Human Folliculogenesis Research Articles

Programmed cell death in the human ovary.

During a woman's reproductive life, only about 400 primordial follicles will develop into a preovulatory follicle and undergo ovulation, releasing an oocyte available for fertilization. The process of formation and selection of these follicles is complex and involves a multistep process characterized by a balance between survival and death of the oocytes and the surrounding follicular cells. Although the mechanisms underlying such process are not completely clarified yet, it is common idea that they can occur through various types of programmed cellular death (PCD). Since atresia is the principal destiny of the ovarian follicles, it is relevant to understand how this process takes place and how it is regulated. In this review, after a summary description of folliculogenesis in humans, the main mechanisms of atresia reported to occur during folliculogenesis from birth to adult age, in the human ovary and in other mammals when appropriate, are described.

Differentiation of human menstrual blood-derived endometrial mesenchymal stem cells into oocyte-like cells.

Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood are a unique stem cell source. Evidence suggests that EnSCs exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. However, the potential of EnSCs to differentiate into germline cells in vitro remains unclear. In this study, EnSCs were induced to differentiate into germ cells in a differentiation medium supplemented with 20% human follicular fluid. Our results demonstrated that EnSCs derived from human menstrual blood form oocyte-like cells and express germ cell markers. The induced cell aggregates contained not only oocyte-like structures but also cells expressing follicle stimulating hormone receptor and luteotropic hormone receptor, and produced estrogen and progesterone regulated by gonodatropin, suggesting that granulosa-like and theca-like cells were also induced. We further found that granulosa cells promote the development of oocyte-like cells and activate the induction of blastocyst-like structures derived from EnSCs. In conclusion, EnSCs may potentially represent an in vitro system for the investigation of human folliculogenesis.

Early Follicular Anti-Mullerian Hormone and Antral Follicle Count as an Indicator of Ovarian Reserve: A First Pilot Study in the Nepalese Population

Background: Anti-Mullerian Hormone (AMH) is produced by the granulosa cells of primary, preantral and greater antral follicles, and plays an imperative role in human folliculogenesis. It is a measure of ovarian function, useful in assessing conditions such as polycystic ovary syndrome and premature ovarian failure. The objective of this study was to evaluate the correlation between chronological age with levels of antral follicle count (AFC), and AMH. Methods: Anti-Mullerian Hormone (AMH) is produced by the granulosa cells of primary, preantral and greater antral follicles, and plays an imperative role in human folliculogenesis. It is a measure of ovarian function, useful in assessing conditions such as polycystic ovary syndrome and premature ovarian failure. The objective of this study was to evaluate the correlation between chronological age with levels of antral follicle count (AFC), and AMH. Results: The correlations between AFC, AMH, and age were statistically significant. The lowest value of AMH and AFC were 0.09 ng/ml and 1 respectively. The highest values of AMH and AFC were 15.90 ng/ml and 14 respectively. The study revealed consistent levels of AMH up to 25 years of age, whereas decline in the levels post 25 years. Conclusion: AFC is being used as a marker of ovarian reserve; AMH levels have a positive correlation with AFC and can be measured on any day of the cycle. Anti-Mullerian hormone is believed to be of paramount importance in its role as an endocrine marker for assessment of decline of the ovarian pool in healthy women; thus, it has a potential ability to predict future reproductive lifespan.

Mutations Analysis of the Growth Differentiation Factor 9 Gene in Syrian Women with Ovarian Failure

Premature ovarian failure (POF) is a primary ovarian defect characterized by absent menarche or premature depletion of ovarian follicles. Growth differentiation factor 9 (GDF9) plays an important role in normal growth, differentiation, and proliferation of granulosa cells surrounding the oocyte in the ovary. The present study was to verify the involvement of GDF9 variations in a POF woman in Syrian. POF cases (n= 80) consist of (primary amenorrhea PA n=55) and (secondary amenorrhea SA n=25) compared with 200 controls. All cases with POF had a normal karyotype analysis (46XX). Genetic analysis of the GDF9 gene were showed, four variants in 23 patients. Two of novel variants were observed in two patients, the first was (c.1231G T) was observed in 17 patients and 15 controls. But the variant (c.546G>A) was showed in one patient and in one control. The variant (c.447C>T) appeared associated with the variant (c.546G>A) in 3 patients (c.447C>T)+(c.546G>A) (compound heterozygous genotype). this compound variant didn't detect in the controls. The researchers' findings are consistent with the critical role played by GDF9 in human folliculogenesis. The presence of these variants might indicate a higher risk for POF,.

