the presentation date, the patients were divided into prepandemic group 1 (March 2018-February 2019), prepandemic group 2 (March 2019-February 2020), and pandemic group (March 2020-February 2021) for comparison. RESULTS: A total of 594 patients were included. There was no significant difference between the three groups in terms of the number of patients who presented for infertility (207, 190, and 197 patients, respectively; p=0.691). The mean age was 36.6±7.2 in the prepandemic group 1, 35.5±7.1 in the prepandemic group 2, and 33.1±6.3 in the pandemic group. Patients who presented during the pandemic were significantly younger (p<0.001). There were no differences between the groups in terms of semen volume (p=0.910) or rates of normospermia and pathological spermiogram findings (p=0.222). CONCLUSIONS: In the first year of the COVID-19 pandemic, there was no significant difference in the number of patients who presented for infertility or in their spermiogram results compared with 2018 and 2019. However, it is noteworthy that the patients were significantly younger during the pandemic than in the previous 2 years.

Assessment of semen parameters in consecutive ejaculates with short abstinence period in oligospermic males.

OBJECTIVE: Human sperm parameters varies widely among men and even between consecutive samples in the same individual with respect to their concentration, motility, morphology, and DNA fragmentation. Less is known about the characteristics of sperm in short abstinence periods. Hence, the current study was conducted to determine the influence of consecutive ejaculate on above parameters after short abstinence period in oligospermic males. METHODS: This observational study was conducted from January 2018 to February 2019 and included 67 men undergoing primary infertility treatment at the SDM Fertility Centre, Dharwad, India. The first semen sample was provided after an abstinence period of 2-7 days, while the second sample was collected 1-3 h after the first. The two consecutive semen samples were analyzed according to the 2010 WHO criteria for semen analysis and their parameters were compared. Sperm DNA fragmentation was also measured. RESULTS: Most of the participants were aged of 31 to 40 years (68.6%). The majority of them had the second sample collected after a 1-hour interval (88%); 10.4% of the subjects had the second sample collected after a 2-hour interval; the remaining 1.4% had the second sample collected after a 3-hour interval. Mean concentration (mill/ml), total motility, and progressive motility (%) were significantly higher in the second sample (p<0.05). The second sample also showed lower DNA fragmentation than the first ejaculate sample. CONCLUSIONS: Our study inferred that consecutive semen samples collected 1-3 hours apart might have a role in managing subfertility in oligospermic males. Further research, possibly a randomized clinical trial, is needed to explore this association.

Semen Analysis of Total Motile Sperm Count Based on the 1999 and 2010 WHO Criteria.

OBJECTIVE: Approximately 15% of the couples suffer from infertility. Half of the cases of infertility are due to male factors. Several sperm function tests have been proposed to evaluate male fertility, but sperm analysis is still the first and most important diagnostic test for male infertility. The prognostic value of semen characteristics such as concentration, morphology and motility markers are often confused with male infertility. Evaluation of seminal parameters and classification for normality remains a frequent topic of discussion. METHODS: This study evaluated 477 semen samples from men undergoing investigation or infertility treatment between 2011 and 2015. RESULTS: The spermograms of 401 patients were deemed abnormal based on the 1999 World Health Organization (WHO) criteria; the number changed to 223 when the spermograms were assessed based on the 2010 WHO criteria and to 200 when Total Motile Sperm Count (TMSC) was used as the criterion. Sperm morphology was the item in the criteria that most significantly changed spermogram classification. Normality parameters became less rigid from 1999 to 2010, thereby significantly changing the proportion of individuals no longer described as infertile/subfertile. CONCLUSIONS: The classification based on TMSC could not differentiate between fertile and infertile subjects for not taking sperm morphology into account. Nevertheless, it may be helpful in cases where intrauterine insemination is indicated.

Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility.

