Analysis Of Ovarian Tissue Research Articles

Mesenchymal stem cells in animal reproduction: sources, uses and scenario.

Studies regarding mesenchymal stem cells turned up in the 1960's and this cell type created a great number of questions about its functions and applicability in science and medicine. When used with therapeutic intent, these cells present an inclination to migrate to sites of injury, inflammation or disease, where they secrete bioactive factors that stimulates the synthesis of new tissue. In this context, studies using rodents reported that MSCs promoted positive effects in the ovarian function in mice with premature aging of follicular reserve. In female bovines, experimental stem cell-based therapies have been used to either generate new oocytes with in vitro quality or stimulate such action in vivo. It is also reported, that the intraovarian application of mesenchymal stem cells generates a greater production of embryos in vitro and the production of early and expanded blastocysts. Additionally, analysis of ovarian tissue in animal subjected to treatment showed an increase in the number of developing follicles. Nevertheless, the treatments involving stem cells with different modes of application, different sources and different species were able to act on the hormonal, tissue, cellular and metabolic levels, generating positive results in the recovery and improvement of ovarian functions.

P-392 Controlled ovarian hyperstimulation applied before unilateral oophorectomy improves fertility preservation in cancer patients

Abstract Study question Can ovarian tissue cryopreservation (OTC) be performed after controlled ovarian hyperstimulation (COH)? Summary answer Unilateral oophorectomy after transvaginal oocyte retrieval is feasible on stimulated ovaries during one surgical step. What is known already In the fertility preservation (FP) field, the timeframe between patient referral and start of curative treatment is limited. Combining oocyte pick-up with ovarian tissue (OT) extraction has been reported to improve FP but COH applied before OT extraction is not currently recommended. Study design, size, duration This retrospective cohort-controlled study involved 58 patients who underwent oocyte cryopreservation immediately followed by OTC between September 2009 and November 2021. The exclusion criteria were a delay between oocyte retrieval and OTC of greater than 24 hours (n = 5) and in vitro maturation (IVM) of oocytes obtained ex-vivo in the ovarian cortex (n = 2). This FP strategy was performed either after COH (stimulated group, n = 18) or after IVM (unstimulated group, n = 33). Participants/materials, setting, methods Oocyte retrieval followed by OT extraction on the same day was performed either without previous stimulation or after COH. Adverse effects of surgery and ovarian stimulation, mature oocyte yield and pathology findings of fresh OT were retrospectively analysed. Thawed OTs were analysed prospectively, for vascularisation and apoptosis using immunohistochemistry, when patient consent was obtained. Main results and the role of chance No surgical complication occurred after OTC surgery in either group. In particular, no severe bleeding was associated with COH. The number of mature oocytes obtained increased after COH (8.5 [5.3–12.0] MII oocytes) compared to the unstimulated group (2.0 [1.0–5.3] MII oocytes, p < 0.001). Neither ovarian follicle density nor cell integrity was affected by COH. Fresh OT analysis showed congestion in half of the stimulated OT which was higher than in the unstimulated OT (3.1%, p < 0.001). COH also increased haemorrhagic suffusion (COH+OTC: 66.7%; IVM+OTC: 18.8%, p=0.002) and oedema (COH+OTC: 55.6%; IVM+OTC: 9.4%, p < 0.001). After thawing, the pathological findings were similar between both groups. No statistical difference in the number of blood vessels was observed between the groups. The oocyte apoptotic rate in thawed OT was not statistically different between the groups (ratio of positive cleaved caspase-3 staining oocytes/total number of oocytes equal to median 0.50 [0.33-0.85] and 0.45 [0.23-0.58] in unstimulated and stimulated groups respectively, p = 0.720). Limitations, reasons for caution The study reports FP from a small number of women following OTC. Follicle density and other pathology findings are an estimate only. Wider implications of the findings This approach could be proposed to post pubertal patients when the number of mature oocytes expected is low or when the risk of residual pathology is high. The reduction of surgical steps for cancer patients also has positive implications for introducing this approach into clinical practice. Trial registration number not applicable

Ovarian tissue cryopreservation can be combined simultaneously with oocyte retrieval after controlled ovarian hyperstimulation.

