The effect of serum and follicular fluid secreted frizzle-related protein-5 on in vitro fertilization outcomes in patients with polycystic ovary syndrome

Zeynep Ozturk Inal1 · Hasan Ali Inal1 · Sami Erdem2

Abstract

In this study, we aimed to investigate serum and follicular fluid (FF) secreted frizzle-related protein-5 (Sfrp-5) levels in nonobese, nonhyperandrogenic patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), in addition to IVF outcomes. In total, 160 patients undergoing IVF treatment were included in the study: 80 patients diagnosed with PCOS according to the Rotterdam criteria (group I, study) and 80 patients with the etiology of male factor infertility (group II, control). There were statistically significant between-group differences in serum estradiol ( E2) levels on the day of hCG administration (2377.00 ± 733.23 vs. 1931.3 ± 1,010.69), the total gonadotropin dose required (2000.63 ± 1,051.87 vs. 1.134.69 ± 286.45), and the total number of retrieved oocytes (8.60 ± 2.06 vs. 11.05 ± 4.39) (p < 0.05). There was also a statistically significant between-group difference in serum and FF Sfrp-5 levels on the day of oocyte retrieval (11.40 ± 2.88 vs. 8.87 ± 1.85, p < 0.001; 11.06 ± 2.30 vs. 9.71 ± 2.15, p = 0.008; respectively). However, there were no between-group differences in fertilization rates, clinical pregnancy rates, and live birth rates (p > 0.05). A correlation analysis showed that serum and FF Sfrp-5 levels were associated with insulin and inflammatory markers (p < 0.05). In a selected population of nonobese, nonhyperandrogenic PCOS patients, there was a significant difference in Sfrp-5 levels of the PCOS group versus those of the control group. Further studies are needed to determine the effects of Sfrp-5 in women with PCOS.

Keywords Invitro fertilization · Polycystic ovary syndrome · Secreted frizzle-related protein-5

Introduction

Polycystic ovary syndrome (PCOS) is a common, complex, and heterogeneous endocrinopathy, affecting approximately 5–10% of reproductive-age women [1, 2]. It is characterized by a proinflammatory state and presents with menstrual irregularities and hyperandrogenism [1, 2]. Although the etiopathogenesis of PCOS is not entirely clear, genetic factors, such as defective steroid biosynthesis and insulin resistance (IR) with hyperinsulinemia, in addition to environmental factors, may play a role [3–5]. Metabolic syndrome is seen in approximately 50% of PCOS patients [3–5]. In addition, 40–70% of patients with PCOS are obese and have an increased risk of type II diabetes mellitus (DM), cardiovascular disease, hyperlipidemia, and hypertension [6, 7]. An unfavorable follicular fluid (FF) microenvironment caused by metabolic and reproductive abnormalities in PCOS may result in decreased fertilization and cause infertility by impairing ovulation and oocyte maturation and quality [2, 7]. The aforementioned factors can also have negative perinatal outcomes, such as miscarriage, preeclampsia, and congenital abnormalities [2, 6, 7].
Previous studies showed that adipose tissue played a role by releasing a number of adipokines and that these adipokines increased cytokines, such as high-sensitive C-reactive protein (hs-CRP) and interleukin (IL)-18, in PCOS patients [3, 7–9]. Research demonstrated that these inflammatory adipokines stimulated or inhibited cell growth, regulated cell differentiation, induced cell chemotaxis, and controlled the release of various other cytokines [8]. Studies reported that some cytokines, such as IL-1, IL-12, and IL-18, improved clinical pregnancy rates by increasing fertilization, with a positive effect on oocyte quality [2, 10]. On the other hand, tumor necrosis factor-α and hs-CRP resulted in lower fertilization rates by reducing oocyte and embryo quality in patients undergoing in vitro fertilization (IVF)–intracytoplasmic sperm injection (ICSI) treatment [10, 11].
Previous studies demonstrated that the wingless-type MMTV integration site family member (Wnt) signaling pathway was involved in cell proliferation, differentiation, motility, and cell development; beta-cell differentiation; pancreatic development and function;and the regulation of adipogenesis, IR, resistance, placental vascularization, and inflammation [12, 13]. Research also showed that this signaling pathway negatively regulated adipogenesis [14].
Recent studies showed that secreted frizzle-related protein-5 (Sfrp-5), a novel antagonist adipokine of the Wnt signaling pathway and one of five known members of the Sfrp family, was secreted by adipocytes [15, 16]. Sfrp-5 regulated lipid metabolism, negatively regulated adipogenesis, decreased IR, and reduced metabolic dysfunction, [15, 16]. It also acted as an anti-inflammatory adipokine and modulated metabolic functions [15, 16]. Sfrp-5exerted these effects by sequestering Wnt proteins in the extracellular compartment and preventing Wnt binding to receptors [14].
There are limited numbers of animal and human studies on the relationship between obesity, insulin resistance, and Sfrp-5. These studies have reported conflicting results, with some studies showing increases in Sfrp-5 in the presence of obesity and IR but others reporting decreases in Sfrp-5 [13, 15, 16]. Experimental studies showed that Wnt signaling was increased in patients with metabolic disorders, which are common among PCOS patents [1, 4].
Thus far, there have been no studies of the relationship of serum and FF Sfrp-5 with invitro fertilization outcomes in patients with PCOS. Therefore, the aim of this study was to investigate the effect of serum and FF Sfrp-5 levels on IVF–ICSI outcomes in patients with PCOS.

