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Hum. Reprod. Advance Access originally published online on December 22, 2007
Human Reproduction 2008 23(3):600-605; doi:10.1093/humrep/dem390
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Suman Rice1,2,4, Kamal Ojha3 and Helen Mason1,2

1 Basic Medical Sciences, Jenner Wing, St George’s University of London, Cranmer Terrace, London SW17 0RE, UK 2 Clinical Development Sciences, St George’s University of London, Cranmer Terrace, London SW17 ORE, UK 3 Department of Obstetrics and Gynaecology, St George’s University of London, Cranmer Terrace, London SW17 ORE, UK

4Correspondence address. Tel: +44-2087251155; Fax: +44-2087252993; E-mail: srice{at}sgul.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
BACKGROUND: 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.

METHODS: 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.

RESULTS: 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 (r2 = –0.986). Microgram quantities of complementary DNA per follicle were synthesized.

CONCLUSIONS: 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.

Key words: human/ovary/biopsy/pre-antral/follicles


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
The underlying basis of female reproduction is the growth and development of the egg-containing structure, the follicle. Our understanding of folliculogenesis informs infertility treatment in women and increases reproductive efficiency of domestic animals and endangered species. Much of the research in this area has been on the antral stages of follicle development and has focused on understanding steroidogenesis in theca and granulosa cells, the arrest and resumption of meiosis in the oocyte, maturation of the oocyte and manipulation of the menstrual cycle to control follicle growth. Our knowledge of the early pre-antral stage is by comparison poor, particularly in humans.

Despite some evidence to the contrary (Johnson et al., 2004Go, 2005Go), it is likely that women are born with their full complement of oocytes, enclosed in a single layer of 4–6 flattened granulose cells and constituting a primordial follicle. These primordial follicles remain in a suspended state until, following puberty, a signal causes a select cohort to initiate growth and development (Gougeon, 1996Go). It is recognized that this pre-antral pool is a potentially valuable resource particularly with respect to fertility preservation (Picton and Gosden, 2000Go; Oktay and Sonmezer, 2004Go; Lee et al., 2006Go). Key to the utilization of this pool is an increased understanding of the processes by which initiation of follicle growth occurs and the factors determining the subsequent survival and growth of the follicle versus atresia. This information would, for example, permit an improvement in culture conditions for follicles and oocytes matured in vitro, particularly those which are grown from the very immature stages. Likewise, this knowledge is also important for assessing the efficacy and safety of ovarian cryopreservation techniques performed to maintain the fertility of cancer patients, and will be beneficial for improving our understanding of medical conditions in which folliculogenesis is disordered. For example, it is becoming clear that the characteristic morphology of the polycystic ovary has its origins in disordered early follicle growth (Hughesdon, 1982Go; Webber et al., 2003Go; Maciel et al., 2004Go).

Human ovarian cortical material is generally obtained from one of two sources: cortical biopsies taken during either Caesarean section or laparoscopic surgery for infertility investigations/crypopreservation of ovarian cortex, or from patients undergoing oophorectomy for various gynaecological disorders. The practical difficulties in studying pre-antral folliculogenesis are the limited amount of human ovarian tissue available, the small size of the follicles (ranging from 30–120 µm) and the random distribution of follicles within the ovarian cortex. These problems have hindered researchers in this area; however, improvements in RNA isolation and PCR amplification techniques mean that even single follicles have the potential to yield informative data.

Our aim was to determine the utility of ovarian biopsy for pre-antral follicle research. We present data on the rates of retrieval of pre-antral follicles from small cortical pieces and the number of follicles obtained at each developmental stage in relation to the age of the woman.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
This study was given ethical approval and all patients gave informed consent. Ovarian cortex was collected at St George’s Hospital, London, by ovarian cortical biopsy from women undergoing elective Caesarean sections (C-sections) (n = 12) or total abdominal hysterectomy (TAH)/bilateral salpingo-oophorectomy (BSO) (n = 7) for a variety of benign gynaecological conditions. The ages of the women ranged from 28 to 46 years for the TAH/BSO group and 30 to 40 years for the C-section group. Patient details are listed in Table I.


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  		Table I. Clinical details of patients and number of follicles retrieved from the ovarian tissue biopsy samples.

