Colony stimulating factor-1 concentrations in blood and follicular fluid during the human menstrual cycle and ovarian stimulation: possible role in the ovulatory process

B. Shinetugs1, E. Runesson2, N.P. Bonello1, M. Brännström2 and R.J. Norman1,3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To evaluate a possible role for colony stimulating factor-1 (CSF-1) in human ovarian function, the peripheral blood CSF-1 concentration throughout the human menstrual cycle and during ovarian stimulation was monitored. Blood was sampled across the menstrual cycle (n = 10) and at specific times during ovarian stimulation. In addition, the CSF-1 concentrations in follicular fluid (FF) during the follicular phase and during the luteinizing hormone (LH) surge of natural cycles, as well as 35–37 h after human chorionic gonadotrophin (HCG) during ovarian stimulation, were determined. There was no significant variation in CSF-1 concentrations during the natural menstrual cycle (median 470, range 212–1364 pg/ml). CSF-1 concentrations in FF (n = 11) were about four-fold higher (P < 0.0001) than those in plasma of the same patients. CSF-1 concentrations in these FF showed some stage dependent variability, with significantly higher values during the ovulatory phase (median of 2017 pg/ml, range 1131–2236 pg/ml), compared to mid-follicular phase (median 961 pg/ml, range 830–1340 pg/ml; P = 0.02). During ovarian stimulation (n = 20), the plasma concentrations were similar to a time prior to stimulation up to and including 35–37 h after HCG. On day 9 after HCG, the values (median 644, range 357–1352 pg/ml) were significantly higher compared to pre-stimulation (median 422, range 253–1598 pg/ml; P < 0.05) and 35–37 h after HCG (median 458, range 250–658 pg/ml; P < 0.01). FF concentrations (n = 27) of CSF-1 at oocyte retrieval (median 3116, range 1824–5883 pg/ml) were about seven-fold higher than blood concentrations (median 472, range 250–1055 pg/ml; P < 0.0001). These results suggest that the intra-ovarian CSF-1, possibly induced by LH/HCG, plays an important role during ovulation and luteinization.

Key words: colony stimulating factor-1/cytokines/follicular fluid/human/ovaries


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
White blood cells (Brännström and Norman, 1993Go; Norman et al., 1997Go) and their secreted cytokines, such as interleukin-1 (IL-1) (Kokia and Adashi, 1993Go; Brännström et al., 1993Go) and tumour necrosis factor-{alpha} (TNF{alpha}) (Terranova et al., 1993Go; Brännström et al., 1995Go) are important modulators of mammalian ovarian function. Colony stimulating factor-1 (CSF-1), also known as macrophage-colony stimulating factor (M-CSF), is a haemopoietic growth factor which is primarily responsible for proliferation, differentiation and survival of the monocyte–macrophage cell lineage (Stanley et al., 1983Go), as well as their recruitment (Wood et al., 1997Go). Monocytes and macrophages have been identified as the most abundant leukocyte subtype within the tissue of the human ovary (Wang et al., 1992Go; Brännström et al., 1994aGo) and are also found in large numbers in human follicular fluid (Loukides et al., 1990Go; Wang et al., 1992Go). The numbers and localization of the macrophages within the ovary vary according to the menstrual cycle (Brännström et al., 1994aGo; Takaya et al., 1997Go).

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., 1997Go). 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., 1995Go).

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., 1996Go), IL-6 (Machelon et al., 1994Go), growth regulated oncogene-{alpha} (GRO-{alpha}) (Karström-Encrantz et al., 1998Go) and leukaemia inhibitory factor (LIF) (Arici et al., 1997aGo) 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was approved by the North-West Adelaide Health Service Ethics of Human Research Committee and the Human Research Ethics Committee of Göteborg University.

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 29–54) with regular cycles (25–35 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 26–41) 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®, 150–300 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 35–37 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 IGo.


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Table I. Sensitivity, inter- and intra-assay coefficients of variation (CV) for assays
 
Statistics
Statistical analysis was performed by non-parametric analysis of variance (ANOVA), Mann–Whitney, Wilcoxon, Dunn and Spearman tests. P values < 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CSF-1 concentrations throughout the menstrual cycle
All subjects (n = 10) demonstrated a normal ovulatory cycle (cycle length 27.7 ± 1.2 days, mean ± SEM) as shown by a mid-cycle LH peak in blood and/or urine samples and a normal luteal phase progesterone profile (data not shown). Serum concentrations of CSF-1 at any stage of the menstrual cycle showed a considerable inter-individual variation (median 470, range 212–1364 pg/ml). No significant variation in circulating CSF-1 concentrations was detected throughout the cycle (Figure 1Go). CSF-1 concentrations in FF measured in the dominant follicle during the natural cycle (n = 11; median 1340, range 830–2714 pg/ml) were at all time points higher than those in plasma (median 296, range 186–438 pg/ml; P < 0.0001). In the subgroup of patients at OVPh, CSF-1 concentrations in FF (median 2017, range 1131–2236 pg/ml) were significantly higher compared to patients at FPh (median 961, range 830–1340 pg/ml; P = 0.02; Figure 2Go).



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Figure 1. Serum concentrations of immunoreactive CSF-1 across the menstrual cycle (n = 10). Each point is the mean value of two replicate measurements by ELISA. Horizontal lines represent median. EF, MF and LF represent early, mid and late follicular stages; LH, time of LH peak; OV, approximate time of ovulation (~36 h after the onset of LH peak); EL, ML and LL, early, mid and late luteal stages. No significant changes in serum concentrations of CSF-1 across the menstrual cycle were detected.

