Ovarian Cortical Tissue Research Articles

Medium and time conservation affect follicular morphology and superoxide dismutase enzyme activity of bovine preantral follicles during storage at 4ºC

The aim of this study was to evaluate the morphology and superoxide dismutase enzyme (SOD) activity of bovine preantral follicles (PFs) preserved in TCM 199, saline solution or PBS at different conservation periods. Cow ovaries (n=6) were divided into 7 fragments. One small piece of each ovarian fragment was randomly removed to evaluate SOD activity, while the remainder was immediately fixed for morphological evaluation as a control group. The other 6 fragments were randomly distributed in tubes containing TCM 199, saline solution, or PBS and maintained at 4ºC for 6 or 24 h. For histological evaluation, the fragments were fixed in Carnoy and stained with PAS-hematoxylin, following being classified PFs in relation to their follicular morphology in normal or degenerated. Determination of SOD activity was based on the ability to inhibit autoxidation of adrenaline in adrenochrome. Evaluation of follicular morphology showed that follicles preserved in TCM 199 for 6 h did not differ from the control (P > 0.05). In contrast, preservation in saline solution and PBS for 6 or 24 h and TCM 199 for 24 h decreased normal PFs compared to the control (P < 0.05). SOD showed a lower activity in ovarian cortical tissue kept in TCM 199 for 6 h and saline solution for 24 h than in the other groups. Our study shows that incubation using TCM 199 at 4°C for 6 h can be used to efficiently conserve female bovine PFs in situ.

Improvement of in situ Follicular Activation and Early Development in Cryopreserved Human Ovarian Cortical Tissue by Co-Culturing with Mesenchymal Stem Cells

Follicular loss and tissue degeneration are great challenges in ovarian tissue culture systems. Mesenchymal stem cells (MSC) secrete a cocktail of growth factors and cytokines which supports adjacent cells and tissues. The aim of the current study was to investigate the impact of human bone marrow (hBM)-MSC, as co-culture cells, on human follicular development in ovarian cortical tissue (OCT) culture. For this purpose, warmed OCT fragments were co-cultured with hBM-MSC for 8 days and compared to monocultured OCT. During the culture period, ovarian follicle survival and development in the OCT were evaluated using histological observation, follicular developmental-related genes expression, and estradiol production. Furthermore, cell proliferation and apoptosis were assessed. The results showed that there were no significant differences in conserved ovarian follicles with a normal morphology between the two groups. However, the percentage of developing follicles, as well as follicular developmental gene expression, significantly increased in the co-culture group compared to the monoculture group. On the other hand, compared with the monoculture group, the co-culture group demonstrated a significant increase in cell proliferation, indicated by Ki67 gene expression, as well as a dramatic decrease in apoptotic cell percentage, revealed by TUNEL assay. These findings indicated that co-culturing of hBM-MSC with OCT could improve follicular activation and early follicular development in human ovarian tissue culture systems.

Heterotopic ovarian allotransplantation in goats: Preantral follicle viability and tissue remodeling

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

Folliculogenesis-Associated Genes Expression in Human Vitrified Ovarian Tissue after Xenotransplantation in γ-Irradiated Mice.

