Expression of interleukin-1ß and its receptor in human granulosa cells and their association with steroidogenesis.

This study aimed to investigate whether interleukin 1ß (IL-1ß) and soluble IL-1 receptor 2 (sIL-1R2) are expressed in human granulosa cells (GCs) and relate to ovarian steroidogenesis. Ninety-six women undergoing in vitro fertilization (IVF) were recruited. RT-PCR and immunocytochemistry were used to detect mRNAs and proteins of IL-1ß and IL-1R2, respectively. The steroidogenesis of primary cultured GCs was evaluated following treatment with either IL-1ß alone or IL-1ß and FSH in combination. There were positive correlations between serum IL-1ß and serum progesterone (r = 0.220, p = 0.032) and follicular fluid (FF) estradiol (r = 0.242, p = 0.018). Additionally, serum and FF sIL-1R2 were negatively and positively correlated with FF estradiol (r = -0.376, p = 0.005) and FF progesterone (r = 0.434, p = 0.001), respectively. The mRNA and protein expression of IL-1ß and IL-1R2 became evident in GCs. IL-1ß alone significantly increased estradiol secretion from GCs, but in the presence of FSH, it could notably promote progesterone secretion in addition to estradiol. In conclusion, IL-1ß and sIL-1R2 are expressed in human GCs and substantially contribute to ovarian steroidogenesis, suggesting that the IL-1ß system may be a potential target for optimizing ovarian hyperstimulation and steroidogenesis in IVF cycles.

Fractalkine and apoptotic/anti-apoptotic markers in granulosa cells of women with polycystic ovarian syndrome.

Owing to the role of fractalkine in regulating cellular apoptosis/proliferation, we investigated fractalkine effects on apoptosis/proliferation signaling of granulosa cells in polycystic ovarian syndrome (PCOS) patients through in vitro and in vivo experiments. In vivo, granulosa cells were collected from 40 women undergoing oocyte retrieval (20 controls and 20 PCOS). The expression levels of fractalkine, BAX, Bcl2, Bcl2-XL, Bad, and TNF-α were assessed using RT-PCR. In vitro, we determined the effect of different doses of fractalkine on the expression of the above mentioned genes in GCs of both groups. We found that the expression levels of fractalkine and Bcl-2 were significantly lower in the GCs of PCOS patients compared to the control group (p < 0.05). In contrast, the expression levels of TNF-α and BAX were higher in the patient's group than in the control group. The results suggested that expression levels of fractalkine were negatively and positively correlated with the number of oocytes and fertilized oocytes respectively. Moreover, fractalkine could dose-dependently increase fractalkine and decrease BAD, BAX, Bcl-xl, and TNF-α expressions in the control GCs. In contrast, GCs collected from PCOS patients revealed an increase in expression of BAD, BAX, and Bcl-xl following fractalkine treatment. Our findings indicated that insufficient expression of fractalkine in PCOS patients is related with elevated apoptotic and inflammatory markers and reduced anti-apoptotic genes in the GCs.

Mating with seminal vesicle-excised male can affect the uterus phospholipid fatty-acids composition during implantation in an experimental mouse model

ABSTRACT Purpose No comprehensive information is available about uterus fatty acid (FA) change during implantation period and possible effects of the seminal vesicle secretion on it. Materials and Methods In this study, we evaluated FA composition of uterus phospholipids during the implantation period in intact and seminal vesicle-excised (SVX) mated female mice. Forty NMRI female mice were divided into control (mated with intact male) and seminal vesicle excised (SVX)-mated (mated with SVX-male) groups. The phospholipid fatty acids composition was monitored during the first five days of pregnancy using gas chromatography and also implantation rate was evaluated on fifth day of pregnancy. Results We found that levels of linoleic acid (LNA) and arachidonic acid (ARA) showed a decreasing trend from the first to the third day of pregnancy and then started to increase on the fourth day and peaked on the fifth day. In contrast, the level of saturated FA (SFA) increased on the second and third day of pregnancy compared to the first (p<0.05) and then decreased on the fourth and fifth. We also found that the seminal vesicle secretion could affect the levels of LNA, ARA, SFA, and PUFA in uterine phospholipids especially on second and third day. Moreover, there was a positive correlation between ARA level and implantation rate in control but not SVX-mated groups. Conclusions It can be concluded that several uterus FA that have important roles in early pregnancy could be affected by seminal vesicle secretion.

