Department of Obstetrics and Gynaecology, Imperial College School of Medicine at St Mary's, London W2 1PG, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: human granulosa/human theca/ovarian morphology/ovarian steroids/pelvic congestion
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The causal relationship between venous stasis and chronic pain is unknown. However, several lines of evidence suggest that ovarian hormones may play a central role in pathogenesis. The condition is found exclusively in women of reproductive age (Taylor, 1954; Beard et al., 1988
; Steege, 1993). Suppression of cyclical ovarian activity, either medically (Reginald et al., 1989
; Farquhar et al., 1989
) or following bilateral oophorectomy (Beard et al., 1991
; Reid and Gangar, 1994
), produces a significant reduction in both venous stasis and chronic pain. In animal studies, changes in uterine blood flow have been noted just before and during oestrus (or ovulation) and during the luteal phase (Greiss and Anderson, 1969
). This may be either a direct effect of oestrogen on the pelvic vasculature (Greiss and Anderson, 1970
) or mediated through the release of prostaglandins and/or nitric oxide (Van Buren et al., 1992
). In the human ovary, where data are limited, selective ovarian vein dilatation has been noted on the side of the developing follicle and venous dilatation increases during the follicular phase to peak at the time of ovulation (Reginald et al., 1986
). In some preliminary studies, no significant differences were found in peripheral gonadotrophins, ovarian steroid concentrations or gonadotrophin pulsatility in women with pelvic congestion compared with asymptomatic controls, matched for ovarian morphologyan observation which weakens the putative link between ovarian hormones and congestion.
An alternative view is that pelvic congestion arises as a result of localized disturbances in ovarian steroid output. The ideal test of this hypothesis would be to take blood samples from the ovarian vein, in both control and affected women, precisely timed according to day of cycle. Such a study has huge practical limitations. An alternative, but more feasible approach is to study ovarian morphology and steroidogenic function in these women. Women with pelvic congestion have been found to have an increased incidence of abnormal ovarian morphology compared with normal controls (Taylor, 1949; Adams et al., 1990
). Using ultrasound as a primary means of defining morphology, it was found (Adams et al., 1990
) that over 50% of women with pelvic congestion had cystic changes in their ovaries. These ovaries fell into two morphologically distinct groups. The first showed the classic polycystic ovary pattern of increased ovarian volume and stroma, with a peripheral distribution of 10 or more small follicles between 2 and 8 mm in diameter. The second were of normal volume, but contained clusters of larger follicles dispersed throughout the stroma and were quite distinct from multicystic ovaries, typically associated with weight loss-related amenorrhoea, in which ovarian and uterine volume are reduced (Adams et al., 1985
). In a parallel morphological study of ovaries removed from women at hysterectomy for chronic pain due to pelvic congestion, two types of ovaries with cystic changes were similarly identified. The first had the classic polycystic ovary characteristics described above, while the second were clearly distinct from both normal and polycystic ovaries by the presence of numerous follicles, 711 mm in diameter throughout a stroma which was of normal density. The latter ovarian morphology has not been previously reported and, since they were only identified in women with pelvic congestion, they are referred to in the present report as pelvic congestion cystic. In the present series of 61 patents undergoing oophorectomy for pelvic congestion, 43% had normal ovaries, 26% had polycystic ovaries, and 23% had pelvic congestion cystic ovaries (C.Gilling-Smith and H.Mason, unpublished observations).
