In vitro culture of mechanically isolated murine primary follicles in the presence of human platelet lysate PLTMax.

OBJECTIVE: To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). METHODS: Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. RESULTS: The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. CONCLUSIONS: PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.

Preservação de fertilidade: cultivo de folículos ovarianos e novo dispositivo para criopreservação seminal / Fertility preservation: ovarian follicle culture and a new device for seminal cryopreservation

INTRODUÇÃO: A oncofertilidade tem o desafio de buscar estratégias para preservar a função reprodutiva. Este estudo explorou duas possibilidades como implementação para as técnicas de preservação da fertilidade feminina e masculina. OBJETIVO: Analisar a eficiência do cultivo de folículos pré-antrais de camundongos suplementado com lisado de plaquetas humanas e desenvolver um protótipo para criopreservação de sêmen humano. MATERIAIS E MÉTODOS: Os folículos pré-antrais foram isolados mecanicamente de ovários de fêmeas de camundongos e foram cultivados individualmente em sistema entre camadas de óleo mineral. Os folículos foram cultivados divididos em 4 grupos, sendo um controle, sem o uso do lisado de plaquetas e três grupos com diferentes concentrações de lisado de plaquetas humanas (PLTMax®). Foram avaliadas a sobrevivência celular, desenvolvimento folicular e características oocitárias. Para o segundo estudo foi desenvolvido e impresso em 3D com filamentos de acrilonitrilo butadieno-estireno (ABS) um protótipo que suporte 10 palhetas com amostras seminais no vapor de nitrogênio líquido (N2L), etapa essencial para criopreservação de sêmen humano. Para os testes foram utilizadas 40 amostras seminais. A temperatura ambiente e no interior das palhetas de envase das amostras foram medidas e estabelecida a curva de resfriamento. Os parâmetros de motilidade, vitalidade e fragmentação do DNA espermático foram avaliados antes do congelamento e após o descongelamento. Foram realizados dois testes, um de posicionamento das palhetas e outro comparativo entre o protótipo e um dispositivo com suporte em poliestireno expandido (EPS). RESULTADOS: O cultivo de 11 dias induziu um aumento no tamanho folicular em todas as condições, sendo maior no grupo controle, seguido do grupo com 10% de PLTMax®, mas com diferença significativa (p<0,001). O grupo controle apresentou maior número de oócitos intactos (>50%) em relação aos demais (<35%). Todos os 4 grupos apresentaram taxas de vitalidade celular acima de 70%. Quanto aos testes com o protótipo em ABS foi verificado que as curvas de refrigeração foram notavelmente reproduzíveis. O material do protótipo resistiu a inúmeros mergulhos (>300) no N2L, sem demonstrar danos ao material. Diferenças significativas (p<0,001) foram observadas para a taxa de recuperação média da motilidade e vitalidade espermática em relação aos dados da amostra 2 fresca em ambos os testes. A motilidade, a vitalidade e a fragmentação do DNA espermático antes do congelamento e após o descongelamento não mostraram diferenças em relação a posição das palhetas. Também não houve diferença quanto ao índice de fragmentação verificada das amostras criopreservadas com uso do protótipo em ABS e o suporte em EPS, mesmo após o cultivo, após 24 horas de cultivo. Contudo, houve diferença em relação a amostra fresca (p<00,1). Quanto a recuperação das taxas de motilidade e vitalidade não houve diferença entre o ABS e EPS após o descongelamento e 24 horas de cultivo. CONCLUSÃO: O PLTMax®, embora tenha apresentado menor desempenho que o HSA, é um candidato de suplementação para o cultivo de folículos pré-antrais que merece ser mais explorado. O protótipo em ABS demonstrou resistência, praticidade e segurança para criopreservação seminal de forma reprodutível e eficiente.

