1 Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville 5011,South Australia, Australia and 2 Department of Obstetrics and Gynaecology, Göteborg University, Sweden
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Abstract |
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Key words: colony stimulating factor-1/cytokines/follicular fluid/human/ovaries
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Introduction |
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Homozygous csfmop/csfmop osteopetrotic mice, that lack functional CSF-1 and thus have reduced ovarian macrophages, have abnormal oestrous cycles and decreased ovulation rates, which can be restored by continuous CSF-1 administration from birth (Cohen et al., 1997). Furthermore, CSF-1 treatment of equine chorionic gonadotrophin/human chorionic gonadotrophin (eCG/HCG) primed immature rats increases ovulation rates, while intrabursal administration of a CSF-1 neutralizing antibody has the opposite effect (Nishimura et al., 1995
).
Several recent studies suggest that there is a selectivity in follicular production of cytokines since only a minority of cytokines detected in follicular fluid (FF), such as IL-8 (Runesson et al., 1996), IL-6 (Machelon et al., 1994
), growth regulated oncogene-
(GRO-
) (Karström-Encrantz et al., 1998
) and leukaemia inhibitory factor (LIF) (Arici et al., 1997a
) have been found in markedly higher concentrations of immuno-activity or bioactivity in FF than in peripheral blood. In line with these observations it was recently shown that human FF from in-vitro fertilization (IVF) patients contains significantly higher concentrations of CSF-1 compared with those in peripheral blood, and granulosa-lutein cells isolated from FF express both CSF-1 and its receptor c-fms (Witt et al., 1997). In an attempt to evaluate the possible role of CSF-1 in human ovarian function, the present study describes the concentrations of immunoreactive CSF-1 in peripheral blood and FF during the menstrual cycle and during ovarian stimulation.
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Materials and methods |
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Menstrual cycle studies
Ten healthy females aged between 23 and 35 years (median 27), who had not been on hormonal contraception for at least 3 months prior to the study, did not suffer from any immunological conditions (asthma, arthritis/rheumatism, colitis, eczema or persistent hyperthermia) and had not received pharmacological treatment during the last month prior to study, were recruited to the study. They had all completed a detailed questionnaire regarding their menstrual history to rule out abnormalities. The women had regular sampling of venous blood throughout one complete menstrual cycle and collected daily urine samples from day 5 until the completion of the study cycle. Plasma oestradiol, progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) as well as urinary LH were analysed by an automated chemiluminescence system (ACS:180 kits; Chiron Diagnostics, Victoria, Australia). The menstrual history and hormone results enabled the grouping of the samples into early (EF, -12.8 ± 1.2, mean ± SEM, days in relation to the day of LH surge considered as day 0), mid (MF, 6.9 ± 0.9) and late (LF, 2.4 ± 0.3) follicular phases, time of LH peak (LH, day 0), approximate time of ovulation (OV, +1 ± 0.3) and early (EL, +3.4 ± 0.3), mid (ML, +7.3 ± 0.5) and late (LL, +11.4 ± 0.3) luteal phases.
FF were obtained at laparoscopy for tubal sterilization. Only women (median age 44, range 2954) with regular cycles (2535 days) who were operated on in the interval between day 5 and up to the time of follicular rupture were included. Based on hormonal (LH, FSH, oestradiol and progesterone) status and size of follicle, the samples were grouped into follicular phase (FPh: oestradiol < 0.6 nmol/l, LH < 8 IU/l, progesterone < 6 nmol/l and follicular diameter < 10 mm), LH surge phase (LHPh: LH > 8 IU/l, progesterone < 10 nmol/l and follicular diameter > 16 mm) and ovulatory phase (OVPh: progesterone > 10 nmol/l, LH < 8 IU/l). At laparoscopy, the FF was aspirated as the first surgical procedure.
Ovarian stimulation
Blood and follicular fluid samples were obtained from patients (median age 32, range 2641) undergoing transvaginal oocyte retrieval (TVOR) as part of the IVF treatment with their informed consent. Patients were down-regulated with leuprolide (Lucrin®, 0.5 mg s.c. daily; Abbott, Sydney, Australia) during the luteal phase of the cycle and ovarian stimulation was induced by daily injection of human menopausal gonadotrophin (HMG; Humegon®, 150300 IU; Organon, Oss, The Netherlands; or Pergonal®; Serono, Rome, Italy) following menstruation and confirmation of basal blood steroid concentrations. When at least two follicles of 16 mm in diameter were seen on ultrasound, HCG (5000 IU; Pregnyl®, Organon or Profasi®, Serono) was administered and the follicular contents aspirated approximately 36 h later under ultrasound guidance. The blood samples were taken at four time-points (before starting ovarian stimulation, 12 h before HCG injection, at the time of operation or 3537 h after HCG and on day 9 after HCG) and collected into tubes with 125 IU heparin separator gel. Plasma was separated by centrifugation at 2000 g for 10 min, divided into aliquots and stored at 20°C until use. Only clear follicular fluids without visible blood contamination were selected for the study. The fluids were measured in a volumetric cylinder, centrifuged, divided into aliquots and stored at 80°C until use. FF concentrations of oestradiol and progesterone were determined as described above. FF androstenedione concentrations were analysed by radioimmunoassay (Diagnostic Systems Laboratories, TX, USA).