In order to explore the hypothesis that a localized disturbance in ovarian steroids may be central to the aetiology of pelvic congestion, in-vitro theca and granulosa cell steroidogenesis and follicular fluid steroid content were compared in three groups of ovaries removed at hysterectomy: normal, polycystic and pelvic congestion cystic. The principal aim was to identify any significant differences in the steroid output of the follicles in the polycystic and pelvic congestion cystic ovaries, as compared with normal ovaries. A further aim was to characterize, for the first time, the endocrine function of the morphologically distinct pelvic congestion ovary.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ovarian dissection
A central section was taken from each ovary for histopathological examination, and the remaining tissue placed in Hanks' balanced salt solution (HBSS) without calcium and magnesium (Gibco BRL, Paisley, UK) for transport to the laboratory and subsequent dissection. Individual follicles were dissected intact from the surrounding ovarian stroma under microscopic vision. Only follicles which appeared healthy were studied, i.e. well-vascularized and yielding translucent, pale yellow follicular fluid. The diameter of each follicle was measured with callipers, and the follicular fluid aspirated using a microsyringe. The follicular fluid was stored at 20°C before measurement of steroids by radioimmunoassay. The follicles were incised and the granulosa cells carefully scraped from the lamina basalis. The theca interna was then peeled from the base of the follicle. Ovarian morphology was defined at dissection according to previously published criteria (Gilling-Smith et al., 1994; Mason et al., 1994
). Ovaries were defined as polycystic if they had at least three of the following four histological features: 10 or more follicles, typically 28 mm in diameter, distributed peripherally in any given cross-section; increased density and amount of ovarian stroma; increased ovarian volume (>9 ml); and thickened tunica (Gilling-Smith et al., 1994
). Ovaries were defined as normal if their volume was <9 ml, there was no evidence of increased quantity and density of stroma, and only one or two follicles could be identified in any given section of the ovary. Criteria for defining an ovary as pelvic congestion cystic at dissection were based on previously published ultrasound data (Adams et al., 1990
) and absence of features characterizing either the normal or polycystic ovary, i.e. evidence of larger follicles (412 mm) distributed in clusters of three to five cysts throughout the ovarian stroma, in ovaries which were of normal volume, had normal stromal density, and no thickening of the tunica.
Theca cell studies
Theca cells were isolated and cultured as previously described (Gilling-Smith et al., 1994). Following enzymatic dispersion, the cells were plated at a density of 1015x104 cells per well in 24 mmx15 mm multi-well plates (Cel cult, Bibby Sterilin, Staffordshire, UK) and incubated in 1 ml serum-free culture medium comprising Medium 199 [(Earle's modified salts with L-glutamine, without phenol red, with extra L-glutamine (0.6 mg/ml), penicillin and streptomycin (50 IU/ml) (all from Gibco BRL)]. Highly purified human pituitary LH (donated by Dr Sé Lynch, Endocrine Services, Bidford-on-Avon, Warwickshire, UK; LH activity 12 700 IU/mg); FSH 7.0 IU/mg; thyroid-stimulating hormone (TSH) 6.5 mIU/mg was added to half the wells at a dose of 2.5 ng/ml (maximum effective dose) at the start of the incubation period. Since theca cell steroid production is increased in large follicles (Gilling-Smith et al., 1994
), only theca from small follicles (
10 mm diameter) was used. All experiments were performed in duplicate or triplicate wells. No substrate was added, and steroid accumulation over 48 h under basal and LH-stimulated conditions was measured. At the end of the culture period, the overlying medium was collected for radioimmunoassay of progesterone, androstenedione and oestradiol content.
Granulosa cell studies
Granulosa cell cultures were performed as previously described (Mason et al., 1994). Cells were pooled from all follicles containing clear follicular fluid. To allow for meaningful comparisons, all follicles of diameter >11 mm were excluded (Mason et al., 1994
). Approximately 5x104 viable cells/well were incubated in a 200 µl volume of serum-free Medium 199 (Gibco BRL) with the addition of antibiotics (penicillin and streptomycin; Gibco) and 200 mmol/l L-glutamine. Incubations were carried out with the addition of 107 mol/l testosterone (Sigma Chemical Co., Poole, UK) as an aromatase substrate, and a range of doses of highly purified human FSH [<16 IU LH (16/40)/mg; kindly supplied by Dr S.Lynch]. All experiments were performed in triplicate wells. Medium was collected after a 48 h incubation period for measurement of oestradiol.
Radioimmunoassay
Androstenedione, progesterone and oestradiol in the stored incubation medium were measured by direct radioimmunoassay of the unextracted culture medium using antibodies and antisera as described previously (Gilling-Smith et al., 1994). Follicular fluid steroids were measured in all follicles containing clear fluid and assayed at dilutions of 1:10 to 1:1000. Parallelism was demonstrated with the standard curve across this range.
