Novel extra cellular-like matrices to improve human ovarian grafting.

PURPOSE: To investigate if human ovarian grafting with pure virgin human recombinant collagen type-1 from bioengineered plant lines (CollPlant™) or small intestine submucosa (SIS) yields better implantation results for human ovarian tissue and which method benefits more when combined with the host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. METHODS: Human ovarian tissue wrapped in CollPlant or SIS was transplanted into immunodeficient mice with/without host/graft treatment. The tissue was assessed by follicle counts (including atretic), for apoptosis evaluation by terminal deoxynucleotidyl transferase assay and for immunohistochemical evaluation of neovascularization by platelet endothelial cell adhesion molecule (PECAM) expression, and for identification of proliferating granulosa cells by Ki67 expression. RESULTS: Human ovarian tissue transplanted with CollPlant or SIS fused with the surrounding tissue and promoted neovascularization. In general, implantation with CollPlant even without additives promoted better results than with SIS: significantly higher number of recovered follicles, significantly fewer atretic follicles, and significantly more granulosa cell proliferation. Moreover, results with CollPlant alone seemed to be at least as good as those after host and graft treatments. CONCLUSIONS: CollPlant is a biomaterial without any potential risks, and grafting ovarian tissue with CollPlant is easy and the procedure may be easily modified, with limited or no foreseeable risks, for auto-transplantation in cancer survivors. Further studies are needed using other novel methods capable of enhancing neovascularization and reducing apoptosis and follicle atresia.

Use of Simvastatin, Fibrin Clots, and Their Combination to Improve Human Ovarian Tissue Grafting for Fertility Restoration After Anti-Cancer Therapy.

Anticancer treatments, particularly chemotherapy, induce ovarian damage and loss of ovarian follicles. There are limited options for fertility restoration, one of which is pre-chemotherapy cryopreservation of ovarian tissue. Transplantation of frozen-thawed human ovarian tissue from cancer survivors has resulted in live-births. There is extensive follicular loss immediately after grafting, probably due to too slow graft revascularization. To avoid this problem, it is important to develop methods to improve ovarian tissue neovascularization. The study's purpose was to investigate if treatment of murine hosts with simvastatin or/and embedding human ovarian tissue within fibrin clots can improve human ovarian tissue grafting (simvastatin and fibrin clots promote vascularization). There was a significantly higher number of follicles in group A (ungrafted control) than in group B (untreated tissue). Group C (simvastatin-treated hosts) had the highest levels of follicle atresia. Group C had significantly more proliferating follicles (Ki67-stained) than groups B and E (simvastatin-treated hosts and tissue embedded within fibrin clots), group D (tissue embedded within fibrin clots) had significantly more proliferating follicles (Ki67-stained) than group B. On immunofluorescence study, only groups D and E showed vascular structures that expressed both human and murine markers (mouse-specific platelet endothelial cell adhesion molecule, PECAM, and human-specific von Willebrand factor, vWF). Peripheral human vWF expression was significantly higher in group E than group B. Diffuse human vWF expression was significantly higher in groups A and E than groups B and C. When grafts were not embedded in fibrin, there was a significant loss of human vWF expression compared to groups A and E. This protocol may be tested to improve ovarian implantation in cancer survivors.

The influence of in vivo exposure to nonylphenol ethoxylate 10 (NP-10) on the ovarian reserve in a mouse model.

AIM: To determine the effect of nonylphenol-ethoxylate-10 (NP-10) on the ovarian reserve in a mouse model. DESIGN: Female mice were maintained on purified water or exposed to NP-10 from 3-7-weeks of age. At 7-weeks they were stimulated, mated and the zygotes were cultured in-vitro. Three and 7-weeks old mice were untreated controls. Identical groups were sacrificed without stimulation. Ovaries were analysed for follicular composition. Respiratory-chain (RC) activity and reactive-oxygen-species (ROS) production were measured in brains and livers. RESULTS: Seven-weeks-old mice produced fewer oocytes/embryos than 3-week-old mice. At 7-weeks, mice exposed to NP-10 produced more oocytes/embryos the controls. Their ovaries contained more primordial/primary follicles, with a lower rate of proliferation and fewer antral follicles. There were no differences in follicular apoptosis, RC-activity or ROS production. CONCLUSIONS: In this model, exposure to NP-10 inhibited the spontaneous follicular recruitment, the first report of successful inhibition of physiologic ovarian aging, to the best of our knowledge.

