In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.

BACKGROUND: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential. METHODS: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 µg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section. RESULTS: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age. CONCLUSIONS: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries.

Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.

The oocyte population is not renewed in transplanted or irradiated adult ovaries.

BACKGROUND: According to conventional theory, the oocyte population is not renewed in mammalian ovaries after birth. A new hypothesis proposes that oocytes are generated continuously from haematopoietic progenitor cells. There is, however, no evidence that they can ovulate, although they may partially restore fertility by organizing 'helper follicles'. The hypothesis that follicles can form de novo in adult ovaries has been tested in a transplant model. METHODS: Ovaries from adult mice were transplanted under the kidney capsule or into the ovarian bursa of histocompatible, transgenic CAG::H2B-EGFP host animals. Some donors were sterilized before transplantation by X-irradiation to ensure 'empty niches' were available for repopulation. The phenotype of follicular oocytes at 2, 4 and 8 weeks post-transplantation was scored by epifluorescence. RESULTS: A total of 819 oocytes were examined in 30 ovarian grafts. None expressed green fluorescence, as would be predicted if they had formed de novo from germ cell progenitors in the systemic circulation of the host. Furthermore, small follicles eliminated by irradiation were not replaced in transplanted ovaries, and the few growing follicles present were apparently survivors of the original population. CONCLUSIONS: No evidence was found to support the hypothesis that progenitor cells from extra-ovarian sources can repopulate the adult ovary. The findings are consistent with the conventional view that a limited number of oocytes are formed before birth and declines with age. The study did not, however, rule out the possibility that germline stem cells may reside in the adult ovary.

A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.

BACKGROUND: A series of monozygotic (MZ) twin pairs discordant for premature ovarian failure presented an unusual opportunity to study ovarian transplantation. METHODS: Ten MZ twin pairs requested ovarian transplantation and eight have undergone transplantation with cryopreservation of spare tissue. Seven had a fresh cortical tissue transplant, one of whom received a second frozen-thawed transplant after the first ceased functioning at three years. One had a fresh microvascular transplant. RESULTS: All recipients reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 77-142 days. Six have already conceived naturally (one twice). Currently, two healthy babies have been delivered, and another three pregnancies are ongoing. The oldest transplant functioned for 36 months, resulting in one child and one miscarriage. She conceived again after a frozen-thawed secondary transplant. There was no apparent difference in return of ovarian function between the eight fresh ovarian grafts and the one frozen graft. CONCLUSIONS: Ovarian transplantation appears to restore ovulatory function robustly. Successful pregnancies, including one after cryopreservation, bode well for application to fertility preservation.

Prevalence of premature ovarian failure in monozygotic and dizygotic twins.

BACKGROUND: Premature ovarian failure (POF) before 40 years of age from natural causes affects approximately 1% of adult women, with minor variations between ethnic groups. A recent case of ovarian transplantation between young monozygotic (MZ) twins in which one had undergone unexplained POF at 14 years has prompted a study of the prevalence of POF. METHODS: Menopausal ages of 832 Australian and UK female twin-pairs were extracted from volunteer national twin registry databases containing medical, reproductive and lifestyle data surveyed by mail questionnaire. Surgical menopause was an exclusion criterion. RESULTS: The prevalence of POF in both MZ and dizygotic (DZ) twins was similar in both registries and 3- to 5-fold greater than the general population at age thresholds 40 and 45 years. No specific factors were found to account for the higher risk of early menopause. Some twins of both zygosities were highly discordant for menopausal age (>or=10 years). Nevertheless, there was significant intra-twin dependence, especially for MZ twins, and the average age difference at last menses was greater in DZ twin-pairs. CONCLUSION: Both MZ and DZ twins are at higher risk of POF. Despite some striking differences within MZ twin-pairs, menopausal ages were more concordant than for DZ twin-pairs, confirming that the timing of menopause has a heritable component.

Expression of mRNAs for DNA methyltransferases and methyl-CpG-binding proteins in the human female germ line, preimplantation embryos, and embryonic stem cells.