 
Follicle isolation was performed according to a well-established protocol with some modifications as previously described (Rice et al., 2007aGo). Briefly, the ovarian tissue matrix was softened using a 30–40 min enzymatic digestion at 37°C with gentle shaking in Krebs–Ringer bicarbonate buffer containing 15 mM HEPES, 180 U/ml deoxyribonuclease I and 1240 U/ml collagenase (all chemicals from Sigma, Poole, Dorset). Follicles were manually dissected free of the partially digested ovarian stroma (Fig. 1a) using fine acupuncture needles under an inverted microscope with Hoffman optics (Leica, UK). Individual follicles were transferred to fresh drops of media using pulled pipettes and photographed using a digital camera (DXM1200) attached to an inverted microscope (Eclipse TE300) with phase contrast and Hoffmann optics (Nikon UK Ltd, Kingston-upon-Thames, UK). The stage of development and an assessment of the morphology of each follicle were made using the image capture analysis software ACTTM (Nikon UK Ltd).


Figure 1
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  		Figure 1: Isolation of follicles from human ovarian cortex biopsy

(a) Partially digested ovarian cortex with stroma and follicles within (indicated by arrowheads), (b) cluster of pre-antral follicles, (c) primordial follicle with flattened granulosa cells. BB, Balbiani’s vitelline bodies (or paranuclear complex), (d) Transitional follicle with partially flattened granulosa cells (fgc) and expanded granulosa cells (egc), (e) primary follicle with one layer of expanded gc, (f) secondary follicle with two layers of gc, (g) follicle showing the start of antrum (A) formation around the eventual cumulus–oocyte complex (COC) (Scale bar = 20 µm).

 
Follicle stages were classified using the following criteria: primordial (one layer of flattened granulosa cells) (Fig. 1c), transitional (one layer of mixed flattened and cuboidal granulosa cells) (Fig. 1d), primary (one layer of fully expanded, cuboidal granulosa cells) (Fig. 1e), primary–secondary transitional (intermediate between one and two layers of granulosa cells), secondary (two distinct layers of expanded, granulosa cells) (Fig. 1f) and multilaminar (multiple layers of granulosa cells without an antrum) (Gougeon, 1996Go; Picton, 2001Go). For this study, the primary–secondary transitional stage was classified as secondary. Owing to the size of the tissue pieces and the fact that they were taken from the outer cortex, early stage antral follicles were rarely retrieved therefore for the purpose of this study only follicles up to the multilaminar stage were included.

RNA extraction
Individual follicles were washed in phosphate-buffered saline/bovine serum albumin to remove any adherent stromal cells, and then placed by direct observation into RNase-free sterile 0.5 ml Eppendorf tubes (Ambion) containing 10 µl of lysis buffer (0.5% IGEPAL CA-630, 10 mM Tris (pH 8.0), 10 mM NaCl and 3 mM MgCl2 (all from Sigma) (Gilliland et al., 1990Go). The lysed follicles were snap-frozen in liquid nitrogen and stored at –80°C prior to reverse transcription using the entire lysate (10 µl total volume). The integrity of the reverse transcription was checked using a nested PCR assay for β-actin (Rice et al., 2007aGo).

Statistics
All data are presented as mean ± SEM. Statistical differences in the number of follicles retrieved per patient between the two types of samples (OV and CB) were compared using {chi}2 test. The difference in distribution of follicle stages between the age groups was compared using analysis of variance (ANOVA). Significance was set at P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
Tissue was collected from the ovaries of seven patients undergoing TAH/BSO (OV) and from 12 patients having cortical biopsy at elective C-sections (CB). The mean age of all patients was 36.9 years, and mean age was similar in the two groups; 37.4 years in the OV group compared with 36.6 years in the CB group. More than 351 follicles were retrieved, and of these, 249 were classifiable into a follicle stage from primordial to multilaminar. The remaining follicles could not be classified as they were either atretic, were denuded oocytes or were clustered in groups which could not be separated (Fig. 1b, Table I).

From the OV samples (n = 7), 84 follicles were retrieved and 59 of these were classifiable into the five stages described earlier. From the CB samples (n = 12), more than 267 follicles were retrieved, of which 190 were classifiable (Table I). The average yield of classifiable follicles per patient was 13.1, with significantly more follicles/patient retrieved from the CB samples (15.8) compared with the OV samples (8.4) (P = 0.01, {chi}2 test). The 249 follicles used from both sources of tissue constituted 80 primordial (32%), 53 transitional (21%), 82 primary (33%), 26 secondary (10%) and 8 multilaminar (3%) follicles. The percentage of primordial (36%) and primary (34%) follicles in the CB group was approximately equal (with 16% transitional), whereas in the OV group, there were less primordial (19%) and more transitional (37%) and primary (31%) follicles (Table II).