Assays
Serum, plasma and follicular fluid concentrations of CSF-1 were determined by human Quantikine M-CSF ELISA kit® (R&D Systems DMC00, Minneapolis, MN, USA). This assay has been validated for CSF-1 concentrations in serum, plasma and culture supernatants and calibrated to the NIBSC/WHO First International Reference Standard for CSF-1 89/512 (one NIBSC 89/512 IU = 0.060 Ï pg/ml). No significant cross-reactivity has been detected with a wide range of human and murine cytokines and growth factors, except murine recombinant CSF-1, which cross-reacts up to 7%. The sensitivity, within (inter) and between (intra) assay percentage coefficients of variation (CV) for all assays are summarized in Table I.
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Results |
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Discussion |
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There now exists increasing evidence of CSF-1 involvement in the cycle-dependent physiological changes of the ovary. Studies on osteopetrotic mice have revealed that these mice exhibit decreased ovulation rates but normal fertilization and implantation rates once oocytes have been released (Cohen et al., 1997). A possible mechanism by which CSF-1 may modulate ovarian function is that CSF-1 produced by the ovary may attract monocytes from circulation, promote their differentiation into macrophages and support the activity of differentiated macrophages, once they have reached their target tissue in the ovary. Activated macrophages are potential sources of a number of ovulatory mediators such as proteases, eicosanoids, cytokines and nitric oxide (Norman et al., 1997
).
Various cytokines, such as IL-1 (Cannon and Dinarello, 1985), granulocyte-colony stimulating factor (G-CSF) (Makinoda et al., 1995
) and TNF
(Brännström et al., 1994b
) show significant variations in peripheral blood during the menstrual cycle. The lack of menstrual cycle-associated variations of CSF-1 expression suggests that this cytokine, as most cytokines, may rather act as an autocrine/paracrine mediator. In the mouse uterus, the concentrations of CSF-1 bioactivity increased about 1000-fold at term pregnancy compared to the non-pregnant state, in contrast to only 1.4-fold increase in serum (Bartocci et al., 1986
), which further supports the local action of CSF-1 in the reproductive organs. Another explanation could be that CSF-1 has a short half life (only 10 min) in circulation (Bartocci et al., 1987
) and consequently, the ovarian contribution (e.g. increased FF concentrations of CSF-1 at OVPh) may not be enough for significant accumulation of circulating CSF-1 in normal conditions. The rise of FF CSF-1 concentrations during OVPh suggests a role of factors involved in ovulation, particularly LH, in ovarian CSF-1 synthesis.
In the situation of ovarian stimulation, CSF-1 concentrations in serum gradually increased during ovarian stimulation in patients with more than 20 follicles with highest concentrations during the mid-luteal phase, independently of the outcome of treatment (pregnancy or not) (Nishimura et al., 1998). In the latter study, there was no detectable change in serum concentrations of CSF-1 in patients who developed less than two follicles (e.g. were non-responders). In the present study, we also found that plasma CSF-1 concentrations were significantly higher on day 9 after HCG injection. These observations suggest a role of CSF-1 during the luteal phase. This may be due either to higher macrophage numbers in the ovary due to their influx into the corpora lutea (Brännström et al., 1993
; Best et al., 1996
; Takaya et al., 1997
) or due to production of CSF-1 by other cells in the corpus luteum, which may attract those macrophages into the ovary.
A variety of cytokines including IL-1ß, IL-6, IL-8, LIF, GRO-, monocyte chemoattractant protein-1 (MCP-1), TNF
and granulocytemacrophage colony-stimulating factor (GMCSF) (Zolti et al., 1990
; Machelon et al., 1994
, 1995
; Jasper et al., 1996
; Runesson et al., 1996
; Arici et al., 1996
, 1997a
, Arici et al., b
; Karström-Encrantz et al., 1998
) have now been identified in human follicular fluid as well as shown to be locally expressed by human ovarian cells. Interestingly, only some of them are present in higher concentrations in FF than in peripheral blood, indicating differences in their production by the ovary or their ability to accumulate in follicular fluid.
Witt and Pollard (1997) reported that immunoreactive CSF-1 concentrations in FF were 2.8-fold higher than those in serum, and cells isolated from FF express both CSF-1 and CSF-1R. In the present study, concentrations in FF, obtained from the dominant follicle during the menstrual cycle and ovarian stimulation, were about four- and seven-fold higher than those in plasma at the same time, respectively. The markedly elevated concentrations in FF of IVF patients and higher concentrations in the ovulatory phase compared to the follicular phase during the natural cycle implies that HCG/LH could be the natural trigger for increased follicular CSF-1 production. However, it should be stated that the samples from the two phases of the natural cycle are from different women, with the limitations of a possible large inter-individual variation.
Furthermore, we found significant correlations between FF and plasma concentrations as well as between FF concentrations of CSF-1 and progesterone. These findings suggest that at least during ovarian stimulation, ovarian synthesis of CSF-1 may contribute to circulating concentrations of CSF-1, which may have systemic impact on immune cell populations. In addition, progesterone and oestrogen have been shown to induce CSF-1 mRNA expression by endometrium during mouse pre-implantation development (Pampher et al., 1991) and human endometrium (Azuma et al., 1990), which may indicate a steroid controlled expression of CSF-1. Increased concentrations of CSF-1 in FF may be important in macrophage chemoattraction and in supporting their activity within the follicle, and it may be linked to folliculogenesis, ovulation, corpus luteum function and regulation of progesterone synthesis via secretion of cytokines such as IL-1ß or TNF
.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 2, 1998; accepted on February 5, 1999.