Cryopreservation Of Ovarian Cortical Tissue Research Articles

P-665 High FSH level impairs VEGF secretion by thawed human ovarian tissue

Abstract Study question How does a high FSH level as related to POI (premature ovarian insufficiency) affect VEGF secretion by thawed human ovarian tissue? Summary answer After thawing, the high FSH concentration of 175mIU/mL (10ng/mL) significantly decreased VEGF secretion by human ovarian tissue by 25.6%. 17.5mIU/mL did not change VEGF secretion. What is known already Cryopreservation of ovarian cortical tissue has become an important method in fertility preservation. However, 70% of the follicles do not survive the reimplantation process due to an insufficient blood supply and oxidative stress. Human ovarian tissue secretes angiogenic factors, including VEGF, spontaneously. Its regulation by FSH is not known although 50% of the reimplantation patients show increased FSH levels (>25mIU/mL) due to POI. Study design, size, duration Provided a signed consent, biopsy punches of ovarian cortical tissue from 8 patients undergoing fertility preservation were studied. Slow freezing and thawing was applied as in the intern routine instructions. Participants/materials, setting, methods Human ovarian cortical tissue was cultured in full medium at 37 °C for 48h after thawing in presence or absence of 17.5mIU/mL (1ng/mL) or 175mIU/mL (10ng/mL) recombinant human FSH. VEGF-A secretion was measured by ELISA. The expression of VEGF-A, HIF-1α and FSHR was assessed by immunohistofluorescence per follicle. The effect of FSH was assessed by one-way-ANOVA with Tukey’s post test. The effect of culture or follicular maturation stage was analyzed by t test. Main results and the role of chance After thawing, VEGF secretion recovers and rises over time. The addition of 175mIU/mL FSH significantly impaired VEGF-A expression compared to untreated ovarian tissue (p = 0.016). The analysis of 481 follicles showed similar results of FSH affecting VEGF-A expression by immunostaining. In particular, primordial follicles expressed significantly less VEGF-A compared to growing follicles (primary+secondary; p < 0.0001). 48h tissue culture significantly increased VEGF-A expression in primordial follicles (p = 0.0041). The addition of the high FSH level significantly decreased VEGF-A expression in growing follicles (p = 0.0005). Limitations, reasons for caution Although VEGF-A is a key factor in angiogenesis, more factors are known to affect angiogenesis. The direct effect on angiogenesis should be evaluated. Moreover, the conditions in tissue culture may not represent those after reimplantation. Wider implications of the findings High FSH levels, as seen in patients with POI, might impair ovarian VEGF expression and angiogenesis. Hormone replacement therapy (HRT) might be endorsed before and during ovarian tissue reimplantation. More clinical and biological data are required to understand the regulators of angiogenesis after ovarian tissue reimplantation adequately. Trial registration number not applicable

Innovative Strategies for Fertility Preservation in Female Cancer Survivors: New Hope from Artificial Ovary Construction and Stem Cell-Derived Neo-Folliculogenesis.

Recent advances in anticancer treatment have significantly improved the survival rate of young females; unfortunately, in about one third of cancer survivors the risk of ovarian insufficiency and infertility is still quite relevant. As the possibility of becoming a mother after recovery from a juvenile cancer is an important part of the quality of life, several procedures to preserve fertility have been developed: ovarian surgical transposition, induction of ovarian quiescence by gonadotropin-releasing hormone agonists (GnRH-a) treatment, and oocyte and/or ovarian cortical tissue cryopreservation. Ovarian tissue cryostorage and allografting is a valuable technique that applies even to prepubertal girls; however, some patients cannot benefit from it due to the high risk of reintroducing cancer cells during allograft in cases of ovary-metastasizing neoplasias, such as leukemias or NH lymphomas. Innovative techniques are now under investigation, as in the construction of an artificial ovary made of isolated follicles inserted into an artificial matrix scaffold, and the use of stem cells, including ovarian stem cells (OSCs), to obtain neo-folliculogenesis and the development of fertilizable oocytes from the exhausted ovarian tissue. This review synthesizes and discusses these innovative techniques, which potentially represent interesting strategies in oncofertility programs and a new hope for young female cancer survivors.

Metabolic and secretory recovery of slow frozen-thawed human ovarian tissue in vitro.