INTRODUCTION: Oncofertility has the challenge of seeking strategies to preserve reproductive function. This study explored two possibilities as implementations for female and male fertility preservation techniques. PURPOSE: To analyze the efficiency of mouse preantral follicle culture supplemented with human platelet lysate and to develop a prototype for human semen cryopreservation. MATERIAL AND METHODS: Preantral follicles were mechanically isolated from female mouse ovaries and were individually cultured using a mineral oil interlayer system. The follicles were cultured divided into 4 groups, one control, without the use of platelet lysate and three groups with different concentrations of human platelet lysate (PLTMax®). Cell survival, follicular development and oocyte characteristics were evaluated. For the second study, a prototype was developed and printed in 3D with acrylonitrile butadiene styrene (ABS) filaments to support 10 straws with seminal samples in liquid nitrogen (N2L) vapor, an essential step for human semen cryopreservation. For the tests 40 seminal samples were used. Ambient and internal temperatures inside the sample straws were measured and the cooling curve was established. The parameters of motility, vitality and sperm DNA fragmentation were evaluated before freezing and after thawing. Two tests were performed, one for positioning the straws and the other comparing the prototype and a device with expanded polystyrene (EPS) support. RESULTS: The 11-day culture induced an increase in follicular size in all conditions, being higher in the control group followed by the group with 10% PLTMax®, but with significant difference (p<0.001). The control group presented a higher number of intact oocytes (>50%) compared to the others (<35%). All 4 groups presented cell vitality rates above 70%. As for the ABS prototype tests, it was verified that the cooling curves were remarkably reproducible. The prototype withstood numerous dips (>300) in N2L without showing damage to the material. Significant differences (p<0.001) were observed for the mean recovery rate of sperm motility and vitality compared to the fresh sample data in both tests. Motility, vitality and sperm DNA fragmentation before freezing and after thawing showed no differences with respect to the position of the straws. There was also no difference in the fragmentation index verified for samples cryopreserved using the ABS prototype and the EPS support, even after 24 hours of culture. However, there was a difference compared to the fresh 4 sample (p<00.1). As for the recovery of motility and vitality rates there was no difference between ABS and EPS after thawing and 24 hours of culture. CONCLUSION: PLTMax®, although it showed lower performance than HSA, is a supplementation candidate for preantral follicle culture that deserves further exploration. The ABS prototype demonstrated strength, practicality and safety for seminal cryopreservation in a reproducible and efficient manner.

Association between terbinafine hydrochloride and sperm DNA fragmentation - case report.

OBJECTIVE: To present the case of a man with normozoospermia and a high level of fragmented spermatozoa, which origin seems to be associated with long-term treatment with terbinafine hydrochloride. CASE DESCRIPTION: A 20-year-old male healthy patient, with no history of disease and addictions, used an antifungal (terbinafine hydrochloride) for one year to treat a toenail. During this treatment, he participated in a study to evaluate a method of sperm DNA fragmentation analysis. He had 99% fragmented sperm, primarily attributed to prolonged abstinence. The samples that were analyzed later indicated that the high fragmentation could be associated with the antifungal treatment and that with a 2-day abstinence and absence of treatment the fragmentation rate was again comparable with that of fertile men (15%). CONCLUSION: Terbinafine hydrochloride is likely to cause problems in male fertility, mainly affecting DNA sperm integrity. Further studies are needed to confirm this observation and to determine at what level of the genitourinary tract the alteration of DNA occurs.

In vitro maturation of Mus musculus mice oocytes after hyperosmotic shock induced by vitrification solutions.