Can ovarian tissue cryopreservation (OTC) be performed after controlled ovarian hyperstimulation (COH)? Unilateral oophorectomy after transvaginal oocyte retrieval is feasible on stimulated ovaries during one surgical step. In the fertility preservation (FP) field, the timeframe between patient referral and start of curative treatment is limited. Combining oocyte pick-up with ovarian tissue (OT) extraction has been reported to improve FP but COH applied before OT extraction is not currently recommended. This retrospective cohort-controlled study involved 58 patients who underwent oocyte cryopreservation immediately followed by OTC between September 2009 and November 2021. The exclusion criteria were a delay between oocyte retrieval and OTC of >24 h (n = 5) and IVM of oocytes obtained ex vivo in the ovarian cortex (n = 2). This FP strategy was performed either after COH (stimulated group, n = 18) or after IVM (unstimulated group, n = 33). Oocyte retrieval followed by OT extraction on the same day was performed either without previous stimulation or after COH. Adverse effects of surgery and ovarian stimulation, mature oocyte yield and pathology findings of fresh OT were retrospectively analysed. Thawed OTs were analysed prospectively, for vascularization and apoptosis using immunohistochemistry, when patient consent was obtained. No surgical complication occurred after OTC surgery in either group. In particular, no severe bleeding was associated with COH. The number of mature oocytes obtained increased after COH (median = 8.5 (25% = 5.3-75% = 12.0)) compared to the unstimulated group (2.0 (1.0-5.3), P < 0.001). Neither ovarian follicle density nor cell integrity was affected by COH. Fresh OT analysis showed congestion in half of the stimulated OT which was higher than in the unstimulated OT (3.1%, P < 0.001). COH also increased haemorrhagic suffusion (COH + OTC: 66.7%; IVM + OTC: 18.8%, P = 0.002) and oedema (COH + OTC: 55.6%; IVM + OTC: 9.4%, P < 0.001). After thawing, the pathological findings were similar between both groups. No statistical difference in the number of blood vessels was observed between the groups. The oocyte apoptotic rate in thawed OT was not statistically different between the groups (ratio of positive cleaved caspase-3 staining oocytes/total number of oocytes equal to median 0.50 (0.33-0.85) and 0.45 (0.23-0.58) in unstimulated and stimulated groups respectively, P = 0.720). The study reports FP from a small number of women following OTC. Follicle density and other pathology findings are an estimate only. Unilateral oophorectomy can be successfully performed after COH with limited bleeding risk and an absence of impact on thawed OT. This approach could be proposed to post pubertal patients when the number of mature oocytes expected is low or when the risk of residual pathology is high. The reduction of surgical steps for cancer patients also has positive implications for introducing this approach into clinical practice. This work was made possible through the support of the reproductive department of Antoine-Béclère Hospital and of the pathological department of Bicêtre Hospital (Assistance Publique Hôpitaux de Paris, France). The authors have no conflict of interest to disclose in this study. N/A.

Transcriptome Sequencing and Bioinformatics Analysis of Ovarian Tissues from Pomacea canaliculata in Guangdong and Hunan.

In this study, the fecundity of Pomacea canaliculata was studied by collecting egg masses from Guangdong and Hunan using field egg collection and indoor propagation. Through high-throughput RNA sequencing (RNA-seq), we analyzed the ovarian tissue of the snails in Guangdong (G_O) and those in Hunan (H_O) using comparative analysis of transcription. Moreover, we used bioinformatics methods to screen the key pathways and genes that affect the fecundity of snails from the two locations. Results. The results showed that the absolute fecundity and weight-relative fecundity of Pomacea canaliculata in Guangdong were significantly higher than those in Hunan. We found 1,546 differential genes through differential gene screening (528 genes upregulated in snails from Guangdong and 1018 in snails from Hunan). The ribosomal signaling pathway and rpl23a, uba52 are critical pathways and essential genes that affect the fecundity of snails. Conclusions. The 27 differential genes in the ribosome signaling pathway, collected from H_O, were all downregulated. As a result, ovarian tissue protein synthesis is impaired, which is an important mechanism that affects snails' ability to reproduce.

Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis.

