Ovarian Fragments Research Articles

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400ng/ml) for 7days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1ng/ml GDF-9. Moreover, 50ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1ng/ml GDF-9. However, the concentration of 50ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

Ovarian tissue cryopreservation and transplantation in patients with central nervous system tumours.

Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours? Malignancy reseeding in cryopreserved ovarian tissue from 20 patients with CNS tumours was not detected by histology, immunohistochemistry (IHC), molecular biology or xenotransplantation. Ovarian metastasis potential has been documented in patients with leukaemia, borderline ovarian tumours, advanced breast cancer and Ewing sarcoma. However, data on the safety of transplanting frozen-thawed ovarian tissue from cancer patients with CNS tumours are still lacking. This prospective experimental study was conducted in an academic gynaecology research laboratory using cryopreserved ovarian cortex from 20 patients suffering from CNS tumours. Long-term (5 months) xenografting was performed in immunodeficient mice. Subjects enrolled in the study were suffering from one of six types of CNS tumours including medulloblastoma, ependymoma, primitive neuroectodermal tumours, astrocytoma, glioblastoma and germinoma. The presence of malignant cells was investigated with disease-specific markers for each patient in cryopreserved and xenografted ovarian tissue by histology, IHC via expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP and ENO2 gene amplification. Serial sections of cryopreserved and xenografted ovarian tissue from 20 patients showed no malignant cells by histology. All samples were negative for NSE and GFAP, although these neural markers were expressed extensively in the patients' primary tumours. Analysis by RT-ddPCR revealed no cancer cells detected in cryopreserved and xenografted ovarian fragments from subjects with astrocytoma, ependymoma, glioblastoma or medulloblastoma. Taken together, the study found no evidence of malignancy seeding in frozen-thawed and xenotransplanted ovarian tissue from patients affected by CNS cancers. This analysis cannot guarantee complete elimination of disseminated disease from all cryopreserved ovarian cortex, since we are unable to examine the fragments used for transplantation. This is the first study to be conducted in patients with CNS cancers undergoing ovarian tissue cryopreservation and transplantation, and clearly demonstrates no tumour seeding in their frozen-thawed and xenografted tissue. This information is vital for doctors to provide patients with meaningful and accurate advice on the possibilities and risks of ovarian tissue reimplantation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique-the Excellence of Science (FNRS-EOS), number 30443682 awarded to M.-M.D. and T.Y.T.N., FNRS grant number 5/4/150/5 and FNRS-PDR Convention grant number T.0077.14 awarded to M.-M.D., grant 2018-042 from the Foundation Against Cancer awarded to A.C., and private donations (Ferrero, de Spoelberch). The authors declare no competing financial interests. N/A.

Protective Effects of Reduced Glutathione and Ulinastatin on Xeno-transplanted Human Ovarian Tissue Against Ischemia and Reperfusion Injury

Recently, transplantation of cryopreserved ovarian tissue is the method for fertility preservation for oncologic and nononcologic reasons. The main challenge of ovarian cryopreservation followed by transplantation is that ischemia reperfusion injury (IRI) induced the loss of follicles. The aim of this study was to evaluate the effects of glutathione (GSH), ulinastatin (UTI) or both (GSH+UTI) on preventing ischemia reperfusion-induced follicles depletion in ovarian grafts. Ovarian fragments were collected from 20 women aged 29±6 years. Frozen-thawed human ovarian tissue was xenografted into SCID mice, at the same time GSH, UTI and GSH+UTI was administrated respectively. The ovarian grafts were collected at the 1st, 3rd, 7th, 14th, 28th, 56th, and 85th day after xenotransplantation. Follicle survival rate was measured by H&E staining and Live/Dead staining. Angiogenic activity and macrophage recruitment was evidenced by immunohistochemical staining. The oxidative stress and inflammatory cytokines in human ovarian xenografts were measured by real-time PCR. The results indicated that after the treatments of GSH, UTI and GSH+UTI in the hosts, follicular survival in ovarian grafts were improved. The level of VEGF, CD31, and antioxidant enzymes superoxide dismutase 1 and superoxide dismutase 2 in ovarian grafts were increased. Accumulation of macrophages, level of IL6 and TNF-α, as well as malondialdehyde was decreased in ovarian grafts from treated groups. In conclusion, administration of GSH, UTI and GSH+UTI decreased the depletion of follicles in human grafts post-transplantation by inhibiting IRI-induced antiangiogenesis, oxidative stress and inflammation.

