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BRIEF REPORT |
Institute of Reproductive and Developmental Biology (L.J.W., S.A.S., K.H., S.F.), Imperial College London, and Reproductive Medicine Unit (R.A.M., G.H.T., S.A.L.), Hammersmith Hospital, London W12 0NN, United Kingdom; and Department of Mathematics (J.S.), Imperial College London, London SW7 2AZ, United Kingdom
Address all correspondence and requests for reprints to: Stephen Franks, Imperial College School of Medicine, Institute of Reproductive and Developmental Biology, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom. E-mail: s.franks{at}imperial.ac.uk.
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Abstract |
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Objective: The aim of this study was to investigate the possibility that loss of preantral follicles by atresia is reduced in PCOS, leading to prolonged follicle survival.
Design: We compared follicle growth in normal and polycystic ovaries using cultures of small ovarian biopsies.
Setting: Tissue samples were obtained at routine laparoscopy from 12 patients with anovulatory PCOS and 16 controls and processed in an ovarian physiology laboratory.
Main Outcome Measures: We performed morphometric analysis of follicle population in tissue fixed at time of biopsy (d 0) or after 5, 10, or 15 d in culture. Analyses included assessment of follicle and oocyte diameter, number and proportion of primordial and growing follicles, and number and proportion of atretic follicles.
Results: In tissue fixed on d 0, the proportion of healthy growing follicles was, as expected, greater in ovaries from PCOS patients than in normal ovaries (64 vs. 28%; P = 0.0005), but there were no differences between PCOS and normal tissue during culture. The rate of atresia throughout the period of culture in follicles was, however, significantly lower in PCOS tissue (P < 0.0001). After culture, 80% of follicles in normal ovarian tissue were atretic compared with 53% in PCOS biopsies.
Conclusion: Follicles from polycystic ovaries demonstrate a decreased rate of atresia in culture, suggesting a mechanism for maintaining a larger follicle pool throughout reproductive life.
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Introduction |
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Little was known, however, about earlier (preantral) follicle development in polycystic ovaries. Using small cortical biopsies obtained at routine laparoscopy, we found an overall increase in the density of preantral follicles and we [and subsequently the group of Erickson and colleagues (4)] confirmed the increased density of primary follicles in polycystic ovaries first observed by Hughesdon (5, 6). Furthermore, we were able to demonstrate for the first time that, in tissue from both anovulatory and ovulatory women with polycystic ovaries, the proportion of primordial (resting) follicles was reduced and the proportion of growing follicles reciprocally increased compared with normal ovaries. It might be expected, therefore, that there would be premature exhaustion of the follicle pool in PCOS, but firstly, we found no reduction in the density of primordial follicles in the adult polycystic ovary compared with normal ovaries (6), and secondly, the age of menopause in women with a history of PCOS appears to be similar to that in a control population (7).
The most obvious explanations for the lack of premature depletion of the follicle pool are that: 1) there are more primordial follicles to start with in the polycystic than in the normal ovary; 2) there is reduced loss of follicles by atresia (i.e. increased survival) during folliculogenesis; and 3) the dynamics of follicle growth (e.g. the rate of progression through the various stages of follicle development) are different between normal and polycystic ovaries (6). These are not mutually exclusive possibilities, but in this study we set out to test the second hypothesis, i.e. that preantral follicles from polycystic ovaries were less likely to be lost by atresia than similar follicles from normal ovaries. Preantral follicle growth cannot easily be studied in vivo, and we therefore used a tissue culture method, well established in our laboratory (8), to examine survival of follicles in vitro over a 15-d period.
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Patients and Methods |
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Patients were recruited from the Hammersmith, Queen Charlotte’s and Chelsea, and St. Mary’s Hospitals in London. Cortical ovarian biopsies were obtained from 12 anovulatory women with PCOS [31.3 ± 1.4 yr (mean ± SE)] and 16 with normal ovarian morphology (32.8 ± 1.3 yr) undergoing laparoscopy for diagnosis or treatment of delayed conception or laparotomy for benign gynecological disease. All of the women with polycystic ovaries had oligomenorrhea or amenorrhea, were known to be anovulatory, and had clinical and/or biochemical evidence of hyperandrogenism [i.e. they fitted the "classic" definition of PCOS (9)]. All of those with normal ovaries had ultrasound and/or endocrine evidence of ovulation (i.e. normal midluteal progesterone concentration). All patients gave informed consent, and the study was approved by the Research Ethics Committees of Imperial College London/Hammersmith, Queen Charlotte’s & Chelsea, and Acton Hospitals and the St. Mary’s Hospital National Health Service Trusts.
Tissue collection and preparation
Cortical biopsies (maximum volume, 5 mm3) were obtained from the surface of the ovary opposite the hilum either using a biopsy device (Cook UK Limited, Hertfordshire, UK) during laparoscopy or with a scalpel during a laparotomy, as previously described (6). In women with regular cycles, the biopsy was, in most cases, taken in the midluteal phase. If a dominant follicle or corpus luteum was present, the contralateral ovary was sampled.