Statistical analysis
Theca cell steroid accumulation is expressed as mean (± SEM) pmol steroid/103 viable cells/48 h of culture unless otherwise stated. As the data were not normally distributed, comparison of steroid production between the three types of ovary was made using the MannWhitney U-test. Granulosa cell responses are expressed in pmol oestradiol/ 103 cells/48 h, and shown as the mean (± SE) of triplicate estimations from each ovary. Cumulative data of FSH doseresponses was analysed using two-way analysis of variance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study are best interpreted in the light of the 2-cell, 2-gonadotrophin theory of folliculogenesis (Armstrong and Dorrington, 1979). During follicular growth, theca and granulosa cells work in synchrony to produce oestradiol. Under the influence of LH, the theca layer converts cholesterol to pregnenolone, which is then converted to androstenedione, the precursor for oestradiol formation. The key enzyme involved in androgen biosynthesis, 17
-hydroxylase cytochrome P450, is found almost exclusively in the theca layer. Aromatase, the enzyme converting androstenedione to oestradiol, is found predominantly in the granulosa layer, although a small amount is present in theca cells (Inkster and Brodie, 1991
; Gilling-Smith et al., 1994
). The majority of the androstenedione diffuses across the lamina basalis to the granulosa layer, where FSH stimulates its aromatization to oestradiol. The relative concentration of androstenedione and oestradiol in follicular fluid reflects the relative steroidogenic activities of the two cell layers. In this study, theca from pelvic congestion cystic and polycystic ovaries both demonstrated increased basal and LH-stimulated androstenedione production compared with the response from normal ovaries. In the pelvic congestion cystic ovary, enhanced theca androgen production almost certainly represents theca cell hyperplasia, which occurs during the process of follicular atresia (Hughesdon, 1982
). This is consistent with finding that the majority of follicles in these ovaries were atretic, as evidenced by the high androgen:oestrogen ratio in the follicular fluid (McNatty, 1981
) and the fact that the oestradiol response to FSH by granulosa cells was poor. By contrast, hyperplasia associated with atresia cannot account for increased theca cell steroidogenesis by polycystic ovaries since the proportion of atretic follicles in these ovaries, as judged by follicular fluid oestradiol content, is no higher than that in normal ovaries (Mason et al., 1994
). Furthermore, granulosa cell responsiveness to FSH is similar to that in normal ovaries in the case of ovulatory subjects, or greater than that of normal ovaries in anovulatory subjects.
Increased theca cell androgen production in the polycystic ovary is thought to reflect an intrinsic abnormality of ovarian androgen biosynthesis rather than a response to atresia, particularly as progesterone production is also enhanced (Gilling-Smith et al., 1994). This concept is backed by in-vivo studies of ovarian androgen secretion (Gilling-Smith et al., 1997
) and recent association and linkage studies which suggest the gene encoding cholesterol side chain cleavage may play a pivotal role in the aetiology of the condition (Franks et al., 1997
; Gharani et al., 1997
).
It was found that the prevalence of pelvic congestion cystic ovaries in our unpublished series of 61 ovaries removed from women with pelvic congestion was 23%, and the prevalence of polycystic ovaries was 21%. The latter is a similar value to that expected in the normal female population (Polson et al., 1988). In terms of abnormal ovarian steroidogenesis, as characterized in this report, just under one-half of the women with pelvic congestion have increased theca cell androgen biosynthesis. One hypothesis leading from these data is that increased local production of androgens, either alone or in combination with physiological concentrations of oestrogen, has a direct effect on the pelvic vasculature. Both testosterone and oestrogen have been found to produce a significant enlargement of ovarian and uterine veins in ovariectomized mice (Forbes and Kapadia, 1976
). However, data linking androgens and venous dilatation in humans are tenuous.
Oestradiol production by theca cells is very low under normal circumstances. Although quite substantial amounts were being produced by theca from two of the pelvic congestion cystic ovaries, overall, there were no significant differences in theca cell oestradiol production between the three groups. The amount of oestradiol secreted by theca is unlikely to represent a significant contribution to the circulating pool of oestrogen.
The majority of women with pelvic congestion cystic ovaries in this series had regularand presumably therefore ovulatorycycles. Since it is not known whether these ovaries are associated exclusively with venous congestion, it is difficult to speculate on how these atretic cysts arise. One possibility is abnormal gonadotrophin stimulation. Preliminary studies on gonadotrophin pulsatility in women with venous congestion have shown abnormalities of LH pulsatility only in women with classic polycystic ovary syndrome (unpublished data). An alternative explanation is that ovarian factors generated during episodes of pain such as nerve growth factor, vasoactive intestinal peptide and noradrenaline result in disordered follicular growth (Ojeda et al., 1989).