Messenger RNA and protein expression of tumor necrosis factor α and its receptors in human follicular granulosa cells.

To evaluate the concentration of tumor necrosis factor α (TNF-α) and its soluble receptors (sTNFR I and II) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of TNF-α and its receptors by luteinized granulosa cells (GCs). In a cross-sectional study and through an in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) program, 81 women undergoing oocyte retrieval were recruited. Serum and FF were obtained from 81 women. GCs were pooled from 20 patients (from six different days of oocyte retrievals, 5-16 follicles per patient). TNF-α and its soluble receptors concentration were determined by enzyme-linked immunosorbent assay and also their expression by immune cytochemistry and reverse-transcription polymerase chain reaction analysis. The median TNF-α concentration in serum was 4.06 pg/ml (interquartile range [IQR], 3.71-6.14) and significantly higher than that in FF with 3.50 pg/ml (IQR, 3.05-5.01), p < 0.001. The sTNFR I and II levels in serum were lower and higher than FF, respectively. The TNF-α levels in serum and FF of good responders were higher than low responders (p = 0.017 and 0.021, respectively). TNF-α cut-off level for low responders versus good responders was 4.174 pg/ml in serum with a pregnancy rate of 25.8% and 40% for below and above of this level, respectively (p = 0.19). For FF, the cut-off value was 3.89 pg/ml. TNF-α and its receptors were expressed by GCs. The presence of TNF-α and its soluble receptors in serum and FF and their expression by GCs suggest an important role for this cytokine in ovarian function.

Mating with seminal vesicle-excised male can affect the uterus phospholipid fatty-acids composition during implantation in an experimental mouse model.

PURPOSE: No comprehensive information is available about uterus fatty acid (FA) change during implantation period and possible effects of the seminal vesicle secretion on it. MATERIALS AND METHODS: In this study, we evaluated FA composition of uterus phospholipids during the implantation period in intact and seminal vesicle-excised (SVX) mated female mice. Forty NMRI female mice were divided into control (mated with intact male) and seminal vesicle excised (SVX)-mated (mated with SVX-male) groups. The phospholipid fatty acids composition was monitored during the fi rst fi ve days of pregnancy using gas chromatography and also implantation rate was evaluated on fi fth day of pregnancy. RESULTS: We found that levels of linoleic acid (LNA) and arachidonic acid (ARA) showed a decreasing trend from the fi rst to the third day of pregnancy and then started to increase on the fourth day and peaked on the fi fth day. In contrast, the level of saturated FA (SFA) increased on the second and third day of pregnancy compared to the fi rst (p<0.05) and then decreased on the fourth and fi fth. We also found that the seminal vesicle secretion could affect the levels of LNA, ARA, SFA, and PUFA in uterine phospholipids especially on second and third day. Moreover, there was a positive correlation between ARA level and implantation rate in control but not SVX-mated groups. CONCLUSIONS: It can be concluded that several uterus FA that have important roles in early pregnancy could be affected by seminal vesicle secretion.

Effects of dietary omega-3 and -6 supplementations on phospholipid fatty acid composition in mice uterus during window of pre-implantation.

Since fatty acid composition of uterus phospholipids is likely to influence embryo implantation, this study was conducted to investigate the effects of dietary omega-3 and -6 fatty acids on implantation rate as well as uterine phospholipid fatty acids composition during mice pre-implantation period. Sixty female mice were randomly distributed into:1) control (standard pellet), 2) omega-3 (standard pellet + 10% w/w of omega-3 fatty acids) and 3) omega-6 (standard pellet + 10% w/w of omega-6 fatty acids). Uterine phospholipid fatty acid composition during the pre-implantation window (days 1-5 of pregnancy) was analyzed using gas-chromatography. The implantation rate on the fifth day of pregnancy was also determined. Our results showed that on days 1, 2 and 3 of pregnancy, the levels of arachidonic acid (ARA) as well as total omega-6 fatty acids were significantly higher and the levels of linolenic acid and total omega-3 fatty acids were statistically lower in the omega-6 group compared to the omega-3 group (p < 0.05). On the fourth day of pregnancy, only the ARA, total omega-6 fatty acids, and poly-unsaturated fatty acids levels were significantly different between the two dietary supplemented groups (p < 0.05). There were positive correlations between the levels of omega-6 fatty acids, especially ARA, with the implantation rate. The present study showed that diets rich in omega-3 and -6 fatty acids could differently modify uterine phospholipid fatty acid composition and uterine levels of phospholipid ARA, and that the total omega-6 fatty acids had a positive association with the implantation rate.

