Abstract
The aim of this study was to measure circulating and intrafollicular concentrations of three inflammatory cytokines from women undergoing ovarian stimulation in order to determine their prognostic value in the outcome of intracytoplasmic sperm injection/embryo transfer cycles. Materials and Methods: A total of 72 women following ovarian stimulation and intracytoplasmic sperm injection were included. Blood serum samples were drawn at the day of chorionic gonadotropin administration. Follicular fluids were collected at the day of oocyte retrieval. The total fractions of tumor necrosis factor α, interleukin (IL)-1β and IL-6 were measured with commercially available immunoassays. Results: The concentrations of IL-1β, both in serum and follicular fluids, were significantly different between ICSI cycles that resulted in pregnancy and those that failed. The concentrations of the other two cytokines did not significantly differ between successful and unsuccessful cycles. Conclusion: The circulating and intrafollicular concentrations of IL-1β seem to be related to the pregnancy outcome in ICSI cycles of healthy women.
The last three decades have been marked by a blooming of assisted reproduction techniques. The advent of in vitro fertilization (IVF), controlled ovarian hyperstimulation (COH) and intracytoplasmic sperm injection (ICSI) are concomitant with an increase in the intensity of research into reproductive physiology. IVF cycles provide unique opportunities to investigate reproductive processes such as folliculogenesis, ovulation and implantation, as it is easy to collect biological samples e.g. blood and follicular fluids (FFs) under controlled and carefully monitored ovarian function.
The imperative necessity for the optimization of IVF attempts has led many scientific teams to study the usefulness of circulating or intrafollicular concentrations of several cytokines and hormones as prognostic markers for the outcome of IVF cycles. Inflammatory cytokines are among the investigated cytokines, yet there is no consensus on their prognostic value for fertilization or pregnancy outcome.
Inflammatory cytokines are present in various ovarian compartments, including the follicles, where they are produced not only by macrophages but also by ovarian tissue (1-13). A large body of evidence suggests that inflammatory cytokines exert direct effects on endocrine ovarian cells, and, on the other hand, the hormonal production of ovarian cells influences the expression of inflammatory cytokines (13-19). Inflammatory cytokines are involved in inflammatory reactions within the ovary and it has been suggested that they play a pivotal role in processes such as follicular atresia, ovulation and possibly to participate in the final maturation of the oocyte (3, 9, 11, 13, 20-22).
In the present study, the concentrations of three inflammatory cytokines in both blood and FFs were studied in women following COH for ICSI and embryo transfer cycles. The studied cytokines were: tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6. The aim was to explore the possible prognostic value of these cytokines in the outcome of ICSI cycles, as well as their relationship with estradiol and progesterone. The women who participated in the study had a normal reproductive system and were undergoing COH, ICSI and embryo transfer due to male factor infertility.
Patients and Methods
Patients, COH, ICSI and embryo transfers. This case-control study was carried out in the Department of Gynecology and Obstetrics, University Clinic of Schleswig-Holstein, Campus Lűbeck. The study population included 72 women without any apparent abnormality of their reproductive system as defined by their medical history, the clinical examination and common hormonal tests. They participated in ICSI cycles exclusively due to male factor infertility. Their age was 32.91±3.82 years (range=25-41 years). Low responders as well as women with a basal follicle-stimulating hormone (FSH) level of ≥10 mIU/ml were excluded.
All patients gave their consent and did not receive any monetary compensation for participating in the study. For each patient, only one cycle was included in the study.
COH was performed with multidose gonadotropin-releasing hormone (GnRH)-antagonist protocol (Lübeck antagonist protocol) as described elsewhere (23, 24). The ovarian stimulation was carried out with recombinant FSH (Gonal-F; Serono International S.A., Geneva, Switzerland) and pituitary suppression with the GnRH-antagonist cetrorelix (Cetrotide; Serono International S.A.). The dosages of gonadotropins were individualized according to serum estradiol concentrations and transvaginal ultrasound measurements of the leading follicles. Final ooocyte maturation was induced by the injection of 10,000 IU human chorionic gonadotropin (hCG) (Pregnyl; N.V. Organon, Oss, the Netherlands) when at least three follicles reached a diameter of 17 mm and the serum estradiol concentration increased to 300-500 pg/ml per follicle. Transvaginal oocyte aspiration with ultrasound guidance was performed 36 hours later.