OBJECTIVE: To evaluate in vitro oocyte maturation rates in embryonic culture medium after induction by hyperosmotic shock caused by exposure to vitrification solutions. METHODS: Bilateral oophorectomy was performed on 20 prepubescent female mice (Swiss). Immature (Prophase I) oocytes (N = 400) were obtained by ovarian dissection, divided into 4 groups, and transferred to culture dishes containing fertilization medium (Sydney IVF Fertilization Medium, Cook® Medical). The control group (CG) did not receive treatment, the test groups (G1, G2, G3) were treated with vitrification solution - 2 (VI-2: 14 M sucrose + ethylene glycol and dimethyl sulfoxide) for 30 seconds and subsequently: G1: 30 seconds in devitrification solution - 2 (DV-2: 0.5M sucrose); G2: 60 seconds DV-2; G3: 60 seconds DV-1(1M sucrose) and 180 seconds DV-2. All groups were cultivated for 24 hours in an incubator at 37ºC and 5% CO2 (Thermo model 3110). After this period, we checked their maturation status. RESULTS: Oocytes exposed to VI-2, DV-1 and DV-2 (G3) showed the highest rate of competence in resuming meiosis and reaching the MII stage; however, there was no statistically significant difference (G3 = 50.5% - 49/97; CG = 27.8% - 10/30). CONCLUSIONS: Oocyte exposure to vitrification solutions, in order to cause osmotic shock, did not interfere with the resumption of meiosis in mice oocytes.

Wistar rats immature testicular tissue vitrification and heterotopic grafting.

OBJECTIVE: To evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation. METHODS: Twenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed. RESULTS: The viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site. CONCLUSION: The vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

Ovarian tissue vitrification and heterotopic autologous transplantation in prepubertal Wistar rats.

OBJECTIVE: To evaluate the efficiency of ovarian tissue heterotopic autografting after vitrification in prepubertal rats. METHODS: Fragments of excised ovaries from prepubertal rats were used after assessing post-warming cellular viability, to determine the best vitrification protocol prior to retroauricular autografting. Pre-pubertal females (N=24) were castrated and divided into three group: Group 1 - fresh ovarian tissue transplantation; Group 2 - vitrified/warmed tissue transplantation; Group 3 - bilateral oophorectomy without transplantation. The ovarian fragments were exposed to solutions from the Ingamed® commercial kit, allocated in bacteriological loops and immersed in liquid nitrogen. Sixty days after transplantation, a vaginal mucus sample was collected for cytology tests, followed by sacrificing the animal, performing a cardiac puncture for collecting a blood sample to determine luteinizing hormone and estradiol levels, and excision of the transplanted fragment for histology tests. RESULTS: Vaginal cytology revealed that 87.5% of females from groups 1 and 2 had estrus while all females in Group 3 remained in diestrus. The mean LH value in groups 1 (0.08 mIU/mL) and 2 (0.34 mIU/mL) were statistically different from that of Group 3 (2.27 mIU/mL). E2 values did not differ between the groups. The histological analysis of Group 1 excised grafts versus those from Group 2 showed a higher percentage of primary follicles (62.5% vs. 12.5%), developing follicles (75% vs. 25%), corpus luteum (37.5% vs. 12.5%) and stromal region (100% vs. 87.5%). CONCLUSION: This study indicated that pre-pubertal ovarian tissue vitrification can be used to preserve fertility and to restore endocrine function in castrated rats.

Effects of energy drinks on biochemical and sperm parameters in Wistar rats

BACKGROUND: The present study evaluates the effects of energy drinks on the reproductive and biochemical parameters of adult male rats. METHODS: A total of 40 male rats (Wistar) were exposed to an energy drink mixed with the drinking water for a period of 120 days. The animals were divided into four groups and exposed to increasing therapeutic doses (DT) of an energy drink, based on allometric extrapolation, resulting in values (mL/day) per animal of 250 g: DT1 2.36 mL, DT3 7.47 mL, and DT6 14.16 mL. The control group (CTRL) consumed water only. During the treatment, the rats were assessed for signs of toxicity. After treatment, the animals were sacrificed and their organs were weighed. Sperm parameters (motility, concentration, and morphology) were evaluated. The biochemical markers alanine eamino transferase, aspartate amino transferase, alkaline phosphatase, lactic dehydrogenase, urea, creatinine, creatine phosphokinase, and creatine kinase MB fraction were measured, in addition to total cholesterol and testosterone. RESULTS: There was a significant decrease (p< 0.05) in the concentration of sperm in the treated groups (DT18.5 ± 0.7; DT3 7.2 ± 0.9; DT6 8.4 ± 0.9) compared to the control group (12.3 ± 1.2). No difference was observed with respect to relative weights of the animals'organs, water consumption, signs of toxicity, behavioral changes, biochemical markers, and sperm motility and morphology. CONCLUSION: The long-term consumption of energy drinks interferes negatively with sperm concentration, without affecting sperm motility and morphology or altering the hepatic, cardiac, or renal functions

Sperm Selection Using Three Semen Processing Techniques.