The Role of Interleukin-18 in Serum and Follicular Fluid during In Vitro Fertilization and Intracytoplasmic Sperm Injection.

Cytokines are key modulators of the immune system and play an important role in the ovarian cycle. IL-18 levels in serum and follicular fluid were analyzed in women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment. The cohort study group consisted of 90 women, who were undergoing IVF or ICSI. The body mass index (BMI) was determined in all patients; IL-18 levels were measured in follicular fluid and serum. IL-18 levels in serum were significantly higher than those in follicular fluid. The median level in serum was 162.75 (80.21) pg/mL and that in follicular fluid, 138.24 (91.78) pg/mL. Women undergoing IVF treatment had lower IL-18 levels in serum (median, 151.19 (90.73) pg/mL) than those treated with ICSI (median, 163.57 (89.97) pg/mL). The correlation between IL-18 levels in serum and BMI was statistically significant, as well as the correlation between IL-18 levels in follicular fluid and ovarian stimulation response (p = 0.003). IL-18 was correlated with the response to ovarian stimulation and was the reason for successful pregnancy after IVF or ICSI treatment. Among other cytokines, IL-18 appears to be a promising prognostic marker of success in reproductive treatment and should be evaluated as such in further prospective studies.

Macrophage colony-stimulating factor (M-CSF) in first trimester maternal serum: correlation with pathologic pregnancy outcome.

PURPOSE: To determine correlations between macrophage colony-stimulating factor (MCSF) levels in maternal blood during first trimester screening with respect to normal and pathological pregnancies. METHODS: This was a prospective single centre study. First trimester screening was performed according to FMF London certificates. Nuchal translucency, PAPP-A and free ß-HCG were obtained as well as M-CSF serum levels in maternal blood. Fetal karyotyping was achieved by chorionic villi sampling. RESULTS: 125 patients were enrolled in this study. 21 pregnancies had confirmed aberrant karyotypes. Trisomy 21 cases showed significantly elevated M-CSF levels of 270 ± 91 pg/ml (p = 0.032), whereas cases of trisomy 13 (183 ± 68 pg/ml) and trisomy 18 (143 ± 40 pg/ml) had low M-CSF levels. Furthermore M-CSF levels tended to be low in preterm deliveries, placental insufficiency and nicotine consumption. In cases with gestational diabetes M-CSF tended to be elevated. Furthermore we found a positive correlation between high free ß-human chorionic gonadotropin (hcg) and MCSF values. There was no correlation between pregnancy associated plasma protein (PAPP-A) and M-CSF. CONCLUSIONS: M-CSF is a cytokine promoting placental growth and differentiation. M-CSF is known to be involved in the process of implantation in pregnancy. The role of M-CSF with respect to disturbed pregnancy outcomes such as placental insufficiency in normal or aberrant karyotypes, for example, is yet subject to further research.

Cut-Off Levels of Anti-Mullerian Hormone for The Prediction of Ovarian Response, In Vitro Fertilization Outcome and Ovarian Hyperstimulation Syndrome.

BACKGROUND: Evaluation of anti-mullerian hormone (AMH) cut-off levels in as- sisted reproductive technology (ART) as predictive factor for individualization of stimulation protocols and to avoid ovarian hyperstimulation syndrome (OHSS). MATERIALS AND METHODS: In a retrospective study, 177 infertile patients were as- sessed for AMH in serum and follicular fluid (FF) on the day of follicular puncture (FP), between 2012 and 2013 in Kiel, Germany. AMH levels and pregnancy rates were compared between low, moderate and high responders and cut-off levels of low and high responders. AMH cut-off levels in pathological cases were evaluated in analysis 1 (OHSS) and in analysis 2 [polycystic ovarian syndrome, (PCOS)] and compared in analysis 3 to normal endocrinological parameters. RESULTS: AMH levels in FF were higher than in serum (P<0.001). AMH levels in serum and FF increased from low through moderate to high responders (P<0.001). Pregnancy rates were 14.7, 23.3 and 44.9% (P=0.009), respectively. AMH cut-off level for poor responders was 0.61 ng/ml in serum with a pregnancy rate of 13.8 and 37.1% for below and above of this level, respectively. For FF, it was 1.43 ng/ml. AMH levels in analysis 1 and 2 were significantly higher than in analysis 3 (P=0.001). AMH cut-off level for OHSS was 1.5 ng/ml in serum with OHSS rates of 80.8 and 19.2 % for above and below of the level, respectively. For FF, it was 2.7 ng/ml. PCOS patients had an AMH cut-off level of 3.9 ng/ml in serum and 6.8 ng/ml in FF, resulting in a PCOS rate of 100% above this level. CONCLUSION: AMH levels can help to assess ovarian response potential and guide ovarian stimulation while avoiding OHSS.