Objective Autograft transplantation of vitrified cortical ovarian tissue is an acceptable clinical technique for fertility preservation in women. Xenograft transplantation into animal models could be useful for evaluating the safety of human vitrified ovarian tissue. This study targeted to evaluate impact of vitrification on expression of the genes associated with folliculogenesis after xenograft transplantation of human vitrified ovarian tissue to γ-irradiated mice. Materials and Methods In this experimental study, ovarian biopsies were gathered from six transsexual persons. The cortical section of ovarian biopsies was separated and chopped into small pieces. These pieces were randomly divided into vitrified and non-vitrified groups. In each group some pieces were considered as non-transplanted tissues and the others were transplanted to γ-irradiated female National Medical Research Institute (NMRI) mice. Before and after two weeks of xenograft transplantation, histological assessment and evaluation of the expression of folliculogenesis- associated genes (FIGLA, GDF-9, KL and FSHR) were performed in both vitrified and non-vitrified groups. Results Percentage of the normal follicles and expression of the all examined genes from transplanted and non- transplanted tissue were similar in both vitrified and non-vitrified groups (P>0.05). After transplantation, the normal follicle rate was significantly decreased and among the folliculogenesis-associated genes, expression of GDF-9 gene was significantly increased, rather than before transplantation in vitrified and non-vitrified tissues (P<0.05). ConclusionThe vitrification method using dimethyl solphoxide and ethylene glycol (EG) had no remarkable effect on the normal follicular rate and expression of folliculogenesis-associated genes after two weeks human ovarian tissue xenografting. In addition, transplantation process can cause a significant decrease in normal follicular rate and expression of GDF-9 gene.

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity

BackgroundIn vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).ResultsIn this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development.ConclusionsOur observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

Do BRCA1 and BRCA2 gene mutation carriers have a reduced ovarian reserve? Protocol for a prospective observational study

IntroductionBRCA1/2 gene mutations increase risk of breast and/or ovarian cancer and may have implications for reproductive health. Indirect biomarkers of the ovarian primordial follicle pool (anti-Müllerian hormone (AMH)) and one...

Reduced Foxo3a, FoxL2, and p27 mRNA expression in human ovarian tissue in premature ovarian insufficiency.

Previous studies have suggested that deletion of Foxo3a, FoxL2, PTEN, p27, and AMH leads to early exhaustion of the primordial follicle pool and premature ovarian insufficiency (POI) in transgenic mice. Our aim was to assess for the first time, to our knowledge, messenger RNA (mRNA) expression of these genes and AMHR2 in human ovarian tissue from women with POI. We hypothesized that these genes would be underexpressed in POI women compared with healthy controls. mRNA levels were evaluated by quantitative reverse transcription-polymerase chain reaction and real-time polymerase chain reaction in cortical ovarian tissue obtained by laparoscopy from Caucasian Greek women with POI (n = 5) and healthy women with normal menstruation (n = 6). Morphological analysis of the ovarian biopsies was also performed to assess the presence of primordial or other types of growing follicles. Ovarian tissue from POI patients showed lower Foxo3a, FoxL2, and p27 mRNA expression compared with controls (p = 0.017, p = 0.017, and p = 0.030, respectively). mRNA expression of AMH, PTEN, and AMHR2 was reduced in ovarian biopsies from POI patients as well. However, these differences were not statistically significant (p = 0.143, p = 0.247, and p = 0.662, respectively). Morphological analysis showed complete lack of follicular structures in all POI biopsies. Our findings suggest a possible role of Foxo3a, FoxL2, and p27 in the pathogenesis of human POI, which may prove to be of great diagnostic-therapeutic value. Further larger studies are needed to identify a similar pattern for AMH, PTEN, and AMHR2 and to investigate gene expression at a protein level.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p<.05. The results showed that the presence of 1,000 and 2,000pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.