Premature ovarian aging in BRCA carriers: a prototype of systemic precocious aging?

PURPOSE: Though former evidence implies a correlation of breast cancer susceptibility gene (BRCA) mutation with reduced ovarian reserve, the data is yet inconsistent. Our aim was to investigate biomarkers of ovarian aging in a cohort of young healthy carriers of the BRCA mutation. We hypothesized that the role played by BRCA genes in aging pathways is not exclusive to the ovary. EXPERIMENTAL DESIGN: Healthy female BRCA carriers, 40 years or younger and healthy male BRCA carriers, 50 years or younger, were enrolled in the study. Serum anti-mullerian Hormone (AMH), fibroblast growth factor-23 (FGF-23), Klotho and IL-1 were measured by enzyme-linked immunosorbent assay (ELISA). Ovarian AMH and protein kinase B (AKT) mRNA from BRCA carriers who underwent prophylactic oophorectomy and from age-matched, healthy, non-carriers who underwent partial oophorectomy due to benign conditions were analyzed by qPCR. RESULTS: Thirty-three female (median age 35y) and 20 male (44y) BRCA carriers were enrolled into the study and matched to control non-carriers (34y and 43y, respectively). Serum AMH level was significantly lower in BRCA female carriers than in both non-carrier controls and age-matched nomograms. The levels of ovarian AMH and AKT mRNA were significantly lower in carriers than in controls. The systemic aging cytokines FGF-23, klotho and IL-1 displayed a differential expression in carriers of both genders. FGF-23 level was higher in carriers (P=0.06). CONCLUSIONS: Our results suggest a link between BRCA mutation, accelerated ovarian aging and systemic aging-related pathophysiology.

Female fertility preservation: past, present and future.

Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation and in vitro maturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined with in vitro maturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependent in vitro system for culturing primordial follicles.

[LAPAROSCOPICALLY-ASSISTED ULTRASOUND-GUIDED PERCUTANEOUS TRANSABDOMINAL OOCYTE COLLECTION: FERTILITY PRESERVATION IN A 17-YEARS-OLD GIRL WITH VAGINAL EWING SARCOMA].

INTRODUCTION: Options for preserving fertility in children and adolescents with cancer depend on patient age, the available time frame, and the treatment regimen. Ovarian stimulation with mature oocyte preservation is often the optimal method in post-menarcheal adolescents. We describe a case of a 17-year-old girl with vaginal soft-tissue Ewing sarcoma in whom transvaginal oocyte collection for fertility preservation was ruled out by the large tumor. To overcome the limitations of the transabdominal approach, we applied a novel method of laparoscopically-assisted ultrasound-guided percutaneous transabdominal oocyte collection. In this manner, we were able to both perform oophorectomy and obtain superficial and deep ovarian follicles for cryopreservation.

[FERTILITY PRESERVATION IN YOUNG CANCER PATIENTS - CAN WE OPTIMIZE THE PATH?]

INTRODUCTION: Advances in cancer therapy have improved the long-term survival of cancer patients. Concerns about fertility represent a major issue for young cancer patients. The emergent discipline of oncofertility, an intersection between oncology and fertility, is a new concept that describes an integrated network of clinical resources that focus on fertility preservation from both clinical and research perspectives. Patients and methods: In this article we describe our designated multidisciplinary program for fertility preservation in pediatric and young adult populations. The program is also designed to serve as a prospective platform for the evaluation of reproductive outcomes in this patient cohort. RESULTS: We have observed considerably higher referral rates following launching the program and earlier referral of chemonaïve patients that concedes maximal fertility preservation. Two hundred and thirty five patients were referred to the program over a period of 3 years. CONCLUSIONS: Our program demonstrates that multidisciplinary programs that encompass relevant specialists, skilled laboratory resources and a facilitated path that drives the process in the shortest time, maximizes the yield.

Attempts to improve human ovarian transplantation outcomes of needle-immersed vitrification and slow-freezing by host and graft treatments.

PURPOSE: To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the "improvement protocol" of host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. METHODS: Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM). RESULTS: Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing + the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups. CONCLUSIONS: Ovarian sample preparation with slow-freezing + the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

Extracellular-like matrices and leukaemia inhibitory factor for in vitro culture of human primordial follicles.

The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen-thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10ngmL-1 and 100ngmL-1), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17ß-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.

Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro.

How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17ß-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17ß-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity.

Aspiration of immature oocytes during cesarean section for fertility preservation.

BACKGROUND: In vitro maturation (IVM) of immature oocytes is an important technology for selected clinical indications. We previously described a pregnant woman with a history of renal transplantation who underwent oocyte aspiration during cesarean section (CS) for fertility preservation and future surrogacy. CASE: A 27-year-old pregnant woman was diagnosed with neck rhabdomyosarcoma at 37 weeks' gestation. CS was performed with direct aspiration of small follicles from one ovary and oophorectomy of the other. Twenty-one identified oocyte-cumulus complexes were cultured, and 12 mature oocytes and 14 ovarian cortex strips were cryopreserved. CONCLUSION: Aspirating competent oocytes during CS may serve as an additional means of fertility preservation in pregnant women. The procedure may also be offered to patients with an IVF pregnancy who are scheduled for elective CS.

Ovarian minimal residual disease in chronic myeloid leukaemia.

The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopreserved-thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report.

Attempted application of bioengineered/biosynthetic supporting matrices with phosphatidylinositol-trisphosphate-enhancing substances to organ culture of human primordial follicles.

PURPOSE: To improve human primordial follicle culture. METHODS: Thin or thick ovarian slices were cultured on alginate scaffolds or in PEG-fibrinogen hydrogels with or without bpV (pic), which prevents the conversion of phosphatidylinositol-trisphosphate (PIP3) to phosphatidylinositol-bisphosphate (PIP2) or 740Y-P which converts PIP2 to PIP3. Follicular growth was evaluated by follicular counts, Ki67 immunohistochemistry, and 17ß-estradiol (E2) levels. RESULTS: BpV (pic) had a destructive effect on cultured follicles. Thawed-uncultured samples had more primordial follicles than samples cultured in basic medium and fewer developing follicles than samples cultured in PEG-fibrinogen hydrogels with 740Y-P. There were more atretic follicles in samples cultured on alginate scaffolds than in PEG-fibrinogen hydrogels, and in samples cultured in PEG-fibrinogen hydrogels with 740Y-P than in PEG-fibrinogen hydrogels with basic medium. Ki67 staining was higher in PEG-fibrinogen hydrogels than on alginate scaffolds. E2 levels were higher in thick than in thin slices. CONCLUSIONS: PEG-fibrinogen hydrogels appear to have an advantage over alginate scaffolds for culturing human primordial follicles. Folliculogenesis is not increased in the presence of substances that enhance PIP3 production or with thin rather than thick sectioning.

Fibroblast growth factor 10 in human ovaries.

The expression of fibroblast growth factor 10 (FGF-10) has not been studied in human ovarian cortical follicles. The aim of the present study was to investigate the expression of FGF-10 in preantral follicles from fetuses, girls and women. Ovarian samples were obtained from 14 human fetuses at 21-33 gestational weeks and from 35 girls and women aged 5-39 years. The specimens were prepared for detection of the FGF-10 protein by immunohistochemistry. Reverse-transcription PCR was applied to ovarian extracts to identify FGF-10 mRNA transcripts. In fetal tissue, the FGF-10 protein was detected in oocytes in 50% of the samples and in granulosa cells in 30%. In ovarian tissue from girls and women, the FGF-10 protein was detected in oocytes and granulosa cells in all samples. FGF-10 mRNA transcripts were present in all adult and fetal samples tested. The identification of FGF-10 at both the protein and mRNA levels suggests that FGF-10 may contribute to human preantral follicle development.

Vasoactive intestinal peptide and its receptors in human ovarian cortical follicles.