OBJECTIVE: This study aimed to assess the efficiency, in terms of recovered motile spermatozoa with normal morphology, of three sperm selection techniques: migration- sedimentation (SS), swim-up from fresh semen (SF), and swim-up from washed (SL) sperm. METHODS: Samples from 20 normozoospermic men were divided into three equal aliquots and processed in parallel. SS was performed in a Jondet tube, using 1 ml of semen and 2.5 ml of Human Tubal Fluid medium (HTF+10% Synthetic Serum Supplement, Irvine, USA). For SF, 1 ml of HTF was layered over 1 ml of fresh semen (SF). For SL, 1 ml of sperm was first centrifuged (300 g, 10 min) and the pellet resuspended in 1 ml of HTF; a second layer of HTF was placed on top. Migration time was 1h (SF and SL) and 1h30' for SS at 37°C. After migration, 200 µl were removed from the top layer (SF, SL) and from the central cone (SS). Concentration, morphology and motility were determined. RESULTS: Recovery rates were 25% for SS, 10.1% for SF and 4.5% for SL. SS recovery rate was significantly higher (P<0.01) than the two swim-up techniques. Total motility was statistically different (P<0.001), with 93.6% for SS, 91.2% for SF, and 77% for SL. Sperm morphology was similar between the three techniques (P= 0.12). CONCLUSION: SS is an efficient technique for the recovery of motile spermatozoa from native semen preparations and yielded better results than SF and SL. Routine use for assisted reproduction remains to be evaluated.

Reproductive toxicology and clastogenic evaluation in mice of a phytotherapeutic formulation obtained from Cinchona calisaya Weddel (Rubiaceae) used in Brazilian folk medicine as female fertility stimulant.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, a phytotherapeutic preparation produced from a standardized tincture of Cinchona calisaya Weddel such that each mL of product contains 400µg of quinine, known in Portuguese as Água Inglesa(®) (English water), is indicated by the manufacturer as a tonic, appetite stimulant, and digestive. However, this preparation has long been used in folk medicine as a female fertility stimulant. Despite its widespread use in folk medicine to stimulate female fertility, no study has been undertaken to assess the potential teratogenic and genotoxic effects of this phytotherapeutic preparation. The aim of the present study was to investigate possible toxic reproductive effects in mice caused by exposure to Água Inglesa(®), either before mating or during the pre- and post-embryo implantation periods. The genotoxic potential was evaluated using the micronucleus assay. MATERIAL, METHODS, AND RESULTS: Virgin female mice, with at least one estrous cycle evidenced by vaginal cytology, were divided into five groups of 15 individuals each (Group I - control, Group II - treated with ethanol solution at 16%, Groups III, IV and V treated with phytotherapeutic preparation at 1.5mL/kg/day, 3.0mL/kg/day and 4.5mL/kg/day, respectively). After the first 28 days of treatment, females were caged individually with adult fertile males. Pregnant females continued to receive treatment for seven days (preimplantation period). Body weight was recorded weekly during treatment. Signs of toxicity (weight loss, food intake, piloerection, apathy, prostration, diarrhea, seizures, behavioral changes, and locomotion) were also observed. The females were sacrificed on the 15th day of pregnancy, uterine horns were evaluated for implantation, and the placental index was recorded. In the micronucleus test, 2000 polychromatic erythrocytes (PCE) per animal, obtained from bone marrow, were scored. Results The results showed that exposure of the females during the pre- and post-implantation periods did not significantly alter the reproductive capacity (p<0.05); however, in higher dose (three times human dose)reduction of fetal weight was observed . There was no difference between the control and phytotherapeutic preparation (p>0.05) in terms of the average number of micronucleated polychromatic erythrocytes. CONCLUSIONS: Although folk medicine suggests that the Água Inglesa(®) preparation is useful as a female fertility stimulant, no such effect was confirmed in mice.

