1 Institut Clinic of Gynecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Faculty of Medicine-University of Barcelona, Hospital Clinic-Institut dInvestigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
3 To whom correspondence should be addressed at: Institut Clinic of Gynecology, Obstetrics and Neonatology, Hospital Clinic, C/Casanova 143, 08036 Barcelona, Spain. e-mail: jbalasch{at}medicina.ub.es
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Abstract |
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Key words: adrenomedullin/follicular fluid/luteinized granulosa cells/macrophages/VEGF
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Introduction |
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Human follicular fluid contains several substances, such as cytokines and growth factors, which may affect follicular growth and maturation, ovulation and follicular atresia (Brännström and Norman, 1993). Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogenic and permeability agent which is considered to be a key factor associated with rapid vascular growth in a variety of physiological and pathological conditions (Ferrara and Davis-Smyth, 1997
; Lebovic et al., 1999
). VEGF is expressed and secreted in the normal premenopausal human ovary in a cyclic manner and regulated by gonadotrophin secretion during the menstrual cycle. VEGF has a paracrine and/or autocrine function necessary for normal ovarian function, mediating angiogenesis and vascular permeability. VEGF has also been implicated in the regulation of angiogenesis in the endometrium (Smith, 2001
) and placenta (Ong et al., 2000
), and embryonic development (Breier, 2000
). Thus, this agent is critical for normal reproductive function (Geva and Jaffe, 2000
). We and others have reported that follicular fluid from IVF patients contains significant concentrations of VEGF (Lee et al., 1997a
; Friedman et al., 1998
; Manau et al., 2000
). VEGF mRNA expression has been detected within the dominant follicle and the corpus luteum of primate species, and both FSH and LH directly stimulate VEGF synthesis by non-human primate granulosa cells (Christenson and Stouffer, 1997
; Lebovic et al., 1999
).
In addition, we first reported that there was a significantly higher amount of adrenomedullin present in follicular fluid relative to plasma (Manau et al., 2000). Adrenomedullin is a potent, long-lasting, multifunctional peptide first isolated from human pheochromocytoma. Subsequent studies have shown that adrenomedullin is expressed in a variety of tissues and has diverse physiological actions (Hinson et al., 2000
). Adrenomedullin has been shown to be present throughout the female reproductive system and, in light of its known roles, it has been suggested that adrenomedullin may have important regulatory roles in reproductive tissues (Hinson et al., 2000
). Recently, adrenomedullin and its receptor have been found in the human granulosa cells at the pre-ovulatory stage and at the midluteal phase (Moriyama et al., 2000
). Adrenomedullin released by the ovary may act as a paracrine/autocrine hormone in regulating follicle growth or maturation, or may be implicated in the luteinization of ovarian follicles (Abe et al., 2000
; Moriyama et al., 2000
). It is also possible that adrenomedullin derived from the ovary may have a role in the secretion of gonadotrophins in the pituitary gland (Montuenga et al., 2000
). Adrenomedullin is also an autocrine growth factor for human endometrial endothelial cells and is involved in endometrial angiogenesis (Rees et al., 1999
; Nikitenko et al., 2000
). During pregnancy, adrenomedullin may be involved in the maternal haemodynamic adaptations to gestation, may play a role during human labour and may be implicated in fetal growth and development (Garayoa et al., 2002
; Yamashiro et al., 2002
; Al-Ghafra et al., 2003
).
Although granulosa luteal cells predominate in follicular fluid, macrophages have been suggested to play important roles in ovarian function and are a source of growth factors and secretory products in the ovary at various stages of follicular development (Brännström and Norman, 1993). The numbers of macrophages are elevated in the periovulatory period and they constitute up to 15% of cells identified in the follicular fluid at the time of oocyte retrieval in IVF patients (Loukides et al., 1990
). Remarkably, recent studies have revealed that monocyte-derived macrophages secrete both VEGF (Ono et al., 1999
; Barbera-Guillem et al., 2002
) and adrenomedullin (Nakayama et al., 1999
; Elsasser and Kahl, 2002
).