Distended jugular lymphatic sacs in fetuses with increased nuchal translucency: correlation with first-trimester findings in aberrant karyotypes.

OBJECTIVE: We investigated the prognostic relevance of ultrasound visibility of distendend jugular lymphatic sacs (JLS) in fetuses with aberrant karyotypes in First-trimester-screening. Furthermore we tried to differentiate between increased nuchal translucency (NT) and cystic hygroma colli. METHODS: We performed a retrospective single center study in 1874 patients presenting for First-trimester-screening between 2009 and 2013. All fetuses with an abnormal risk calculation and NT > 2.5 mm (95th percentile) were reviewed for ultrasound visibility of JLS. A group of 30 fetuses with normal risk calculation served as control. Karyotyping was performed by chorionic-villi-sampling or amniocentesis, respectively. RESULTS: In a total of 2030 fetuses 70 (3.44%) with pathologic first-trimester-screening results showed either aberrant karyotypes or severe ultrasound pathologies. Main aberrant karyotypes were trisomy 21 (25), trisomy 18 (16), trisomy 13(six), Monosomy X (four), 47, XYY or 47, XXX (three) and Noonan' syndrome (two). Distended JLS were visible in 47% of all cases. Statistical anaylsis found a significant correlation between NT and JLS size for the fetuses with trisomies 21, 18 and 13 (r = 0.53, p < 0.002). Cystic hygroma colli was present in all Turner and Noonan syndromes. CONCLUSIONS: Distended JLS have a strong correlation with abnormal karyotypes and increased nuchal translucency. Karyotyping should be offered in these cases.

Oocyte morphology on day 0 correlates with aneuploidy as detected by polar body biopsy and FISH.

PURPOSE: For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. METHOD: Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4-13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. RESULT: Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern "1, 2, 3 and fine" in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). CONCLUSION: There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.

Sham-controlled implantation after preimplantation genetic screening by polar body biopsy and FISH.

PURPOSE: Preimplantation genetic screening wants to improve artificial reproductive technologies, primarily by raising the rates of pregnancy, implantation and birth. We investigated if embryos derived from oocytes detected euploid for five chromosomes implant better than those which were biopsied but where the genetic detection failed. They were nevertheless transferred, thus serving as a sham control. METHOD: From 2004 to 2008 we performed 104 cycles of PGS with laser biopsy of the first polar body and FISH with five chromosomes. It was offered to all patients with eight or more oocytes, free of charge. The average female age was 36 years. If no euploid oocytes were available, not detected oocytes were transferred. RESULT: In 104 cycles 99 embryo transfers (95 %) were performed, resulting in 28 pregnancies (27 %), 20 births (71 %) and 8 miscarriages (29 %). The implantation rate in the euploid group was 19 vs. 13 % in the not detected group (n.s.). This trend was the same independent of age and embryo morphology. CONCLUSION: The pregnancy rate does not differ significantly from the national average. The trend in better implantation rates of euploid oocytes justifies a continuation of studies in this matter.

Combined surgical and hormone therapy for endometriosis is the most effective treatment: prospective, randomized, controlled trial.

