Optimizing the process of fertility preservation in pediatric female cancer patients - a multidisciplinary program.

BACKGROUND: Current evidence indicates sub-optimal incidence of fertility preservation (FP) in eligible patients. We present herein our designated multidisciplinary program for FP in pediatric and adolescent population and present our data on FP in female patients. METHODS: Pediatric patients (age 0-18) who were candidate for highly gonadotoxic treatments were referred to FP program for a multidisciplinary discussion and gonadal risk-assessment followed by either oocyte cryopreservation or ovarian cryopreservation (OCP) for female patients, and sperm banking for male patients. The OCP protocol consists of aspiration of oocytes from small antral follicles and in-vitro maturation followed by cryopreservation, as well as ovarian tissue cryopreservation. RESULTS: The establishment of a designated FP program resulted in a significant increase in referral and subsequent FP procedures of all eligible patients. Sixty-two female patients were referred for FP discussion during a period of 36 months; 41 underwent OCP; 11 underwent oocyte cryopreservation and six were declined due to parental decision. The median age was 13.2y (range 18 months-18y). Thirty-two (51.6 %) were chemotherapy-naïve. Seventeen patients (27 %) had sarcoma, 16 patients (26 %) had acute leukemia. The mean number of mature oocytes that were eventually vitrified was significantly higher in chemotherapy-naïve patients compared with chemotherapy-exposed patients (mean 12 oocytes (1-42) versus 2 (0-7)). CONCLUSION: Multidisciplinary programs that encompass experts of all relevant fields, skilled laboratory resources and a facilitated path appear to maximize the yield. We observed a considerable higher referral rates following launching a designated program and earlier OCP in chemo-naïve patients that culminated in a better fertility preservation procedure.

Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

STUDY QUESTION: Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? SUMMARY ANSWER: Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. WHAT IS KNOWN ALREADY: Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. STUDY DESIGN, SIZE, DURATION: A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. MAIN RESULTS AND ROLE OF CHANCE: Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). LIMITATIONS, REASONS FOR CAUTION: There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. WIDER IMPLICATIONS OF THE FINDINGS: Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. STUDY FUNDING/COMPETING INTERESTS: The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. TRIAL REGISTRATION NUMBER: N/A.

The importance of neuronal growth factors in the ovary.

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase ß receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research.

Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women.

Keratinocyte growth factor (KGF) promotes growth of rat pre-antral follicles. There is limited information regarding its presence or that of its unique receptor (KGFR) in human ovaries, specifically in pre-antral follicles. The aim of the study was to investigate the expression of KGF and KGFR in ovarian samples from human fetuses and girls/women. The samples were prepared for immunohistochemical study of the KGF protein and for in situ hybridization to localize mRNA transcripts of KGFR. Total RNA was extracted from frozen ovarian samples, and the expression of KGF mRNA transcripts was investigated by reverse transcriptase polymerase chain reaction. In both fetuses and girls/women, the protein for KGF was detected from primordial stages in oocytes, granulosa cells (GCs) and stroma cells. Its mRNA transcripts were also detected in all extracts. The mRNA transcripts for KGFR were detected mainly in stroma cells in ovarian samples from both sources; in 10% of the samples, follicular staining was noted also in oocytes and GCs. Further studies adding KGF to the culture medium are needed to elucidate its putative role in human primordial follicle activation.

Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries.

There is no information regarding the presence of platelet-derived growth factors (PDGFs) and their receptors in human ovaries. The expression of PDGF-A, -B and their two receptors, PDGFR-alpha and -beta, was investigated in ovarian samples from women/girls and from human fetuses, at the protein and mRNA levels. The samples were prepared for immunohistochemical staining for PDGF-A and -B and their two receptors and in situ hybridization for the detection of the mRNA transcripts of the receptors. Total RNA was extracted from frozen ovarian samples, and the expression of PDGF-A and -B was investigated by reverse transcription-polymerase chain reaction. The proteins for PDGF-A and -B were detected in oocytes, and in granulosa cells (GC) of 50% of the follicles from women/girls. The proteins and mRNA transcripts for the two receptors were detected in oocytes (mRNA for PDGFR-beta only in 25% of the oocytes). PDGFR-alpha mRNA was expressed in GC of a minority of the samples from women/girls, whereas PDGFR-beta protein and mRNA were identified in over 50% of the GC from this source. PDGF-A and -B transcripts were identified in all the extracts. The presence of the receptors in GC suggests that PDGFs might be involved in the activation of primordial follicles.

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation.

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.

Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults.