Therefore, the present study examines the relative contribution of macrophages and granulosa cells in the production of VEGF and adrenomedullin in the human ovulatory follicle.
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Materials and methodS |
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Controlled ovarian stimulation for IVF was performed according to a protocol previously reported, including gonadotrophin ovarian stimulation under pituitary suppression with a GnRH agonist (Balasch et al., 2001). Briefly, daily s.c. leuprolide acetate was started in the midluteal phase of the previous cycle, and gonadotrophin stimulation of the ovaries was started 1314 days later when the serum estradiol concentration declined to <50 pg/ml and a vaginal ultrasonographic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, six and four ampoules/day of recombinant FSH (Gonal-F, Serono S.A., Madrid, Spain), respectively, were administered s.c. On days 3, 4 and 5 of ovarian stimulation, two ampoules/day of FSH were administered to each patient. From day 6 onwards, FSH was administered on an individual basis according to the ovarian response.
Sequential transvaginal ultrasonography and serum estradiol measurements were carried out to assess follicular development. Ultrasonic scans were performed with a 5 mHz vaginal transducer attached to an Aloka sector scanner (model SSD-620, Aloka, Tokyo, Japan). Finally, HCG (5000 IU) (Profasi; Serono S.A.) was administered i.m. when a consistent rise in serum estradiol concentration was associated with the presence of two or more follicles of 18 mm diameter. Oocyte aspiration was performed by vaginal ultrasonography 3536 h after HCG injection. The maturational status of the oocytes was recorded according to published criteria (Veeck, 1986
).
Blood samples were obtained from each subject on the day of oocyte retrieval for comparative purposes with follicular fluid. Also, we assessed the relationship of both circulating and follicular fluid concentrations of VEGF and adrenomedullin and parameters of ovarian response (age, basal FSH levels, peak estradiol serum concentration, oocytes retrieved and oocyte maturiry), to stimulation with gonadotrophins as well as the IVF outcome (fertilization rate and clinical pregnancy).
Specimen collection and cell cultures
Human follicular fluid macrophages were obtained from pooled follicular fluid of individual patients (n = 20) following a method previously described to isolate peritoneal macrophages from ascites of cirrhotic patients (Pérez-Ruiz et al., 1999). In brief, pooled clear follicular fluid (
35 ml obtained from
1317 different follicles) from each individual patient was centrifuged at 200 g for 10 min at 4°C and the resulting supernatant was stored at 20°C until further measurements. Then, 2 x 104 cells/well (n = 10) or 105 cells/well (n = 10) were seeded on 96-well plates (BD Falcon, Bedford, MA) in their own follicular fluid. After incubation for 90 min at 37°C in a humidified atmosphere (95% air and 5% CO2), the non-adherent cells were removed from the wells by three washes with 300 µl of pre-warmed Dulbeccos phosphate-buffered saline (DPBS), and the remaining adherent cells were incubated with 300 µl of phenol red-free RPMI 1640 medium supplemented with 2% heat-inactivated fetal calf serum (FCS), penicillin/streptomycin (50 U/ml and 50 µg/ml) and L-glutamine (2 mmol/l) at 37°C in a humidified atmosphere (95% air and 5% CO2). About 98 ± 2% of the adherent cells were non-specific esterase-positive which is the most commonly used marker for monocytes (Auger and Ross, 1992
), and had the morphological appearance of macrophages (large cells, spread on the plate with a marked ruffing of the membrane border) when examined after Giemsa staining. In addition, preliminary experiments showed positive immunoreactivity for CD68 in the isolated cells. Accumulation of VEGF and adrenomedullin in the culture medium of the isolated human follicular fluid macrophages seeded on 96-well plates was determined at variable time intervals ranging between 0 and 48 h.