Recent evidence indicates that mammalian gametogenesis and preimplantation development may be adversely affected by both assisted reproductive and stem cell technologies. Thus, a better understanding of the developmental regulation of the underlying epigenetic processes that include DNA methylation is required. We have, therefore, monitored the expression, by PCR, of the mRNAs of DNA methyltransferases (DNMTs), methyl-CpG-binding domain proteins (MBDs), and CpG binding protein (CGBP) in a developmental series of amplified cDNA samples derived from staged human ovarian follicles, oocytes, preimplantation embryos, human embryonic stem (hES) cells and in similar murine cDNA samples. Transcripts of these genes were detected in human ovarian follicles (DNMT3A, DNMT3b1, DNMT3b4, DNMT1, MDBs1-4, MeCP2, CGBP), germinal vesicle (GV) oocytes (DNMT3A, DNMT3b1, DNMT1, MDBs1-4, MeCP2, CGBP), mature oocytes (DNMT3A, DNMT3b1, DNMT1, CGBP), and preimplantation embryos (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD2, MDB4, CGBP). Differential expression of DNMT3B gene transcripts in undifferentiated (DNMT3b1) and in vitro differentiated human ES cells (DNMT3b3) further demonstrated an association of the DNMT3b1 transcript variant with totipotent and pluripotent human cells. Significantly, whilst the murine Dnmt3L gene is both expressed and essential for imprint establishment during murine oogenesis, transcripts of the human DNMT3L gene were only detected after fertilisation. Therefore, the mechanisms and/or the timing of imprint establishment may differ in humans.

Oocyte biology and genetics revelations from polar bodies.

The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while viable they possess the genetic potential to support normal embryonic development after transfer to a cytoplast. In addition to the theoretical possibility of using this non-cloning technique to generate more embryos, polar bodies can be used for genetic testing. By cytogenetic analysis of both PB using fluorescent in-situ hybridization (FISH) or chromosome painting, partial or full chromosomal status in the oocyte can be predicted; this approach finds particular application for women of advanced reproductive age as well as with maternally inherited translocations and single gene defects. By studying both of the PB, potential problems of interpretation arising from allele dropout can be reduced; a heterozygous first polar body provides the least ambiguous result. Mitochondria segregate randomly during meiotic cleavages providing an opportunity also to use the PB to screen for mitochondrial mutations and deletions. Thus, the PB can serve useful diagnostic purposes, especially where pre-fertilization screening or avoidance of embryo biopsy is desirable.

Gonadal tissue cryopreservation and transplantation.

Transplantation of ovarian and testicular tissue has been practised for over a century, mainly for experimental purposes. It is now being considered as a potential strategy for preserving fertility in young patients, including children, undergoing sterilizing treatment for cancer and other diseases. Ovarian tissue biopsies can be stored at liquid nitrogen temperatures indefinitely so that, after thawing, they can be returned as either ortho- or heterotopic grafts to the original patient. A different approach is needed for preserving male germ cells to restore fertile potential. Experimental studies have shown that spermatogonial stem cells injected into the rete testis/seminiferous tubules can re-initiate spermatogenesis after sterilizing treatment with alkylating agents; alternatively, in prepubertal cases, testicular biopsies that have been cryopreserved can be grafted subcutaneously to generate enough spermatozoa for intracytoplasmic sperm injection (ICSI). These strategies have been demonstrated in animal models and are now undergoing clinical testing.

Oogenesis as a foundation for embryogenesis.

The majority of oocytes in postnatal ovaries are small, non-growing and reside in primordial follicles. They have to undergo a prolonged phase of growth and differentiation before nuclear and cytoplasmic maturation enables them to resume meiosis and undergo fertilization. A better knowledge of this phase of oogenesis is essential for understanding causes of oocyte pathology and optimizing methods for growing oocytes in vitro and for cryopreservation. There could also be spin-off discoveries for contraceptive strategies and pharmacologically controlling oocyte maturation. During oocyte growth, a molecular programme for development is assembled for the timely expression of mRNAs, some of which are expressed throughout oogenesis while others are 'masked' until or after meiotic maturation. Masking and stability in storage are largely due to a truncated poly(A) tail, controlled by regulatory sequences on the 3'-untranslated region (UTR) of the mRNA. Most maternal RNAs are degraded early in cleavage, there being a narrow overlap between persisting maternal mRNAs and activation of the embryonic genome. Accumulation of RNAs and proteins are not, however, the only major changes taking place during oogenesis. Cytoplasmic organelles multiply and redistribute, and there are epigenetic modifications of DNA for monoallelic expression of imprinted genes. The granulosa cells are obligatory for they provide physical support, nutrients and mediate the regulatory influences of gonadotrophic hormones. On the other hand, the oocyte actively influences the growth and differentiation of its granulosa cells. Thus, healthy embryos reflect the quality of both the oocyte and the granulosa cells.

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization.

OBJECTIVE: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body. DESIGN: Prospective case study. SETTING: Research laboratories, university hospital. PATIENT(S): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI. MAIN OUTCOME MEASURE(S): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro. RESULT(S): Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X -15 cht -19 cht -22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21. CONCLUSION(S): The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.

Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma.