It is accepted that the populations of ovaries studied in the theca and granulosa experiments were different, since the methodology for culturing theca cell cultures was established at a later date than that for granulosa cells. However, the conclusions drawn from this study are justified since they are based on within-group comparisons of theca and granulosa cell function in the three different types of ovaries. A further point is that one of the most important variables controlling steroidogenic output from either cell type is follicle diameter, and all follicles studied were 10 mm in diameter.
The characterization of the pelvic congestion ovary as a distinct entity, morphologically and functionally, as described in this report, paves the way for further studies. The morphological definition is purely descriptive, and precise histological and ultrasound morphological criteria are needed if these findings are to have a practical clinical application. A preliminary histological analysis of dissected ovaries removed at oophorectomy was performed in which 41 full-width histology sections were analysed from 29 ovaries designated as pelvic congestion cystic by the descriptive criteria used in this report. Morphometric analysis revealed that each section contained an average of five cysts with a mean diameter of 8 mm (range 410.8 mm). It was observed that, unlike classic polycystic ovaries, the cysts in these ovaries tended to be arranged in clusters of three to five, often centrally distributed in a stroma which appeared loose and often oedematous. When compared with 23 previously stored histology sections of anovulatory polycystic ovaries, the cyst diameter and the percentage section occupied by the cysts were significantly greater in pelvic congestion cystic ovaries. A prospective histological analysis with larger numbers is clearly needed to establish strict morphological criteria for defining the pelvic congestion cystic ovary, and this should be performed in conjunction with both two- and three-dimensional pelvic ultrasound analysis of these ovaries before oophorectomy to enable ultrasound criteria to be likewise defined. With these criteria established, future studies should address the prevalence of pelvic congestion ovaries in asymptomatic women as well as in those with pelvic congestion to evaluate the full clinical significance of these ovaries.
Pelvic congestion ovaries are clearly distinguishable from classic polycystic ovaries on two-dimensional ultrasound (Adams et al., 1990). Figure 3
shows the ultrasonographic features of normal, multicystic, polycystic and pelvic congestion ovaries, and illustrates the difficulties that ultrasonographers face when reporting ovarian morphology. Confusion between pelvic congestion ovaries and multicystic ovaries is inevitable unless close attention is paid to additional features present on the scan, such as uterine size and endometrial thickness. Women with multicystic ovaries have low oestrogen status, and consequently a small uterus and thin endometrium. By contrast, women with pelvic congestion have normal oestrogen concentrations, and the uterus and endometrium are either of normal or increased size (Adams et al., 1990
). Clinically, multicystic ovaries are associated with oligomenorrhoea or amenorrhoea, while pelvic congestion ovaries are not usually associated with cycle disturbance.
|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J., Reginald, P.W., Franks, S. et al. (1990) Uterine size and endometrial thickness and the significance of cystic ovaries in women with pelvic pain due to congestion. Br. J. Obstet. Gynaecol., 97, 583587.[ISI][Medline]
Armstrong, D.T. and Dorrington, J.H. (1979) Estrogen biosynthesis in the ovaries and testes. In Thomas, J.A. and Singhal, R.L. (eds), Regulatory Mechanisms Affecting Gonadal Hormone Action. University Park Press, Baltimore, pp. 217258.
Beard, R.W., Highman, J.W., Pearce, S. et al. (1984) Diagnosis of pelvic varicosities in women with chronic pelvic pain. Lancet, ii, 946949.
Beard, R.W., Reginald, P.W. and Wadsworth, J. (1988) Clinical features of women with chronic lower abdominal pain and pelvic congestion. Br. J. Obstet. Gynaecol., 95, 153161.[ISI][Medline]
Beard, R.W., Kennedy, R.G., Gangar, K.F. et al. (1991) Bilateral oophorectomy and hysterectomy in the treatment of intractable pelvic pain associated with pelvic congestion. Br. J. Obstet. Gynaecol., 98, 988992.[ISI][Medline]
Farquhar, C.M., Rogers, V., Franks, S. et al. (1989) A randomized controlled trial of medroxyprogesterone acetate and psychotherapy for the treatment of pelvic congestion. Br. J. Obstet. Gynaecol. 96, 11531162.[ISI][Medline]
Forbes, R.T. and Kapadia, S.E. (1976) Specific responses of ovarian and uterine veins of mice to sex hormones. Am. J. Anat., 147, 325328.[ISI][Medline]
Franks, S., Gharani, N., Waterworth, D. et al. (1997) The genetic basis of polycystic ovary syndrome Hum. Reprod., 12, 26412648.[Abstract]
Gharani, N., Warteworth, D.M., Batty, S. et al. (1997) Association of the steroid synthesis gene CYP11a with polycystic ovary syndrome and hyperandrogenism. Hum. Mol. Genet., 6, 397402.