STUDY OBJECTIVE: To evaluate 3 therapy strategies: hormone therapy, surgery, and combined treatment. DESIGN: Prospective, randomized, controlled study (Canadian Task Force classification I). SETTING: University-based teaching hospital. PATIENTS: Four hundred fifty patients with genital endometriosis, aged 18 to 44 years, before first laparoscopy. INTERVENTIONS: Patients were randomly assigned to 1 of 3 treatment groups: hormone therapy, surgery, or combined treatment. Patients were reevaluated at second-look laparoscopy, at 2 to 2 months after 3-month hormone therapy in groups 1 and 3 and at 5 to 6 months in group 2 (surgical treatment alone). Outcome data were focussed on the endometriosis stage, recurrence of symptoms, and pregnancy rate. MEASUREMENTS AND MAIN RESULTS: All treatment options, independent of the initial Endoscopic Endometriosis Classification stage, achieved an overall cure rate of ≥50%. A cure rate of 60% was achieved with the combined treatment, 55% with exclusively hormone therapy, and 50% with exclusively surgical treatment. Recurrence of symptoms was lowest in patients who received combined treatment. Significant benefit was achieved for dysmenorrhea and dyspareunia. An overall pregnancy rate of 55% to 65% was achieved, with no significant difference between the therapeutic options. CONCLUSION: In the quest to find the most effective treatment of genital endometriosis, this clinical randomized study shows the lowest incidence of recurrence with combined surgical and medical treatment and improved pregnancy rate in any medically treated patients with or without surgery. The highest cure rate (Endoscopic Endometriosis Classification stage 0) for endometriosis was also achieved in the combined treatment group.

Circulating concentration of stem cell factor in serum of stimulated IVF patients.

Stem cell factor (SCF) plays a major role in haematopoiesis and spermatogenesis, and possibly female fertility. This study investigated the role of changes in SCF concentrations in 74 assisted conception patients. In group 1 (n=74) SCF concentration was assessed in serum and follicular fluid (FF) on the day of follicular puncture (FP) and compared in serum and FF in response to ovarian stimulation between low (n=25), moderate (n=26) and high (n=14) responders. In group 2 (n=30) serum for SCF assessment was collected throughout the menstrual cycle until gestation. SCF concentration related to the number of follicles in serum and in FF decreased from low to moderate and high responders (P<0.001); pregnancy rates were 20.0%, 34.6% and 50.1%, respectively (P=0.05). SCF in serum increased from stimulation days 6-8 to 9-11 and peaked on the day of human chorionic gonadotrophin injection (P=0.03). The SCF concentrations dropped slightly on the day of FP, increased significantly to the day of pregnancy confirmation and reached highest concentration (P=0.02) during gestation. SCF is involved in follicle development and may be a predictor of IVF outcome.

Apoptosis resistance in endometriosis.

INTRODUCTION: In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. METHODS: Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling) and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5). RESULTS: Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. CONCLUSION: A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.

Circulating level of macrophage colony-stimulating factor can be predictive for human in vitro fertilization outcome.

OBJECTIVE: To evaluate the level of macrophage colony-stimulating factor (M-CSF) in serum in response to ovarian stimulation (group 1) in low-response (n = 26), moderate-response (n = 40), and high-response (n = 29) patients and to compare its changes (n = 23, group 2) throughout the menstrual cycle between pregnant and nonpregnant patients. DESIGN: Randomized controlled trial. SETTING: University IVF program. PATIENT(S): Ninety-five women undergoing IVF. INTERVENTION(S): Serum and FF collection from 95 women. MAIN OUTCOME MEASURE(S): The M-CSF concentration was determined by ELISA. RESULT(S): The M-CSF levels in FF were higher than in serum. The M-CSF levels in serum increased from low-, through moderate-, to high-response patients; pregnancy rates were 11.5%, 22.5%, and 51.7%, respectively. Levels of M-CSF in serum increased throughout stimulation until the day of oocyte retrieval and decreased until ET. During the postretrieval days, from the day of ET, through implantation, to the day of confirmation of pregnancy, the M-CSF levels of those patients who became pregnant (n = 13) increased significantly and reached their highest level. After implantation the M-CSF level decreased slightly and reached a plateau during gestation. CONCLUSION(S): Macrophage colony-stimulating factor is involved in follicle development and ovulation and could be an additional predictor for IVF outcome.

Differential interleukin-6 messenger ribonucleic acid expression and its distribution pattern in eutopic and ectopic endometrium.