Within the options available for fertility preservation, cryopreservation of ovarian cortical tissue has become an important technique. Freezing and thawing procedures have been optimized to preserve tissue integrity and viability. However, the improvement of the tissue retransplantation is currently of great interest. Rapid angiogenesis is needed at the retransplantation site to accomplish sufficient blood supply to provide oxygen and nutrients. Many studies address this issue. However, we need to understand the physiology of the thawed tissue to gain further understanding of the complexities of the procedure. As freezing and thawing generally impairs cellular metabolism, we aimed to characterize the changes in metabolic activity and secretion of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) of frozen-thawed ovarian cortical tissue over time. Biopsy punches of ovarian cortical tissue from patients undergoing fertility preservation were maintained in culture without freezing or after a slow-freezing and thawing procedure. VEGF-A secretion was measured after 48 h by ELISA. To examine temporary changes, metabolic activity was assessed for both fresh and frozen-thawed tissue of the same patient. Metabolic activity and VEGF-A secretion were measured at 0, 24 and 48 h in culture. Thawed ovarian cortical tissue secreted significantly less VEGF-A compared to fresh ovarian cortical tissue within 48 h of culture. After thawing, metabolic activity was significantly reduced compared to fresh ovarian cortex but over the course of 48 h, the metabolic activity recovered. Similarly, VEGF-A secretion of thawed tissue increased significantly over 48 h. Here, we have shown that it takes 48 h for ovarian cortical tissue to recover metabolically after thawing, including VEGF-A secretion.

THE THERAPEUTIC POTENTIAL OF IMMATURE OOCYTES FROM FERTILITY PRESERVATION PROCEDURES AND THEIR IN-VITRO MATURATION AT DIFFERENT AGES

Cryopreservation of ovarian cortical tissue (OTC) followed by auto-transplantation has emerged as a promising fertility preservation option . Immature oocyte can be found in the dissection medium during tissue handling and the ability to mature them in vitro and cryopreserve them as mature oocytes, provides an additional option for fertility preservation. In this work we aim to evaluate the developmental potential and epigenetic function of these premature oocytes, in different age groups.

In vitro maturation of immature oocytes from ovarian tissue prior to shipment to a cryobank.

Female fertility preservation prior to gonadotoxic therapies can be achieved by the cryopreservation of ovarian cortical tissue. Immature oocytes may be recovered during the preparation, matured in vitro and lead to live births, thereby providing an additional option for fertility preservation. The purpose of this study was to test the feasibility of this approach in a setting with unilateral biopsy of a small piece of ovarian tissue and minimal tissue preparation prior to shipment to an external cryobank. A prospective observational clinical study in an academic center was performed from January 2018 through December 2019. Ovarian tissue was obtained laparoscopically. Immature oocytes were recovered by minimal preparation of the tissue before shipment to an external cryobank for cryopreservation. In vitro maturation was performed on recovered immature oocytes. Twelve patients were enrolled. Immature oocytes could be recovered for all. The maturation rate was 38.9% (n = 14/36). Metaphase II (MII) were either directly used for intracytoplasmic sperm injection (ICSI) with a fertilization rate of 66.6% (n = 4/6) or vitrified (n = 8). PNs were cryopreserved (n = 4). Vitrified MII were warmed with a post-warming vitality rate of 75.0% (n = 3/4) and used for ICSI with a fertilization rate of 33.3% (n = 1/3). Immature oocytes can be successfully retrieved from ovarian tissue through minimal tissue preparation prior to shipment to a cryobank, matured in vitro, fertilized and cryopreserved for potential future fertility treatments. The total number of oocytes available for fertility preservation can be increased even without controlled ovarian stimulation in a situation where only ovarian biopsy for cryopreservation is performed. German Clinical Trials Register (DRKS), DRKS00013170. Registered 11 December 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013170 .

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.

Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture

Neutral red (NR) may assist identification of preantral follicles in pieces of cortical tissue prior to cryopreservation in cancer patients requesting fertility preservation. This study is the first to analyze this effect by follicle growth rate after long-term culture in primates. Ovarian cortex was obtained from adult rhesus macaques, was cut into fragments, and was incubated with NR. Secondary follicles were readily visualized following NR staining and then were encapsulated into alginate beads and cultured individually for 4weeks in αMEM media supplemented with 10ng/ml FSH at 5% O2. The survival rates of secondary follicles during culture were similar between those derived from control tissue (71±13%) and those treated with NR (68±9%). The proportion of surviving follicles that formed an antrum were also similar in both groups (70±17% control; 48±24% NR-treated). Follicle diameters were not different between control follicles (184±5μm) and those stained with NR (181±7μm) on the day of isolation. The percentages of surviving follicles within three cohorts based on their diameters at week 4 of culture were similar between the control group and NR-stained tissue group, fast-grow follicles (24±6% vs. 13±10%), slow-grow follicles (66±5% vs. 60±9%), or no-grow (10±9% vs. 27±6%), respectively. There were no differences in follicle diameters between groups during the culture period. Pre-exposure of secondary follicles to NR diminished their capacity to produce both estradiol and androstenedione by week 4 of culture, when follicles are exhibiting an antrum. Inhibitory effects of NR on steroid production by slow-grow follicles was less pronounced. NR does not affect secondary follicle survival, growth, and antrum formation during long-term culture, but steroid hormone production by fast-grow follicles is compromised. NR can be used as a non-invasive tool for in situ identification of viable secondary follicles in ovarian cortex before tissue cryopreservation without affecting follicle survival and growth in vitro. Whether maturation or developmental competence of oocytes derived from antral follicles in 3D culture that were previously isolated from NR-stained tissue is normal or compromised remains to be determined. Likewise, the functional consequences of pre-exposure to NR prior to ovarian cortical tissue cryopreservation and transplantation are unknown.

