Maximizing Success: An Overview of Optimizing the Ovarian Tissue Transplantation Site.

Ovarian tissue cryopreservation and transplantation (OTCT) has emerged in recent years as a potential method for reversing abnormal endocrine and reproductive functions, particularly in patients receiving gonadotoxic cancer treatments having longer survival rates. From its first rodent experiments to human trials, OTCT has evolved tremendously, opening new windows for further utilization. Since then, significant progress has been achieved in terms of techniques used for surgical removal of the tissue, optimal fragment size, freezing and thawing procedures, and appropriate surgical sites for the subsequent reimplementation of the graft. In addition, various approaches have been proposed to decrease the risk of ischemic injury, which is the leading cause of significant follicle loss during neo-angiogenesis. This review aims to discuss the pros and cons of ovarian and retroperitoneal transplantation sites, highlighting the justifications for the viability and efficacy of different transplantation sites as well as the potential advantages and drawbacks of retroperitoneal or preperitoneal area.

Erythropoietin effects on cryopreserved/transplanted cat ovarian tissue: A comparison of two incubation methods.

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.

Modeling delay of age at natural menopause with planned tissue cryopreservation and autologous transplantation.

BACKGROUND: Ovarian tissue cryopreservation has been proven to preserve fertility against gonadotoxic treatments. It has not been clear how this procedure would perform if planned for slowing ovarian aging. OBJECTIVE: This study aimed to determine the feasibility of cryopreserving ovarian tissue to extend reproductive life span and delay menopause by autotransplantation near menopause. STUDY DESIGN: Based on the existing biological data on follicle loss rates, a stochastic model of primordial follicle wastage was developed to determine the years of delay in menopause (denoted by D) by ovarian tissue cryopreservation and transplantation near menopause. Our model accounted for (1) age at ovarian tissue harvest (21-40 years), (2) the amount of ovarian cortex harvested, (3) transplantation of harvested tissues in single vs multiple procedures (fractionation), and (4) posttransplant follicle survival (40% [conservative] vs 80% [improved] vs 100% [ideal or hypothetical]). RESULTS: Our model predicted that, for most women aged <40 years, ovarian tissue cryopreservation and transplantation would result in a significant delay in menopause. The advantage is greater if the follicle loss after transplant can be minimized. As an example, the delay in menopause (D) for a woman with a median ovarian reserve who cryopreserves 25% of her ovarian cortex at the age of 25 years and for whom 40% of follicles survive after transplantation would be approximately 11.8 years, but this extends to 15.5 years if the survival is 80%. As another novel finding, spreading the same amount of tissue to repetitive transplants significantly extends the benefit. For example, for the same 25-year-old woman with a median ovarian reserve, 25% cortex removal, and 40% follicle survival, fractionating the transplants to 3 or 6 procedures would result in the corresponding delay in menopause (D) of 23 or 31 years. The same conditions (3 or 6 procedures) would delay menopause as much as 47 years if posttransplant follicle survival is improved to 80% with modern approaches. An interactive Web tool was created to test all variables and the feasibility of ovarian tissue freezing and transplantation to delay ovarian aging (here). CONCLUSION: Our model predicts that with harvesting at earlier adult ages and better transplant techniques, a significant menopause postponement and, potentially, fertile life span extension can be achieved by ovarian tissue cryopreservation and transplantation in healthy women.

Síndrome do ovário remanescente em cadela ­ relato de caso / Ovarian remnant syndrome in bich ­ case report / Síndrome de ovario restante en perra ­ reporte de caso

A síndrome do ovário remanescente é caracterizada pela remoção incompleta do ovário durante o procedimento de castração em fêmeas. O presente trabalho relata o caso de uma cadela, sem raça definida, submetida a ovário-histerectomia há um ano, mas continuava demonstrando sinais de estro pós castração. Foi necessário a realização de exames de exames complementares como ultrassonografia abdominal e citologia vaginal, sendo identificado uma estrutura vascularizada em topografia de ovário e células em fase de estro. A cadela foi submetida a uma laparotomia exploratória para retirada de tecido ovariano remanescente.