Mutations Analysis of the Growth Differentiation Factor 9 Gene in Syrian Women with Ovarian Failure

Premature ovarian failure (POF) is a primary ovarian defect characterized by absent menarche or premature depletion of ovarian follicles. Growth differentiation factor 9 (GDF9) plays an important role in normal growth, differentiation, and proliferation of granulosa cells surrounding the oocyte in the ovary. The present study was to verify the involvement of GDF9 variations in a POF woman in Syrian. POF cases (n= 80) consist of (primary amenorrhea PA n=55) and (secondary amenorrhea SA n=25) compared with 200 controls. All cases with POF had a normal karyotype analysis (46XX). Genetic analysis of the GDF9 gene were showed, four variants in 23 patients. Two of novel variants were observed in two patients, the first was (c.1231G T) was observed in 17 patients and 15 controls. But the variant (c.546G>A) was showed in one patient and in one control. The variant (c.447C>T) appeared associated with the variant (c.546G>A) in 3 patients (c.447C>T)+(c.546G>A) (compound heterozygous genotype). this compound variant didn't detect in the controls. The researchers' findings are consistent with the critical role played by GDF9 in human folliculogenesis. The presence of these variants might indicate a higher risk for POF,.

Transcriptional profiling of five isolated size-matched stages of human preantral follicles

Little is known of the early stages of human follicular development and the complex processes that regulate follicular growth. To identify genes of potential importance, we analysed follicle-related transcripts in five populations of isolated size-matched human preantral follicles by microarray analysis. Oocyte-specific genes were found to be the most abundant and differentially expressed transcripts and included germ cell transcription factors LHX8 and SOHLH2 which were significantly down-regulated during preantral follicle development. Differentially expressed genes also included transcription factors of NOTCH signalling, IGF2, orphan nuclear receptor LRH-1, and homeobox gene HOXA7, indicating potentially important regulatory roles for these genes during early human folliculogenesis. We also found that FSHR mRNA and protein were present in the earliest stages of preantral follicles, whereas LHR was absent. In conclusion, our data identify specific oocyte and somatic genes in small human follicles that impact early follicle growth, and provide foundation for further analysis of the signalling pathways involved in early human folliculogenesis.

Expression and regulation of the tumor suppressor, SEF, during folliculogenesis in humans and mice.

Similar expression to FGF (Sef or IL17-RD), is a tumor suppressor and an inhibitor of growth factors as well as of pro-inflammatory cytokine signaling. In this study, we examined the regulation of Sef expression by gonadotropins during ovarian folliculogenesis. In sexually immature mice, in situ hybridization (ISH) localized Sef gene expression to early developing oocytes and granulosa cells (GC) but not to theca cells. Sef was also expressed in mouse ovarian endothelial cells, in the fallopian tube epithelium as well as in adipose tissue venules. SEF protein expression, determined by immunohistochemistry (IHC), correlated well with Sef mRNA expression in GC, while differential expression was noticed in oocytes. High Sef mRNA but undetectable SEF protein levels were observed in the oocytes of primary/secondary follicles, while an inverse correlation was found in the oocytes of preantral and small antral follicles. Sef mRNA expression dropped after pregnant mare's serum gonadotropin (PMSG) administration, peaked at 6-8 h after human chorionic gonadotropin (hCG) treatment, and declined by 12 h after this treatment. ISH and IHC localized the changes to oocytes and mural GC following PMSG treatment, whereas Sef expression increased in mural GC and declined in granulosa-lutein cells upon hCG treatment. The ovarian expression of SEF was confirmed using human samples. ISH localized SEF transcripts to human GC of antral follicles but not to corpora lutea. Furthermore, SEF mRNA was detected in human GC recovered from preovulatory follicles. These results are the first to demonstrate SEF expression in a healthy ovary during folliculogenesis. Hormonal regulation of its expression suggests that SEF may be an important factor involved in intra-ovarian control mechanisms.

