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.

Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9.

CONTEXT: The signals initiating growth of primordial follicles are unknown. Bone morphogenetic protein 15 (BMP15) and growth differentiating factor 9 (GDF9) are promising candidates. OBJECTIVE: The objective of the study was to evaluate for the first time the effects of human recombinant BMP15 and human recombinant GDF9 on the in vitro development of human primordial follicles. DESIGN AND SETTING: This was a controlled culture study performed in a major tertiary university-affiliated medical center. MATERIALS: Materials included ovarian tissue from 17 girls/women and three aborted human fetuses. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURE: Histological and immunohistochemical (proliferating cell nuclear antigen, BMP15, and GDF9) studies and an endocrine assay of 17ß-estradiol were conducted. RESULTS: In the samples from girls/women, the number of developing follicles was greater with GDF9 or BMP15 alone than with no BMP15 or GDF9. Higher 17ß-estradiol secretion was noted after treatment with GDF9 than with BMP15 or with GDF9+anti-GDF9. The number of atretic follicles was greater with BMP15 than with GDF9. Proliferating cell nuclear antigen expression was greater with the higher dose of both growth factors than the lower dose. Expression of BMP15 and GDF9 was identified in samples cultured without BMP15 or GDF9. Results for the fetal follicles yielded no distinguishable pattern. CONCLUSIONS: Although both BMP15 and GDF9 promoted activation of human primordial follicles from girls/women (but not human fetuses) in a dose-dependent manner, GDF9 seems more beneficial.

Improving posttransplantation survival of human ovarian tissue by treating the host and graft.

OBJECTIVE: To improve posttransplantation survival of frozen-thawed human ovarian tissue in immunodeficient mice. DESIGN: Histologic study of transplanted human ovaries after treating the host and graft. SETTING: Infertility unit, university-affiliated tertiary medical center. PATIENT(S): Ovarian tissue from six girls/women (aged 5-23 years) who had undergone ovarian laparoscopy for fertility preservation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Thawed ovarian samples were transplanted into the back muscle of immunodeficient mice divided into four groups: A) no treatment; B) host treatment with vitamin E and gonadotropins before and after grafting; C) graft incubation with vascular endothelial growth factor A (VEGF-A) and vitamin E before transplantation; and D) host as in B, graft as in C. Ungrafted thawed samples served as control. Assessment of graft survival was conducted by follicle counts, apoptosis evaluation, immunohistochemical stainings for proliferating cell nuclear antigen (PCNA) and VEGF-A expression. RESULT(S): Only grafts incubated before transplantation (groups C and D) retained their original size. Follicle number was low in all grafts. PCNA expression was found in most grafts. Apoptosis was significantly lower in the untreated and treated grafts transplanted into treated hosts (groups B and D) than in ungrafted-thawed samples and group A grafts. All grafted groups had significantly higher expression of VEGF-A than ungrafted-thawed samples. CONCLUSION(S): Survival of transplanted human ovarian tissue may be improved by treatment of the host and graft. Further studies to evaluate treatments with a potential benefit in human ovarian autotransplantation are needed.

WITHDRAWN: Bone Morphogenetic Protein 15 (BMP15)/Growth Differentiating Factor 9 Heterodimer Is More Potent than BMP15 Alone in in Vitro Activation of Human Primordial Follicles.

This manuscript was withdrawn at the request of the authors.

Occasional involvement of the ovary in Ewing sarcoma.

BACKGROUND: Ewing sarcoma (EWS) is a highly metastatic malignancy in young patients. Ovarian cryopreservation is often an option for fertility preservation in cancer patients of reproductive age, specifically in minors. Thus, the possibility of ovarian involvement in EWS needs to be elucidated. METHODS: Eight patients aged 13-20 years with EWS participated in the study. Ovarian samples were fixed and prepared for light microscopy, and frozen in liquid nitrogen for RNA extraction followed by RT-PCR. Histological studies, including immunostaining for the adhesion receptor CD99, were used to detect histopathological features. Sensitive molecular methods were used to detect translocations causing the formation of tumor-specific EWS-Friend leukemia virus integration site 1 fusion gene (EWS-FLI1). RESULTS: In seven patients, there was no evidence of EWS in the ovaries from pathological/molecular studies. However, in one patient, the RT-PCR showed the EWS translocation, although there was no pathological evidence. CONCLUSIONS: Ovarian involvement is possible in EWS. Therefore, in patients with EWS ovarian tissue should be examined for traces of malignancy at both the pathological and molecular levels prior to the grafting of cryopreserved tissue in order to minimize the risk of reseeding the cancer.

Bone morphogenetic protein 15 expression in human ovaries from fetuses, girls, and women.

OBJECTIVE: To investigate, for the first time, the protein expression of bone morphogenetic protein (BMP) 15 in human ovaries from fetuses, girls/women as well as its mRNA transcripts in ovaries from fetuses and girls. DESIGN: Controlled immunohistochemical and in situ hybridization study of expression of BMP-15 protein and mRNA transcripts in human ovaries. SETTING: Major tertiary care academic center. PATIENT(S): Nine patients that underwent pregnancy terminations at 21-33 gestational weeks and 18 girls and women aged 5-39 years that underwent ovarian laparoscopies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunohistochemistry (protein detection) in all specimens and in situ hybridization (mRNA detection) in specimens from fetuses and girls. Both procedures were conducted on paraffin sections. RESULT(S): The expression of the BMP-15 protein and its mRNA was identified already from primordial stages. Protein expression was detected in all oocytes and stroma cells from both ovarian sources, and in granulosa cells of specimens from girls and women. The mRNA transcripts were detected in the oocyte, granulosa, and stroma cells from fetuses and girls. CONCLUSION(S): The BMP-15 protein is expressed already at primordial stages in fetuses, girls, and women, and its mRNA transcripts in fetuses and girls. Further studies should be conducted to elucidate if indeed BMP-15 is involved in the activation of human primordial follicles.

Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles.

OBJECTIVE: To evaluate whether basic fibroblast growth factor (FGF) benefits the in vitro development of human primordial follicles. DESIGN: Human ovarian tissue was placed in organ culture for 4 weeks with basic FGF and either fetal calf serum or a serum-free combination. Control groups were cultured with a neutralizing antibody against basic FGF. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Fourteen women/girls undergoing various gynecological operations and two fetuses from women undergoing pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular counts, immunohistochemistry for proliferating cell nuclear antigen and bromodeoxyuridine incorporation and measurement of 17beta E(2) production. RESULT(S): Only in the serum-free culture system was the number of developing follicles in samples cultured with basic FGF significantly higher than in uncultured specimens. The E(2) production increased significantly in the second week, and there was a significant reduction in E(2) secretion with the addition of the neutralizing antibody against basic FGF. The percentage of granulosa cells (GCs) that stained for proliferating cell nuclear antigen or bromodeoxyuridine was significantly higher in developing follicles than in primordial follicles, regardless of treatment. CONCLUSION(S): Basic FGF apparently plays a role in the E(2) production of early follicles. High doses of basic FGF enhanced follicular development in serum-free media.

Selection of patients before and after anticancer treatment for ovarian cryopreservation.

BACKGROUND: Although ovarian cryopreservation in patients with cancer should ideally be performed before the initiation of therapy, cryopreservation from such patients often becomes an option only later. The justification for the procedure needs to be elucidated. METHODS: Eighteen cancer patients before chemotherapy and 23 others after chemotherapy participated in the study. Freshly dissected ovarian samples were prepared for light microscopy to demonstrate follicular numbers and apoptosis, transmission electron microscopy to enhance intracellular changes, and staining with fluorescent markers (calcein AM, rhodamin 123 and ethidium homodimer) to test for viability. RESULTS: High numbers of preantral follicles were detected in ovaries of patients < or =20 years. No antral follicles were detected. All the follicles were viable and not apoptotic. Deterioration in follicular quality was observed after chemotherapy, manifested mainly as an increase in abnormal granulosa cell nuclei (P < 0.05-0.0001) and in oocyte vacuolization (P < 0.0001). CONCLUSIONS: Our study stresses the importance of prechemotherapy ovarian cryopreservation. However, the large number of viable, non-apoptotic follicles in ovaries of younger patients (age < or = 20 years) indicates that ovarian cryopreservation might be considered after treatment in this age group. Further studies of ovarian samples from women aged 20-30 years are needed to determine the exact age margin wherein postchemotherapy ovarian cryopreservation can be suggested.

Expression of bone morphogenetic proteins 4 and 7 and their receptors IA, IB, and II in human ovaries from fetuses and adults.

OBJECTIVE: To investigate the immunocytochemical and mRNA expression of bone morphogenetic proteins 4 (BMP-4) and 7 (BMP-7) and their three receptors (BMPR-IA, BMPR-IB, and BMPR-II) in ovaries from human adults and fetuses. DESIGN: Immunocytochemical and in situ hybridization study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Sixteen adolescents/adults aged 13-38 years and 31 women undergoing second and third trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and adults. RESULT(S): The expression of the proteins for BMP-4 and BMP-7 and their receptors was detected in oogonia/oocytes and stroma cells from both sources (fetuses and women/adolescents). BMP-7 and the three receptors were identified in all of these granulosa cells, and BMP-4 was detected only in the granulosa cells of women/adolescents. Transcripts for all five ligands were identified in stroma cells of all samples and in fetal oogonia/oocytes. BMPR-IA and BMPR-IB mRNA expression was also identified in oocytes from women/adolescents. BMP-7 and BMPR-IA mRNA staining was detected in fetal granulosa cells. CONCLUSION(S): This is the first report of the expression of BMP-4 and BMP-7 and their receptors in human ovaries from fetuses as well as adults. However, to elucidate whether indeed BMP-4 or BMP-7 are involved in the initiation of human primordial follicular growth, they should be added to the culture medium.

Growth hormone and its receptor in human ovaries from fetuses and adults.

OBJECTIVE: To investigate the presence of growth hormone (GH) and its receptor (GH-R) in early developing follicles. DESIGN: Immunocytochemical and in situ hybridization study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Ten ovarian samples from adults/girls aged 6-38 years and from 10 fetuses of women undergoing second and third trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and women/girls. RESULT(S): The proteins and the mRNA transcripts for GH and GH-R were detected in oocytes, granulosa (GC), and stroma cells from both sources (fetuses and women/girls), with low staining intensity only in a portion of the fetal GC. CONCLUSION(S): This is the first report of the expression of GH-R in human ovaries from fetuses as well as women/girls and of GH in human fetal ovaries. Be that as it may, further experiments should be conducted to elucidate if indeed GH is involved in the initiation of human primordial follicular growth.

Survival of frozen-thawed human ovarian fetal follicles in long-term organ culture.

Ovarian follicles obtained from second and third-trimester human fetuses survived 4 weeks in organ culture and secreted 17-beta estradiol (E(2)).