OBJECTIVE: To evaluate the concentration and distribution pattern of interleukin-6-producing cells in ectopic and eutopic endometrium. DESIGN: Collection of both tissues at the proliferative cycle phase. SETTING: A university endocrinology and laparoscopy program. PATIENT(S): Twelve samples from six women were obtained at operative laparoscopy in the proliferative phase of each woman's cycle. INTERVENTION(S): Paired sampling of ectopic (n = 6) and eutopic endometrium (n = 6). MAIN OUTCOME MEASURE(S): Concentration of IL-6 messenger RNA (mRNA) was measured by real-time RT-PCR, and its distribution was determined by in situ RT-PCR. RESULT(S): In vitro RT-PCR analysis showed distinct bands at 234 bp and 115 bp for IL-6 mRNA in eutopic and ectopic endometrium and in placenta as positive control. Real-time RT-PCR indicated significantly higher expression of IL-6 in tissue with endometriosis than in tissue without. In all 12 samples subjected to in situ RT-PCR, positive signals of IL-6 mRNA were observed in cytoplasm of epithelial and stroma cells of ectopic and eutopic endometrium tissue. CONCLUSION(S): The significant IL-6 mRNA increase in endometriosis compared with in eutopic endometrium may play a role in the development of endometriosis and may provide new directions for endometriosis research.

Expression of mRNA and protein of macrophage colony-stimulating factor and its receptor in human follicular luteinized granulosa cells.

OBJECTIVE: To evaluate the concentration of macrophage colony-stimulating factor (M-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of M-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF- intracytoplasmic sperm injection (ICSI) program. PATIENT(S): Serum and FF were obtained from 85 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 85 women. The GCs were pooled from 15 (3 x 5) patients (3-14 oocytes each). MAIN OUTCOME MEASURE(S): The M-CSF concentration was determined by ELISA, the expression of M-CSF and its receptor by the immunocytochemical technique and reverse transcription polymerase chain reaction analysis. In addition, M-CSF expression was investigated by cell culture time course studies. RESULTS: The median M-CSF concentration in FF (2,409.2 pg/mL) was significantly higher than that in serum (242.5 pg/mL). The M-CSF and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of M-CSF in FF than in serum and the expression of M-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.

Identification of the M-CSF receptor in endometriosis by immunohistochemistry and RT-PCR.

PROBLEM: The aim of this paper is to provide further evidence that the dystopic proliferation of endometriotic epithelia is caused by the stimulation of peritoneal macrophages. It is essential to show that endometriotic epithelial cells express the macrophage colony-stimulating factor receptor (M-CSFR) which binds the M-CSF produced by the peritoneal macrophages. METHOD OF STUDY: For the detection of M-CSFR, samples of ectopic endometrium (n = 79) and eutopic endometrium (n = 18) were compared. The specimens were gained at operative laparoscopy in the proliferative phase of the cycle. Cryostat sections were used for immunohistochemical detection. For in vitro reverse transcriptase polymerase chain reaction (RT-PCR) tests, the tissue was immediately shock frozen on paraffin sections. For the in situ RT-PCR technique the specimens were placed in a para-formaldehyde solution, embedded in paraffin and later processed. The Gene Amp 1000 in situ PCR system (Perkin Elmer) was used as the thermal cycler. RESULTS: M-CSF and the M-CSF receptor are present in eutopic and ectopic endometrium. Qualitatively, with both PCR techniques we found the M-CSF receptor to be present in all samples examined. Using the histochemical detection technique, the M-CSF receptor was found in nearly 70% of endometriosis patients compared with a statistically significant lower percentage in normal endometrium. CONCLUSIONS: The in situ RT-PCR technique and immunohistochemistry elaborated the need to trace the cellular sources of the M-CSF receptor. The identification of the M-CSF receptor in endometriotic tissue and in endometrium is apt to open a new experimental field in endometriosis research.

Detection of granulocyte colony-stimulating factor and its receptor in human follicular luteinized granulosa cells.

OBJECTIVE: To evaluate concentration of granulocyte colony-stimulating factor (G-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of G-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF-ICSI program. PATIENT(S): Serum and FF were obtained from 82 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 82 women. Granulosa cells were pooled from 15 patients (three experiments with five patients each; 3-14 oocytes each). MAIN OUTCOME MEASURE(S): Granulocyte colony-stimulating factor concentration was determined by ELISA, the expression of G-CSF, and its receptor by the immunocytochemical technique and reverse transcriptase polymerase chain reaction analysis. Additionally, G-CSF expression was investigated by cell culture time course studies. RESULT(S): The median G-CSF level in FF (117.98 pg/mL) was significantly higher than that in serum (67.5 pg/mL). Granulocyte colony-stimulating factor and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of G-CSF in FF than in serum and the expression of G-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.