Remnant ovary syndrome is characterized by incomplete removal of the ovary during the castration procedure in females. The present work reports the case of a dog, of no defined breed, who underwent ovariosalpingohysterectomy 1 year ago and continued to show signs of heat after castration. It was necessary to perform imaging tests such as abdominal ultrasound, which identified a vascularized structure in ovarian topography and vaginal cytology, which identified cells in the estrus phase. The dog required an exploratory laparotomy to remove remaining ovarian tissue.

El síndrome de ovario remanente se caracteriza por la extirpación incompleta del ovario durante el procedimiento de castración en las mujeres. El presente trabajo reporta el caso de una perra, sin raza definida, a quien se le realizó ovariosalpingohisterectomía hace 1 año y continuó presentando signos de celo luego de la castración. Fue necesario realizar pruebas de imagen como ecografía abdominal, que identificó una estructura vascularizada en la topografía ovárica y citología vaginal, que identificó células en fase de estro. La perra requirió una laparotomía exploratoria para extirpar el tejido ovárico restante.

Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers.

The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Effects of erythropoietin on ischaemia-reperfusion when administered before and after ovarian tissue transplantation in mice.

RESEARCH QUESTION: Is the optimal timing for administering erythropoietin to minimize ischaemic injury in ovarian tissue transplantation before ovary removal for cryopreservation and subsequent transplantation or after transplantation? DESIGN: Thirty Swiss mice (nu/nu) were divided into three groups: treatment control group (n = 10); erythropoietin before harvesting group (EPO-BH) (n = 10) and erythropoietin after transplantation group (EPO-AT) (n = 10). Animals underwent bilateral ovariohysterectomy and their hemiovaries were cryopreserved by slow freezing. At the same time, previously cryopreserved hemiovaries were transplanted subcutaneously in the dorsal region. Erythropoietin (250 IU/kg) and sterile 0.9% saline solution were administered every 12/12 h over 5 consecutive days in the EPO-AT and EPO-BH groups, respectively. RESULTS: Administration of erythropoietin in the EPO-AT group improved the viability of ovarian follicles, reducing degeneration and increasing the number of morphologically normal growing follicles at 14 days after transplantation compared with the EPO-BH group (P = 0.002). This group also showed higher percentages of proliferative follicles at 7 days after transplantation (P ≤ 0.03), increased blood vessel count (P ≤ 0.03) and greater tissue area occupied by blood vessels at days 7 and 14 after transplantation (P ≤ 0.03), compared with hormone administration before cryopreservation (EPO-BH group) and the treatment control group. Additionally, treatment with erythropoietin before or after transplantation reduced fibrotic areas at 7 days after transplantation (P = 0.004). CONCLUSION: Erythropoietin treatment after transplantation reduced ischaemic damage in transplanted ovarian tissue, increased angiogenesis, maintenance of ovarian follicle proliferation and reduced fibrosis areas in the grafted tissue.

A Holistic Approach To Oncofertility In A Patient With Rectal Cancer: A Case Report.

OBJECTIVE: Cryopreservation techniques are used to preserve fertility before cancer treatment with gonadotoxic agents. Herein, we report a case of fertility preservation involving a 29-year-old G0P0 woman, married for one year, who was referred to our hospital for fertility preservation before starting rectal cancer treatment. CASE DESCRIPTION: Ovarian tissue and embryo cryopreservation were performed. Before the procedure, ovarian reserve was evaluated, and antral follicle counts were determined. Laparoscopic ovarian tissue cryopreservation was performed from the left side with a lower antral follicle count. Thus, we were able to keep the number of oocytes obtained in the following controlled ovarian hyperstimulation cycle at the highest level. Subsequently, the right ovary was transposed into the lateral wall of the abdomen under the peritoneum. Conventionally controlled ovarian hyperstimulation was initiated on the first postoperative day, depending on the menstrual cycle phase. Intracytoplasmic sperm injection was performed on four mature oocytes obtained, and one embryo was cryopreserved. Controlled ovarian hyperstimulation was initiated on the first postoperative day, and the process was repeated on the seventh postoperative day, yielding a total of seven viable embryos for cryopreservation. CONCLUSIONS: There is usually only one chance of controlled ovarian hyperstimulation in patients requiring a fertility-sparing approach due to malignancy. In the combined technique, performing ovarian tissue resection from the ovary with a lower number of antral follicles can keep the number of oocytes at the highest level in the following controlled ovarian hyperstimulation cycle.

Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers

The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.(AU)

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.

Genistein Blunted Detrimental Effects of Polycystic Ovary Syndrome on the Ovarian Tissue of Rats by Improving Follicular Development and Gonadotropin Secretion.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a common cause of female infertility worldwide. It has been shown that genistein, a natural isoflavone, may influence follicular competence via the production of gonadotropins in women with PCOS. The current study aims to evaluate the effects of genistein on the ovarian tissue of rats with PCOS. METHODS: Thirty female Wistar rats were randomly divided into the following four groups: Control; PCOS (rats received 2 mg/kbW estradiol valerate); Genistein (rats given 1 mg/kg BW of genistein for 14 days); and Genistein + PCOS. All animals were slaughtered under anesthesia and blood samples were collected for biochemical analysis. Follicular morphology was analyzed based on histologic examination. RESULTS: Histologic examination exhibited enhanced follicular atresia at various stages in the rats with PCOS compared to controls (p<0.001). Induction of PCOS caused significant reduction in gonadotropin levels and steroid hormone levels consistent with insulin resistance (p<0.01). Data showed that 14-day administration of genistein might improve follicular morphology in rats with PCOS (p<0.001). Genistein treatment increased the production of gonadotropins and steroid hormones and alleviated insulin resistance in Rats with PCOS (p<0.001). CONCLUSIONS: This study indicated that genistein treatment exerted a beneficial effect on the ovarian tissue of rats with PCOS by improving follicular growth and hormone balance.

Biobanking and use of gonadal tissues - a promising strategy for conserving wildlife from the Caatinga biome.

Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.

Detailed Morphological Analysis of Cryoinjury in Human Ovarian Tissue Following Vitrification or Slow Freezing.

Cryopreservation of human ovarian tissue represents a key procedure for fertility preservation. The two most widely used cryopreservation methods for human ovarian cortex samples are slow freezing\thawing (SF\T) and vitrification\warming (V\W). The aim of the present study was to analyze the effects of SF\T and V\W using a metal chamber, on specific follicle and oocyte structures and on the stromal organization post-cryopreservation. We did histology analysis of SF\T and V\W ovarian fragments from nine healthy subjects. Overall results showed that cryopreserved tissues presented significant rates of damage in primordial and primary follicles. Altered nuclear structure of primordial follicles and cell detachment from primordial and primary follicles were the main injuries observed after V/W and SF/T. The stromal components were similarly well preserved after cryopreservation. We conclude that both cryopreservation methods may be used for fertility preservation purposes with similar outcomes in terms of follicular and stromal integrity. Detachment of follicle cells from basal membrane represents an important cryoinjury that deserves further investigation.

Biobanking and use of gonadal tissues - a promising strategy for conserving wildlife from the Caatinga biome

Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.(AU)

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.

Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

Aloe vera increases mRNA expression of antioxidant enzymes in cryopreserved bovine ovarian tissue and promotes follicular growth and survival after in vitro culture.

The aims of the present study were to evaluate the effects of Aloe vera extract on expression of mRNA for antioxidant enzymes in bovine ovarian tissue after vitrification, as well as on follicular morphology, viability, activation and extracellular matrix in cultured ovarian tissues that had been previously vitrified. Fragments from bovine ovarian cortical tissue were cryopreserved in a vitrification solution alone or supplemented with two concentrations of Aloe vera (10 or 50%). After thawing, the cryopreserved tissues were analyzed by histological techniques, as well as the levels of mRNA for SOD, CAT, PRDX6 and GPX1 were investigated. Furthermore, cryopreserved fragments were then culture in vitro in α-MEM for 6 days. Histological evaluation of cultured tissues was performed to determine the percentages of normal and developing follicles. The results showed that, after vitrification, the presence of Aloe vera in both concentrations was able to maintain percentages of collagen fibers similar to fresh tissues (P < 0.05). Aloe vera in both concentrations significantly increased mRNA levels for PRDX6 and GPX1 in cryopreserved tissues, while 10% Aloe vera increased mRNA levels for SOD (P < 0.05). In parallel, after in vitro culture, fragments vitrified in the presence of 10% Aloe vera had significantly higher levels of morphologically healthy follicles when compared to tissue that were vitrified without Aloe vera. In fragments vitrified with Aloe vera, the rate of developing follicles was significantly higher than in tissues vitrified without Aloe vera. Tissues vitrified with 10% Aloe vera and cultured in vitro maintained percentages of collagen fibers similar to fresh tissues. In conclusion, 10% Aloe vera increases the expression of mRNA for PRDX6, GPX1 and SOD in vitrified ovarian tissues, maintains follicular survival and promotes activation and development of follicles after in vitro culture of vitrified bovine ovarian tissue.