The preparation of oocytes, ICSI and the culture of fertilized oocytes were performed as previously described (25). Only fresh semen samples obtained by masturbation after 3-4 days of abstinence were used.
Embryo transfers were performed 48 hours after oocyte aspiration under ultrasound guidance. Up to three cleaving embryos were transferred, according to the German Embryo Protection Law. Before embryo transfers, the cumulative embryo score (CES) was calculated as described elsewhere (26).
The luteal phase was supported daily with 600 mg natural progesterone administrated vaginally (Utrogestan; Besins-Iscovesco, Paris, France). Pregnancies were defined by the presence of positive fetal heart beats. In these cases, the administration of progesterone was continued up to week 12 of gestation.
Sampling and measurements. On the day of hCG administration, a peripheral blood sample was taken from each woman, serum was extracted and aliquots stored at −75°C.
During oocyte aspiration, from each patient, FF samples were collected among the first one to three mature follicles containing metaphase II (MII) oocytes and were placed in sterile tubes. FFs mixed with flushing fluids or with obvious blood contamination were excluded. In cases where two or three FF samples were collected from a patient, FF samples were pooled. The pooled FF samples were mildly centrifuged immediately after the collection and the supernatants were pooled and stored at −75°C in aliquots for further analysis.
In serum and FF samples, the levels of the total fractions of the following cytokines were measured: IL-1β, IL-6 and TNFα. Estradiol was measured in both serum and FFs. Additionally progesterone and testosterone levels were measured in serum samples.
In serum samples, estradiol, progesterone and testosterone were measured with Elecsys immunoanalyzer (Roche Diagnostics, Mannheim, Germany) having the following intra- and inter-assay variation (AV): <5% and <10% for estradiol, <3% and <5% for progesterone and <5% and <7% for testosterone, respectively. All the other measurements were made with commercial enzyme immunoassay kits as follows. Estradiol: DSL-10-4300 Active Estradiol EIA (DSL, Webster, Texas, USA), intra-AV: 3.3-4.8%, inter-AV: 6.5-8.2%, minimum detectable dose (MDD): 7 pg/ml. IL-1β: Quantikine DLB50 (R&D Systems Inc, Minneapolis, USA), intra-AV: 2.8-8.5%, inter-AV: 4.1-8.4%, MDD: 1 pg/ml. IL-6: Quantikine D6050 (R&D Systems Inc), intra-AV: 1.6-4.2%, inter-AV: 3.3-6.4%, MDD: 0.7 pg/ml. TNFα: Quantikine DTA00C (R&D Systems Inc), intra-AV: 4.2-5.2%, inter-AV: 4.6-7.4%, MDD: 0.5 pg/ml.
As the above kits are validated for serum samples but not for FFs, before running FF samples, the recovery rate of at least three spiked FF samples was assessed and the linearity of the results after multiple dilutions determined. The results were satisfactory, with recovery rates ranging from 83% to 118%. All samples were measured in duplicate, according to the manufacturers' instructions. In cases of very high or low results, the measurements were repeated.
Statistical analysis. The cycles that resulted in pregnancy are referred to as group A, while the cycles that failed to result in pregnancy are referred to as group B. The normality of all studied parameters was checked with the Shapiro–Wilks' W test and an additional evaluation of kurtosis and skewness. The statistical analysis included descriptive statistics for both studied groups. The comparisons of clinical data as well as of hormonal and cytokine concentrations between the groups were performed either with the t-test, or with two nonparametric tests: Mann-Whitney U-test and Kolmogorov-Smirnov. The number of transferred embryos (range: 1-3) was treated as a categorical variable and the comparison between the two study groups was made with Pearson's chi-square test. Correlations were evaluated with Spearman's rank test. The two-tailed significant level was set at p<0.05. The software used for statistical analysis was STATISTICA 6.0 (StatSoft Inc., Tulsa, OK, USA). All values are presented as the mean±standard deviation.