Ovarian tissue was collected into HEPES-buffered MEM (Life Technologies, Inc., Invitrogen Life Technologies, Paisley, UK) for immediate transfer at 37 C to the laboratory, where it was divided into pieces of approximately 1 mm in diameter under sterile conditions. Samples were anonymized and coded so that the observer (L.J.W.) was unable to identify the type of ovary from which the biopsy was taken until completion of the data collection.
Tissue culture
At least one piece of each biopsy was fixed immediately in Bouin’s solution and stored in alcohol as previously described (6); this was designated d 0. The remaining pieces (approximately 1 mm3) were put into culture for up to 15 d. For each patient, three wells of a 24-well culture plate (Nunclon, Roskilde, Denmark) were prepared as follows: a culture plate insert (Millicell-CM, 12 mm diameter, 0.4-µm pore size; Millipore, Bedford, MA), which has a semipermeable membrane at its base, was placed into each well that was to be used and coated with artificial extracellular matrix (Matrigel; Becton Dickinson, Bedford, MA) as previously described (8). The Matrigel was diluted 1:3 with serum-free Earle’s balanced salt solution (Life Technologies, Inc.), and 100 µl was laid onto the base of each well insert. Culture medium was made up from 
-MEM (Life Technologies, Inc.), supplemented with human serum albumin (2.5% final concentration, Zenalb 20; Bio Products Laboratory, Elstree, UK), 1% insulin/transferrin/selenium (Life Technologies, Inc.; 10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 ng/ml sodium selenite), 300 mIU/ml FSH, (Puregon; Organon, Cambridge, UK), and antibiotics (antibiotic/antimycotic solution, Life Technologies, Inc.; 50 U/ml penicillin G, 50 µg/ml streptomycin sulfate, 0.125 µg/ml amphotericin B). Preconditioned medium (0.1 ml) was placed into each insert and another 0.3 ml was placed around the outside (8). Up to four pieces of tissue were randomly assigned to each of the three inserts, and the plates were incubated at 37 C under 5% CO2 for 5, 10, or 15 d.
Medium was completely removed every 2 d, and tissue was washed with 0.4 ml of fresh medium, which was discarded and then replaced. After 5, 10, and 15 d in culture, all the tissue pieces from one well were removed, fixed in Bouin’s solution, and stored in 70% alcohol. Tissue from d 0, 5, 10, and 15 was then dehydrated, blocked in paraffin wax, sectioned serially at 5 µm, mounted on microscope slides, and stained with hematoxylin and eosin.
Assessment of follicle development and survival
Follicle stage and survival were assessed microscopically on serial sections (6, 10). Coded anonymized slides were examined on an E600 microscope (Nikon UK Ltd., Kingston-Upon-Thames, UK). Follicles were classified as healthy or atretic. Atretic follicles were defined by a degenerated oocyte nucleus, uneven or folded nuclear membrane, vacuoles in the oocyte, or pyknotic nuclei in several granulosa cells (6). Each follicle was examined in every section in which it appeared and matched with the same follicle on adjacent sections to avoid double counting, thus ensuring that each and every follicle was only counted once, regardless of its size. Using a x60 objective, images of every follicle at its widest point were captured on a DXM 1200 digital camera (Nikon UK). For each healthy follicle, the stage of development was recorded, and the maximum diameter was measured using the Lucia image analysis program (Nikon) (see below).
Classification of follicles was as previously described (6): primordial (resting) follicles comprised an oocyte enclosed by a single layer of granulosa cells in which more than 50% were flattened "pregranulosa" cells; primary follicles were defined by a single layer of granulosa cells where more than 50% had undergone transformation to a cuboidal phenotype, and secondary follicles had two or more layers of granulosa cells. A follicle was defined as "growing" if it was at the primary stage or beyond. Follicle and oocyte diameters were measured only in healthy follicles, at the center of the follicle as previously described (10). This was taken to be the section containing the oocyte nucleolus if present; otherwise measurements were taken at the widest part identified. Follicle diameter was recorded from edge to edge of granulosa cell basement membrane, or from the outside edge of the theca cell layer when present. Oocyte diameter was recorded from edge to edge of the oocyte membrane. Two perpendicular diameters were recorded for each, and the mean of each pair was used for statistical analysis. A second observer (S.A.S.) reanalyzed a number of sections to confirm accuracy and reproducibility of the analysis.
Statistical analysis
Proportions of atretic follicles were calculated as a percentage of all (healthy plus atretic) follicles. Healthy follicles included both primordial and growing follicles. Proportions of growing follicles before and during culture were calculated as a percentage of healthy follicles only. Estimates of proportions and the comparison of proportions between different types of ovary need to take account of both the number of follicles in each sample and the variability between samples (i.e. between subjects). For this reason we analyzed proportions using binomial regression allowing for overdispersion (Stata 8 program; Stata Corporation, College Station, TX), as previously described (6). Where appropriate, the change in the proportion of follicles at any particular stage throughout the culture period was compared in normal and polycystic ovaries by regression analysis using a general linear model, binomial family, identity link. The effects of time in culture on follicle and oocyte diameters were compared in normal and polycystic ovaries by the Kruskal-Wallis test (nonparametric ANOVA). Where P was less than 0.05, paired comparisons were made using the Mann Whitney U test. Apart from the regression analyses, all comparisons were made using GraphPad Instat version 3.0 for Macintosh (GraphPad Software Inc., San Diego, CA).