A Review on Biomaterials for Ovarian Tissue Engineering

Considerable challenges in engineering the female reproductive tissue are the follicle's unique architecture, the need to recapitulate the extracellular matrix, and tissue vascularization. Over the years, various strategies have been developed for preserving fertility in women diagnosed with cancer, such as embryo, oocyte, or ovarian tissue cryopreservation. While autotransplantation of cryopreserved ovarian tissue is a viable choice to restore fertility in prepubertal girls and women who need to begin chemo- or radiotherapy soon after the cancer diagnosis, it is not suitable for all patients due to the risk of having malignant cells present in the ovarian fragments in some types of cancer. Advances in tissue engineering such as 3D printing and ovary-on-a-chip technologies have the potential to be a translational strategy for precisely recapitulating normal tissue in terms of physical structure, vascularization, and molecular and cellular spatial distribution. This review first introduces the ovarian tissue structure, describes suitable properties of biomaterials for ovarian tissue engineering, and highlights recent advances in tissue engineering for developing an artificial ovary.

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

Anti-Müllerian Hormone (AMH) regulates BRCA1 and BRCA2 gene expression after ovarian cortex transplantation

Objective To test whether recombinant anti-Müllerian hormone (rAMH) could exert an inhibitory function on BRCA1/2 expression in human ovarian cortex. Methods Pilot study on ovariectomized nude mice xenotransplanted with human vitrified/warmed ovarian cortex and treated with rAMH via infusion pump. Twelve nude mice were ovariectomized and Alzet pumps delivering 1.23 mcg rAMH/day to reach a serum concentration of 17.5 ng/mL, or placebo (controls), were inserted intraabdominally. Previously vitrified/warmed 2x2 mm ovarian cortex fragments were transplanted on day 7 and then harvested on day 14 after pump placement. PCR analyses determined mRNA levels for BRCA1 and BRCA2 in the human ovarian cortex. Results In mice treated with rAMH, BRCA1 expression was significantly lower (0.196 fg/µg RNA, IQR 0.158, 0.236) than in controls (0.544 fg/µg RNA, IQR 0.458, 0.554; p = .030), while BRCA2 expression remained similar in rAMH mice (5.355 fg/µg RNA, IQR 4.479, 6.230) and in controls (4.011 fg/µg RNA, IQR 3.650, 4.182; p = .327). Conclusion Administration of rAMH in the peri-transplant period caused downregulation of BRCA1, but not of BRCA2 expression, in human ovarian cortex. These results help our understanding of DNA repair mechanism in the ovarian cortex and identify AMH’s possible protective effect on ovarian reserve in BRCA1 mutation carriers.

Whole Ovary Cryopreservation and Transplantation: A Systematic Review of Challenges and Research Developments in Animal Experiments and Humans.