OBJECTIVE: To investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility. DESIGN: Diagnostic test reliability study. SETTING: Andrology laboratories. PATIENT(S): A total of 219 men with infertility. INTERVENTION(S): Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions. MAIN OUTCOME MEASURE(S): Cohen's κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%-29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland-Altman plots). RESULT(S): The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546-0.718). A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812-0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, -0.72 to 2.23), indicating no systematic difference between paired observations. CONCLUSION(S): Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp ) kept in captivity

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane – EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

In vitro sperm characteristics and in vivo fertility of sex-sorted and conventional semen in suckled Nelore cows at a traditional schedule for timed-AI with estrus detection / Características espermáticas in vitro e fertilidade in vivo de sêmen convencional e sexado em vacas Nelore pósparto submetidas à IATF tradicional com detecção de estro

The aim of this study was to assess in vitro sperm characteristics and pregnancies/AI (P/AI) of conventional and sex-sorted semen at timed-AI of suckled, multiparous Nelore cows. All cows (n=348) were submitted to a traditional estradiol/progesterone (P4)-based protocol. At 48h after P4-device removal, the estrous behavior was recorded, and AI was performed with conventional or sex-sorted semen from two bulls. The following sperm assessments were performed: CASA, Hyposmotic Test, sperm morphometry and chromatin structure by TB staining. P/AI were reduced (P<0.001) for sex-sorted compared to conventional semen in cows expressing estrus (27vs47%) or not (11vs.37%). Membrane integrity (Bull1: 30.3±9.6 vs. 52.3±12.4%, P=0.01; Bull2: 24.5±3.0 vs. 48.7±1.6%, P=0.006) and sperm concentration (Bulll: 23.2±0.6 vs. 43.0±0.8x10 sperm/mL, P<0.001; Bull2: 25.1+2.8 VS. 42.1±0.7x10 sperm/mL; P<0.001) were reduced in sex-sorted compared to conventional semen, for both bulls. Total and progressive motility were reduced in sex-sorted semen for Bull1 (TM: 49.7±15.9 vs. 94.9±1.9%, P=0.007; PM: 16.7±3.4 vs. 44.1±13.2%, P=0.009) and no differences were detected for Bull2 (TM: 45.0±17.5 vs. 68.2±19.1%, P=0.098; PM: 12.8±4.7 vs. 30.0±13.0%, P=0.065). Sperm ellipticity from sex-sorted was lower than conventional semen for Bull2 (0.306±0.01 vs. 0.342±0.02, P=0.02) and no difference was detected for Bull1 (0.332±0.01 vs. 0.330±0.01, P=0.55). Reduced in vivo fertility was observed for sex-sorted semen, regardless of estrous behavior. In vitro sperm quality of sex-sorted semen was compromised for both bulls, but differently affected for each sire.

O estudo teve como objetivo avaliar características espermáticas in vitro e a taxa de concepção (TC) de sêmen convencional e sexado em um programa de IATF tradicional de vacas Nelore pós-parto. Todas as vacas (n=348) foram submetidas ao mesmo protocolo de IATF à base de estradiol e de progesterona. Após 48 horas da retirada do implante, foi determinada a expressão de estro dos animais e a IA foi realizada com sêmen convencional e sexado de dois touros Angus. As seguintes características espermáticas foram avaliadas: análise computadorizada do sêmen, teste hiposmótico, morfometria espermática e estrutura cromatinica por meio da coloração com azul de toluidina. A TC foi menor (P<0,001) para sêmen sexado comparado ao convencional, em vacas que expressaram estro (27 vs. 47%) e que não apresentaram estro (11 vs. 37%). A integridade da membrana plasmática (Touro 1: 30,319,6 vs. 52,3+12,4%, P=0,010; Touro 2: 24,5+3,0 vs. 48,7±1,6%, P=0,006) e a concentração espermática (Touro 1: 23,2±0,6 vs. 43,0±0,8x10 sperm/mL, P<0,001; Touro 2: 25,1+2,8 vs. 42,1 +0,7x10'sperm/mL, P<0,001) foram menores no sêmen sexado comparado ao convencional, para ambos os touros. Motilidades total e progressiva foram menores no sêmen sexado comparado ao convencional para o Touro 1 (MT: 49,7±15,9 vs. 94,9±1,9%, P=0,007; MP: 16,7+3,4 vs. 44,1+13,2%, P=0,009), enquanto diferenças não foram detectadas no Touro 2 (MT: 45,0±17,5 vs. 68,2±19,1%, P=0,098; MP: 12,8±4,7 vs. 30,0±13,0%, P=0,065). Elipticidade espermática do sêmen sexado foi menor do que do sêmen convencional no Touro 2 (0,306±0,01 vs. 0,342±0,02, P=0,020), mas não houve diferença no Touro 1 (0,332±0,01 vs. 0,330±0,01, P=0,552). Reduzida fertilidade in vivo foi observada para o sêmen sexado em relação ao convencional, independentemente da expressão de cio das vacas. A qualidade seminal in vitro do sêmen sexado foi comprometida para ambos os touros, mas diferentemente afetada para cada reprodutor.