Can ovarian biopsying per se and/or autotransplantation of fragmented ovarian cortical tissue activate dormant follicles and increase the number of recruitable follicles for IVF/ICSI in women with diminished ovarian reserve (DOR)? Ovarian biopsying followed by immediate autotransplantation of fragmented cortical tissue failed to increase the number of recruitable follicles for IVF/ICSI 10weeks after the procedure either at the graft site or in the biopsied ovary, but 12 of the 20 women subsequently had a clinical pregnancy during the 1-year follow-up. Infertile women with DOR constitute a group of patients with poor reproductive outcome mainly due to the low number of mature oocytes available for IVF/ICSI. Recent studies have shown that in vitro activation of residual dormant follicles by both chemical treatment and tissue fragmentation has resulted in return of menstrual cycles and pregnancies in a fraction of amenorrhoeic women with premature ovarian insufficiency. This is a prospective clinical cohort study including 20 women with DOR treated at the fertility clinic, Rigshospitalet, Denmark, during April 2016-December 2017. Non-pregnant patients were on average followed for 280days (range 118-408), while women who conceived were followed until delivery. Study follow-up of non-pregnant patients ended in September 2018. The study included infertile women aged 30-39years with preserved menstrual cycles, indication for IVF/ICSI and repeated serum measurements of anti-Müllerian hormone (AMH) ≤ 5pmol/L. Patients were randomized to have four biopsies taken from either the left or the right ovary by laparoscopy followed by fragmentation of the cortical tissue to an approximate size of 1mm3 and autotransplanted to a peritoneal pocket. The other ovary served as a control. Patients were followed weekly for 10weeks with recording of hormone profile, antral follicle count (AFC), ovarian volume and assessment for ectopic follicle growth. After 10weeks, an IVF/ICSI-cycle with maximal ovarian stimulation was initiated. No difference in the number of mature follicles after ovarian stimulation 10weeks after the procedure in the biopsied versus the control ovaries was observed (1.0 vs. 0.7 follicles, P= 0.35). In only three patients, growth of four follicles was detected at the graft site 24-268days after the procedure. From one of these follicles, a metaphase II (MII) oocyte was retrieved and fertilized, but embryonic development failed. Overall AMH levels did not change significantly after the procedure (P= 0.2). The AFC increased by 0.14 (95% CI: 0.06;0.21) per week (P< 0.005), and the biopsied ovary had on average 0.6 (95% CI: 0.3;-0.88) follicles fewer than the control ovary (P= 0.01). Serum levels of androstenedione and testosterone increased significantly by 0.63nmol/L (95% CI: 0.21;1.04) and 0.11nmol/L (95% CI: 0.01;0.21) 1week after the procedure, respectively, and testosterone increased consecutively over the 10weeks by 0.0095nmol/L (95% CI: 0.0002;0.0188) per week (P= 0.045). In 7 of the 20 patients, there was a serum AMH elevation 5 to 8weeks after the procedure. In this group, mean AMH increased from 2.08pmol/L (range 1.74-2.34) to 3.94pmol/L (range 3.66-4.29) from Weeks 1-4 to Weeks 5-8. A clinical pregnancy was obtained in 12 of the 20 (60%) patients with and without medically assisted reproduction (MAR) treatments. We report a cumulated live birth rate per started IVF/ICSI cycle of 18.4%. Limitations of the study were the number of patients included and the lack of a non-operated control group. Moreover, 9 of the 20 women had no male partner at inclusion and were treated with donor sperm, but each of these women had an average of 6.8 (range 4-9) unsuccessful MAR treatments with donor sperm prior to inclusion. Although 12 out of 20 patients became pregnant during the follow-up period, the current study does not indicate that biopsying, fragmenting and autotransplanting of ovarian cortical tissue increase the number of recruitable follicles for IVF/ICSI after 10weeks. However, a proportion of the patients may have a follicular response in Weeks 5-8 after the procedure. It could therefore be relevant to perform a future study on the possible effects of biopsying per se that includes stimulation for IVF/ICSI earlier than week 10. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication. None of the authors have a conflict of interest. NCT02792569.

Do Bioreactor Designs with More Efficient Oxygen Supply to Ovarian Cortical Tissue Fragments Enhance Follicle Viability and Growth In Vitro?