Investigation of some genetic variations in BMP15 accompanied with premature ovarian failure (POF) in Syrian women

Abstract Background Premature ovarian failure (POF) is a primary ovarian defect characterized by absent menarche or premature depletion of ovarian follicles. Bone morphogenetic protein 15 (BMP15) is an oocyte-derived growth factor acting as a major player in follicle differentiation in mammals. Mutations in this gene, lead to defective secretion of bioactive dimmers. The present study was to verify the involvement of BMP15 variations in POF women in Syria. Results Genetic screening of 65 patients with POF, 55 primary amenorrhea (PA), 10 secondary amenorrhea (SA) and 100 controls of Syrian origin was done. Five variants in six patients were observed, including two missense substitutions: p. N103S (308A > G), p. A180T (538G > A), one silent variation p.S284S (852C > T), one insertion of three nucleotides (788 insTCT), and one novel intronic variant c. (851 + 13G > A). Variant (308A > G) was observed at the heterozygous state in one patient, variant (538G > A) was found at the heterozygous state in another patient, variant (852C > T) was identified in four patients: two patients at the heterozygous, the third patient had the variant (852C > T) associated with the insertion of three nucleotides [788insTCT]/(+)[852C > T]. This compound variant was detected for the first time in our study, and the fourth patient who had the variant (852C > T) was associated with novel intronic variant (851 + 13G > A) [851 + 13G > A](+)[852C > T]. In our controls the variant (852C > T), was observed in three controls, but the variant (308A > G) was detected in one control. Conclusion Several data indicate that POF has a strong genetic component; these findings are consistent with the critical role played by BMP15 in human folliculogenesis.

The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders.

A large number of studies have contributed to understanding the general mechanisms driving ovarian folliculogenesis in humans and show a complex endocrine dialog between the central nervous system, the pituitary and the ovary, integrated by various intraovarian paracrine messages. The role of intraovarian paracrine regulation has acquired more relevance in the recent years owing to the discovery of previously unknown factors, such as the oocyte-derived bone morphogenetic protein (BMP)15. A thorough literature search was carried out in order to summarize what has been reported so far on the role of BMP15, and the BMP15 paralog, growth and differentiation factor 9 (GDF9), in ovarian function and female fertility. Research articles published in English until March 2014 were included. The biological actions of BMP15 include: (i) the promotion of follicle growth and maturation starting from the primary gonadotrophin-independent phases of folliculogenesis; (ii) the regulation of follicular granulosa cell (GC) sensitivity to FSH action and the determination of ovulation quota; (iii) the prevention of GC apoptosis and (iv) the promotion of oocyte developmental competence. The existence of biologically active heterodimers with GDF9, and/or the synergistic co-operation of BMP15 and GDF9 homodimers are indeed relevant in this context. Experimental disruption of the bmp15 gene in mice resulted in a mild fertility defect limited to females, whereas natural missense mutations in ewes cause variable phenotypes (ranging from hyperprolificacy to complete sterility) depending on a fine gene dosage mechanism also involving GDF9. Strong evidence supports the concept that such a mechanism plays an important role in the regulation of ovulation rate across mammalian and non-mammalian species. Following the discovery of sheep fecundity genes, several research groups have focused on alterations in human BMP15 associated with primary ovarian insufficiency (POI) or polycystic ovary syndrome. Several variants of BMP15 are significantly associated with POI supporting their pathogenic role, but the underlying biological mechanism is still under investigation and of great interest in medicine. BMP15 maps to the Xp locus involved in the determination of the ovarian defect in Turner syndrome and significantly contributes to the determination of ovarian reserve. Pioneering studies in women undergoing controlled ovarian stimulation indicate that BMP15 may represent a marker of ovarian response or oocyte quality. BMP15, an oocyte-derived growth and differentiation factor, is a critical regulator of folliculogenesis and GC activities. Variations in BMP15 gene dosage have a relevant influence on ovarian function and can account for several defects of female fertility. The modulation of BMP15 action may have interesting pharmacological perspectives and the analysis of BMP15 may become a useful marker in IVF procedures. Recent outcomes indicate that the close interactions of BMP15/GDF9 have a critical biological impact that should be taken into account in future studies.

Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro.

