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INTRODUCTION: Despite recent advances in assisted reproduction techniques and recent knowledge regarding embryo and endometrium quality, implantation and birth rates remain low. The objective of this study was to investigate whether clomiphene citrate alters endometrial maturation in infertile patients. METHODS: In a prospective self-matched cohort study, we assessed the ovulation of women in spontaneous and stimulated cycles (with clomiphene citrate). We determined the ovulation day by ultrasound scanning. In both cycles, we took four blood samples (BS1 - at early proliferative phase, BS2 - at mid proliferative phase, BS3 - after ovulation and BS4 - at mid luteal phase) to determine the serum concentrations of FSH, LH, estradiol and progesterone. We retrieved an endometrial biopsy five days after ovulation, followed by blinded analysis and classification according to Noyes criteria, in both cycles. RESULTS: Twenty-two participants completed the study. There were significant differences in FSH BS3 (p=0.001), in LH BS3 and BS4 (p<0.001 and p=0.049, respectively), in estradiol BS2, BS3 and BS4 (p<0.001, p=0.024 and p<0.001, respectively) and in progesterone BS3 and BS4 (p=0.028 and p<0.001, respectively). Considering Noyes criteria, there was a one-day delay when comparing the stimulated cycle with the spontaneous cycle (p=0.004), and a two-day delay when comparing the stimulated cycle with the biopsy day. CONCLUSION: This study indicates that ovarian stimulation with clomiphene citrate delays the endometrial maturity, and could possibly impair the implantation process due to asynchrony.

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OBJECTIVE: The aim of this study was to investigate the efficacy of protocols for mice ovary cryopreservation to compare the differences in Mouse Vasa Homologue expression (a germline cell marker) and ovarian viability after vitrification or slow freezing. METHODS: Female CF1 mice aged 40-45 days were randomly divided into three groups: Control, vitrification or slow freezing. Their ovaries were surgically removed, rinsed in saline solution and cryopreserved. For vitrification, we used a commercial protocol and for slow freeze, we used 1.5 M ethylene glycol (EG) as cryoprotectant. After that, the ovaries were processed for histological an immunohistochemical analysis, and counting of primordial, primary, pre-antral and antral follicles. RESULTS: No significant difference was found in the proportion of high-quality primordial, primary and pre-antral follicles after thawing/warming in the slow freezing and vitrification groups. The immunohistochemistry for MVH antibody demonstrated that the slow freeze group had a higher number of unmarked cells (p=0.012), indicating a harmful effect on the MVH expression in the ovarian tissue, where the cell structure is complex. CONCLUSION: Although both protocols indicated similar results in the histological analysis of follicular counts, the vitrification protocol was significantly better to preserve ovarian stem cells, an immature germ cell population. These cells are able to self-renew having regeneration potential, and may be effective for the treatment of ovarian failure and consequently infertility.

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Purpose: To evaluate the use of adipose-derived stem cells (ADSC) in reducing the necrosis area in an experimental model of cutaneous ischemic flap in rats submitted to subcutaneous nicotine injection to simulate a smoker patient. Methods: In an experimental study, 30 rats were enrolled and divided into two experimental groups of 15 animals all submitted to a subcutaneous nicotine injection to create ischemic cutaneous flaps on their backs. Other 10 animals were used only to obtain adipose tissue derived stem cells (ADSC). The first group (n=15) received ADSC treatment at the end of surgery while the other group, the control (n=15), received no other interventions. After euthanasia, a decal was performed on the whole area of the flap, accurately defining the transition from necrosis to healthy region. Photos of all animals were collected and evaluated by scales standardized by Paint-Autocad- 2015 software to define the area of flap necrosis in each rat. Student T test was performed to compare the groups, considering a p< 0.05 significant. Data were analyzed using SPSS IBM® 18 version. Results: Through the analysis of the images by the program Paint-Autocad-2015 and the area of decal obtained by the transparent sheet, we obtained a mean of 46% necrosis of the total area of the flap in the treatment group and 69.4% in the control group. In the descriptive analysis, a mean of 3.7 cm of necrosis CI 95% (3.2 - 4.2) was evident in the treatment group whereas a mean value of 5.56 CI 95% (5.2 - 5.9) was found in control group, with p value <0.001 for this comparison. Conclusion: The application of adipose-derived stem cells reduces the percentage of necrosis in an experimental model of randomized cutaneous flap in rats submitted to subcutaneous nicotine injection.(AU)