Simple SummaryFreshwater fish populations are in global decline, with many Australian freshwater species expected to become extinct in the next twenty years. The storage of reproductive cells and tissues at extremely cold temperatures in bio-banks known as “Frozen Zoos”, allows for the indefinite storage of genetic material, meaning that in the event of an extinction, we have a genetic blueprint available to produce new individuals and reintroduce a species into the wild. Here we have developed a cryopreservation protocol for the storage of ovarian tissue from the threatened Murray River Rainbowfish. Many Australian freshwater fish species are threatened with extinction, our methodology provides a framework for the conservation of other fish species in Australia and globally.Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.

Effects of different energy diets on DNA methylation and mRNA expression in follicle stimulating hormone receptor gene promoter region of Duolang sheep during estrus.

The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. In this study, polyembryo estrus Duolang sheep under different energy levels were selected as the experimental subjects. Dietary nutrient level reference (NY/T 816-2004), medium energy level was 10.88MJ/day, high and low energy groups were increased and decreased by 15% on the basis of medium energy level, respectively 12.51MJ/day, 9.25MJ/day. Through RNA and DNA extraction, qPCR, bisulfitegenomicse-quencing PCR (BSP), sequence matching and other analysis of ovarian tissue of Duolang sheep. The difference of DNA methylation level and mRNA expression of FSHR gene during estrus in Duolang sheep fed with different energy diets was detected. The results showed the expression level of FSHR in high energy group was significantly higher than that in low energy group (P < 0.01), the expression level of FSHR in high energy group was significantly higher than that in medium energy group (P < 0.05), and the expression level of FSHR in medium energy group was significantly higher than that in low energy group (P < 0.05). In the target fragment of the promoter region of the FSHR gene, the methylation rate was 25% in the high energy group, 50% in the normal group, and 75% in the low energy group. This study revealed that different dietary energy levels had certain effects on the FSHR gene DNA methylation level and mRNA expression, and the expression level was negatively correlated with methylation level.

Fecundity pattern and spawning dynamics of the common cuttlefish Sepia officinalis

ABSTRACT The fecundity pattern of common cuttlefish, Sepia officinalis, was assessed in Thermaikos Gulf, one of the species’ main fishing grounds in the eastern Mediterranean. Female samples, collected with coastal fishing gears during the population spawning season (January–July), were analysed using a combination of histology and whole mount analysis of ovarian tissue. The potential fecundity was shown to vary seasonally, showing the highest values at the beginning of spawning, gradually dropping thereafter. Previtellogenic oocytes (<0.5 mm) were the most prevalent group occurring throughout the spawning period in all maturity stages. Low or null levels of oocyte recruitment during the spawning period in combination with seasonal drop in fecundity and clutch-specific egg production suggest that the fecundity pattern of cuttlefish displays similarities with the so-called ‘determinate fecundity’ pattern of fishes. Spent females end up having small reserves of secondary growth oocytes (pre- and vitellogenic) that will never be spawned. Potential annual fecundity was thus estimated as the difference in total fecundity between pre-spawners and spent females and equalled 2569 oocytes/eggs. For a spawning period of four months, the ratio between potential fecundity and clutch size (100–200 eggs) indicated 13–25 different spawning events with a mean interval of 5–9 days.

P-461 Pre-selected for an award: A 16-year bicentric retrospective analysis of ovarian tissue cryopreservation (OTC) in paediatric patients: indications, results and outcome