Human granulosa cells were isolated from follicular fluid obtained during oocyte retrieval. After removing the oocytes, the remaining cumulus cells from each individual patient (n = 20) were collected under the microscope from fluids without obvious blood contamination, pooled and washed twice with Hams-HEPES F-10 medium supplemented with 2% FCS, penicillin/streptomycin (50 U/ml and 50 µg/ml) and L-glutamine (2 mmol/l). Then, cells were transferred to a 12 ml tube containing 2 ml of supplemented Hams-HEPES F-10 medium and cell clusters were mechanically separated by vigorous pipetting. After centrifugation at 160 g (10 min, room temperature), the resulting pellet was gently resuspended in 0.5 ml of supplemented Hams-HEPES F-10 medium. Cell number and viability were assessed by the trypan blue exclusion test using a haemocytometer. Morphological examination of isolated samples revealed typical granulosa cell morphology and the purity of the cell culture was assessed by positive -inhibin but negative CD68 immunohistochemical staining, with <1% being CD68 positive cells. Then 2 x 104 cells/well (n = 10) or 105 cells/well (n = 10) were seeded in 96-well plates and incubated for 24 h at 37°C under humidified conditions (95% air, 5% CO2). Thereafter, cells were washed with 300 µl of pre-warmed DPBS to remove cells in suspension; attached cells were cultured in 300 µl of supplemented Hams-HEPES F-10 medium at 37°C in a humidified environment (95% air, 5% CO2). Accumulation of VEGF and adrenomedullin in the culture medium of the isolated human granulosa cells seeded on 96-well plates was determined at variable time intervals ranging between 0 and 48 h.
Peripheral monocytes were chosen to assess whether other cells of the monocyte/macrophage linage exhibit a secretion pattern of VEGF and adrenomedullin similar to that of macrophages isolated from the follicular fluid. Peripheral monocytes can be obtained easily by venipuncture, thus avoiding unnecessary interventions in the subject. Cells were isolated as previously reported (Pérez-Ruiz et al., 1999) from fresh blood by venipuncture using acid citrate dextrose as anticoagulant. Blood was centrifuged at 80 g (15 min, room temperature), and leukocytes were fractionated by FicollHypaque gradient centrifugation. Then monocytes were obtained from the mononuclear cell layer according to the method of Denholm and Wolber (1991
). Briefly, mononuclear cells were resuspended in DPBS plus 0.1% bovine serum albumin, added to a Percoll:10x Hanks balanced salt solution (HBSS) mixture (10:1.65) in a 10 x 1.5 cm round-bottom polypropylene tube and centrifuged at 160 g, 25°C for 30 min. Monocytes were collected from the upper 5 mm of the gradient, counted in a haemocytometer and viability was estimated by trypan blue staining. Cytospin preparations of monocytes stained with Giemsa confirmed a 9094% pure monocyte population. Then, 105 viable cells/well (n = 5) were seeded on 96-well plates and incubated overnight with supplemented phenol red-free RPMI 1640 medium under standard conditions of humidity and temperature (37°C, 95% air, 5% CO2). Finally, cells were washed with 300 µl of pre-warmed DPBS and the remaining adherent cells were cultured for 48 h in 300 µl phenol red-free RPMI 1640 medium supplemented with 2% heat-inactivated FCS, penicillin/streptomycin (50 U/ml and 50 µg/ml) and L-glutamine (2 mmol/l) at 37°C in a humidified atmosphere (95% air, 5% CO2). Culture samples obtained at different intervals between 0 and 48 h were stored at 20°C for analysis of VEGF and adrenomedulllin concentrations.