BACKGROUND: Ovarian cryopreservation is one option for fertility preservation in patients with cancer. The danger of reseeding malignancies could be eliminated by in vitro maturation of primordial follicles from the frozen-thawed tissue. However, the development of this system is hindered by uncertainties regarding factors that activate primordial follicles. Neuronal growth factors such as vasoactive intestinal peptide (VIP) play important roles in early mammalian folliculogenesis. There are no data on the expression of VIP and its vasoactive intestinal peptide pituitary adenylate cyclase 1 and 2 receptors (VPAC1-R and VPAC2-R) in human preantral follicles. METHODOLOGY/PRINCIPAL FINDINGS: Tissue samples from 14 human fetal ovaries and 40 ovaries from girls/women were prepared to test for the expression of VIP, VPAC1-R, and VPAC2-R on the protein (immunohistochemisty) and mRNA (reverse transcription polymerase chain reaction) levels. Immunohistochemistry staining was mostly weak, especially in fetal samples. The VIP protein was identified in oocytes and granulosa cells (GCs) in the fetal samples from 22 gestational weeks (GW) onwards. In girls/women, VIP follicular staining (oocytes and GCs) was identified in 45% of samples. VPAC1-R protein was identified in follicles in all fetal samples from 22GW onwards and in 63% of the samples from girls/women (GC staining only in 40%). VPAC2-R protein was identified in follicles in 33% of fetal samples and 47% of the samples from girls/women. The mRNA transcripts for VIP, VPAC1-R, and VPAC2-R were identified in ovarian extracts from fetuses and women. CONCLUSIONS: VIP and its two receptors are expressed in human ovarian preantral follicles. However, their weak staining suggests they have limited roles in early follicular growth. To elucidate if VIP activates human primordial follicles, it should be added to the culture medium.

Possible improvements in human ovarian grafting by various host and graft treatments.

BACKGROUND: Anticancer treatment poses a high risk of ovarian failure. In many cases cryopreservation of ovarian tissue is the only option for fertility preservation. Although autologous transplantation of cryopreserved-thawed ovarian tissue has resulted in live births, slow graft revascularization and ischemia after transplantation leads to substantial follicular loss. Therefore, methods to improve and hasten graft vascularization are needed. The aim of the study was to examine the benefits of host and graft treatments with melatonin, hyaluronan (HA), vascular endothelial growth factor A (VEGF-A) and vitamin E with regard to the outcome of human ovarian tissue grafting. METHODS: Five young cancer patients who underwent laparoscopic ovarian surgery for fertility preservation donated ovarian tissue. Thawed ovarian samples were transplanted into immunodeficient mice divided into seven groups: (A) no treatment; (B) host treatment with melatonin before and after grafting; (C) graft incubation with HA-rich biological glue before transplantation; (D) host as in (B), graft as in (C); (E) host as in (B), graft incubation with VEGF-A and vitamin E; (F) graft as in (C) combined with VEGF-A and vitamin E; (G) host as in (B), graft as in (F). Graft survival was assessed by follicle counts, apoptosis assay and immunohistochemical staining for proliferating cell nuclear antigen and VEGF-A expression. RESULTS: Only grafts implanted in melatonin-treated hosts and grafts incubated with HA-rich biological glue retained their original size. Apoptosis was significantly lower after host treatment with melatonin and graft incubation with HA-rich biological glue plus VEGF-A and vitamin E than in untreated grafts; apoptosis was specifically low in Group G. There were significantly more atretic follicles in the untreated group than in most treated groups. CONCLUSIONS: The findings suggest that host treatment with melatonin or graft incubation with HA-rich biological glue, especially when combined with VEGF-A and vitamin E improves graft survival. This protocol can be applied and holds promise in ovarian autotransplantation for fertility restoration.

Alginate scaffold for organ culture of cryopreserved-thawed human ovarian cortical follicles.

PURPOSE: To compare macroporous alginate scaffolds with Matrigel for culturing frozen-thawed human primordial follicles in organ culture. METHODS: Twelve girls/women donated ovarian tissue. One tissue sample was fixed immediately after thawing (uncultured samples). Slices were cultured for 2 weeks on either Matrigel or on alginate scaffolds with a serum-free culture medium. Growth evaluation consisted of follicular counts and classification, immunohistochemistry and measurement of 17ß-Estradiol (E(2)) production. RESULTS: The number of developing follicles was significantly higher in alginate scaffold-cultured samples than on Matrigel with a concomitant decrease in the number of primordial follicles in alginate scaffold-cultured samples than uncultured samples. The number of atretic follicles after 1 week was significantly higher in the Matrigel-cultured samples than in the alginate scaffold cultured samples. E(2) production was similar in both groups. CONCLUSIONS: Three dimensional alginate scaffolds are a promising putative in vitro technology for developing human primordial follicles.