Gilling-Smith, C., Willis, D.S., Beard, R.W. et al. (1994) Hypersecretion of androstenedione by isolated thecal cells from polycystic ovaries. J. Clin. Endocrinol. Metab., 79, 11581165.[Abstract]
Gilling-Smith, C., Story, E.H., Rogers, V. et al. (1997) Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin. Endocrinol., 47, 9399.[ISI][Medline]
Greiss, F.C. and Anderson, S.G. (1969) Uterine vascular changes during the ovarian cycle. Am. J. Obstet. Gynecol., 103, 629640.[ISI][Medline]
Greiss, F.C. and Anderson, S.G. (1970) Effects of ovarian hormones on the uterine vascular bed. Am. J. Obstet. Gynecol., 107, 829836.[ISI][Medline]
Hughesdon, P.E. (1982) Morphology and morphogenesis of the Stein-Leventhal ovary and of so called `hyperthecosis'. Obstet. Gynaecol. Surv., 37, 5977.[Medline]
Inkster, S.E. and Brodie, A.M.H. (1991) Expression of aromatase cytochrome P450 in premenopausal and postmenopausal human ovaries: an immunocytochemical study. J. Clin. Endocrinol. Metab., 73, 717726.[Abstract]
Mason, H.D., Willis, D.S., Beard, R.W. et al. (1994) Estradiol production by granulosa cells of normal and polycystic ovaries (PCO): relationship to menstrual cycle history and to concentrations of gonadotrophins and sex steroids in follicular fluid. J. Clin. Endocrinol. Metab., 79, 13551360.[Abstract]
McNatty, K.P. (1981) Hormonal correlates of follicular development in the human ovary. Aust. J. Biol. Sci., 34, 249268.[ISI][Medline]
Ojeda, S.R., Lara, H. and Ahmed, C.E. (1989) Potential relevance of vasoactive intestinal peptide to ovarian physiology. Semin. Reprod. Endocrinol., 7, 5260.[ISI]
Polson, D.W., Adams, J., Wadsworth, J. et al. (1988) Polycystic ovaries a common finding in normal women. Lancet, i, 870872.
Reginald, P.W., Adams, J., Franks, S. et al. (1986) The aetiology of pelvic varicosities in women with pelvic pain syndrome. Proceedings, 24th British Congress of Obstetrics and Gynaecology: 81.
Reginald, P.W., Adams, J., Franks, S. et al. (1989) Medroxyprogesterone acetate in the treatment of pelvic pain due to venous congestion. Br. J. Obstet. Gynaecol., 96, 11481152.[ISI][Medline]
Reid, B. and Gangar, K.F. (1994) Oophorectomy in young women: can it ever be justified ? Contemp. Rev. Obstet. Gynaecol., 6, 4145.
Steege, J.F., Stout, A.L. and Somkuti, S.G. (1993) Chronic pelvic pain in women: toward an integrative model. Obstet. Gynecol. Survey, 48, 95110.[Medline]
Stones, R.W., Rae, T., Rogers, V. et al. (1990) Pelvic congestion in women: evaluation with transvaginal ultrasound and observation of venous pharmacology. Br. J. Radiol., 63, 710711.[Abstract]
Taylor, H.C. (1954) Pelvic pain based on a vascular and autonomic nervous system disorder. Am. J. Obstet. Gynecol., 57, 11771196.
Taylor, H.C. (1949) Vascular congestion and hyperaemia. Their effect on structure and function in the female reproductive system. Am. J.. Obstet. Gynecol., 57, 211230.[ISI]
Van Buren, G.A., Yang, D. and Clark, K. (1992) Estrogen-induced uterine vasodilation is antagonized by L-nitroarginine methyl ester, an inhibitor of nitric oxide synthesis. Am. J. Obstet. Gynecol., 167, 828833.[ISI][Medline]
Submitted on July 13, 2000; accepted on September 15, 2000.