Ovarian tissue cryopreservation and transplantation is the only fertility preservation option that enables both restoration of fertility and resumption of ovarian endocrine function, avoiding the morbidity associated with premature menopause. It is also the only technique available to prepubertal patients and those whose treatment cannot be delayed for life-threatening reasons. Ovarian tissue cryopreservation can be carried out in two different ways, either as ovarian cortical fragments or as a whole organ with its vascular pedicle. Although use of cortical strips is the only procedure that has been approved by the American Society for Reproductive Medicine, it is fraught with drawbacks, the major one being serious follicle loss occurring after avascular transplantation due to prolonged warm ischemia. Whole ovary cryopreservation involves vascular transplantation, which could theoretically counteract the latter phenomenon and markedly improve follicle survival. In theory, this technique should maintain endocrine and reproductive functions much longer than grafting of ovarian cortical fragments. However, this procedure includes a number of critical steps related to (A) the level of surgical expertise required to accomplish retrieval of a whole ovary with its vascular pedicle, (B) the choice of cryopreservation technique for freezing of the intact organ, and (C) successful execution of functional vascular reanastomosis upon thawing. The aim of this systematic review is to shed light on these challenges and summarize solutions that have been proposed so far in animal experiments and humans in the field of whole ovary cryopreservation and transplantation.

Patient with ovarian insufficiency: baby born after anticancer therapy and re-transplantation of cryopreserved ovarian tissue

BackgroundThe second major cause of death is cancer. In fact, the effectiveness of anticancer treatments and positive long-term prognosis for young women has increased. However, the problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic and lead to the functional death of ovaries. There is potential key solution to this problem: cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence. Data regarding cryopreservation and re-transplantation of ovarian tissue from patients with ovarian insufficiency is limited. The aim of this treatment was the re-transplantation of cryopreserved ovarian tissue after anticancer therapy of patient with ovarian insufficiency (56 IU/l FSH, 8 ng/l β-estradiol, < 1.1 ng/ml anti-Mullerian hormone, 1 primary follicle per 10mm3).Case presentationAfter the operation, four tissue fragments (10–16 × 8–13 × 1.0–1.2 mm) were cooled to 5 °C in the freezing medium (culture medium+ 6% ethylene glycol+ 6% dimethyl sulfoxide+ 0.15 M sucrose) for 24 h, frozen and thawed. Freezing was performed in four standard 5 ml cryo-vials with ice formation at − 9 °C, cooling from − 9 to − 34 °C at a rate of − 0.3 °C/min and plunging at − 34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min. exposure in sucrose and 30 min. stepping rehydration. After thawing of one cryo-vial, part (5 mm3) of experimental ovarian tissue after 7 day in vitro culture was histological evaluated and two ovarian fragments (8 × 7 × 1.0 mm and 7 × 6 × 1.0 mm) were re-transplanted. The quantity of follicles after cryopreservation and in vitro culture was not increased (P > 0.1): it was found 1 primordial follicle in 5 mm3 of tissue. Thirty seven days after the re-transplantation of ovarian tissue, the restoration of the menstrual cycle of Patient W. was noted. Three months after the transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term.ConclusionsDescribed protocol of conventional cryopreservation of ovarian tissue can be used for treatment of patients with ovarian insufficiency.

Combining fertility preservation procedures to spread the eggs across different baskets: a feasibility study.