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  		Table II. Distribution of follicle numbers classified by follicle stage from each patient.

 
The total yield of classifiable follicles was then analysed according to the age of the patients which was stratified into three groups: under 30 years (≤30, n = 3); 31–39 years (31–39, n = 11) and over 40 years (≥40, n = 5). A total of 71 follicles were retrieved from the ≤30 group, 167 from the 31–39 group and 11 from the patients of ≥40. There was a wide variation in total number of follicles retrieved from the 31–39 group with total numbers from the ≤30 and ≥40 group tending to cluster closer together (Fig. 2). There was a significant inverse correlation between age and mean follicle numbers/patient from 23.7 ± 1.7 (≤30), reducing to 15.2 ± 3.1 (31–39) and 2.2 ± 0.95 in those patients over 40 years (values expressed as mean ± SEM) (r2 = –0.986).


Figure 2
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  		Figure 2: Graph showing the distribution of the total numbers of classifiable follicles from all samples relative to the age (years) of the patient

There is a wide variation in follicle numbers in the 31–39 year group, whereas the total follicle numbers in the other two categories tend to cluster together. There is a significant inverse correlation with age (r2 = –0.986)

 
There was a significantly lower number of follicles of all stages per patient in the oldest group compared with the younger groups (ANOVA P = 0.01). There was a trend (not statistically significant) for a greater number of primordial follicles (expressed as a percentage of the total) collected from the ≤30 group (35%) compared with the 31–39 group (31%) and the ≥40 group (27%). Conversely, more primary follicles tended to be obtained from the oldest group (45.5%) compared with the 31–39 group (33.5%) and the ≤30 group (29.6%) (not statistically significant).

It was not possible to measure RNA from the individual lysed follicles but using the Nanodrop Spectrophotometer (Nanodrop Technologies, USA) complementary DNA (cDNA) levels of individual follicles were determined. The average cDNA level measured per follicle was 1.3 µg/µl with a distinct increase from the primordial to the transitional stage and onwards (data not shown). A test for integrity of the reverse transcription reaction using a nested PCR assay showed that 91% of follicles were positive for β-actin expression, with approximately equal numbers of positive results in the OV and CB samples (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
We have shown that in spite of the well-documented heterogeneity in distribution of pre-antral follicles in the ovarian cortex (Lass et al., 1997Go; Kohl et al., 2000Go; Qu et al., 2000Go; Poirot et al., 2002Go; Schmidt et al., 2003Go), cortical biopsies can provide a useful source of pre-antral follicles for research purposes. Unlike other studies in which ovarian biopsies were used to estimate follicular reserve by counting follicles in tissue slices after fixation, our rationale was to obtain whole intact single follicles for use in research. Once the tissue has been enzymatically digested, it begins to deteriorate fairly rapidly, and hence the follicle isolation must be performed as rapidly as possible. Even given this time constraint, cortical tissue proved to be highly productive, with all but 2 out of the 19 samples yielding follicles. The majority (70%) of the pre-antral follicles retrieved were classifiable into their different stages.

As expected, there was a significant age-related decline in the number of follicles retrieved per patient, with two of the samples from patients over 40 years old yielding no follicles at all. This reduction in follicle yield correlates well with the accelerated loss in follicles shown to occur in women from 38 years of age onwards (Faddy et al., 1992Go; Gougeon et al., 1994Go). Interestingly, there was a wide variation in the total number of follicles obtained from patients in the 30–39 year group which in practical terms made it difficult to predict the follicle yield in this group. This unpredictability in numbers is a reflection of the heterogeneous distribution of primordial follicles within the ovarian cortex (Kohl et al., 2000Go; Qu et al., 2000Go; Poirot et al., 2002Go; Schmidt et al., 2003Go), and has serious implications for the accuracy of using cortical biopsies to assess ovarian reserve and for the successful harvesting and maturation of pre-antral follicles from fresh/cyropreserved ovarian cortical samples (Lass, 2004Go; Revel and Schenker, 2004Go).

