Bilateral inguinal hernia repair and male fertility: a randomized clinical trial comparing Lichtenstein versus laparoscopic transabdominal preperitoneal (TAPP) technique.

BACKGROUND: The effects of hernia repair on testicular function remain uncertain, regardless of the technique used. Studies that analyze testicular volume and flow after hernia repair or hormonal measurements are scarce and show contradictory results. This study aimed to evaluate the impact of bilateral inguinal hernia repair on male fertility in surgical patients in whom the Lichtenstein and laparoscopic transabdominal preperitoneal (TAPP) techniques were used. METHODS: A randomized clinical trial comparing open (Lichtenstein) versus laparoscopic (TAPP) hernia repair using polypropylene mesh was performed in 48 adult patients (20 to 60 years old) with primary bilateral inguinal hernia. Patients were evaluated preoperatively and 90 and 180 postoperative (PO) days. Sex hormones (Testosterone, FSH, LH and SHGB) analysis, testicular ultrasonography, semen quality sexual activity changes and quality of life (QoL) were performed. Postoperative pain was evaluated using the visual analog scale (VAS). RESULTS: Thirty-seven patients with aged of 44 ± 11 years were included, 19 operated on Lichtenstein and 18 operated on TAPP. The surgical time was similar between techniques. The pain was greater in the Lichtenstein group on the 7th PO day. The biochemical and hormonal analyses, testicular ultrasonography (Doppler, testicular volume, and morphological findings) and sperm quality were similar between groups. However, the sperm morphology was better in the Lichtenstein group after 180 days (p < 0.05 vs. preoperative) and two patients who underwent Lichtenstein hernia repair had oligospermia after 180 days. The QoL evaluation showed a significant improvement after surgery in the following domains: physical function, role emotional, bodily pain and general health (p < 0.05). On comparison of Lichtenstein vs. TAPP none of the domains showed statistically significant differences. No patient reported sexual changes. CONCLUSION: Bilateral inguinal hernia repair with polypropylene mesh, whether using Lichtenstein or TAPP, does not impair male fertility in terms of long-term outcomes. TRIAL REGISTRATION: Approved by the Ethics Committee for the Analysis of Research Projects (CAPPesq) of the HC/FMUSP, Number 2.974.457, in June 2015, Registered on Plataforma Brasil in October 2015 under Protocol 45535015.4.0000.0068. Registered on Clinicaltrials.gov, NCT05799742. Enrollment of the first subject in January 2016.

Gene expression profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells.

PURPOSE: Gelfoam scaffold is a feasible and safe non-invasive technique for Adipose tissue-derived Stem Cell (ASC)-delivery in the treatment of frozen-thawed ovarian autografts. This study seeks to analyze the genes expression profile of rat frozen-thawed ovarian autografts treated with scaffold-based delivery of adipose tissue-derived stem cells. METHODS: Eighteen adult Wistar rats were distributed into three groups: Control (frozen-thawed only); Group 1 (G1) and Group 2 (G2) (frozen-thawed ovaries treated with culture medium or ASC, respectively). Both treatments were performed immediately after autologous retroperitoneal transplant with scaffold-based delivery. The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for apoptosis, angiogenesis, and inflammatory cytokines (84 genes in each pathway) were evaluated by RT-PCR. Graft morphology (HE), apoptosis (cleaved-caspase-3), neoangiogenesis (VEGF), and cellular proliferation (Ki-67) were assessed. RESULTS: In grafts treated with ASC, the apoptosis pathway showed the highest number of genes over-regulated - 49 genes - compared to inflammation cytokines and angiogenesis pathway - 36 and 23 genes respectively, compared to grafts treated with culture medium. Serpinb5 family was highlighted in the angiogenesis pathway and Cxcl6 in the inflammation cytokines pathway. In the apoptosis pathway, the most over-regulated gene was Capsase14. ASC treatment promoted the reduction of cleaved caspase-3 in the theca internal layer and increased cell proliferation by Ki-67 in the granulosa layer without altering VEGF. A mild inflammatory infiltrate was observed in both groups. CONCLUSION: ASC therapy in rat frozen-thawed ovarian autografts promoted an abundance of genes involved with apoptosis and inflammatory cytokines without compromising the ovary graft morphology and viability for short time. Further studies are necessary to evaluate the repercussion of apoptosis and inflammation on the graft in the long term.