Background: Autotransplantation of cryopreserved ovarian tissue is currently the main option to preserve fertility for cancer patients. To avoid cancer cell reintroduction at transplantation, a multi-step culture system has been proposed to obtain fully competent oocytes for in vitro fertilization. Current in vitro systems are limited by the low number and health of secondary follicles produced during the first step culture of ovarian tissue fragments. To overcome such limitations, bioreactor designs have been proposed to enhance oxygen supply to the tissue, with inconsistent results. This retrospective study investigates, on theoretical grounds, whether the lack of a rational design of the proposed bioreactors prevented the full exploitation of follicle growth potential. Methods: Models describing oxygen transport in bioreactors and tissue were developed and used to predict oxygen availability inside ovarian tissue in the pertinent literature. Results: The proposed theoretical analysis suggests that a successful outcome is associated with enhanced oxygen availability in the cultured tissue in the considered bioreactor designs. This suggests that a rational approach to bioreactor design for ovarian tissue culture in vitro may help exploit tissue potential to support follicle growth.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24hours followed by 24hours of 1mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N= 241) and human (N= 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. Incubation in 0.1% SDS for 18-24hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). N/A. Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

Variation in follicle health and development in cultured cryopreserved ovarian cortical tissue: a study of ovarian tissue from patients undergoing fertility preservation

This study investigated how follicle health and development in human ovarian tissue cryopreserved for fertility preservation varied between patients before and after 6 d of in vitro culture. Ovarian tissue from 12 patients (9–25 years) was used. In 3 patients, a 1hr neutral red (NR) incubation was used to identify tissues with viable follicles. Tissues were fixed, sectioned and follicles staged and graded for health. Inter-patient differences were observed in the non-cultured tissue in the number of both healthy follicles (p = 0.005) and growing follicles (p = 0.005). After culture there was significant variation in the number of transitional, primary and secondary follicles between patients (p < 0.001). Asymmetric primary follicles with a single complete layer of granulosa cells plus two or more additional partial layers were 5.5 times more likely to be observed in cultured compared to non-cultured tissue (p = 0.0063). Non-cultured (p = 0.0125) and cultured (p < 0.001) tissue selected using NR had more healthy follicles compared to tissue not selected using NR. Non-cultured and cultured tissue selected using NR had more healthy follicles compared to tissue not selected using NR (p = 0.0125; p < 0.001). We demonstrate that inter-patient variation exists in the health and development of follicles before and after culture. Culture systems need to be optimized to support cryopreserved ovarian tissue and these findings should prompt researchers to consider patient variation when evaluating culture systems.

Transcriptional profiling of long noncoding RNAs and their target transcripts in ovarian cortical tissues from women with normal menstrual cycles and primary ovarian insufficiency.

Previous studies have shown that long noncoding RNAs (lncRNAs) show a highly tissue- and disease-specific expression pattern and that they regulate the expression of neighboring genes. Because lncRNAs have been shown to be secreted into the general circulation, they may be used as diagnostic tools for some diseases. Primary ovarian insufficiency (POI) is a disease in which women have menstrual cessation before the age of 40, accompanied by elevated follicle stimulating hormone and decreased estrogen levels. In this study, ovarian cortical tissues from five women with normal menstrual cycles and from five POI patients were used for next-generation RNA sequencing. We found 20 differentially expressed lncRNAs with 12 upregulated and eight downregulated lncRNAs in cortical tissues of POI ovaries, compared with normal controls (fold change ≥ 2 and false discovery rate[FDR] ≤ 0.05). We also found 52 differentially expressed messenger RNA transcripts, with 33 upregulated and 19 downregulated ones (foldchange ≥ 2 and FDR ≤ 0.05). Functional annotation showed that these differentially expressed transcripts were associated with follicular development and granulosa cell function. Thirteen differentially expressed lncRNAs and their targeted neighboring transcripts were coregulated in ovarian cortical tissues, including lnc-ADAMTS1-1:1/ADAMTS1, lnc-PHLDA3-3:2/CSRP1, lnc-COL1A1-5:1/COL1A1, lnc-SAMD14-5:3/COL1A1, and lnc-GULP1-2:1/COL3A1. Furthermore, serum levels of these lncRNAs in POI patients were significantly different from those in normal patients ( p < 0.05), and expression differences were consistent with those in ovarian cortical tissues. This study showed that key lncRNAs were differentially expressed in both ovarian cortical tissues and serum samples between women with normal menstrual cycles and POI patients. Further studies on the regulation of ovarian lncRNAs during follicular development are critical in understanding the etiologies of POI. Analyses of lncRNA expression in serum samples might provide a basis for early diagnosis and treatment of POI.