Impacto da qualidade de sêmen sobre os resultados da IATF / Impact of sperm quality on IATF results

O Brasil vive um cenário de grande demanda pelo produto carne, tanto para o mercado interno quanto para o mercado externo. Para atender a esta demanda, há o envolvimento de toda a cadeia da carne, desde os programas de melhoramento genético com o fornecimento de touros melhoradores para tais características, como as centrais de colheita de sêmen desses animais para uso em massa por meio da Inseminação Artificial em Tempo Fixo (IATF). Para que haja sucesso no uso desta biotécnica, é de vital importância a qualidade do produto sêmen aliado às outras características como ambiência e nutrição.(AU)

Brazil is now with a great demand of beef products, for the domestic and exportation markets. To delivery the demand, the entire meat chain must be involved, included genetics programs to provide and separate superior bulls, and the collection centers that collect this superior genetics bulls to be used in the Fixed Timed Artificial Insemination (FTAI) biotechnique in a large scale. In order to be successful using this biotechnology, the quality of the semen combined with other characteristics such as ambience and nutrition are extremely important.(AU)

Presencia de SARS-COV-2 en semen de hombres con COVID-19: revisión sistemática / Presence of SARS-CoV-2 in semen of men with COVID-19: a systematic review

RESUMEN La pandemia de la COVID-19 ha causado un impacto aún desconocido en la salud reproductiva masculina. Por ello, el objetivo de esta revisión fue recolectar evidencia para descubrir si el agente causante de la COVID-19 puede encontrarse en el semen. La búsqueda se realizó en las bases de datos PubMed, BVS y Google Scholar, e incluyó los artículos que mostraban los resultados del análisis de semen realizado para encontrar el virus SARS-CoV-2 en hombres diagnosticados con COVID-19. Once estudios fueron seleccionados para esta revisión que aportaron 296 muestras. De ellas, solo el 2,70 % fueron positivas. Además, se encontraron resultados mixtos respecto a la morfología de los espermatozoides, y también pacientes con azoospermia u oligospermia. Este último hallazgo podría ser consecuencia del elevado número de receptores de la enzima convertidora de angiotensina 2 (ECA 2) que existen en los testículos, lo que hace a estos órganos un blanco del virus SARS-CoV-2. Se concluye que debido al limitado número de estudios y muestras aún no es posible responder la pregunta de estudio planteada.

ABSTRACT The COVID-19 pandemic has caused an unknown impact on the male reproductive health. Therefore, this article aimed to collect evidence to discover if the causative agent of COVID-19 is found in semen. A search was carried out in the PubMed, BVS and Google Scholar databases. Articles with results of semen analyses to determine the presence of SARS-CoV-2 in men diagnosed with COVID-19 were considered. Eleven studies with a total of 296 samples were collected in this review, out of which only 2.70 % showed positive results. In addition, mixed results regarding the morphology of the spermatozoa, as well as patients with azoospermia or oligospermia, were reported. The latter could be a consequence of the high levels of the angiotensin converting enzyme 2 (ACE2) in the testes, making them a target organ for SARS-CoV-2. It is concluded that, due to the limited number of studies and samples, it is still not possible to answer this study question.