Gene expression profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells

Abstract Purpose: Gelfoam scaffold is a feasible and safe non-invasive technique for Adipose tissue-derived Stem Cell (ASC)-delivery in the treatment of frozen-thawed ovarian autografts. This study seeks to analyze the genes expression profile of rat frozen-thawed ovarian autografts treated with scaffold-based delivery of adipose tissue-derived stem cells. Methods: Eighteen adult Wistar rats were distributed into three groups: Control (frozen-thawed only); Group 1 (Gl) and Group 2 (G2) (frozen-thawed ovaries treated with culture medium or ASC, respectively). Both treatments were performed immediately after autologous retroperitoneal transplant with scaffold-based delivery. The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for apoptosis, angiogenesis, and inflammatory cytokines (84 genes in each pathway) were evaluated by RT-PCR. Graft morphology (HE), apoptosis (cleaved-caspase-3), neoangiogenesis (VEGF), and cellular proliferation (Ki-67) were assessed. Results: In grafts treated with ASC, the apoptosis pathway showed the highest number of genes over-regulated — 49 genes — compared to inflammation cytokines and angiogenesis pathway — 36 and 23 genes respectively, compared to grafts treated with culture medium. Serpinb5 family was highlighted in the angiogenesis pathway and Cxcl6 in the inflammation cytokines pathway. In the apoptosis pathway, the most over-regulated gene was Cap-sasel4. ASC treatment promoted the reduction of cleaved caspase-3 in the theca internal layer and increased cell proliferation by Ki-67 in the granulosa layer without altering VEGF. A mild inflammatory infiltrate was observed in both groups. Conclusion: ASC therapy in rat frozen-thawed ovarian autografts promoted an abundance of genes involved with apoptosis and inflammatory cytokines without compromising the ovary graft morphology and viability for short time. Further studies are necessary to evaluate the repercussion of apoptosis and inflammation on the graft in the long term. HIGHLIGHTS The scaffold-based delivery therapy with adipose tissue-derived stem cells in the rat ovarian autografts seems to be the best option when compared to direct injection or systemic route. Ovarian grafts treated with adipose tissue-derived stem cells showed the highest number of genes over-regulated in the apoptosis pathway, compared to inflammation cytokines and angiogenesis pathway. Capsase14 was the most over-regulated gene in the apoptosis pathway. The treatment with adipose tissue-derived stem cells in ovarian grafts treated didn't compromise the ovary graft morphology and viability for short time.

Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation.

BACKGROUD: Melatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole-containing molecule may protect ovarian grafts during the process of cryopreservation. Therefore, we aimed to determine whether melatonin pretreatment improves rat ovarian graft quality. METHODS: Twenty-six female rats were allocated to two study groups of thirteen animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Ten rats of each group were submitted to 24-h freezing, and whole ovaries autologous and avascular transplantation with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, the animals were euthanized and ovarian grafts were recovered for histological and immunohistochemical (Ki-67, cleaved caspase-3, TUNEL, von Willebrand factor, estrogen, and progesterone receptors) analyses. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess the effects of cryopreservation. ANOVA and Tukey's tests were used and the rejection level of the null hypothesis was set at 0.05 or 5% (p < 0.05). RESULTS: Melatonin promoted faster restart of the estrous cycle and increased the expression of mature follicles, collagen type I, von Willebrand factor, Ki-67, and cleaved caspase-3 on corpora lutea and estrogen receptors in the ovaries as compared to control. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III. CONCLUSION: Based on the evaluated parameters, melatonin may promote the quality of ovarian grafts. Reproductive function enhancement should be further studied.

Does a bilateral polypropylene mesh alter the duct deferens morphology, testicular size and testosterone levels? Experimental study in rats.

PURPOSE: To evaluate the effect of a PP mesh on duct deferens morphology, testicular size and testosterone levels. METHODS: Forty adult male rats were distributed into groups: 1) no surgery; 2) inguinotomy; 3) mesh placed on the duct deferens; and 4) mesh placed on the spermatic funiculus. After 90 postoperative days, the inguinal region was resected, and blood samples were collected for the measurement of serum testosterone (pg/dl). The ducts deferens were sectioned in three axial sections according to the relationship with the mesh - cranial, medial and caudal. The wall thickness and duct deferens lumen area were measured. RESULTS: The morphology of the duct deferens was preserved in all groups. The mesh placement did not alter this morphology in any of the analyzed segments. Surgery, with or without mesh placement, did not alter the morphology, wall thickness or lumen area (p>0.05). In all operated groups, serum testosterone levels were similar (p>0.05) but there was a decrease in testicle size (p<0.05). CONCLUSION: Surgery, with or without mesh placement, did not alter the morphology of the duct deferens and, although this treatment resulted in testicular size reduction, it did not affect serum testosterone levels.