Future developments: In vitro growth (IVG) of human ovarian follicles

Removal and storage of ovarian cortical tissue is currently offered to young female cancer patients undergoing potentially sterilizing chemotherapy and/or radiotherapy. For patients at high risk of reintroduction of malignancy through auto-transplantation, the ultimate aim is to achieve complete oocyte development from this tissue in vitro. The ability to develop human oocytes from the earliest follicular stages through to maturation and fertilization in vitro would revolutionize fertility preservation practice. This has been achieved in mice where in vitro grown oocytes from primordial follicles have resulted in the production of live offspring. Systems that support growth and development of oocytes from human ovarian cortex are being developed by several groups. This review focuses on the steps required to recapitulate in vitro the process of human oocyte development from the primordial stage and the systems currently available to support this.

Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner

To test if ovarian microenvironmental cues affect oogonial stem cell (OSC) function in a species-specific manner. Animal and human study. Research laboratory. Human ovarian cells obtained from cryopreserved ovarian cortical tissue of reproductive-age women, and ovarian cells and tissues from female C57BL/6 mice. Mouse ovarian tissue, mouse OSCs (mOSCs) and human OSCs (hOSCs) were analyzed for extracellular matrix (ECM) protein expression, and OSCs isolated from adult mouse and human ovaries were cultured in the absence or presence of ECM proteins without or with an integrin signaling inhibitor. Gene expression and invitro derived (IVD) oocyte formation. Culture of mOSCs on a collagen-based ECM significantly elevated the rate of differentiation of the cells into IVD oocytes. Mouse OSCs expressed many integrins, including Arg-Gly-Asp (RGD)-binding subunits, and ECM-mediated increases in mOSC differentiation were blocked by addition of integrin-antagonizing RGD peptides. In comparison, hOSCs expressed a different pattern of integrin subunits compared with mOSCs, and hOSCs were unresponsive to a collagen-based ECM; however, hOSCs exhibited increased differentiation into IVD oocytes when cultured on laminin. These data, along with in silico analysis of ECM protein profiles in human ovaries, indicate that ovarian ECM-based niche components function in a species-specific manner to control OSC differentiation.

Steroids and miRNAs in assessment of ovarian tissue damage following cryopreservation.

Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue's survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific microRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovarian steroidal production and microRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P<0.05) both steroid production (oestradiol and progesterone) and expression of microRNA-193b and 320A in spent culture media over 5 days, however, expression of microRNA-24 increased (P<0.05). The number of primordial follicles were also reduced (P<0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of microRNA-193b and microRNA-320A together with upregulation of microRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production. Thus, there appears to be an interplay between these microRNAs, ovarian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.

Fertility and hormone preservation and restoration for female children and adolescents receiving gonadotoxic cancer treatments: A systematic review