BACKGROUND: Infertility is a common late effect of chemotherapy and radiotherapy, and has a substantial effect on the quality of life for young survivors of cancer. For men, semen cryopreservation is a simple way of preserving reproductive potential but for women, storage of mature eggs rarely proves successful, and the alternative-immediate in vitro fertilisation with cryopreservation of embryos-is not always appropriate. Reimplantation of cryopreserved ovarian tissue has been shown to restore natural fertility in animals. We applied this technique in a woman who had received sterilising chemotherapy for lymphoma. METHODS: A 36-year-old woman underwent a right oophorectomy with cryopreservation of ovarian cortical strips before receiving high-dose CBV chemotherapy for a third recurrence of Hodgkin's lymphoma. 19 months later, when serum sex steroid analysis confimed a postmenopausal state, two ovarian cortical strips were thawed and reimplanted-one onto the left ovary and another at the site of the right ovary. FINDINGS: 7 months after reimplantation of ovarian cortical strips, the patient reported resolution of hot flashes and, for the first time, oestradiol was detected in the serum. This finding was associated with a decrease in the concentrations of follicle-stimulating hormone and luteinising hormone, and ultrasonography revealed a 10 mm thick endometrium, a poorly visualised left ovary, and a 2 cm diameter follicular structure to the right of the midline. The patient had one menstrual period, but by 9 months after the implantation, her sex steroid concentrations had returned to those seen with ovarian failure. INTERPRETATION: Orthotopic reimplantation of frozen/thawed ovarian cortical strips is a well tolerated technique for restoring ovarian function in women treated with sterilising chemotherapy for cancer.

Administration of cyclophosphamide at different stages of follicular maturation in mice: effects on reproductive performance and fetal malformations.

This study assessed reproductive performance, fetal viability and teratogenicity in female mice exposed to cyclophosphamide across a timeline corresponding to different stages of follicle maturation. Pregnancies were established in female Balb/c mice 1-4 weeks after administration of a non-sterilizing dose of cyclophosphamide (75 mg/kg). Each mating group represented a different stage of follicular growth at the time of cyclophosphamide exposure. The number of corpora lutea, pregnancies and fetal resorptions were determined. Surviving fetuses were evaluated for gross malformations. Results indicated that conceptions attributable to follicles exposed to cyclophosphamide at a mature stage had a significantly lower number of implantation sites, 4.82 +/- 1.01 versus 8.27 +/- 0.81 in controls (P = 0.001) and a high resorption rate, 56% +/- 0.11 versus 34% +/- 0.07 in controls (P = 0.05). The proportion of corpora lutea in this group which resulted in viable fetuses was extremely low, 0.2 +/- 0.06 versus 0.51 +/- 0.07 in controls (P = 0.001). Malformation rate was more than 10 times higher in all treated groups (P < 0.05) and a particularly high incidence of 33% (P = 0.0014) was observed in conceptions attributable to oocytes exposed to cyclophosphamide at the earliest stages of follicle growth. With an extended interval between exposure and mating the malformation rate gradually decreased towards normal values in the 12th week group. This study suggests that the effect of cyclophosphamide on female gametes and subsequently on future reproduction is influenced by the stage of oocyte maturation at the time of exposure. Early fertilization post-chemotherapy can result in a high rate of pregnancy failure and high malformation rate. This should be taken into account when considering the use of oocyte retrieval, IVF and embryo cryopreservation in patients currently undergoing chemotherapy.

Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation.

OBJECTIVE: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient's testes after thawing. DESIGN: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. SETTING: Laboratory research environment. PATIENT(S): Men undergoing routine surgery in a urology department. INTERVENTION(S): Testicular biopsy. MAIN OUTCOME MEASURE(S): Cellular viability and infusion of seminiferous tubules. RESULT(S): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. CONCLUSION(S): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization.

In vitro growth of human primordial follicles from frozen-banked ovarian tissue.

The rarity of human oocytes frequently limits the success of assisted reproductive technology and delays research progress. Development of technologies to grow mature oocytes from the more abundant small follicles, perhaps after long-term storage at low temperatures, is a theoretically attractive solution to both problems. The length of the follicular growth span from the primordial to Graafian stage and changes in the trophic requirements of the cells, cellular interactions, morphogenesis and the sheer increase in bulk as the antrum forms are major challenges for cell culture technology. Even so, much progress has been made with animal follicles, and has begun with human tissue. A multi-step procedure, which reflects these changes, is perhaps the most likely to succeed. At present, the best strategy appears to be to initiate follicle growth in situ and isolate the follicles or granulosa-oocyte complexes once they have progressed to preantral stages. The final step is to mature the oocytes within their cumulus cells. The prospects of succeeding at each stage, and producing a fertile gamete at the end, are likely to be greater by preserving cellular interactions and the phenotype of follicle cells as these provide the physiological environment in which oocytes develop.