Does a bilateral polypropylene mesh alter the duct deferens morphology, testicular size and testosterone levels? Experimental study in rats

Abstract Purpose To evaluate the effect of a PP mesh on duct deferens morphology, testicular size and testosterone levels. Methods Forty adult male rats were distributed into groups: 1) no surgery; 2) inguinotomy; 3) mesh placed on the duct deferens; and 4) mesh placed on the spermatic funiculus. After 90 postoperative days, the inguinal region was resected, and blood samples were collected for the measurement of serum testosterone (pg/dl). The ducts deferens were sectioned in three axial sections according to the relationship with the mesh — cranial, medial and caudal. The wall thickness and duct deferens lumen area were measured. Results The morphology of the duct deferens was preserved in all groups. The mesh placement did not alter this morphology in any of the analyzed segments. Surgery, with or without mesh placement, did not alter the morphology, wall thickness or lumen area (p>0.05). In all operated groups, serum testosterone levels were similar (p>0.05) but there was a decrease in testicle size (p<0.05). Conclusion Surgery, with or without mesh placement, did not alter the morphology of the duct deferens and, although this treatment resulted in testicular size reduction, it did not affect serum testosterone levels.

Cell-free therapy with the secretome of adipose tissue-derived stem cells in rats' frozen-thawed ovarian grafts.

The use of secretome may be a new strand of cell therapy, which is equal to or even superior to the injection of live cells, called cell-free therapy. In ovarian transplantation, this approach may be a therapeutic possibility for the ovarian graft in hypoxia. We designed the present study to evaluate whether the cell-free therapy with the secretome of adipose tissue-derived stem cells (ASCs) in rat frozen-thawed ovarian grafts could protect a graft against ischemic injury. A single dose of rat ASCs secretome or vehicle was injected into the bilateral frozen-thawed ovaries of 18 adult female rats immediately after an autologous transplant. Nine animals were used to control the cryopreservation protocol and were evaluated before and after the cryopreservation process. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability by trypan blue, graft morphology by HE, and apoptosis by TUNEL and cleaved-caspase-3 were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (p > 0.05). However, compared with the vehicle-treated grafts, the morphology of the secretome-treated grafts was impaired, showing reduced follicular population and increased apoptosis (p < 0.05). ASC secretome impaired the rat frozen-thawed ovarian graft from ischemic injury. However, more studies are needed to evaluate the factors involved and the possibility of applying the secretome in scaffolds to optimize its use.

Progressive Evaluation of Apoptosis, Proliferation, and Angiogenesis in Fresh Rat Ovarian Autografts Under Remote Ischemic Preconditioning.

This study evaluated the remote ischemic preconditioning (R-IPC) early and late repercussion on fresh ovarian transplants, aiming to assess a probable protective effect in ovarian follicular pool. Sixty Wistar EPM-1 rats were used, divided in 2 study groups: ovarian transplantation (Tx) and Tx + R-IPC, submitted to ovary transplant with or without R-IPC, respectively. These groups were subdivided according to the date for euthanasia: 4th, 7th, 14th, 21st, and 30th days of the postoperatory period. Morphology, morphometry, neoangiogenesis (vascular endothelial growth factor [VEGF]), proliferative activity (Ki-67), and apoptosis (cleaved caspase-3) were evaluated. Remote ischemic preconditioning was performed in the common iliac artery. Fresh autologous ovarian tissue was implanted integrally in the retroperitoneum. All animals showed resumption of estrous phase after ovary transplantation. Remote ischemic preconditioning attenuated the lesions progressively from the 7th day, with greater number of the immature follicles (14 days, P < .05), but didn't affect mature follicles and corpora lutea (P > .05). Immunohistochemical analyzes, taken as a whole, show that R-IPC benefic effect is more evident in the later periods of evaluation, when a greater proliferative activity (14, 21, and 30 days, P < .05) and lesser cell apoptotic activity (21 and 30 days, P < .05). The VEGF expression was similar in all times (P > .05). Remote ischemic preconditioning could have a benefic effect in the progressive evaluation of freshly grafted ovarian, especially on the latest phases of the posttransplant period. The 14th day was a landmark in the recuperation of the graft. Further investigations are necessary to determine the role of R-IPC in this scenario and its effect in frozen-thawed ovarian tissue.