Biopsies obtained during C-section yielded many more follicles/patient than samples taken from oophorectomys. In the first instance, it appeared that this difference was not due to age since the mean age of the two groups was similar. However, there were two young women (≤30 years) in the OV group and their cortical samples yielded most of the follicles in this group. Without these two women, the mean age of this group is 41 years, with a much lower follicle yield (2.1 follicles/patient) as would be predicted.

Overall, there were equal distributions of primordial and primary follicles from all the samples. When the data were analysed by age, the ≤30 group had more primordial than primary follicles, the numbers were more or less equal in the 31–39 age group (with a slight bias towards the primary follicles), whereas there were significantly more primary follicles than primordial follicles in the ≥40 group. This finding is supported by other studies showing that there is a greater proportion of growing follicles in samples from older women compared with younger women which also reflects the accelerated decrease in the primordial pool with age (Block, 1952Go; Gugeon et al., 1994Go; Schmidt et al., 2003Go).

In spite of the low numbers of follicles obtained from some samples, a key point is that with improvements in genomic technology even single follicles could in theory yield informative data. Microgram quantities of cDNA per reverse transcribed follicle were obtained, which was well within the sensitivity range of the nested primers used in a recent study from our group analysing the stage-specific expression of receptors in individual, isolated human follicles (Rice et al., 2007aGo). In addition, we have also successfully amplified RNA from small pools of follicles (n = 3) at the primordial to secondary stage and from single multilaminar follicles and used microarray hybridization to analyse differential gene expression during pre-antral folliculogenesis (Rice et al., 2007bGo).

We have shown that ovarian cortical biopsies, particularly from younger women, provide a useful source of pre-antral follicular material for research purposes, since the improved sensitivities of downstream genomic technology can be applied to even a limited amount of starting material. It is also reassuring that minor resections of one ovary have been shown to have no affect on the timing of menopause (Faddy et al., 1992Go) thereby making this a valid research option.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
The Wellcome Trust (WT073572MA, 081420/Z/06/Z to S.R); St George’s Charitable Foundation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
We would like to thank the patients who kindly donated tissue for this study and the surgeons and midwives of the Obstetric and Gynaecology Department of St George’s Hospital, London, for their time and help in collecting biopsies.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
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Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod (1992) 7:1342–1346.[Abstract/Free Full Text]

Gilliland G, Perrin S, Bunn HF. PCR protocols: a guide to methods and applications. (1990) London, UK: Academic Press Ltd.

Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev (1996) 17:121–155.[Abstract/Free Full Text]

Gougeon A, Ecochard R, Thalabard JC. Age-related changes of the population of human ovarian follicles: increase in the disappearance rate of non-growing and early-growing follicles in aging women. Biol Reprod (1994) 50:653–663.[Abstract]

Hughesdon PE. Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called "hyperthecosis". Obstet Gynecol Surv (1982) 37:59–77.[Medline]

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Lass A, Silye R, Abrams D-C, Krausz T, Hovatta O, Margara R, Winston RML. Follicular density in ovarian biopsy of infertile women: a novel method to assess ovarian reserve. Hum Reprod (1997) 12:1028–1031.[Abstract/Free Full Text]

Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, Beck LN, Brennan LV, Oktay K. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol (2006) 24:2917–2930.[Abstract/Free Full Text]

Maciel GAF, Baracat EC, Benda JA, Markaham SM, Hensinger K, Chang JR, Erickson GF. Stockpiling of transitional and classic primary follicles in ovaries of women with polycystic ovary syndrome. JCEM (2004) 89:5321–5327.[Abstract/Free Full Text]

Oktay K, Sonmezer M. Ovarian tissue banking for cancer patients: fertility preservation, not just ovarian cryopreservation. Hum Reprod (2004) 19:477–480.[Abstract/Free Full Text]

Picton HM. Activation of follicle development: the primordial follicle. Theriogenology (2001) 55:1193–1210.[CrossRef][Web of Science][Medline]

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Rice S, Ojha K, Whitehead S, Mason H. Stage-specific expression of androgen receptor, follicle-stimulating hormone receptor, and anti-Müllerian hormone type II receptor in single, isolated, human pre-antral follicles: relevance to polycystic ovaries. JCEM (2007) a 92:1034–1040.[Abstract/Free Full Text]

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Submitted on July 12, 2007; resubmitted on October 29, 2007; accepted on November 14, 2007.


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