What is the reproductive potential following combinations of ovarian stimulation, IVM and ovarian tissue cryopreservation (OTC) in female patients seeking fertility preservation (FP)? In selected patients, combining different FP procedures is a feasible approach and reproductive outcomes after FP in patients who return to attempt pregnancy are promising. FP is increasingly performed in fertility clinics but an algorithm to select the most suitable FP procedure according to patient characteristics and available timeframe is currently lacking. Vitrification of mature oocytes (OV) and OTC are most commonly performed, although in some clinical scenarios a combination of procedures including IVM, to spread the sources of gametes, may be considered in order to enhance reproductive options for the future. Retrospective, observational study in a university-based, tertiary fertility centre involving all female patients who underwent urgent medical FP between January 2012 and December 2018. Descriptive analysis of various FP procedures, either stand-alone or combined, was performed, and reproductive outcomes of patients who attempted pregnancy in the follow-up period were recorded. In total, 207 patients underwent medical FP. Patient-tailored strategies and procedures were selected after multidisciplinary discussion. When deemed feasible, FP procedures were combined to cryopreserve different types of reproductive tissue for future use. The main primary outcome measure was the number of mature oocytes. Live birth rates were evaluated in patients who returned for reproductive treatment. Among patients seeking FP, 95/207 (46%) had breast cancer, 43/207 (21%) had haematological malignancies and 31/207 (15%) had a gynaecological tumour. Mean ± SD age was 27.0 ± 8.3 years. Eighty-five (41.1%) patients underwent controlled ovarian stimulation (COS), resulting in 10.8 ± 7.1 metaphase II (MII) oocytes for vitrification. Eleven (5.3%) patients had multiple COS cycles. Transvaginal oocyte retrieval for IVM was performed in 17 (8.2%) patients, yielding 9.2 ± 10.1 MII oocytes. Thirty-four (16.4%) patients underwent OTC combined with IVM of oocytes retrieved from ovarian tissue 'ex vivo' (OTO-IVM), yielding 4.0 ± 4.3 MII oocytes in addition to ovarian fragments. Seventeen (8.2%) patients had OTC combined with OTO-IVM and transvaginal retrieval of oocytes for IVM from the contralateral ovary, resulting in 13.5 ± 9.7 MII oocytes. In 13 (6.3%) patients, OTC with OTO-IVM was followed by controlled stimulation of the contralateral ovary, yielding 11.3 ± 6.6 MII oocytes in total. During the timeframe of the study, 31/207 (15%) patients have returned to the fertility clinic with a desire for pregnancy. Of those, 12 (38.7%) patients had preserved ovarian function and underwent ART treatment with fresh oocytes, resulting in nine (75%) livebirth. The remaining 19 (61.3%) patients requested warming of their cryopreserved material because of ovarian insufficiency. Of those, eight (42.1%) patients had a livebirth, of whom three after OTO-IVM. To date, 5/207 patients (2.4%) achieved an ongoing pregnancy or livebirth after spontaneous conception. Our FP programme is based on a patient-tailored approach rather than based on an efficiency-driven algorithm. The data presented are descriptive, which precludes firm conclusions. Combining different FP procedures is likely to enhance the reproductive fitness of patients undergoing gonadotoxic treatment but further follow-up studies are needed to confirm this. No external funding was used for this study and the authors have no competing interests. N/A.

Surgical approaches of drug-free in vitro activation and laparoscopic ovarian incision to treat patients with ovarian infertility

To demonstrate our procedures of drug-free invitro activation (IVA) for treating patients with premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR), as well as the laparoscopic ovarian incision (LOI) procedure for treating patients with resistant ovary syndrome (ROS). Step-by-step video demonstration of the surgical procedures. Fertility clinic and reproductive medicine department. Women were diagnosed with POI based on recent amenorrhea before 40 years of age or with DOR according to the Bologna criteria, showing growth of a few antral follicles after ovarian stimulation. ROS patients were diagnosed based on amenorrhea with hypergonadotropic hypoestrogenism but showing age-appropriate number of antral follicles under transvaginal ultrasound. The drug-free IVA consists of the following 4 steps: removing a part of the cortex from one or both ovaries; cutting ovarian cortical pieces into small cubes invitro; making pockets for ovarian tissue grafting; and grafting ovarian cortical cubes. The LOI procedure consisted of only one step: cutting ovarian cortex in situ. Both procedures were followed by ovarian hyperstimulation for at least 1 year. Informed consent was obtained from patients and approval was granted by the Biomedical Ethics Committee of the International University School of Medicine and the Rose Ladies Clinic. The present clinical trial was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Follicle growth. These procedures can be completed within 1 hour under laparoscopic surgery. There were no complications. In 13 of 15 patients treated with drug-free IVA, increases in antral follicle numbers were found, followed by a higher number of retrieved oocytes for invitro fertilization. In addition to one spontaneous pregnancy, embryo transfer allowed four live births and one ongoing pregnancy. Five additional patients and one miscarriage patient have cryopreserved embryos for future transfer. We also found follicle growth to the preovulatory stage in seven of 11 ROS patients who have not responded to any endogenous and exogenous follicle-stimulating hormone stimulations for follicle growth prior to LOI treatment, allowing the retrieval of mature oocytes for invitro fertilization. Four ROS patients became pregnant, followed by the delivery of three healthy infants and one ongoing pregnancy. A drug-free IVA approach provided an infertility treatment for recent POI or DOR patients. This procedure promoted growth of residual ovarian follicles following ovarian tissue fragmentation invitro, leading to Hippo signaling disruption. Although ROS patients exhibited symptoms of hypergonadotropic hypoestrogenism similar to that of POI patients, they still had multiple secondary follicles. Hippo signaling disruption invivo based on cutting ovarian cortex using LOI could promote follicle growth. UMIN000029807.