Avaliação do comportamento de ratos alojados em caixas de cores diferentes / Evaluation of the behavior of rats housed in boxes of different colors

A adequação do ambiente físico quanto aos hábitos da espécie em relação ao bem-estar animal e o refinamento das pesquisas, ultimamente vem ganhando importância investigativa. Com o objetivo de avaliar o comportamento de ratos alojados em caixas de diferentes cores, utilizaram-se 48 animais machos com 3 meses de idade divididos em seis grupos, sendo que três grupos foram alojados em caixas brancas e outros três em caixas pretas, durante 4 semanas. Empregando-se o labirinto em cruz elevado (LCE), os resultados mostraram maior tempo de permanência nos braços fechados dos animais alojados nas caixas brancas. No teste do campo aberto (CA), houve maior ambulação do grupo mantido em caixas pretas, e na esquiva inibitória, os dados indicaram um aumento significativo no tempo de permanência na plataforma do grupo alojado em caixas pretas. Assim, os animais alojados em gaiolas de cor preta apresentaram maior tendência exploratória (menor ansiedade) e melhor aprendizado.(AU)

Lately, the adequacy of a physical ambient to the specie’s habits relating to their well being and refined researches is getting important for researchers. With the aim of evaluate the behavior of rats housed in boxes of different colors, it was used 48 male animals, with 3 months of age, divided in 6 groups, three groups in white boxes and the other three in black boxes, for 4 weeks. Observing on the elevated plus maze, the results showed that the animals from white boxes stayed more time in the closed arms. On the open field test, there was more walking of the group kept in Black boxes, and on the inhibitory avoidance, data indicated a significant increase on the time spent on the platform by the group housed in the black boxes. Thus, the animal housed in Black cages have shown a greater exploratory tendency (decreased anxiety), and better learning.(AU)

Avaliação do comportamento de ratos alojados em caixas de cores diferentes / Evaluation of the behavior of rats housed in boxes of different colors

A adequação do ambiente físico quanto aos hábitos da espécie em relação ao bem-estar animal e o refinamento das pesquisas, ultimamente vem ganhando importância investigativa. Com o objetivo de avaliar o comportamento de ratos alojados em caixas de diferentes cores, utilizaram-se 48 animais machos com 3 meses de idade divididos em seis grupos, sendo que três grupos foram alojados em caixas brancas e outros três em caixas pretas, durante 4 semanas. Empregando-se o labirinto em cruz elevado (LCE), os resultados mostraram maior tempo de permanência nos braços fechados dos animais alojados nas caixas brancas. No teste do campo aberto (CA), houve maior ambulação do grupo mantido em caixas pretas, e na esquiva inibitória, os dados indicaram um aumento significativo no tempo de permanência na plataforma do grupo alojado em caixas pretas. Assim, os animais alojados em gaiolas de cor preta apresentaram maior tendência exploratória (menor ansiedade) e melhor aprendizado.

Lately, the adequacy of a physical ambient to the specie’s habits relating to their well being and refined researches is getting important for researchers. With the aim of evaluate the behavior of rats housed in boxes of different colors, it was used 48 male animals, with 3 months of age, divided in 6 groups, three groups in white boxes and the other three in black boxes, for 4 weeks. Observing on the elevated plus maze, the results showed that the animals from white boxes stayed more time in the closed arms. On the open field test, there was more walking of the group kept in Black boxes, and on the inhibitory avoidance, data indicated a significant increase on the time spent on the platform by the group housed in the black boxes. Thus, the animal housed in Black cages have shown a greater exploratory tendency (decreased anxiety), and better learning.