The signals initiating the growth of primordial follicles are unknown. Growth factors such as neurotrophin 4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) may play a role in this process. To investigate the expression of NT-4/5 and BDNF and their receptor tyrosine kinase B (TrkB) in the early developing follicles, we fixed and froze 12 ovarian samples from adolescents/adults and 31 ovaries from human fetuses. The fixed samples were prepared for immunohistochemical staining for NT-4/5, BDNF and the TrkB receptor. Total RNA was extracted from the frozen ovarian samples, and the expression of NT-4/5, BDNF and the TrkB receptor (full length and two truncated isoforms) was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunohistochemical staining revealed the expression of NT-4/5 and BDNF mainly in oocytes and, in a minority of samples, also in the granulosa cells (GCs); TrkB receptor was identified in oocytes and GCs. Transcripts of NT-4/5, BDNF and all forms of TrkB receptor were identified in the samples. To elucidate whether indeed NT-4/5 and BDNF are involved in growth initiation of human primordial follicles, they should be added to the culture medium.

Presence of NGF and its receptors in ovaries from human fetuses and adults.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13-39 and 33 ovaries from human fetuses aged 19-33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT-PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.

Immunocytochemical detection and RT-PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as leukaemia inhibitory factor (LIF) may play a role in this process. To investigate the expression of LIF and its receptor in early developing follicles, nine ovarian samples from adolescents/adults aged 13-43 years and 23 ovaries from human fetuses aged 19-33 gestational weeks were immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of LIF and its two receptor units (LIF-R and gp 130). mRNA was extracted from the frozen ovarian samples, and the expression of LIF, LIF-R and gp 130 was investigated by RT-PCR. Products were resolved by 10% polyacrylamide gel electrophoresis and image analysis. There was strong to moderate immunocytochemical staining for LIF and LIF-R in oocytes from the primordial follicular stages onwards, and very weak to moderate staining for gp 130. LIF-R was also detected in granulosa cells of primary and secondary follicles from adolescents/adults. Transcripts of LIF, LIF-R and gp 130 RNA were identified by RT-PCR in all samples. The immunocytochemical staining and mRNA expression of LIF and its receptor are consistent with the concept that LIF might be involved in growth initiation of human primordial follicles through its receptor.

Turner's syndrome and fertility: current status and possible putative prospects.

Women with Turner's syndrome should be carefully followed throughout life. Growth hormone therapy should be started at age 2-5 years. Hormone replacement therapy for the development of normal female sexual characteristics should be started at age 12-15 years and continued for the long term to prevent coronary artery disease and osteoporosis. Most women with Turner's syndrome have ovarian dysgenesis; therefore, they are usually infertile, and in very rare cases have spontaneous menses followed by early menopause. Only 2% of the women have natural pregnancies, with high rates of miscarriages, stillbirths and malformed babies. Their pregnancy rate in oocyte donation programmes is 24-47%, but even these pregnancies have a high rate of miscarriage, probably due to uterine factors. A possible future prospect is cryopreservation of ovarian tissue containing immature follicles before the onset of early menopause, but methods of replantation and in-vitro maturation still need to be developed. Should these autologous oocytes indeed be used in the future, affected women would need to undergo genetic counselling before conception, followed by prenatal assessment.

Soluble L-selectin levels during controlled ovarian hyperstimulation.

We sought to determine whether neutrophil activation, as reflected by soluble L-selectin levels, plays a role in controlled ovarian hyperstimulation (COH) and the possible correlation between soluble L-selectin and serum sex steroid levels. The study population consisted of 14 consecutive patients undergoing our routine in vitro fertilization (IVF) long gonadotropin-releasing hormone (GnRH) analog protocol. Blood was drawn three times during the COH cycle: (1) on the day when adequate suppression was obtained (Day-S); (2) on the day of, or the day prior to, human chorionic gonadotropin (hCG) administration (Day-hCG); and (3) on the day of ovum pick-up (Day-OPU). Levels of sex steroids and plasma soluble leukocyte selectin (L-selectin) were compared among the three time points. Soluble L-selectin was measured with a commercial sandwich enzyme-linked immunosorbent assay (ELISA). The results showed significantly higher levels of soluble L-selectin on Day-OPU than on Day-S and Day-hCG, and significantly lower levels on Day-hCG than Day-S. Though no significant correlations were found between soluble L-selectin and serum estradiol or hCG levels, soluble L-selectin positively correlated with serum progesterone levels. We conclude that hCG administration leads to neutrophil activation, which correlates with the degree of luteinization. Further studies are required to elucidate the relationship between the immune system and COH. These may lead to new strategies for predicting and preventing complications of COH.