Evidence of metabolic activity during low-temperature ovarian tissue preservation in different media.

Although ovarian tissue transportation has been validated for up to 24h, there is no standard protocol to date. We aimed to elucidate how existing media currently used for ovarian tissue transportation affect ovarian tissue metabolism and cell viability. Cow ovarian fragments were immersed in 0.9% NaCl solution, IVF medium, Leibovitz 15 medium (L-15), or PBS for 1, 4, or 24h at 4°C. Media were analyzed for pH, lactate dehydrogenase (LDH) activity, and glucose, pyruvate, and lactate concentrations, while apoptosis was assessed by TUNEL assays in fixed fragments. Viability rates were assessed by flow cytometry (FACS). There were lower pH levels in NaCl at all time points compared with other media. LDH activity increased with time and was lowest in NaCl at 1 and 4h. There was no significant difference in glucose levels, but a significant pyruvate decrease in L-15 and a significant lactate increase in all media. TUNEL showed apoptosis rates ranging from 0 to 5%. FACS showed a mean of 4% necrotic cells and 15-19% apoptotic cells after 1h of incubation, but less than 1% necrotic cells and 2-6% apoptotic cells after 24h in all media. Our results indicate marked metabolic activity in ovarian tissue at 4°C and suggest that cells use internal sources of energy, which may influence transplantation outcomes. This highlights the importance of better understanding whole tissue dynamics to develop a standard protocol for ovarian tissue transportation. Graphical abstract.

Модификация этапов технологии интраовариальной витрификации ооцитов Sus Scrofa Domesticus

Abstract. Intraovarian vitrification of oocytes of farm animals today is a very promising direction. The composition of cryoprotective solutions requires further improvement. Vitrification of ovarian fragments (FО) allows tо save primordial follicles, a structure that is more stable, due to the lack of follicular fluid, to destructive processes provoked by ultra-low temperatures.The high reproductive potential of pigs is due to their physiological characteristics of growth and development. Silicon is a minor trace element in its content; it is increasingly used in pharmacology, veterinary medicine, and clinical practice. Nitrogen silicon dimethylglycerolate (SDMGC) are antimicrobial, wound healing and anti-inflammatory in nature. The presence of a compact cumulus surrounding the oocyte is one of the important indicators of the reliability of the female gamete for extrafollicular maturation. Purpose of this study: to was to evaluate the effect of introducing 0.2 % silicon dimethylglycerolate into cryoprotective solutions during intraovarian vitrification and the medium for maturation of devitrified oocyte-cumulus complexes of pigs on the morphology of cumulus cells and the state of nuclear material. Ovarian fragments of pigs (15mmx 20mm) to vitrification are exposed prior for 25 minutes and 15 minutes direct in cryoprotective solutions of the following composition: CPA 1 – 7.5 % ethylene glycol (EG), 7.5 % dimethyl sulfoxide (DMSO), 65 % phosphate-buffered saline (PBS), 20 % fetal bovine serum (PBS); CPA 2 – 20 % EG, 20 % DMSO, 60 % FSB, 0.5 mol / L sucrose. The experimental group was treated for 10 minutes in a solution of phosphate-saline buffer with 0.2 % SDMGC. Hoechst 33258 was used to evaluate chromatin status, samples were analyzed using a ZEISS Axio Imager Imager A 2 m microscope.The result. Incubation of ovarian fragments of pigs in a solution containing 0.2 % SDMGC before vitrification, as well as the introduction of 0.2 % SDMGC in the composition of oocyte maturation media, had a positive effect on the morphology of cumulus cells (degree of expansion) after the thawing procedure. The proportion of compact cumulus devitrified cells pretreated with 0.2 % SDMGC increased compared with the control group (64 % vs 56 %, P &lt; 0.05 (χ2-test). The level of oocytes with cumulus is in a high degree of expansion after 44 hours of cultivation with 0.2 % SDMGC in the experimental group was 75 % vs 54 % in the control group, P &lt; 0.05 (χ2-test). The use of SDMGC in the cultivation of devitrified oocyte-cumulus complexes of pigs for 44 hours increased the proportion of matured oocytes from 41 % in the control group to 64 % in the experimental group P &lt; 0.05 (χ2-test). The scientific novelty: the stages of the technology of intraovarian vitrification of porcine oocytes by means of preventive incubation of ovarian fragments in a solution of 0.2 % SDMGC and its introduction into the medium for culturing oocyte-cumulus complexes were modernized.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals Possible Peripheral Regulation of the Ovary.