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200ng/ml FGF-2 (p<0.05). However, the percentage of apoptotic cells only decreased (p<0.05) in ovarian tissues cultured in 1 or 10ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.

Expression of TGF-beta superfamily growth factors, their receptors, the associated SMADs and antagonists in five isolated size-matched populations of pre-antral follicles from normal human ovaries

In mammals, members of the transforming growth factor-beta (TGF-β) superfamily are known to have key roles in the regulation of follicular growth and development. The aim of the study was to evaluate the expression of TGF-β superfamily growth factors, their receptors, downstream SMAD signalling molecules and TGF-β/bone morphogenetic protein (BMP) antagonists during early human folliculogenesis. Human pre-antral follicles were enzymatically isolated from surplus ovarian tissue obtained from women having ovarian cortical tissue frozen for fertility preservation. A total of 348 human pre-antral follicles, ranging from 40 to 200 µm in diameter, were isolated from ovarian tissue obtained from 15 women, aged 24-34 years. Isolated pre-antral follicles were grouped according to diameter in five size-matched populations spanning the primordial, primary and secondary stage follicles and analysed by whole-genome microarray analysis. Selected proteins/genes were analysed by immunocytochemistry and quantitative RT-PCR. TGF-β superfamily genes with overall highest mRNA expressions levels included growth differentiation factors 9 (GDF9), BMP15, BMP6, BMP-receptor-2 (BMPR2), anti-Müllerian hormone receptor 2 (AMHR2), TGFβR3, inhibin-α (INHA) and intracellular SMAD3 and SMAD4. Moreover, genes which were differentially expressed from the primordial to the late secondary stage follicles included GDF9, BMP15, AMH, INHBB, TGFβR3, SMAD4 and antagonists Follistatin (FST) and GREM1. Collectively, these data indicate that the active TGF-β superfamily pathways in early human folliculogenesis consist of primarily GDF9 combined with possible synergistic effects of BMP15 through the BMPR2 and intracellular activation of SMAD3 and SMAD4, and that AMH and INHBB are engaged in intrafollicular events from the onset of follicular growth. Moreover, the presence of multiple TGF-β/BMP antagonists imply that certain growth factors are subjected to local regulation on different levels that address another important level of intraovarian regulation of follicle development in humans.

Differentiation of Human Embryonic Stem Cells Into Functional Ovarian Granulosa-like Cells

Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1), AMH, the type 2 AMH receptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36%, 12%, and 34%, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.

Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction

The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells

The present study correlated concentrations of activin A and follistatin in follicular fluid (FF) from human small antral follicles to FF concentrations of AMH, inhibin B, progesterone, and oestradiol and to the mRNA expression of FSH-receptor (FSHR), LH-receptor (LHR), AMH-receptor2 (AMHR2), CYP19a, and androgen-receptor (AR) in the corresponding granulosa cells (GC). FF from 144 follicles (3–12mm in diameter) was included whereas mRNA expression profiles were established in GC from 66 of the 144 follicles.Levels of follistatin remained constant in relation to follicular diameter, whereas activin A levels increased in follicles exceeding 10mm in diameter. Levels of activin A and inhibin B showed a highly significant inverse association. Follistatin showed highly significant positive associations with AMH and inhibin B levels and with FSHR and AR gene expression in GC.This study revealed unexpected associations that probably reflect the complicated regulatory mechanisms governing human folliculogenesis.

Variable expression of the Fragile X Mental Retardation 1 (FMR1) gene in patients with premature ovarian failure syndrome is not dependent on number of (CGG)n triplets in exon 1