Scaffold-based delivery of adipose tissue-derived stem cells in rat frozen-thawed ovarian autografts: preliminary studies in a rat model.

PURPOSE: This study aimed to evaluate whether a gelatin-based Gelfoam sponge is feasible as a scaffold for adipose tissue-derived stem cell (ASC) therapy in rat frozen-thawed ovarian autografts. METHODS: Two sets of studies were performed. The in vitro set evaluated ASCs' viability in the Gelfoam scaffold at different times of co-culturing (after 24, 48, 72, 96, and 120 h). The in vivo set used 20 12-week-old adult female Wistar rats. Frozen-thawed ovarian grafts were treated with ASCs delivered in Gelfoam scaffolds immediately after an autologous retroperitoneal transplant (ASCs-GS, n = 10). The controls received Gelfoam with a culture medium (GS, n = 10). Assessment of graft quality was conducted by vaginal smears (until euthanasia on the 30th postoperative day), histological analyses, follicular density, and viability and fibrosis. Immunohistochemical staining for VEGF-A expression, vascular network (vWF), apoptosis (caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)), cell proliferation (Ki-67), and hormone receptors (estrogen and progesterone) were performed. RESULTS: The cells remained viable in Gelfoam for up to 120 h of co-culturing. The graft morphology was similar among the groups. ASC therapy promoted the earlier resumption of the estrous phase (GS 16.6 ± 3 vs. ASCs-GS 12.8 ± 1.3 days) and enhanced estrogen receptors compared with the controls (p < 0.05) without interfering with the quantity and viability of the ovarian follicles, fibrosis, endothelial cells, VEGF immunoexpression, apoptosis, or cell proliferation (p > 0.05). CONCLUSION: The Gelfoam scaffold could be a feasible and safe non-invasive technique for ASC delivery in the treatment of frozen-thawed ovarian autografts. Future studies should evaluate the real benefit of this treatment on the survival and endocrine activity of the graft.

Polypropylene and polypropylene/polyglecaprone (Ultrapro®) meshes in the repair of incisional hernia in rats.

PURPOSE: To compare the inflammatory response of three different meshes on abdominal hernia repair in an experimental model of incisional hernia. METHODS: Median fascial incision and skin synthesis was performed on 30 Wistar rats. After 21 days, abdominal hernia developed was corrected as follows: 1) No mesh; 2) Polypropylene mesh; and, 3) Ultrapro(r) mesh. After 21 days, the mesh and surrounding tissue were submitted to macroscopic (presence of adhesions, mesh retraction), microscopic analysis to identify and quantify the inflammatory and fibrotic response using a score based on a predefined scale of 0-3 degrees, evaluating infiltration of macrophages, giant cells, neutrophils and lymphocytes. RESULTS: No significant difference was seen among groups in adherences, fibrosis, giant cells, macrophages, neutrophils or lymphocytes (p>0.05). Mesh shrinkage was observed in all groups, but also no difference was observed between polypropylene and Ultrapro mesh (7.0±9.9 vs. 7.4±10.1, respectively, p=0.967). Post-operatory complications included fistula, abscess, dehiscence, serohematic collection and reherniation, but with no difference among groups (p=0.363). CONCLUSION: There is no difference between polypropylene (high-density) and Ultrapro(r) (low-density) meshes at 21 days after surgery in extraperitoneal use in rats, comparing inflammatory response, mesh shortening, adhesions or complications.

