Ronit Abir

Morphological study of fully and partially isolated early human follicles

OBJECTIVE To compare the development of fully and partially isolated human follicles by using various culture systems. DESIGN Human ovarian material was incubated with collagenase and deoxyribonuclease. Fully and partially isolated follicles (30-50 microm) were dissected and studied under light and electron microscopy. The follicles were then cultured on and within various matrices. Fully isolated follicles were also cocultured with stromal cells. SETTING Rabin Medical Center, a major care and referral center. PATIENT(S) Women undergoing laparoscopy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Microscopy studies, follicular measurements. RESULT(S) Electron microscopy studies revealed an excess of lipid droplets in the granulosa cells of freshly isolated follicles. An increase in follicular size and granulosa cell number was observed only in the fully isolated follicles cultured within collagen gels for 24 hours. Most of the partially isolated follicles detached from the collagen gels. When cultured on collagen, extracellular matrix, and poly-L-lysine, both the fully and the partially isolated follicles deteriorated within the first 24 hours; coculture with stromal cells had no beneficial effect. CONCLUSION(S) The excess in lipid droplets in granulosa cells of isolated follicles might suggest that the isolation process does not yield completely healthy follicles. However, despite this finding, our studies show that fully isolated follicles, but not partially isolated follicles, can grow within, but not on, a culture matrix.

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.

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.

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 17β-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 17β-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.

Preliminary studies on apoptosis in human fetal ovaries

OBJECTIVE To evaluate if apoptosis occurs in human germ cells between 19 and 33 gestational weeks (GW). DESIGN Human fetal ovaries were obtained from aborted fetuses aged 19-33 GW. SETTING Rabin Medical Center, a major tertiary care and referral center. PATIENT(S) Twenty-seven women undergoing pregnancy termination. The abortions were mostly because of fetal anatomical or chromosomal abnormalities. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Microscopy studies, terminal deoxynucleotidyl transferase (TdT) assay (TUNEL), and immunocytochemistry for B-cell lymphoma/leukemia-2 (bcl-2). RESULT(S) TUNEL assay revealed a slight increase in apoptotic oocytes in fetuses from 23 GW, with a peak at 27 GW. Overexpression of bcl-2 was detected in all ovarian components, regardless of fetal age. CONCLUSION(S) There seems to be a slight increase in apoptosis in oocytes from 23 GW with a peak at 27 GW. However, it is very unlikely that these low apoptotic rates could be the cause of the extensive germ cell loss throughout human pregnancy. The overexpression of bcl-2 possibly suggests either that this gene is necessary to overcome extensive apoptotic activity or that it is responsible for the low apoptosis rates. However, these results should be considered with caution, since the ovaries were mostly from abnormal fetuses after feticide.

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.

Preservation of Fertility in Women Undergoing Chemotherapy: Current Approach and Future Prospects

Purpose:Anticancer treatment causes ovarian failure.Methods:Some hormones may have a protective effect on the ovary. Cryopreservation (freezing) of oocytes has had very limited success, and therefore, currently its use before chemotherapy is not a feasible option. However, cryopreservation of embryos is possible. Another solution is oocyte donation followed by in vitro fertilization (IVF).Results:Ovarian cortical slices containing primordial follicles have been cryopreserved successfully. To restore fertility, cryopreserved–thawed tissue taken from cancer patients before therapy could be replanted after recovery. The possible risk of malignancy restoration could be eliminated by obtaining unilaminar follicles from cryopreserved–thawed tissue and growing them in vitro, followed by routine IVF.Conclusions:Although women who undergo chemotherapy face limited options for fertility preservation, intensive studies in cryopreservation and in vitro maturation of follicles harbor hope for brighter prospects in the future.

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.

Parameters affecting successful transplantation of frozen-thawed human fetal ovaries into immunodeficient mice

OBJECTIVE To compare the development and survival of human fetal follicles frozen-thawed with dimethylsulfoxide (DMSO) and propandiol (PROH) in immunodeficient mice, to study the effects of host treatment with FSH, and to compare kidney and subcutaneous transplantation. DESIGN Controlled histologic study. SETTING Major tertiary care and referral academic center.Twenty-one women undergoing second-trimester pregnancy termination. Microscopic morphometric analysis and immunocytochemistry for proliferating-cell nuclear antigen in human fetal ovaries grafted into immunodeficient mice. RESULTS Renal grafts that were frozen-thawed with DMSO rather than PROH survived better in the hosts (79.6% compared with 58.8%), but significantly more follicles were identified in grafts frozen-thawed with PROH (P<.001). Follicular development was observed only in FSH-treated hosts, and follicular survival and development was better in the kidney than the subcutaneous site. CONCLUSION(S) This is the first report showing development of human fetal follicles in immunodeficient mice. Freezing-thawing with PROH seems to support development and survival better than with DMSO. The kidney is a better transplantation site than the subcutaneous site, probably because of its superior vascularization. Administration of FSH to the host is essential for follicular development. Follicular development and growth was better in ovarian grafts from older fetuses, as they contained more formed follicles.