Fertility Preservation in Children and Adolescents With Cancer

Advancements in oncologic therapy have increased long-term survival rates for children with childhood cancers. As survival has increased, the secondary effects of treatment have come into focus for patients and family. Infertility preservation in prepubertal children is a particularly difficult task as options are limited compared to adult counterparts with mature gametes. A systematic review of the published literature was conducted using keywords relevant to fertility preservation in the pediatric population undergoing oncologic treatment. We review the impact of cancer therapy upon gonadal function and identify the risk factors for future infertility in the prepubertal population. Treatment modifications that could modify the degree of potential damage to reproductive organs yet maintain oncologic principles were highlighted. Pubertal males and females have the opportunity to donate mature sperm or oocytes as do their adult counterparts; however, for the prepubertal child this is not the case. The options for these patients are considered investigational at this point and center on testicular tissue cryopreservation in males and oophorectomy vs ovarian cortical tissue cryopreservation in females. Infertility is an unfortunate side effect of oncologic treatment. Options are limited in the prepubertal population but tissue preservation and potential fertility should be discussed with all at-risk patients and their parents.

Acute onset of ovarian dysfunction in young females after start of cancer treatment

Female childhood cancer survivors are at risk of ovarian failure and premature ovarian insufficiency. We hereby present an interim analysis of a prospective observational study of ovarian function during cancer treatment of young females in relation to clinical factors. Thirty-four consecutive female cancer patients aged 0-18 year were included after informed consent. Serum/Plasma levels of anti-Müllerian hormone (AMH), inhibin B, FSH, LH, and oestradiol (E2) were measured at diagnosis and every 3-4 months during and after treatment. All patients had detectable AMH levels at diagnosis. Eleven patients had reached menarche (mean age 14½ years) and the remaining patients had a mean age of 6½ years. They all showed a rapid decline in AMH after 3 months of treatment, regardless of AMH at diagnosis, age, menarche, or treatment given. Those given radiotherapy below the diaphragm and/or stem cell transplantation (SCT) (n = 9) had no ovarian recovery during or 1½-year after treatment. However, recovery was observed in those given standard treatment for acute lymphatic leukemia (n = 7) already during maintenance chemotherapy. For the remaining patients, longer follow-up is required for analysis of ovarian recovery after treatment. Rapid ovarian dysfunction is observed in all females after initiation of cancer treatment as measured by AMH and inhibin B. Our data regarding those who require abdominal radiotherapy and/or SCT confirms the recommendations in the Nordic countries where these patients are eligible for cryopreservation of ovarian cortical tissue before start of cancer treatment.

Fertility and pregnancy aspects in Turner syndrome

In Turner syndrome, about a third of the diagnosed girls undergoes at least some pubertal development, and up to 5% are fertile. About 50% of Turner syndrome girls have follicles in their ovaries. The likelihood to have them is highest among mosaic Turner syndrome girls who have signs of spontaneous puberty, but also 25% of the non-mosaic Turner girls have them. Hence, cryopreservation of ovarian cortical tissue for infertility treatment in the future is possible. Oocyte donation is an effective option for Turner Syndrome women to obtain children. Pregnancy rates of 30–60% per embryo transfer have been reported. Single embryo transfer is a requirement, because twin pregnancies bear higher risk of pre-eclampsia and impaired glucose tolerance, and Turner women are already a high risk group for such problems. To avoid complications during pregnancies, a cardiology control including magnetic resonance imaging has to be carried out before planned pregnancies.

Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: A tool for evaluating cryopreservation utility

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n = 2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8+/-8.7% of group A follicles compared to 48.3+/-11.2% of group B follicles (p < 0.05). Apoptosis was demonstrated in 25.4+/-8.4% of group A follicles compared to 60.9+/-6.0% of group B follicles (p < 0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.

Treatment of gonadal damage in recipients of allogeneic or autologous transplantation for haematological malignancies.

Management of iatrogenic gonadal reproductive failure and sexual morbidity assumes a priority, especially in young recipients of high-dose chemotherapy and stem cell transplantation (SCT). Hormone replacement treatment (HRT) is beneficial for correction of sexual symptoms and osteoporosis in both sexes, especially in females. Sperm banking is the standard technique for preservation of fertility in adult and sexually mature adolescent males. Testicular tissue cryopreservation has a place in well-selected azoospermic adults and in mentally and sexually competent adolescents. In vitro fertilisation using superovulation with embryo-cryopreservation (for future embryo transfer) is the most tried method in female SCT recipients with good results. In mentally and sexually competent adolescents and adults without a partner, ovarian cortical tissue cryopreservation has a place for subsequent re-implantation to orthotopic or heterotopic sites. Gonadotrophin releasing hormone (GnRH) co-treatment during chemotherapy, is a promising method for the future. Although generally reassuring, continued monitoring of the offspring of SCT survivors and follow-up of all recipients of SCT is important for return of spontaneous or induced fertility.

Cryopreservation and culture of human primordial and primary ovarian follicles

Cryopreservation of ovarian cortical tissue containing high numbers of primordial and primary follicles would benefit young women who are going to undergo chemotherapy or radiotherapy, or anticipated premature ovarian failure. Human ovarian tissue has been successfully cryopreserved using dimethyl sulphoxide, propanediol and ethylene glycol as cryoprotectants. The viability after thawing has been shown morphologically, using viability tests, by transplanting the tissue to immunodeficient mice, and by culturing them in vitro. Maturation of oocytes in in vitro cultures from early follicles would be better than replantation for girls with malignancies which could be replanted with the tissue. For the time being we have managed to culture cryopreserved human primordial and primary follicles to secondary, and occasionally to early antral stages in organ culture within slices of cortical tissue in extracellular matrix. The culture conditions have to be improved to get systematically early antral follicles for a second step of maturation of cumulus-oocyte-complexes.

Maturation of mouse primordial follicles by combination of grafting and in vitro culture.

Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j x CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-microliter droplets of alpha-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO(2) in air at 37 degrees C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14-16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 +/- 4.45 micrometer) was similar to that of oocytes from the control group (70.5 +/- 2.35 micrometer). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.

Reproductive technologies 1998: options available for the cancer patient.

Recent advances in the field of reproduction have potentially opened opportunities for the preservation of the reproductive potential of young cancer patients with good long-term prognosis for survival. In the postpubertal male, cryopreservation of ejaculated sperm is both feasible and potentially successful. Semen parameters at the time of procurement are of minor significance; intracytoplasmic sperm injection (ICSI) can bypass sperm concentration and motility problems and can lead to successful fertilization. For the prepubertal male there are no clinically applicable options insofar as extraction and cryopreservation of postmeiotic sperm cells (mature spermatozoa or round spermatids) is not feasible. To date, efforts for culture of testicular tissue and in vitro maturation of male germ cells have not been successful. In both pre- and postpubertal females, cryopreservation of ovarian cortical tissue or enzymatically extracted follicles and the in vitro maturation of preantral follicles are of potential clinical use, but, to date, these approaches have been successful only in laboratory animals. An additional option available to the postpubertal female is the stimulation of ovaries with exogenous gonadotropins and retrieval of mature oocytes for cryopreservation. The recent application of ICSI in previously cryopreserved human mature oocytes has improved fertilization rates and has resulted in live births. Unfortunately, a shortcoming of this approach is the limited number of oocytes that can be harvested after stimulation of the ovaries. Further, all these approaches potentially harbor the risk of the cryopreservation of malignant cells with their subsequent reintroduction in the patient at a later date. This is a more realistic concern for patients suffering from hematologic or gonadal tumors. Finally, even though cryopreservation of embryos has been successfully used for many years, this option is not available to the pediatric and adolescent patient. It should not be forgotten that, even if the patients' own gametes are not available in the future, donor sperm and eggs provide the option for offspring and can give the opportunity to the females to carry a pregnancy as long as their uterus has not been affected by the cancer treatment. Given the rapid progress we are witnessing in the field of reproductive medicine, it is probable that in the very near future most of the options described and newer ones will be clinically available.