The contraceptive potential of Carica papaya seed on oestrus cycle, progesterone, and histomorphology of the Utero-ovarian tissue of adult wistar rats.

OBJECTIVE: The study's goal was to ascertain the contraceptive effects of Aqueous extract of Carica papaya on female rats by assessing changes in the body weight, estrous cycle, serum progesterone level and the cyto-architecture of the Utero-ovarian tissue. METHODS: We used twenty (20) healthy young Adult Female Albino rats. The study ran for 7 and 21 days, respectively. Each study group has their Experimental (treated 200mg/kg aqueous extract of Carica papaya seed extract) and Control group (n=5). We determined daily the phases and frequencies of the estrous cycles of the rats during the administration of the extract. We processed the utero-ovarian tissue for histological analysis, and we assessed serum progesterone level and the oestrus cycle pattern. RESULTS: There was a significant increase in body and Ovarian weights after 21 days of treatment when compared to controls and those treated for 7 days. However, uterine weight reduced significantly (p<0.05), serum progesterone level decreased (p<0.05) in the treated rats, mostly in those submitted to 21 day-treatments; the ovary showed marked degeneration of the theca cells, granulosa and corpus luteum, and loss of mucin granules in the uterine tissues. Carica papaya administered for 7 and 21 days caused the animals to have more proestrus and diestrus phases as compared to the control animals. The estrous cycle became irregular, with prolonged diestrous and proestrus phase. CONCLUSION: The aqueous extract of Carica papaya seeds caused antifertility, anti-implantation, by a reduction in progesterone level, disruption of oestrus pattern and histological alteration of utero-ovarian tissue.

Maternal protein restriction impairs nutrition and ovarian histomorphometry without changing p38MAPK and PI3K-AKT-mTOR signaling in adult rat ovaries.

AIMS: Because an adequate protein supply is detrimental for the maintenance of folliculogenesis and ovulation, we evaluated the impact of maternal low protein diet on nutritional parameters, estrous cycle, ovarian histomorphometry, and on the expression of metabolic and survival signaling molecules in different follicular stages. MAIN METHODS: Twenty Wistar pregnant rats were divided into two groups: the normoprotein (NP) group, composed of animals that received 17% protein, and a low-protein (LP) group, composed of animals that received 6% protein during gestation and lactation period. After weaning, female rats were fed with standard diet until the 120-days-old. KEY FINDINGS: LP animals showed reduced body mass index, total body weight, energy intake, feed efficiency, and visceral fat. The ovarian tissue presented vascular congestion and fat accumulation in the medulla, followed by a significant reduction in the amount of primordial and primary follicles. In addition, the number of atretic follicles was higher in LP than in NP animals. Maternal undernutrition also resulted in increased levels of estradiol (E2) and progesterone (P4) while testosterone (T) was unchanged in the offspring. Although discrete changes in p38MAPK and in PI3K-AKT-mTOR immunostaining were observed in the ovarian follicles and corpus luteum in LP, no differences were found at their protein levels. SIGNIFICANCE: Maternal protein restriction alters estrous cycle and histomorphometry of the offspring's ovary without changing the levels of intracellular regulatory molecules in adulthood. These morphofunctional changes may alter reproductive performance in female offspring, highlighting maternal dietary conditions as an important factor for offspring reproductive health.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.