Laboratory methods
The laboratory methods for VEGF and adrenomedullin measurement used have been reported previously (Manau et al., 2000). Culture supernatants, follicular fluid and plasma VEGF concentrations were quantified by enzyme-linked immunosorbent assay (ELISA; Quantikine Human VEGF Immunoassay, R&D Systems Inc., Minneapolis, MN) that recognizes the soluble isoforms VEGF121 and VEGF165 which are the most frequently expressed spliced variants of the VEGF A gene. Intra- and inter-assay coefficients of variation (CVs) in plasma samples were 7.1 and 9.2%, respectively. In follicular fluid, these values were 4.7 and 7.5%, respectively. The portion of inhibition produced by serial dilution of follicular fluid samples (n = 5) paralleled the standard curve (data not shown). The recovery of 400 pg of recombinant human VEGF165 added to follicular samples was 108%, and the recovery of 100 pg was 106%.
The adrenomedullin concentrations of plasma, follicular fluid and culture supernatants were measured by radioimmunoassay (Phoenix Pharmaceuticals, Mountain View, CA) after extraction of adrenomedullin on Sep-Pack C18 cartridges (Waters Associates, Mildford, MA). Briefly, fluid samples (2 ml) were acidified with 4% acetic acid (3 ml) and applied twice to cartridges pre-activated with methanol, distilled water and 4% acetic acid. Cartridges were then washed with distilled water and 25% ethanol, and adrenomedullin was eluted with 4 ml of glacial acetic acid in 86% ethanol. The eluted adrenomedullin was then dried and reconstituted for radioimmunoassay. The recovery rate for the extraction procedure was 67, 78 and 85%, as determined by the addition of 125I-labelled adrenomedullin to plasma, follicular fluid or culture supernatant, respectively. Maximum binding of the anti-adrenomedullin antibody in the radioimmunoassay was 40.6%. The sensitivity of the assay was 5 pg/tube and intra- and inter-assay CVs were 12.4 and 13.6%, respectively. These values were similar to those reported in other investigations (Cuzzola et al., 2001; Honshi et al., 2003
). Dilution curves obtained from plasma, follicular extracts or culture supernantants paralleled the standard curve.
Statistical analysis
Data were analysed using SPSS statistical software using the non-parametric MannWhitney U-test. Results are expressed as median and range and were considered significant at a P-value <0.05.
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Results |
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Preliminary experiments performed in our laboratory demonstrated that concentrations lower than 105 cells per well do not allow enough sensitivity to measure the adrenomedullin released by the cells accurately. For this reason, we were unable to perform time course production experiments of adrenomedullin either in follicular fluid macrophages or in luteinized granulosa cells since the total amount of cells obtained from each patient precluded this type of experiment. However, this was not the case regarding VEGF where concentrations of 2 x 104 cells per well resulted in sufficient amounts of VEGF to be measured.
The time course of production of VEGF in follicular fluid macrophages from five patients is shown in Figure 1. VEGF secreted by follicular fluid macrophages increased progressively within 48 h of cell culture. A similar response pattern was observed with the culture medium of luteinized granulosa cells (Figure 2). However, the production rate of this substance was higher in cultured macrophages compared with cultured granulosa cells.
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Discussion |
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The present study adds new evidence favouring the concept that, in the ovary, the immune system contributes to the regulation of gonadal function (Mori et al., 1996; Bukulmez and Arici, 2000
) by showing that both follicular fluid macrophages and luteinized granulosa cells are ovarian sources of VEGF and adrenomedullin. The following facts support this contention. First, there was a significantly higher amount of both VEGF and adrenomedullin present in follicular fluid relative to plasma. Secondly, it is accepted that the ovary is not an immunologically privileged site and there is a traffic of leukocytes in and out of ovarian tissues concomitant with marked morphological and functional changes during the ovarian cycle. Thus, monocytes of the blood may differentiate into macrophages as they enter ovarian tissues (Brännström and Norman, 1993
). However, VEGF and adrenomedullin production is a characteristic of follicular fluid macrophages as we showed that circulating monocytes obtained from the same patient on the day of follicular puncture did not produce these substances.