Increased expression of the Fragile X Mental Retardation 1 (FMR1) gene in blood cells has been claimed to be associated with variable (CGG)(n) triplet numbers in the 5' untranslated region of this gene. Increased CGG triplet numbers, including that of the so-called premutation range (n= 55-200), were shown to have a risk of <26% to impair ovarian reserve leading to primary ovarian insufficiency and premature ovarian failure (POF). DNA and RNA samples were isolated from 74 patients with idiopathic POF to evaluate quantitatively the expression of FMR1 in leukocytes and CGG triplet number on FMR1 gene alleles. mRNA levels were normalized and compared with those of control women. Expression of the encoded protein (FMRP) was analysed by immunohistochemistry on ovarian biopsy tissue sections. A large variance of the FMR1 transcript level was found in the leukocyte RNA samples, but only in patients with POF, and this variability did not correlate to variance of CGG triplet numbers found on both FMR1 alleles (19 < n > 90). During normal folliculogenesis, FMRP is predominantly expressed in granulosa cells. Our data suggest that FMR1 expression during human folliculogenesis is probably a quantitative trait. Proper function of FMRP in granulosa cells seems to depend on an optimal transcript level. All women with CGG triplet numbers outside the range associated with normal folliculogenesis (26 < n > 34) are therefore expected to have a relaxed FMR1 transcription control. FMR1 transcript levels in leukocytes might therefore be diagnostic for altered FMRP levels in granulosa cells, which will affect the process of folliculogenesis.

The role of gonadotropins in the follicular phase

Folliculogenesis in humans is a lengthy process that involves several regulators. Pituitary gonadotropins play crucial roles in the late stages, particularly in the last 15 days of follicle maturation. During the intercycle rise of follicle-stimulating hormone (FSH), selection of the dominant follicle takes place. This is a complex process that also involves locally produced substances. At the same time, luteinizing hormone (LH) stimulates the synthesis of androgens, which serve as the substrate for the production of estrogens. During the second half of the follicular phase, the follicle becomes dependent on LH. Induction of multiple follicular development by exogenous FSH results in a marked suppression of endogenous LH. For normal follicle maturation, both an LH threshold and an LH ceiling have been considered. In the context of an in vitro fertilization program, application of protocols for ovarian stimulation that will prevent the marked suppression of endogenous LH secretion might provide a better approach to treatment optimization.

Human sFRP4 expression is down-regulated by hCG in granulosa cells

OBJECTIVE: Ovarian follicular development and ovulation in mammals is a highly regulated process. Genes involved in this processes, have been the subject of growing interest. WNT signaling was shown to be involved in reproduction of animal models. Among the WNT components, the Secreted Frizzled-Related Protein 4 (SFRP4) was shown to be expressed in a hormonal dependent manner. However, nothing is known about its expression in human folliculogenesis.

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9. rhBMP-15, in particular, is an excellent substrate for G-CK. In each protein a single residue is phosphorylated by G-CK, corresponding to the serine residue at the sixth position of the mature region of both rhBMP-15 and rhGDF-9, whose phosphorylation is required for biological activity.

Expression of forkhead transcription factors in human granulosa cells

Members of the forkhead box O1 (FOXO) family of transcription factors are expressed in granulosa cells during various stages of follicle development, and evidence from rodent and other model systems suggests that they may be involved in regulating follicular activation and oocyte maturation. In this report, we show that FOXO1, FOXO3, and FOXO4 are expressed in human luteinized mural granulosa cells, which may suggest that these transcription factors are also involved in human folliculogenesis and luteinization.

Human ovarian biopsies as a viable source of pre-antral follicles

Our knowledge of the early pre-antral stage of human folliculogenesis is still poor due to small follicle size and the limited availability of human ovarian tissue. Our aim was to determine the utility of ovarian biopsy for pre-antral follicle research. Ovarian cortical biopsies were obtained from women (28-46 years old) undergoing elective Caesarean sections or total abdominal hysterectomy/bilateral salpingo-oophorectomy for a variety of benign gynaecological conditions. Follicle isolation and staging was performed according to a well-established protocol, involving enzymatic digestion, isolation using fine needles and image capture analysis software. RNA was also isolated for reverse transcription. More than 351 follicles were retrieved from 19 patients and 249 were classifiable into follicle stages: 80 primordial, 53 transitional, 82 primary, 26 secondary and 8 multilaminar. All samples, except two from women aged over 40 years, yielded follicles. The average yield of classifiable follicles/patient was 13. There was an age-related decline in mean follicle numbers/patient (r(2) = -0.986). Microgram quantities of complementary DNA per follicle were synthesized. Despite the heterogeneous distribution of follicles throughout the cortex and the significant age-related decline in the numbers of follicles retrieved, biopsy samples of ovarian cortical tissue provide a useful source of pre-antral follicles. This, coupled with the sensitivity of genomic technology, makes this method a viable research approach.