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -β, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.

Fertility preservation in borderline ovarian tumor patients and survivors.

Borderline ovarian tumors (BOTs) represent around 15% of all epithelial ovarian cancer. Around one third of those patients is under 40 and has not completed childbearing when the tumor is diagnosed. Cancer survivors are more and more concerned about their future fertility since a large proportion of those with BOTs are young. Whatever the tumor stage, information regarding future fertility after treatment and fertility preservation (FP) options must be delivered to all patients before treatment. A multidisciplinary team will discuss and propose personalized treatment and FP strategies. Nowadays, the FP options offered to patients with BOT are the followings: i) minimal invasive conservative surgery, ii) oocyte cryopreservation after controlled ovarian stimulation (COS) or in vitro maturation (IVM) and iii) ovarian tissue cryopreservation. Generally, the most common strategy to preserve future fertility is represented by minimal invasive conservative surgery. However, with the remarkable success and evolution of assisted reproductive technologies (ART) -notably progress and efficiency in COS and oocyte vitrification -have led to offer another potential approach for FP consisting in oocyte cryopreservation. Several COS protocols, such as random start or dual stimulation associating tamoxifen or aromatase inhibitors with gonadotropins provide similar results when compared to standard protocols while providing safety by minimizing the risk of high estrogen exposure. When COS is contraindicated, oocyte cryopreservation can still be possible throw IVM. Even though, oocyte competence after IVM is lower than that obtained after COS. A less used approach is cryopreservation of ovarian tissue, consisting in freezing ovarian cortex fragments for a future thawing and graft. Some concerns and limitations regard the ovarian cortex graft and the risk of reintroducing malignant cells once performed. Nonetheless, the latter it is the only option in prepubertal patients.

BMP-15 activity on in vitro development of collared peccary (Pecari tajacu Linnaeus, 1758) preantral follicles.

This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6days in Tissue Culture Medium 199 (TCM199+ ) supplemented with BMP-15 at rates of 0, 1, 25 or 50ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67%±0.69) when compared to other treatments (p<.05); however, this result is similar to 1ng/ml BMP-15 (74.00%±1.90, p>.05). Moreover, 25 and 50ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1day of culture when compared to fresh fragments or the control samples (p<.05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p>.05). In summary, the addition of 25ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6days in culture.