Análisis de las variables del espermograma en pacientes de la consulta de infertilidad del municipio de Cienfuegos / Spermogram variables analysis in the infertility consultation patients of the Cienfuegos municipality

RESUMEN Fundamento El análisis de la calidad del semen es considerado un elemento básico en la evaluación de la fertilidad masculina. Objetivo analizar los valores del espermograma de pacientes atendidos en la consulta municipal de Infertilidad de Cienfuegos. Métodos estudio descriptivo, que incluyó 128 muestras resultantes de espermogramas realizados a pacientes atendidos en la consulta municipal de Infertilidad de Cienfuegos, entre los años 2019 y 2020. Los espermogramas registran las variables: edad, volumen, concentración, movilidad, morfología y vitalidad. Estas variables fueron comparadas con los valores de referencia propuestos por la OMS. Resultados en cuanto a la concentración, resaltó que los valores oscilaron en un rango amplio, desde 0 hasta 350 mmol/L-1, con una media de 89,1; mientras que la movilidad de los espermatozoides mostró valores muy bajos, incluso algunos con 0 %. La movilidad total (movilidad rápida más movilidad lenta) fue de un 57,7 %. El porcentaje de espermatozoides con una morfología adecuada fue de 46,3, mientras que la vitalidad fue de 71,8 %. Conclusión La mayoría de las muestras analizadas mostraron valores normales en todos los parámetros. Resultó significativo el número de pacientes con leucospermia, parámetro que probablemente tenga relación directa con la infertilidad en estos sujetos.

Background: Semen quality analysis is considered a basic element in the evaluation of male fertility. Objective to analyze the spermogram values in patients treated at the Cienfuegos Municipal Infertility Clinic. Methods descriptive study, which included 128 samples resulting from spermograms performed on patients treated at the Cienfuegos municipal infertility clinic, between 2019 and 2020. Spermograms record the variables: age, volume, concentration, mobility, morphology, and vitality. These variables were compared with the reference values proposed by the WHO. Results regarding the concentration, it highlighted that the values oscillated in a wide range, from 0 to 350 mmol / L-1, with a mean of 89.1; while the mobility of the spermatozoa showed very low values, even some with 0%. Total mobility (fast mobility plus slow mobility) was 57.7%. The percentage of sperm with an adequate morphology was 46.3, while the vitality was 71.8%. Conclusion Most of the analyzed samples showed normal values in all parameters. The number of patients with leukospermia was significant, a parameter that is probably directly related to infertility in these subjects.

Decline of semen quality over the last 30 years in Uruguay.

BACKGROUND: Over the last years, there has been an increasing concern about a global decline in men's fertility. Specifically, some evidence indicates that sperm quality has decreased over the last years. However, reports showing the changes in sperm quality with time are inconsistent. Part of the contradictions between studies is attributed to geographical differences. Surprisingly, few studies include data from South American countries, creating a bias in the conclusions. This study aims to determine how sperm quality has evolved over the past 30 years in Uruguay. For this purpose, 317 medical records from allegedly healthy sperm donor candidates, aged between 18 and 36 years old, who voluntarily requested to be considered as sperm donors between 1988 and 2019, were analyzed. The studied variables were the following sperm parameters: semen volume, sperm cell concentration, total sperm number, progressive motility, vitality, and sperm morphology. A correlative statistical analysis was performed between seminal parameter values and the year data were collected. RESULTS: We found a statistically significant decrease in sperm concentration and normal sperm morphology during the studied period. There was no decrease in vitality, seminal volume, and total progressive motility. Semen parameters were not associated with tobacco, drugs, or alcohol consumption. CONCLUSIONS: We conclude that the sperm quality of donor candidates in Uruguay decreased during this period. Further studies should be carried out to verify the occurrence of this phenomenon in the general population and find its possible causes.