Abstract Study question What is the outcome of ovarian tissue cryopreservation (OTC) in paediatric patients from the beginning of its setting in two different French centres? Summary answer In our cohort of 75 paediatric patients who underwent OTC, the mean age, malignancy rate and survival rate were 9.7 years, 70.7% and 77.3% respectively. What is known already Cancer treatments of last decades improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. The preservation of fertility is now an integral part of care of children requiring gonadotoxic treatments. Currently OTC represents the only possibility of preserving the potential fertility in prepubertal girls. OTC is an effective fertility preservation option which allows long-term storage of primordial follicles, subsequent transplantation restores endocrine function and fertility. The efficacy of these techniques is well-demonstrated within adult population but the data are poor for paediatric patients. Study design, size, duration This is a retrospective study of OTC practice of two French centres from January 2004 to May 2020. Participants/materials, setting, methods A total of 75 patients from paediatrics units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or benign diseases. The ovarian cortex was cut into fragments and the number of follicles per square millimeter was evaluated histologically. The long-term follow-up includes survival rate, hormonal and fertility status. Main results and the role of chance The mean age at OTC of 75 patients was 9.7 years [0.2 – 20], 32% were postpubertal. 53 had malignant disease and 22 had non-malignant disease. The most frequent diagnoses in this cohort included acute leukemia, hemoglobinopathies and neuroblastoma. Indication for OTC was stem cell transplantation for 78.7% (n = 59) girls. A third of each ovary was collected for 62,7% (n = 47) patients, a whole ovary for 33,3% (n = 25) patients and a third of one ovary alone for 4,0% (n = 3) patients. An average of 17 fragments [5-35] per patient was cryoconserved. A correlation was found between age and the number of fragments (p &amp;lt; 0.001). More fragments were obtained from partial bilateral harvesting than from whole ovary harvesting (p &amp;lt; 0.05). Histological analysis of ovarian tissue showed a median of 6.0 primordial follicles/mm2 [0.0–106.5] and no malignant cells were identified. A negative correlation was found between age and follicular density (p &amp;lt; 0.001). Median post-harvest follow-up was 92 months [1–188] 17 girls had died, 12 were still treated for their pathology and 46 were in complete remission. Of all patients, 29 have been subject to hormonal status evaluation and 26 were diagnosed with premature ovarian insufficiency (p &amp;lt; 0.001). One patient had undergone thawed ovarian tissue transplantation. Limitations, reasons for caution This study is a retrospective analysis. The cohort was not compared with a control group who did not undergo OTC or with an adult population. Furthermore, many of these girls are still young and do not intend to use the transplantation of thawed ovarian tissue yet. Wider implications of the findings OTC should be proposed to all girls with high risk of developing premature ovarian insufficiency following gonadotoxic therapies in order to give them the possibility of fertility and endocrine restoration. Trial registration number Not applicable

P–461 A 16-year bicentric retrospective analysis of ovarian tissue cryopreservation (OTC) in paediatric patients: indications, results and outcome

Abstract Study question What is the outcome of ovarian tissue cryopreservation (OTC) in paediatric patients from the beginning of its setting in two different French centres? Summary answer In our cohort of 75 paediatric patients who underwent OTC, the mean age, malignancy rate and survival rate were 9.7 years, 70.7% and 77.3% respectively. What is known already Cancer treatments of last decades improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. The preservation of fertility is now an integral part of care of children requiring gonadotoxic treatments. Currently OTC represents the only possibility of preserving the potential fertility in prepubertal girls. OTC is an effective fertility preservation option which allows long-term storage of primordial follicles, subsequent transplantation restores endocrine function and fertility. The efficacy of these techniques is well-demonstrated within adult population but the data are poor for paediatric patients. Study design, size, duration This is a retrospective study of OTC practice of two French centres from January 2004 to May 2020. Participants/materials, setting, methods A total of 75 patients from paediatrics units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or benign diseases. The ovarian cortex was cut into fragments and the number of follicles per square millimeter was evaluated histologically. The long-term follow-up includes survival rate, hormonal and fertility status. Main results and the role of chance The mean age at OTC of 75 patients was 9.7 years [0.2 – 20], 32% were postpubertal. 53 had malignant disease and 22 had non-malignant disease. The most frequent diagnoses in this cohort included acute leukemia, hemoglobinopathies and neuroblastoma. Indication for OTC was stem cell transplantation for 78.7% (n = 59) girls. A third of each ovary was collected for 62,7% (n = 47) patients, a whole ovary for 33,3% (n = 25) patients and a third of one ovary alone for 4,0% (n = 3) patients. An average of 17 fragments [5–35] per patient was cryoconserved. A correlation was found between age and the number of fragments (p &amp;lt; 0.001). More fragments were obtained from partial bilateral harvesting than from whole ovary harvesting (p &amp;lt; 0.05). Histological analysis of ovarian tissue showed a median of 6.0 primordial follicles/mm2 [0.0–106.5] and no malignant cells were identified. A negative correlation was found between age and follicular density (p &amp;lt; 0.001). Median post-harvest follow-up was 92 months [1–188]: 17 girls had died, 12 were still treated for their pathology and 46 were in complete remission. Of all patients, 29 have been subject to hormonal status evaluation and 26 were diagnosed with premature ovarian insufficiency (p &amp;lt; 0.001). One patient had undergone thawed ovarian tissue transplantation. Limitations, reasons for caution This study is a retrospective analysis. The cohort was not compared with a control group who did not undergo OTC or with an adult population. Furthermore, many of these girls are still young and do not intend to use the transplantation of thawed ovarian tissue yet. Wider implications of the findings: OTC should be proposed to all girls with high risk of developing premature ovarian insufficiency following gonadotoxic therapies in order to give them the possibility of fertility and endocrine restoration. Trial registration number Not applicable

Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

BackgroundEquine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).ResultsR-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment.The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.ConclusionsThe present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

Analysis of histology and expression of sex hormone receptors er and pr in the ovaries at polycystic ovary syndrome

Histologic analysis of ovarian tissue at polycystic ovary syndrome (PCOS) identified specific markers that are pathogenic factors in the case of chronic anovulation. Immunohistochemical research of expression sex hormones receptors in the ovarian tissue in PCOS showed characteristic changes of the expression of estrogen (ER) and progesterone receptors (PR) in the stroma and follicles There was revealed that changes of the expression of sex hormones in the follicles and ovarian stroma can play the important role in the process of folliculogenesis and fertility for women with PCOS.

Ovarian ageing, follicle depletion, and cancer: a hypothesis for the aetiology of epithelial ovarian cancer involving follicle depletion

The association between ovarian cancer risk and reproductive factors has been well established, and two main theories, incessant ovulation and gonadotropin stimulation, have been proposed to explain the mechanism. Recent studies using animal models of ovarian tumorigenesis, and analysis of ovarian tissues from prophylactic oophorectomies, suggest that depletion of ovarian follicles might underlie the epidemiological findings linking reproductive history and ovarian cancer risk.

Changes in germ cells population and in vitro oocyte maturation in female mice with imperforated vagina

This study characterized female mice with a congenital defect in their reproductive tract. In females derived from an outbred colony maintained in the Department of Genetics and Evolution, the frequency of the imperforated vaginae was approximately 2%. A consequence of this defect is infertility. Affected animals developed hydrometrocolpos (the uterus and vagina were greatly distended by fluid). Morphology of the ovary and oviduct was normal and similar to that of control mice. In the females with an imperforated vagina numerous corpora lutea were observed. Accordingly, we have found that both spontaneous as well as exogenous gonadotrophin-induced ovulation occurs in such females. Nevertheless, oocytes derived from ovaries of occluded females exhibited a partial block during in vitro maturation. Histological analysis of ovarian tissue revealed an increase in the number of primary follicles (type I follicles) and a decrease in the number of secondary and antral follicles (type IV and type VI follicles) when compared with control mice. The concentration of androgens in the ovarian tissue was higher in the affected females. Our data show that females with an imperforated vagina can be a useful model for studying the mechanism of genetic control of the development of the urogenital tract in mammals.

Inhibition of p53 during physiological angiogenesis in the hamster ovary does not affect extent of new vessel formation but delays vessel maturation

Transcription factor p53 regulates the cell cycle and apoptosis and may impair angiogenesis by the deregulation of pro-angiogenic factors and the activation of anti-angiogenic factors. Our aim has been to elucidate further the role of p53 in physiological angiogenesis. By treating hamsters with the wildtype p53 inhibitor pifithrin-alpha (PFT) versus equivalent volumes of the vehicle dimethylsulfoxide, we showed a reduced p53 tissue protein level, a reduction of poly(ADP-ribose) polymerase and cleaved caspase-3 products, and a slightly increased proliferation of cell nuclear antigen and cyclin D1 by Western blot protein analysis of ovarian tissue. PFT further increased platelet-derived growth factor and did not influence vascular endothelial growth factor in female reproductive tissue. Despite these differences in tissue levels of proteins potentially involved in angiogenesis, in vivo fluorescence-microscopic analysis of freely transplanted ovarian follicles revealed comparable kinetics and an extent of revascularization with almost identical densities of network microvessels in both groups. However, follicles of PFT-treated animals exhibited enlarged diameters and higher volumetric blood flow within the newly formed microvessels. Less-dense basement membranes with unclear laminar structure and only a loose contact of pericytes to endothelial cells were also occasionally found, providing evidence of delayed maturation and impaired diameter control of microvessels. Thus, inhibition of wildtype p53 during physiological angiogenesis does not affect the extent of new vessel formation but may delay the maturation of newly formed microvessels.