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PURPOSE: To evaluate the use of adipose-derived stem cells (ADSC) in reducing the necrosis area in an experimental model of cutaneous ischemic flap in rats submitted to subcutaneous nicotine injection to simulate a smoker patient. METHODS: In an experimental study, 30 rats were enrolled and divided into two experimental groups of 15 animals all submitted to a subcutaneous nicotine injection to create ischemic cutaneous flaps on their backs. Other 10 animals were used only to obtain adipose tissue derived stem cells (ADSC). The first group (n=15) received ADSC treatment at the end of surgery while the other group, the control (n=15), received no other interventions. After euthanasia, a decal was performed on the whole area of the flap, accurately defining the transition from necrosis to healthy region. Photos of all animals were collected and evaluated by scales standardized by Paint-Autocad- 2015 software to define the area of flap necrosis in each rat. Student T test was performed to compare the groups, considering a p< 0.05 significant. Data were analyzed using SPSS IBM® 18 version. RESULTS: Through the analysis of the images by the program Paint-Autocad-2015 and the area of decal obtained by the transparent sheet, we obtained a mean of 46% necrosis of the total area of the flap in the treatment group and 69.4% in the control group. In the descriptive analysis, a mean of 3.7 cm of necrosis CI 95% (3.2 - 4.2) was evident in the treatment group whereas a mean value of 5.56 CI 95% (5.2 - 5.9) was found in control group, with p value <0.001 for this comparison. CONCLUSION: The application of adipose-derived stem cells reduces the percentage of necrosis in an experimental model of randomized cutaneous flap in rats submitted to subcutaneous nicotine injection.

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Abstract Purpose To evaluate the use of adipose-derived stem cells (ADSC) in reducing the necrosis area in an experimental model of cutaneous ischemic flap in rats submitted to subcutaneous nicotine injection to simulate a smoker patient. Methods In an experimental study, 30 rats were enrolled and divided into two experimental groups of 15 animals all submitted to a subcutaneous nicotine injection to create ischemic cutaneous flaps on their backs. Other 10 animals were used only to obtain adipose tissue derived stem cells (ADSC). The first group (n=15) received ADSC treatment at the end of surgery while the other group, the control (n=15), received no other interventions. After euthanasia, a decal was performed on the whole area of the flap, accurately defining the transition from necrosis to healthy region. Photos of all animals were collected and evaluated by scales standardized by Paint-Autocad- 2015 software to define the area of flap necrosis in each rat. Student T test was performed to compare the groups, considering a p< 0.05 significant. Data were analyzed using SPSS IBM® 18 version. Results Through the analysis of the images by the program Paint-Autocad-2015 and the area of decal obtained by the transparent sheet, we obtained a mean of 46% necrosis of the total area of the flap in the treatment group and 69.4% in the control group. In the descriptive analysis, a mean of 3.7 cm of necrosis CI 95% (3.2 - 4.2) was evident in the treatment group whereas a mean value of 5.56 CI 95% (5.2 - 5.9) was found in control group, with p value <0.001 for this comparison. Conclusion The application of adipose-derived stem cells reduces the percentage of necrosis in an experimental model of randomized cutaneous flap in rats submitted to subcutaneous nicotine injection.

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Cell therapy has been linked to an unexplained return of ovarian function and fertility in some cancer survivors. Studies modeling this in mice have shown that cells transplantation generates donor-derived oocytes in chemotherapy-treated recipients. This study was conducted to further clarify the impact of cell transplantation from different sources on female reproductive function after chemotherapy using a preclinical mouse model. Methods. Female mice were administered 7.5 mg/kg cisplatin followed by cell transplantation (one week later) using GFP+ female cell donors. For cell tracking, adipose derived stem cell GFP+ (ADSC), female germline stem cell GFP+/MVH+ (FGSC), or ovary cell suspension GFP+ mice were transplanted into cisplatin-treated wild-type recipients. After 7 or 14 days animals were killed and histological analysis, IHQ for GFP cells, and ELISA for estradiol were performed. Results. Histological examinations showed that ADSC, ovary cell suspension, and FGSC transplant increase the number of follicles with apparent normal structure in the cells recipient group euthanized on day 7. Cell tracking showed GFP+ samples 7 days after transplant. Conclusion. These data suggest that intraovarian injection of ADSCs and FGSC into mice with chemotherapy-induced ovarian failure diminished the damage caused by cisplatin.

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OBJECTIVE: The aim of this study was to determine the most efficient protocol for cryopreservation of ovarian tissue using the automatic Freeze Control® system and to test two different cooling curves combined with two different cryoprotectants: dimethyl sulfoxide (DMSO) and ethylene glycol (EG). METHODS: In this study, 20 female Wistar rats underwent bilateral oophorectomy. The ovaries were divided into two groups: one cryopreserved in 1.5M DMSO and the other in 1.5M EG. Two cooling curves, slow (1h 50min) and rapid (35min) were analyzed. Tissue samples were frozen, thawed, fixed, and stained with hematoxylin and eosin to analyze oocyte integrity. Follicular analysis was performed under optical microscopy (400x magnification) and preantral follicles were classified as primordial or primary according to developmental stage. ANOVA was performed, and Tukey's test was used for comparison between means, with P<0.05 defined as significant. RESULTS: In cryopreserved tissue, the follicles with preserved integrity in each ovary were 79% primordial and 29% primary. In non-frozen (control) tissue, all follicular types were observed (primordial, primary, secondary, preantral, and antral). Reversible changes included cytoplasmic vacuolization and irregular cell outline. Irreversible changes included pyknosis. EG was more efficient than DMSO, preserving a greater number of viable primordial and primary follicles. Comparison of both cooling curves revealed no statistically significant differences between them. CONCLUSION: The EG is more effective as a cryoprotectant than DMSO for obtaining higher viable numbers of primordial and primary follicles from rat ovarian tissue. Further studies are needed to demonstrate ovarian functionality, such as detection of hormone levels.