RéSUMé: CONTEXTE: Au cours des dernières années, on s'est de plus en plus inquiété d'une baisse de la fertilité masculine. Plus précisément, certaines données indiquent que la qualité du sperme a diminué au cours des dernières années. Cependant, les rapports montrant les changements dans la qualité du sperme avec le temps sont incohérents. Une partie des contradictions entre les études est attribuée à des différences géographiques. Étonnamment, peu d'études ont inclue des données provenant de pays d'Amérique du Sud, ce qui crée un biais dans les conclusions. Cette étude vise à déterminer comment la qualité du sperme a évolué au cours des 30 dernières années en Uruguay. À cette fin, 317 dossiers médicaux de candidats donneurs de sperme prétendument en bonne santé, âgés de 18 à 36 ans, qui ont volontairement demandé à être considérés comme donneurs de sperme entre 1988 et 2019, ont été analysés. Les variables étudiées étaient les paramètres suivants du sperme: volume de sperme, concentration de cellules spermatiques, nombre total de spermatozoïdes, motilité progressive, vitalité, et morphologie des spermatozoïdes. Une analyse statistique corrélative a été effectuée entre les valeurs des paramètres séminaux et l'année de recueil des données. RéSULTATS: Nous avons trouvé une diminution statistiquement significative de la concentration de spermatozoïdes et de la morphologie normale des spermatozoïdes pendant la période étudiée. Il n'y a eu aucune diminution de la vitalité, du volume de sperme, et de la motilité progressive totale. Les paramètres du sperme n'étaient pas associés à la consommation de tabac, de drogues ou d'alcool. CONCLUSIONS: Nous concluons que la qualité du sperme des candidats donneurs en Uruguay a diminué au cours de cette période. D'autres études devraient être menées pour vérifier l'apparition de ce phénomène dans la population générale et en trouver les causes possibles.

Infertilidad masculina: causas y diagnóstico / Male infertility: causes y diagnosis

En una pareja con infertilidad, la evaluación masculina es fundamental por dos razones principales. En primer lugar, es la única causa de infertilidad en el 20% de las parejas y en el 50% se encuentra asociada a una causa de infertilidad femenina; en segundo lugar, existe evidencia de la relación entre infertilidad masculina y comorbilidades, como enfermedades cardiovasculares, oncológicas, reumatológicas e incluso con aumento de la mortalidad. Por esto, los pacientes deben ser evaluados por urólogos-andrólogos entrenados que permitan llegar al diagnóstico etiológico, como también buscar comorbilidades asociadas. Una correcta historia clínica, examen físico, espermiograma y exámenes complementarios permitirán obtener el diagnóstico etiológico y por lo tanto el tratamiento adecuado. Las causas genéticas de infertilidad son al menos el 15% de las etiologías, aumentando hasta el 25% en casos de azoospermia. A través del desarrollo y avance en biología molecular, en el futuro se podrán identificar otras causas genéticas que actualmente son categorizadas como infertilidad de origen idiopático.

When treating infertility, a study of the male partner is necessary for two main reasons: 1) In 20% of cases of infertility there is only a male root cause and in addition, in 50% of the cases the root cause is associated with the male and the female. 2) There is supporting and growing evidence that male infertility is related to comorbidities, including cardiovascular disease, cancer, rheumatologic disease, and even mortality. A thorough clinical history, physical examination, semen analysis and auxiliary tests will help us identify the cause and the correct treatment. Near 15% of male infertility are attributed to genetic causes, and this goes up to 25% in cases of azoospermia. With evolving advances and development of molecular biology, some causes of male infertility currently classified as idiopathic, will be specifically identified and categorized.