Epigallocatechin-3-gallate (EGCG) reduces apoptosis of preantral follicles through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway after in vitro culture of sheep ovarian tissue

The aims of this study were to analyze the effects of different concentrations of epigallocatechin-3-gallate (EGCG) on the primordial follicle survival and development after in vitro culture of ovarian tissue, and to verify the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway in the EGCG actions in the sheep ovary. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or with different concentrations of EGCG (0.01; 0.1; 1; 10 or 100 μg/mL) for 7 days. Inhibition of PI3K activity was performed in fragments cultured with 1 μg/mL EGCG plus LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3 and AKT phosphorylation (p-AKT). The results showed that 1 μg/mL EGCG maintained the follicular survival similar (P > 0.05) to that of the fresh control and higher (P < 0.05) than that of the α-MEM+ and other EGCG treatments. No difference (P > 0.05) in the follicular activation was observed. However, both follicle and oocyte diameters increased after in vitro culture with 1 μg/mL EGCG compared to other treatments (P < 0.05), except for 10 μg/mL EGCG (P > 0.05). After PI3K inhibition, there was an increase (P < 0.05) of the follicular apoptosis and a reduction of p-AKT immunolocalization. In conclusion, EGCG at 1 μg/mL reduces apoptosis of preantral follicles through the PI3K/AKT pathway after in vitro culture of sheep ovarian tissue.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Restoration of estrous cycles by co-transplantation of mouse ovarian tissue with MSCs.

This study investigates the effect of bone marrow (BM-MSCs) and visceral peritoneum (VP-MSCs)-derived mesenchymal stem cells on the transplanted ovary. VP-MSCs and BM-MSCs were obtained from green fluorescent protein-expressing mice (GFP+). Six- to eight-week-old female NMRI mice were divided into four experimental groups, autograft ovarian tissue fragments (AO), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel (AO-H), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing BM-MSCs (AO-HB) and autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing VP-MSCs (AO-HP). Intact ovary (IO) was the control group. The estrous cycles resumption time was monitored and at the third estrous cycle, the blood samples and grafted ovaries were evaluated using hormonal, histological and gene expression analysis. Onset of estrous cycles, especially at the second cycle, was earlier in AO-HB and AO-HP groups than in the AO-H group (P < 0.05). Moreover, E2 and FSH levels in AO-HB and AO-HP groups were returned to those of the intact group. However, folliculogenesis was still retarded as compared with the IO group. The gene expression of theca (Lhcgr, Cyp17a1, Gli2, Gli3 and Ptch1), granulosa (Amh and Fshr), oocyte (Zp3 and Gdf9), germ cells (Stella and Prdm1), angiogenesis (VEGF and bFGF) and apoptosis (Bax/Bcl2 and Caspase3) markers was similar in both AO-HB and AO-HP groups. Expression of Amh, Fshr, Gdf9 and VEGF increased only in the AO-HP group whereas expression of Ptch1 increased only in the AO-HB group, as compared with the AO group (P < 0.05). In conclusion, BM-MSCs or VP-MSCs can improve ovarian autotransplantation in mice with no superiority over each other.

Novel aspects on gonadotoxicity and fertility preservation in lymphoproliferative neoplasms.

The topic of fertility preservation in patients with a lymphoproliferative disease offers new aspects of debate, due to the introduction of novel chemotherapeutic regimens and small molecules in the clinical landscape. Cancer related infertility is mostly dependent on gonadotoxic treatments and fertile female patients are today addressed to the oocyte cryopreservation or to ovarian cortex fragment cryopreservation. These methods present advantages and disadvantages, which will be discussed in the present review, together with the options for male patients. The recent discovery of functional ovarian stem cells (OCSs) in woman ovarian cortex, opens new avenues offering a innovative procedure for fertility preservation through as model of regenerative medicine. Here, we review the gonadotoxic potential of "classical" chemotherapeutic treatments as well as of "novel" targeted therapies actually employed for lymphoproliferative neoplasms in young patients and revisit both the today available and future chances to preserve and restore fertility after the cancer healing.