 


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Figure 2. Plasma and follicular fluid (FF) concentrations of CSF-1 during the natural cycle in patient samples obtained during laparoscopy. Each point represents individual sample value (mean value of two replicate measurements by ELISA; plasma concentrations are indicated by open circles and FF concentrations by filled circles) and horizontal lines represent medians. FPh = follicular phase, LHPh = time of LH surge and OVPh = ovulatory phase. *P = 0.02 (Mann–Whitney test).

 
CSF-1 concentrations during ovarian stimulation
Overall, the peripheral blood concentrations of CSF-1 in ovarian stimulation patients (n = 20) were not significantly different from those of the menstrual cycle but showed less inter-individual variation. During ovarian stimulation, the concentrations in plasma showed significant variation but were not different when pre-stimulation values (median 422, range 253–1598 pg/ml), values at 12 h pre-HCG (median 522, range 219–900 pg/ml), and values at 35–37 h after HCG (median 458, range 250–659 pg/ml) were compared. Significantly higher concentrations were seen on day 9 after HCG (P < 0.01; median 644, range 357–1352 pg/ml) compared with pre-stimulation and 35–37 h after HCG (P < 0.05; Figure 3Go). FF concentrations of CSF-1 (median 3116, range 1824–5883 pg/ml) were about seven-fold higher than those in blood samples (n = 27) taken 35–37 h after HCG (P < 0.0001; median 472, range 250–1055 pg/ml; Figure 4AGo). At this time, there were significant correlations between FF and plasma concentrations of CSF-1 (n = 28) 35–37h after HCG (r = 0.56, P < 0.005; Figure 4BGo), between FF concentrations of CSF-1 and progesterone (Figure 5AGo), as well as between two individual FF concentrations from the same patient independent of follicular volumes (Figure 5BGo). No significant correlations were found between FF concentrations of oestradiol or androstenedione. Also, no statistical relations were found between FF or blood CSF-1 concentrations and patients' age, body mass index, polycystic ovarian morphology (5/20), follicular volumes and number of retrieved oocytes or resulting pregnancies.



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Figure 3. Plasma concentrations of CSF-1 during ovarian stimulation (n = 20, P < 0.005). Individual values are shown and horizontal lines represent medians. *P < 0.01, **P < 0.05 (Dunn's multiple comparison test).

 


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Figure 4. (A) Concentrations of CSF-1 in plasma and FF in samples obtained from IVF patients 35–37 h after HCG. Each point is the mean value of two replicate measurements by ELISA. Horizontal lines represent median. FF concentrations were significantly higher than those in plasma (P < 0.0001, Wilcoxon test). (B) Correlation between plasma and FF concentrations of CSF-1 in samples obtained during transvaginal oocyte retrieval from IVF patients (n = 28, r = 0.56, P < 0.005, Spearman rank correlation).

 


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Figure 5. (A) Correlation between FF concentrations of CSF-1 and progesterone (n = 34, r = 0.49, P < 0.005; Spearman rank correlation). (B) Correlation between the concentrations of CSF-1 in the two individual FF obtained from same patient. Each point is the mean value of two replicate measurements by ELISA (n = 23, r = 0.77, P < 0.0001; Spearman rank correlation).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The striking similarities between the ovulatory process and a local inflammatory reaction as first recognized by Espey (Espey, 1980Go) have led to suggestions that white blood cells play important roles in ovarian physiology (Norman and Brännström, 1994Go; Norman et al., 1997Go). Macrophages have been identified as the most abundant leukocyte subtypes within ovaries and their numbers correlate with the numbers of developing follicles (Katabuchi et al., 1996Go) and ovulated ova (Nishimura et al., 1995Go). Proliferation, differentiation, survival of monocytes and macrophages are largely dependent on CSF-1. This cytokine is a homodimeric protein, which due to alternative splicing occurs in at least two molecular forms of secreted and membrane bound glycoproteins. CSF-1 is expressed by a number of cell types including monocyte–macrophages, fibroblasts, endothelial cells and endometrial–epithelial cells. The receptor for CSF-1, fms, is found mostly on monocytes and macrophages, but also on epithelial cells of the female reproductive tract (reviewed in Pollard and Stanley, 1996Go; Stanley et al., 1997Go).

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., 1997Go). 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., 1997Go).

Various cytokines, such as IL-1 (Cannon and Dinarello, 1985Go), granulocyte-colony stimulating factor (G-CSF) (Makinoda et al., 1995Go) and TNF{alpha} (Brännström et al., 1994bGo) 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., 1986Go), 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., 1987Go) 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., 1998Go). 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., 1993Go; Best et al., 1996Go; Takaya et al., 1997Go) 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-{alpha}, monocyte chemoattractant protein-1 (MCP-1), TNF{alpha} and granulocyte–macrophage colony-stimulating factor (GM–CSF) (Zolti et al., 1990Go; Machelon et al., 1994Go, 1995Go; Jasper et al., 1996Go; Runesson et al., 1996Go; Arici et al., 1996Go, 1997aGo, Arici et al., bGo; Karström-Encrantz et al., 1998Go) 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., 1990Go), 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{alpha}.


    Acknowledgments
 
This study was supported by grants from NHMRCAustralia (to R.J.N.) and the Swedish Medical Research Council (11607 to M.B.); B.S. is an AusAID scholar. We thank Drs Madaleine Buchholz, Lou Warnes, Mr Fred Amato, and IVF laboratory staff of the QEH, South Australia and Göteborg University, Sweden.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 2, 1998; accepted on February 5, 1999.