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Results |
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The diameter of healthy follicles from both polycystic and normal ovaries increased significantly during culture. In tissue fixed before culture, median follicle diameters were similar in the two groups, but the changes in follicle diameter that occurred during the culture periods were different in the two groups (P < 0.0001, Kruskal-Wallis test) (Fig. 1A
). This difference was due principally to the greater follicle diameter after 5 d in culture in tissue from PCOS subjects.
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The number of healthy primordial and growing follicles in each biopsy at d 0 (i.e. in tissue fixed without culture) and at 5, 10, and 15 d of culture are shown in Table 1 and proportions are shown in Fig. 1B
. As expected from previous studies (6, 10) the proportion of healthy growing follicles at d 0 (i.e. percentage of total healthy follicles that are at primary stage or above) was greater in PCOS than in normal ovaries (P = 0.0005). After 5 d of culture, the majority of follicles had started to grow and there were no differences between types of ovary during the subsequent 10 d of culture (Fig. 1B). At each time point between d 5 and 15 d in culture, 60–70% of healthy follicles were at the primary stage, and 20–30% were classified as secondary or (occasionally) multilayered preantral follicles, but, as with the overall proportion of growing follicles, there were no significant differences between normal and PCOS ovaries.
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The number of healthy and atretic follicles in each biopsy and the proportions (percentage) of atretic follicles before culture or after 5, 10, or 15 d in culture are shown in Table 2 and (proportions) in Fig. 1C. In both types of ovary, the proportion of atretic follicles increased during culture. However, there was a striking difference between normal and PCOS ovarian tissue, so that in normal tissue, there was a much steeper increase in the proportion of atretic follicles with time in culture (i.e. a significant difference between the slopes of the two curves; intercepts P = 0.017 and slopes P = 0.00001). After 15 d in culture, 53% of follicles in PCOS tissue were atretic compared with 80% in normal ovarian tissue pieces (Fig. 1C).
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Discussion |
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Primordial follicles can initiate growth in vitro in a number of different species, as shown using organ culture in the mouse (11) and tissue-slice culture for pieces of ovarian cortex from the cow (12), goat (13), baboon (14), and human (15). There are advantages in using tissue or whole organ culture rather than isolated follicles; the structural integrity of the follicle is maintained, as well as the preservation of interactions both between neighboring follicles and with the surrounding stroma. This system was therefore chosen as a model to examine in vitro the initiation and early stages of growth of small preantral follicles within tissue from normal and polycystic ovaries.
Culture medium was supplemented with FSH and growth factors, and the rate of initiation of follicle growth in culture is predictably greater than in vivo (8, 16). The medium chosen for the model was designed to support maximal follicle growth and survival over a lengthy culture period. The majority of follicles were recruited into the growing phase after only the first 5 d in culture. The rapidity with which this transition occurred almost certainly reflects the nature of the culture conditions rather than the velocity of natural follicle development (17), as has been observed in previous studies of cultured ovarian tissue from several species (18). Although the evidence is scant, the early stages of preantral follicle development in vivo probably take several months (17). The complex medium -MEM supports follicle growth over extended periods of culture more successfully than a simple medium, and the addition of FSH reduces atresia (8). Growth and survival of preantral follicles can be influenced by FSH but are not dependent on it. Human serum albumin supplemented with insulin, transferrin, and selenium (the latter two being free-radical scavengers) also supports growth and survival more effectively than human serum (16). Follicle survival is also supported by the use of an artificial extracellular matrix (15). Nevertheless, despite being exposed to the same in vitro environment of hormones and growth factors, follicles from polycystic ovaries demonstrated a lower rate of atresia and hence prolonged survival [perhaps because of an extended residence in the primary pool (4, 6)] compared with those from normal ovarian cortex.
The mechanism underlying the lower rate of atresia (and, by implication, prolonged survival) of follicles from polycystic ovaries remains to be determined, but it is likely that endogenous growth factors within polycystic ovary follicles play a part. Whatever the reason for enhanced survival, this finding helps to explain why, despite increased recruitment of follicles from the primordial pool into the growing phase, there is no evidence for premature depletion of follicles and reduced reproductive lifespan in women with PCOS.
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Footnotes |
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None of the authors has anything to declare with regard to relevant conflicts of interest.
First Published Online March 6, 2007
Abbreviation: PCOS, Polycystic ovary syndrome.
Received July 5, 2006.
Accepted February 22, 2007.
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  S. Franks, J. Stark, and K. Hardy Follicle dynamics and anovulation in polycystic ovary syndrome Hum. Reprod. Update, May 22, 2008; (2008) dmn015v1. [Abstract] [Full Text] [PDF]
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