Results
The ovarian stimulation was successful in all patients. On average, 9.55±5.31 MII oocytes were retrieved (range: 1-28). Embryo transfer was performed in all patients. There were no cases of ovarian hyperstimulation syndrome. Among the 72 cycles, 20 resulted in clinical pregnancies. The clinical characteristics, as well as the measurements of the cycles that resulted in pregnancy (group A) and those that failed (group B), are presented in Table I. The two groups were similar in terms of age, body mass index (BMI) and the quality of transferred embryos. Group A received lower amounts of gonadotropins, had slightly lower peak serum estradiol concentrations and a slightly higher fertilization rate, in comparison to group B, although these differences were not statistically significant.
Clinical characteristics, hormonal and cytokines concentrations of the studied groups
Among the three cytokines, IL-6 was detected in all samples. In the majority of serum samples, IL-6 concentrations were <2 pg/ml, with a median of 1.64 pg/ml and a mean of 8.66±43.61 pg/ml. In general, the concentrations of IL-6 in FF samples (FF-IL-6) were higher than in serum samples, most of them ranging between 1.48 and 12 pg/ml, with a median of 7.4 pg/ml and a mean of 14.30±31.51 pg/ml. In two patients, the concentrations of IL-6 in both serum and FF samples exceeded 100 pg/ml.
The concentration of IL-1β was detectable in 51 serum and 47 FF samples. In serum samples, the median concentration was 0.22 pg/ml, whereas in FF samples it was 0.6 pg/ml. No sample had a concentration higher than 25.5 pg/ml.
The concentration of TNFα was undetectable in 26 serum and 54 FF samples. In serum samples, the mean concentration was 0.72±1.66 pg/ml whereas in FF samples (FF-TNFα), it was 17.70±41.22 pg/ml.
The concentrations of IL-1β were significantly associated with the pregnancy outcome. Serum IL-1β concentrations in group A were significantly lower than those in group B. On the other hand, intrafollicular concentrations (FF-IL-1β) in group A were significantly higher than those in group B.
The correlations among the concentrations of the inflammatory cytokines and those of hormones as well as the other studied parameters are presented in Tables II and III. The age of patients was negatively correlated with the intrafollicular concentration of IL-1β (FF-IL-1β). The intrafollicular TNFα (FF-TNFα) concentration was negatively correlated with intrafollicular estradiol (FF-E) concentration, whereas serum TNFα concentration was negatively correlated with serum IL-1β concentration.
The fertilization rate and CES were not correlated either with serum or with intrafollicular cytokine concentrations.
Discussion
Ovulation resembles an inflammatory process and inflammatory cytokines seem to play a pivotal role in this process. The expression of inflammatory cytokines increases during certain phases of the cycle and in turn they induce the production of factors contributing to the weakening of follicular wall, factors promoting angiogenesis and adhesion molecules promoting implantation. Souter et al. showed that during controlled ovarian simulation, TNFα concentrations increase at the mid follicular phase, whereas IL-6 concentrations increase at the luteal phase of the cycle (27). It is known that TNFα increases the production of collagenases (22) whereas IL-1β induces the production of prostaglandins, nitric oxide, vascular endothelial growth factor and adhesion molecules (28). Karagouni et al. reported that gonadotropins administrated during controlled ovarian stimulation induce both local and systemic production of Il-1 (10). There is also evidence indicating that IL-1β stimulates progesterone production by luteal cells (16, 19).
Correlations among the clinical characteristics of the cycles and the serum concentrations of hormones and cytokines on the day of hCG administration. Spearman's R is given. *p<0.05.
Correlations among the clinical characteristics of the studied cycles and the concentrations of hormones and cytokines in follicular fluid (FF) samples. Spearman's R is given. *p<0.05.