The role of VEGF in ovarian physiology and pathology has been reviewed recently (Geva and Jaffe, 2000). In the corpus luteum, luteinized granulosa and thecal cells exhibited staining for VEGF. However, with further maturation, lutein cell staining for VEGF protein became more variable. The theca lutein cell staining was less intense than exhibited by most granulosa lutein cells (Geva and Jaffe, 2000
). This notwithstanding, a significant proportion of the non-luteal cells present in the corpus luteum are constituents of the immune system, such as lymphocytes and macrophages (Brännström and Norman, 1993
). In the human corpus luteum, the macrophage is the predominant leukocyte, comprising at least 10% of the total cells (Brännström and Fridén, 1997
). As well as being primarily phagocytic in nature, immune cells are now believed to be directly involved in regulating luteal function through the integration of immunological and endocrinological mechanisms throughout the life-span of the corpus luteum (Brännström and Fridén, 1997
). Macrophages are terminally differentiated cells demonstrating wide heterogeneity in biological function. They produce a number of potent angiogenic cytokines and growth factors and modulate events in the extracellular matrix-degrading enzymes and modulating enzymes which may be important in angiogenesis and tissue reorganization (Brännström and Fridén, 1997
; Ono et al., 1999
). This study adds further evidence in this regard by showing for the first time that follicular fluid macrophages produce significant amounts of VEGF, at an even higher rate than that of luteinized granulosa cells. The potential implications of these findings for corpus luteum formation warrant further investigation.
Adrenomedullin elicits a number of physiological actions, including a regulatory function on the release or activity of several hormones (Hinson et al., 2000). During the normal menstrual cycle, it has been shown that plasmatic adrenomedullin levels fluctuate with the cyclic changes in gonadotrophins and ovarian steroids (Marinoni et al., 2000
). Moreover, adrenomedullin and its receptor have been found in human granulosa cells at the pre-ovulatory stage and at the midluteal phase, demonstrating that adrenomedullin acts as a local factor to enhance progesterone production by these cells (Moriyama et al., 2000
). The identification of adrenomedullin and its receptors in human granulosa cells suggests that at least part of them may be produced by the ovary itself. Consistently, immunohistochemical staining for adrenomedullin showed that it was most abundant in granulosa lutein cells at the midluteal phase, less abundant in granulosa cells in dominant follicles, and least abundant in granulosa cells in primordial and pre-antral follicles in the human ovary (Moriyama et al., 2000
). This is inconsistent with the previous study by Abe et al. (1998
) who found that the maturation and luteinization of rat granulosa cells induced by gonadotrophins were associated with a significant suppression in adrenomedullin mRNA expression in those cells cultured in vitro. The reason for the difference in the adrenomedullin expression in the human and rat ovary is not known. However, the apparent difference between the data obtained with rat granulosa cells in vitro and human granulosa cells in vivo may be due to species differences or to differences in the environment in which granulosa cell specimens were luteinized, i.e. in vivo or in vitro (Moriyama et al., 2000
). The present study adds new information on the subject by showing that both granulosa lutein cells and follicular fluid macrophages have a contributory role in adrenomedullin production by the ovary.
We (Manau et al., 2000) and others (Friedman et al., 1998
) previously have reported that follicular fluid concentrations of VEGF and adrenomedullin can be useful markers of the ovarian response in patients undergoing IVF. This could not be confirmed in the present study because only relatively young patients with normal basal FSH and adequate ovarian response to gonadotrophin stimulation were included in this investigation. In fact, discrete age populations were chosen arbitrarily in those previous studies in an effort to exaggerate potential physiological differences among groups of women based on age.
In conclusion, this study suggested for the first time that both luteinized granulosa cells and macrophages are actively secreting VEGF and adrenomedullin into follicular fluid in the human ovary.
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Acknowledgments |
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Submitted on December 19, 2003; accepted on February 10, 2004.
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