Polypropylene and polypropylene/polyglecaprone (Ultrapro(r)) meshes in the repair of incisional hernia in rats

PURPOSE: To compare the inflammatory response of three different meshes on abdominal hernia repair in an experimental model of incisional hernia. METHODS: Median fascial incision and skin synthesis was performed on 30 Wistar rats. After 21 days, abdominal hernia developed was corrected as follows: 1) No mesh; 2) Polypropylene mesh; and, 3) Ultrapro(r) mesh. After 21 days, the mesh and surrounding tissue were submitted to macroscopic (presence of adhesions, mesh retraction), microscopic analysis to identify and quantify the inflammatory and fibrotic response using a score based on a predefined scale of 0-3 degrees, evaluating infiltration of macrophages, giant cells, neutrophils and lymphocytes. RESULTS: No significant difference was seen among groups in adherences, fibrosis, giant cells, macrophages, neutrophils or lymphocytes (p>0.05). Mesh shrinkage was observed in all groups, but also no difference was observed between polypropylene and Ultrapro mesh (7.0±9.9 vs. 7.4±10.1, respectively, p=0.967). Post-operatory complications included fistula, abscess, dehiscence, serohematic collection and reherniation, but with no difference among groups (p=0.363). CONCLUSION: There is no difference between polypropylene (high-density) and Ultrapro(r) (low-density) meshes at 21 days after surgery in extraperitoneal use in rats, comparing inflammatory response, mesh shortening, adhesions or complications. .

Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts.

The preliminary results of ovarian transplantation in clinical practice are encouraging. However, the follicular depletion caused by ischemic injury is a main concern and is directly related to short-term graft survival. Cell therapy with adipose tissue-derived stem cells (ASCs) could be an alternative to induce early angiogenesis in the graft. This study aimed to evaluate ASCs therapy in rat cryopreserved ovarian grafts. A single dose of rat ASC (rASCs) or vehicle was injected into the bilateral cryopreserved ovaries of twelve adult female rats immediately after an autologous transplant. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability, graft morphology and apoptosis were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (P>0.05). However, compared with the vehicle-treated grafts, the morphology of the ASCs-treated grafts was impaired, with diffuse atrophy and increased apoptosis (P<0.05). ASCs direct injected in the stroma of rat cryopreserved ovarian grafts impaired its morphology although may not interfere with the functional resumption on short-term. Further investigations are necessary to evaluated whether it could compromise their viability in the long-term.

N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats.

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.

Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function.

BACKGROUND: Cryopreservation of the ovarian tissue has shown promising results. However, there remain controversial issues such as the short half-life of grafts. In this aspect, there are some evidences that preconditioning the ovarian tissue before transplantation is beneficial. OBJECTIVE: To determine the effect of hypoxic preconditioning in vitro on ovarian tissue prior to transplantation. METHODS: Eighteen female adult Wistar rats, were sorted into three experimental groups. Ovaries were maintained in DMEM low glucose serum free at 37°C with 5% CO2, at atmospheric oxigen concentration (normoxia) or 1% O2 (hypoxia) for 16 hours. Oxigen concentration was determined by injection of nitrogen in the incubator. Animals submitted to ovarian transplantation immediately after oophorectomy were the Control Group (C). After this, the ovaries were implanted in the retroperitoneum with nonabsorbable suture and animals evaluated for thirty days after transplantation. Beginning on postoperative (PO) day 11, a daily collection of vaginal smear was carried out. Analyses comprised morphological, morphometric (counting ovarian follicles and corpora lutea) and immunohistochemistry for cleaved caspase-3 (apoptosis). RESULTS: In normoxia and control groups all animals recovered their estrous cycles, while in the hypoxia group, two animals did not ovulate but, among those which did, resumption took longer than in the other groups (p < 0.05). The number of ovarian follicles and corpora lutea decreased significantly in the hypoxia group when compared to the other two groups (p < 0.001) and apoptosis was increased in the few ovarian follicles which remained viable (p < 0.001). CONCLUSION: The hypoxic preconditioning in vitro was not beneficial to the graft and worsened their viability, compromising its functionality or delaying the return of this.

N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle. .

Study on the vaginal smear of rats submitted to autologous ovarian transplant: impact of remote ischemic preconditioning.

PURPOSE: To evaluate the remote ischemic preconditioning (R-IPC) impact on the quality of the ovarian graft by means of vaginal smear of transplanted rats. METHODS: Sixty rats were used divided in six groups: Control; Fresh transplant (TxF); Cryopreserved transplant (TxC); R-IPC; R-IPC + fresh transplant (TxF+R-IPC); R-IPC + cryopreserved transplant (TxC+R-IPC). R-IPC was performed in the common iliac artery. Autologous ovarian tissue was implanted integrally in the retro peritoneum. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. RESULTS: R-IPC showed a tendency to an early estrus re-initiation (p>0.05) as well as increase the number of cycles in the fresh transplanted group while in the cryopreserved transplant the number of cycles was similar, regardless of the stimulus R-IPC (p>0.05). The animals which had undergone fresh grafts had a longer estrous period than the ones which had undergone cryopreserved grafts, with or without R-IPC (p<0.05). CONCLUSION: R-IPC promoted earlier re-initiation of ovarian activity in the PO and greater estrous frequency, with more consistent results in the fresh grafts than in the cryopreserved ones.