Background/PurposeThe purpose of this systematic review by the American Pediatric Surgical Cancer Committee was to summarize evidence from the current medical literature regarding fertility restoration and hormone replacement for female children and adolescents treated with gonadotoxic treatments. MethodsUsing PRISMA guidelines, questions were addressed by searching Medline, Cochrane, Embase Central and National clearing house databases using relevant search terms. Eligible studies included those that addressed ovarian tissue cryopreservation (OTC), oocyte harvest, ovarian transposition, and ovarian tissue auto-transplantation for females under the age of 20. Four reviewers independently screened studies for eligibility, extracted data and assessed the risk of bias. Study outcomes were summarized in a narrative synthesis. ResultsTwo thousand two hundred seventy-six studies were identified by database search and manual review and 2185 were eliminated based on defined exclusion criteria. Ninety-one studies served as the basis for the systematic review. There were 1019 patients who underwent OTC with ages ranging from 0.4 to 20.4 years old, with 298 under the age of 13. Twenty patients aged 13–20 years old underwent successful oocyte harvest. Thirty-seven children underwent ovarian transposition as a means of fertility preservation. Eighteen patients underwent auto-transplantation of thawed ovarian cortical tissue that was harvested before the age of 21 years resulting in 10 live births. ConclusionsClinically accepted and experimental fertility preservation options such as OTC, oocyte cryopreservation, and ovarian transposition are available to females aged 20 years and younger who are at risk for premature ovarian insufficiency and infertility due to gonadotoxic treatments. There is a large cohort of pediatric-aged patients, with a wide variety of diagnoses and treatments, who have undergone fertility preservation. Currently, fertility and hormone restoration experience for patients who were 20- years of age or younger at the time of fertility preservation remains limited. Level of EvidenceIV.

Fertility preservation for female cancer patients by manipulating ovarian stem cells that survive oncotherapy

Infertility and premature ovarian failure are unwanted side effects of oncotherapy in females; however, a large number of patients survive cancer due to recent advances in their management. One of the available options to restore fertility in cancer survivors is to transplant ovarian cortical tissue slices at orthotopic sites which has resulted in the birth of 130 babies. Spontaneous pregnancies have also been reported after heterotopic transplantation of cortical tissue slices which can only be explained by the presence of stem cells and paracrine support provided by transplanted ovarian slices to the nonfunctional ovary. The ovary harbors two populations of stem cells, including very small embryonic-like stem cells (VSELs) and slightly bigger ovarian stem cells (OSCs) that divide and undergo clonal expansion to form germ cell nests in adult ovary before undergoing neo-oogenesis and primordial follicle assembly. Being relatively quiescent, VSELs survive oncotherapy and can regenerate the nonfunctional ovary. Stem cells niche gets affected by oncotherapy and transplanting autologous bone marrow mesenchymal stem cells (MSCs, which provide paracrine support) have shown to normalize ovarian function in rodents with the birth of healthy pups. Similarly, transplanting of autologous MSCs in human ovary with premature ovarian failure resulted in the birth of a baby. These advances in the field of OSCs need to be put in proper context before considering making transplantation of ovarian cortical tissue at orthotopic sites as method of standard care. Transplanting autologous MSCs is safe, and efficacy to regenerate nonfunctional ovaries needs to be evaluated in clinical settings.

Female Fertility Preservation through Stem Cell-based Ovarian Tissue Reconstitution In Vitro and Ovarian Regeneration In Vivo.

Historically, approaches designed to offer women diagnosed with cancer the prospects of having a genetically matched child after completion of their cytotoxic treatments focused on the existing oocyte population as the sole resource available for clinical management of infertility. In this regard, elective oocyte and embryo cryopreservation, as well as autologous ovarian cortical tissue grafting posttreatment, have gained widespread support as options for young girls and reproductive-age women who are faced with cancer to consider. In addition, the use of ovarian protective therapies, including gonadotropin-releasing hormone agonists and sphingosine-1-phosphate analogs, has been put forth as an alternative way to preserve fertility by shielding existing oocytes in the ovaries in vivo from the side-effect damage caused by radiotherapy and many chemotherapeutic regimens. This viewpoint changed with the publication of now numerous reports that adult ovaries of many mammalian species, including humans, contain a rare population of oocyte-producing germ cells—referred to as female germline or oogonial stem cells (OSCs). This new line of study has fueled research into the prospects of generating new oocytes, rather than working with existing oocytes, as a novel approach to sustain or restore fertility in female cancer survivors. Here, we overview the history of work from laboratories around the world focused on improving our understanding of the biology of OSCs and how these cells may be used to reconstitute “artificial” ovarian tissue in vitro or to regenerate damaged ovarian tissue in vivo as future fertility-preservation options.

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold