Shmuel Nitke

Morphological study of fully and partially isolated early human follicles

OBJECTIVEnTo compare the development of fully and partially isolated human follicles by using various culture systems.nnnDESIGNnHuman 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.nnnSETTINGnRabin Medical Center, a major care and referral center.nnnPATIENT(S)nWomen undergoing laparoscopy.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nMicroscopy studies, follicular measurements.nnnRESULT(S)nElectron 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.nnnCONCLUSION(S)nThe 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.

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

OBJECTIVEnTo evaluate whether basic fibroblast growth factor (FGF) benefits the in vitro development of human primordial follicles.nnnDESIGNnHuman 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.nnnSETTINGnMajor tertiary care and referral academic centers.nnnPATIENT(S)nFourteen women/girls undergoing various gynecological operations and two fetuses from women undergoing pregnancy terminations.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nFollicular counts, immunohistochemistry for proliferating cell nuclear antigen and bromodeoxyuridine incorporation and measurement of 17beta E(2) production.nnnRESULT(S)nOnly 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.nnnCONCLUSION(S)nBasic 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

BACKGROUNDnAlthough 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.nnnMETHODSnEighteen 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.nnnRESULTSnHigh 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).nnnCONCLUSIONSnOur 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

OBJECTIVEnTo 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.nnnDESIGNnControlled histologic study.nnnSETTINGnMajor 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.nnnRESULTSnRenal 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.nnnCONCLUSION(S)nThis 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.

Possible Direct Cytoxicity Effects of Cyclophosphamide on Cultured Human Follicles: An Electron Microscopy Study

AbstractPurpose: To evaluate the direct effect of cyclophosphamide on cultured human ovarian follicles.nMethods: Human ovarian cortical slices from premenopausal women were incubated with medium containing cyclophosphamide (0.0005–0.5 mg/mL) for 2–48 h and assessed by transmission electron microscopy. Noncultured specimens and samples cultured without cyclophosphamide were used as controls.nResults: There were significantly more damaged granulosa cell nuclei after incubation with 0.5 mg/mL cyclophosphamide for at least 4 h. There were also more changes in the basement membrane after incubation with cyclophosphamide at concentrations of 0.05 and 0.5 mg/mL.nConclusions: Although the cyclophosphamide dose that caused damage to the granulosa cell nuclei was above the pharmacological level, our results suggest that cyclophosphamide, and not only its active metabolite phospharamide mustard, might have a destructive effect on human follicles, as it remains in the circulation longer. This effect could be mediated by damage to the granulosa cells and perhaps the basement membrane.

Short Communication: Development of Human Fetal Follicles in an Immunodeficient Mouse

growth from secondary stages only (1). Therefore, PETAH TIKVA, ISRAEL before clinical use, the growth and maturation capacity of frozen-thawed fetal oocytes need to be tested. The Development of Human Fetal Follicles in aim of the present study was to examine the ability of an Immunodeficient Mouse* human fetal oocytes to develop in an immunodeficient nu/nu mouse and to assess their growth by immunocytochemical expression of proliferating cell nuclear antiSubmitted: November, 24, 1999 gen (PCNA). PCNA is a protein involved in cell cycle Accepted: January 29, 2000 found to correlate with in initiation of human adult (2), bovine fetal (3), and baboon (4) folliculogenesis and not expressed in primordial follicles. INTRODUCTION

Tramadol-metoclopramide or remifentanil for patient-controlled analgesia during second trimester abortion: a double-blinded, randomized controlled trial ☆

STUDY OBJECTIVEnTo compare patient-controlled analgesia (PCA) with tramadol with PCA with remifentanil in second trimester abortion.nnnDESIGNnProspective, randomized double-blinded studynnnSETTINGnUniversity-affiliated medical center.nnnPATIENTSn30 ASA physical status 1 and 2 patients undergoing a second trimester abortion.nnnINTERVENTIONSnPatients received PCA with either tramadol or remifentanil. Analgesia was initiated in the tramadol group by an initial loading dose of tramadol 1.0 mg/kg with 10 mg of metoclopramide followed by a PCA bolus of 0.3 mg/kg/mL of tramadol every 5 minutes. For remifentanil, which does not require a loading dose, a placebo of 100 mL of 0.9% normal saline was given followed by PCA of 0.4 μg/kg/mL every two minutes.nnnMEASUREMENTSnWomen were evaluated for pain via verbal analog score (VAS; 0-100), sedation, nausea, blood pressure, pulse, and respiratory rate. On the day of discharge, women were analyzed for overall satisfaction. Primary outcome was pain scores and general satisfaction.nnnMAIN RESULTSnAnalysis by time yielded no statistically significant difference in VAS scores between the groups at any point except 16-20 hours after induction of labor, when pain was lower in the tramadol group (11.3 ± 18.1 vs. 36.7 ± 27.4; P = 0.04). The average VAS score was low in both groups, with no significant differences noted between groups (P = 0.74). Satisfaction scores were high in both groups, with no significant differences noted between them (P = 0.89).nnnCONCLUSIONnBoth drugs are acceptable choices for pain control in patients undergoing second trimester abortions.