Study on the vaginal smear of rats submitted to autologous ovarian transplant: impact of remote ischemic preconditioning / Estudo do esfregaço vaginal de ratas submetidas a transplante autólogo de ovário: impacto do precondicionamento isquêmico remoto

PURPOSE: To evaluate the remote ischemic preconditioning (R-IPC) impact on the quality of the ovarian graft by means of vaginal smear of transplanted rats. METHODS: Sixty rats were used divided in six groups: Control; Fresh transplant (TxF); Cryopreserved transplant (TxC); R-IPC; R-IPC + fresh transplant (TxF+R-IPC); R-IPC + cryopreserved transplant (TxC+R-IPC). R-IPC was performed in the common iliac artery. Autologous ovarian tissue was implanted integrally in the retro peritoneum. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. RESULTS: R-IPC showed a tendency to an early estrus re-initiation (p>0.05) as well as increase the number of cycles in the fresh transplanted group while in the cryopreserved transplant the number of cycles was similar, regardless of the stimulus R-IPC (p>0.05). The animals which had undergone fresh grafts had a longer estrous period than the ones which had undergone cryopreserved grafts, with or without R-IPC (p<0.05). CONCLUSION: R-IPC promoted earlier re-initiation of ovarian activity in the PO and greater estrous frequency, with more consistent results in the fresh grafts than in the cryopreserved ones.

OBJETIVO: Avaliar o impacto do precondicionamento isquêmico remoto (PCI-R) na qualidade do enxerto ovariano através dos esfregaços vaginais dos animais transplantados. MÉTODOS: Foram utilizadas 60 ratas, distribuídas em seis grupos de estudo: Controle; Transplante fresco; Transplante criopreservado; PCI-R; PCI-R + Transplante fresco; PCI-R + Transplante criopreservado. O PCI-R foi realizado na artéria ilíaca comum. O tecido ovariano foi implantado íntegro no retroperiônio. No 1º dia de pós-operatório (PO) foram coletados esfregaços vaginais diariamente. Após 30 dias foram considerados para análise o dia de PO de retorno do estro, assim como o número de dias em estro e de ciclos estrais. RESULTADOS: PCI-R mostrou tendência em reinício mais precoce do cilo estral (p>0,05), assim como aumento no número de cilcos estrais nos grupos com transplante fresco enquanto que no criopreservado o número de ciclos foi semelhante, independente do PCI-R (p>0,05). Os animais que receberam enxertos frescos apresentaram mais dias na fase estro do que os criopreservados, com ou sem PCI-R. CONCLUSÃO: O PCI-R promoveu retorno mais precoce da atividade ovariana no PO e maior freqüência de estro, sendo os resultados mais consistentes nos enxertos frescos do que nos criopreservados.

Indirect evaluation of estrogenic activity post heterotopic ovarian autograft in rats / Avaliação indireta da atividade estrogênica após transplante heterotópico de ovário em ratas

PURPOSE: To morphologically evaluate the estrogenic effect on the uterus and vagina of rats submitted to ovarian autografts. METHODS: Twenty Wistar EPM-1 adult rats were bilaterally ovariectomized, followed by ovarian transplants in retroperitoneal regions. The animals were divided in four groups of five animals, according to the day of euthanasia: G4, G7, G14 and G21, corresponding to the 4th, 7th, 14th and 21st day after surgery, respectively. Vaginal smears were collected from the first day of surgery until euthanasia day. After that, the vagina and uterus were removed, fixed in 10 percent formaldehyde and submitted to histological analysis and stained with hematoxiline and eosine. RESULTS: All animals showed estrous cycle changes during the experiment. In 4th day, the uterus showed low action of estrogen with small number of mitosis and eosinophils as well as poor development. On the 7th day, the endometrium was atrophic without mitotic signals and presented a small number of eosinophils. On the 14th and 21th days the histological findings were similar, with the presence of mitosis in the endometrial glands and intense leucocyte infiltration with a large number of eosinophils. Morphometric results showed that the endometrial and myometrial thickness as well as the number of eosinophils presented the highest values during the 14th and 21th days of the evaluation. The 7th day group also presented the lowest eosinophil numbers. Vaginal epithelium features were: 4th and 7th day groups presented non-keratinized stratified epithelium with 5 and 2 cell layers, respectively. The 14th and 21st day groups presented non-keratinized stratified epithelium with 14 and 15 cell layers. CONCLUSION: Experimental ovarian autografts in the evaluated organs presented maximum estrogen activity after the 21st day of surgery, according to morphological and morphometric data.