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.

Controlled ovarian hyperstimulation--a state of endothelial activation.

PROBLEM: To aim of the study was to investigate whether controlled ovarian hyperstimulation (COH) causes endothelial activation and whether there is a correlation between endothelial activation and serum sex-steroid levels. METHOD OF STUDY: The study population consisted of 14 consecutive patients undergoing our routine IVF long gonadotropin-releasing hormone-analog protocol. Blood was drawn three times during the COH cycle: (1) day when adequate suppression was obtained (Day-S); (2) on the day of or the day prior to hCG administration (Day-hCG); and (3) on the day of ovum pick-up (Day-OPU). The levels of sex steroids and plasma soluble endothelial (E)-selectin were compared among the time points. Soluble E-selectin was measured with a commercial sandwich enzyme-linked immunosorbent assay. RESULTS: Soluble E-selectin levels were significantly higher on Day-OPU than Day-S and Day-hCG, whereas no difference was observed between Day-hCG and Day-S. No significant correlations were found between soluble E-selectin level and patient age, number of gonadotropin ampoules used, number of oocytes retrieved, or serum estradiol, progesterone and human chorionic gonadotropin levels. CONCLUSIONS: Human chorionic gonadotropin administration leads to endothelial activation regardless of the degree of ovarian response. Further studies are required to elucidate the relationship between COH and endothelial activation. These findings may lead to new strategies for predicting and preventing complications of COH, such as severe ovarian hyperstimulation syndrome.

Primate and bovine immature oocytes and follicles as sources of fertilizable oocytes.

This review discusses the mechanisms underlying ovarian follicle development and the potential of immature follicles and oocytes from non-rodent mammalian species particularly human and bovine to serve as sources of oocytes for the in-vitro production of embryos. Factors that regulate growth and differentiation of unilaminar (primordial and primary) and multilaminar (secondary) follicles and the maturation of oocytes are highlighted. We conclude that many obstacles must still be overcome before fertilizable oocytes can be obtained from human and bovine ovaries, and more research on the quality of and culture conditions for immature oocytes and follicles is required before these can be considered as a source for in-vitro production.

Controlled ovarian hyperstimulation: a state of neutrophil activation.

PROBLEM: To investigate if controlled ovarian hyperstimulation (COH) affects the expression of neutrophil adhesion molecules and if a correlation exists between neutrophil activation and serum sex-steroid levels. METHOD OF STUDY: The pilot study was carried out in the in vitro fertilization (IVF) unit of our department, and required no modification of our routine IVF protocol. Four patients arriving for baseline hormonal profile on day 1 of the menstrual cycle before initiation of COH (control group) and 11 patients admitted for oocyte recovery (study group) were included. Venous blood was obtained from all patients and examined for hormonal profile and neutrophil activation. The latter was performed by staining for the surface adhesion molecules beta2 integrin and L-selectin. Positive cell count and mean fluorescence intensity were determined by flow cytometry. RESULTS: While neutrophil L-selectin was significantly lower in the study group than in the control group, neutrophil beta2 integrin was nonsignificantly higher. Though no significant correlations were found between neutrophil adhesion molecules and patient age, serum estradiol level, and human chorionic gonadotropin level; neutrophil L-selectin was negatively correlated with serum progesterone levels. CONCLUSIONS: COH leads to neutrophil activation, which correlates with the degree of luteinization. Further studies are required to elucidate the relationship between the immune system and COH. These may lead to new strategies for promoting fertility and preventing complications of COH.

The role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome. A pilot experimental study.

OBJECTIVE: To evaluate the role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome (OHSS). DESIGN: A pilot experimental study. MATERIAL AND METHODS: Ovarian hyperstimulation was induced in 5 rabbits using human menopausal gonadotropin/human chorionic gonadotropin, after pretreatment without (control group) or with bovine serum albumin (BSA group). RESULTS: Despite an increase in serum protein levels, the BSA group showed comparable delta increase in body weight and degree of ascites formation. CONCLUSIONS: Intravenous albumin did not prevent severe OHSS in a rabbit model despite the observed increase in serum oncotic pressure.

Pilot study of isolated early human follicles cultured in collagen gels for 24 hours.

The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen-thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen-thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.

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.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE: To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN: Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING: Patients giving informed consent in hospitals. PATIENT(S): Women undergoing laparotomy or oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular size, E2, histology. RESULT(S): Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S): It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.