Cytokines have a paracrine and autocrine mode of action that does not require high concentrations. In the present study, the circulating and the intrafollicular concentrations of the three cytokines, especially those of IL-1β and TNFα were low, while in a number of samples they were undetectable. This finding is in agreement with previous studies which reported low or undetectable IL1β and TNFα concentrations in many serum and FF samples (10-11, 15, 25, 29, 30). Elevated concentrations of both cytokines have been reported in cases of endometriosis, polycystic ovarian syndrome and immunological disorders (31-34).
The present study investigated the concentrations of the three inflammatory cytokines within two critical time-points: just before the administration of hCG to induce ovulation and luteinization of follicles and secondly, 36 hours later when luteinization has occurred, the follicular walls are weakened by enzymes and the oocytes, which have entered metaphase II, are ready to be extruded. In general, the intrafollicular concentrations of the three cytokines were higher than the circulating ones. Taking into consideration that inflammatory cytokines actively participate in the ovulation process, it is reasonable to have higher concentrations in FFs which were collected on the day of ovulation compared to their concentrations in serum samples, which were collected before luteinization.
This study showed that among the three inflammatory cytokines, IL-1β is of special interest as its concentration both in serum and FFs was associated with the pregnancy outcome of the ICSI/ET cycles. Namely, the statistical analysis revealed that women who became pregnant, compared with those who failed to achieve pregnancy, had lower concentrations of IL-1β in blood on the day of hCG but higher concentrations in FFs on the day of oocyte retrieval.
At first sight, these findings appear contradictory. However, it is necessary to bear in mind that the constitutive production of inflammatory cytokines is usually low or absent, whereas the expression of IL-1β in the ovary is enhanced around the time of ovulation (3, 4). In FFs, IL-1β is released not only by leukocytes and activated tissue macrophages but also by granulosa and cumulus cells at the time of ovulation (4, 15). Thus, during the periovulatory period, the levels of IL-1β are elevated (4). Furthermore, serum concentrations of IL-1β may reflect its production in other sites than the ovary. It could be postulated that elevated serum concentrations of IL-1β before the induction of ovulation are possibly indicative of pathological inflammatory reactions. On the other hand, elevated intrafollicular concentrations of IL-1β after ovulation induction are probably indicative of a healthy and competent follicular environment.
Our results are in agreement with those of Karagouni et al. who found that intrafollicular IL-1β concentrations in IVF patients who conceived were significantly higher than in IVF patients who failed to conceive (10). It is also worth noting that in a previous study including women with various reasons for infertility, we found higher intrafollicular IL-1β concentrations in ICSI cycles that resulted in pregnancy compared to those that failed, although the difference was not statistically significant (35). However, other investigators have failed to find a relation between intrafollicular IL-1β concentrations and pregnancy outcome (7, 16, 29).
The statistical analysis of the correlations between the concentrations of cytokines and hormones showed that intrafollicular TNFα was negatively correlated with intrafollicular estradiol. A similar result was also reported by Souter et al. (27) and it is in line with a previous study suggesting inhibition of TNFα production by estrogens (17). Furthermore, the significant positive correlation between serum IL-6 concentrations and BMI is in agreement with previous reports (36). Another interesting finding requiring further investigation is the strong negative correlation between serum concentrations of TNFα and IL-1β.
As a conclusion, the present study showed that in women with no apparent pathology undergoing COH, IL-1β, TNFα and IL-6 are usually present in peripheral blood and FFs in low concentrations. Among the three cytokines, IL-1β seems to be related to the achievement of pregnancy in ICSI/ET cycles. Although the results of the present study are not enough to establish IL-1β as a prognostic marker in ICSI/ET cycles, they highlight IL-1β as a promising candidate for future research. Further studies with a higher number of cases are needed to confirm the value of serum and intrafollicular IL-1β concentrations as prognostic markers for the pregnancy outcome in ICSI cycles of healthy women.
Acknowledgements
The study was partially supported by Deutscher Akademischer Austausch Dienst (DAAD) and the Greek Scholarships Foundation (IKY) through the program IKYDA.
- Received December 16, 2009.
- Revision received April 19, 2010.
- Accepted April 23, 2010.
- Copyright © 2010 The Author(s). Published by the International Institute of Anticancer Research.
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