OBJETIVO: Avaliar morfologicamente a atividade estrogênica no útero e vagina de ratas submetidas a transplante autólogo de ovário. MÉTODOS: Foram utilizadas 20 ratas adultas, Wistar EPM-1, submetidas à ooforectomia bilateral seguida de transplante de ovário em retroperitônio, distribuídas em grupos, conforme a data pré-estabelecida para eutanásia, com cinco animais cada: G4, G7, G14 e G21 - eutanásia no 4º, 7º, 14º e 21º dia de pós-operatório, respectivamente. No 1º dia após a cirurgia foi iniciada coleta de esfregaço vaginal diariamente até o dia pré-estabelecido para eutanásia. Após a eutanásia foi realizada exérese em bloco da vagina e útero, que foram fixados em formol 10 por cento para processamento histológico e coloração pela Hematoxilina-Eosina. RESULTADOS: Todos os animais mostraram alterações do ciclo estral no decorrer dos dias do experimento. No 4º dia, o útero mostrava sinais de baixa atividade estrogênica, o endométrio apresentava poucas figuras de mitose, pouco desenvolvimento e pequena quantidade de eosinófilos. Já no 7º dia, o endométrio encontrava-se atrofiado, sem atividade mitótica e com raros eosinófilos. No 14º e 21º dias os achados foram semelhantes, com presença de mitose nas glândulas endometriais e intenso infiltrado leucocitário, com predomínio de eosinófilos. Os resultados da morfometria mostraram que tanto as espessuras endometrial e miometrial quanto o número de eosinófilos foram maiores no 14º e 21º dias de avaliação, com menores valores no 7º dia. As características do epitélio vaginal foram: epitélio pavimentoso não queratinizado no 4º e 7º (com cerca de cinco e duas camadas celulares, respectivamente) e pavimentoso queratinizado em no 14º e 21º dias (com cerca de 14 e 15 camadas celulares, respectivamente) CONCLUSÃO: O transplante experimental autólogo de ovário manteve a atividade estrogênica nos órgãos avaliados, com atividade estrogênica máxima ao final de 21 dias, conforme parâmetros morfológicos...

Indirect evaluation of estrogenic activity post heterotopic ovarian autograft in rats.

PURPOSE: To morphologically evaluate the estrogenic effect on the uterus and vagina of rats submitted to ovarian autografts. METHODS: Twenty Wistar EPM-1 adult rats were bilaterally ovariectomized, followed by ovarian transplants in retroperitoneal regions. The animals were divided in four groups of five animals, according to the day of euthanasia: G4, G7, G14 and G21, corresponding to the 4th, 7th, 14th and 21st day after surgery, respectively. Vaginal smears were collected from the first day of surgery until euthanasia day. After that, the vagina and uterus were removed, fixed in 10% formaldehyde and submitted to histological analysis and stained with hematoxiline and eosine. RESULTS: All animals showed estrous cycle changes during the experiment. In 4th day, the uterus showed low action of estrogen with small number of mitosis and eosinophils as well as poor development. On the 7th day, the endometrium was atrophic without mitotic signals and presented a small number of eosinophils. On the 14th and 21th days the histological findings were similar, with the presence of mitosis in the endometrial glands and intense leucocyte infiltration with a large number of eosinophils. Morphometric results showed that the endometrial and myometrial thickness as well as the number of eosinophils presented the highest values during the 14th and 21th days of the evaluation. The 7th day group also presented the lowest eosinophil numbers. Vaginal epithelium features were: 4th and 7th day groups presented non-keratinized stratified epithelium with 5 and 2 cell layers, respectively. The 14th and 21st day groups presented non-keratinized stratified epithelium with 14 and 15 cell layers. CONCLUSION: Experimental ovarian autografts in the evaluated organs presented maximum estrogen activity after the 21st day of surgery, according to morphological and morphometric data.