Materials and methods

This was a prospective sequential cross-sectional study of 160 women with primary infertility performed between January and July 2017 in the reproductive endocrinology department of Konya Research and Education Hospital. The 160 patients were divided into two groups: 80 nonobese and nonhyperandrogenic patients diagnosed with PCOS (group I) and 80 patients diagnosed with male factor infertility (group II, control group). PCOS was defined according to the Rotterdam criteria devised by a consensus workshop in May 2003 [17]. According to the Rotterdam criteria, PCOS includes menstrual disorders, such as oligoovulation/anovulation, clinical or biochemical hyperandrogenism, and polycystic ovaries on an ultrasonographic examination (at least 10 follicles 2–9 mm in size or ovarian volume greater than 10 ml). IR was determined as follows, using the homeostatic model assessment of IR (HOMA-IR): fasting insulin (µIU/ml) × fasting glucose (mmol/l)/22.5) [5]. A 160 cycles, 160 serum samples, and 160 FF samples were analyzed, and demographic, hormonal, and biochemical parameters were recorded.
The exclusion criteria were as follows: patients with evidence of clinical hyperandrogenemia, a body mass index (BMI) > 30 kg/m2, diminished ovarian reserves, a history of ovarian surgery, and autoimmune diseases. The exclusion criteria were selected to exclude the effects of BMI and IR on serum and FF Sfrp-5 levels.
This study was approved by the local ethics committee and the institutional review board (Necmettin Erbakan University Medical Faculty, Ethical Commity, reference number: 2016/4149). Written informed consent was obtained from all the participants for future data use. The ethical principles for medical research involving human subjects stipulated in the 18th World Medical Association Declaration of Helsinki were applied.

In vitro fertilization stimulation

The long luteal down regulation protocol was applied, with all patients receiving the GnRH agonist leuprolide acetate (Lucrin, Abbot, Turkey) in the mid-luteal phase of the previous cycle until the day of hCG administration. After satisfactory pituitary desensitization was achieved (serum estradiol [E2] level < 50 pg/ml, endometrial thickness < 5 mm, and serum luteinizing hormone [LH] levels < 5 IU/ml), the GnRH agonist dose was reduced to half. Recombinant follicle stimulating hormone (FSH) (Gonal-F; Merck Serono, Istanbul, Turkey; Puregon; Organon, Istanbul, Turkey) administration was then started, according to the patient’s age, baseline serum FSH concentration on day 3, and BMI. The gonadotropin dose was adjusted according to the ovarian response monitored by serial E 2 measurements and transvaginal ultrasonography. Recombinant hCG (250 µg) (Ovitrelle; Serono, Istanbul, Turkey) was administered subcutaneously when there were at least three follicles with a mean diameter of 18 mm. Oocyte retrieval was done 36 h after the hCG injection by the guidance of transvaginal ultrasonography. ICSI was used for all IVF-embryo transferred patients. Fresh single-embryo transfers were performed on day 3 in all cases. Luteal support was given by vaginal progesterone (Crinone 8% gel; Serono), and supplementation was started on the day of hCG administration Pregnancy was determined by blood levels of β-hCG levels in tests performed 14 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac, accompanied by a fetal heartbeat.

Collection of blood serum and FF samples

Venous blood samples were collected from the antecubital veins of all subjects from 8:00 a.m. and 10:00 a.m., between the second and thirth days of the menstrual cycle after a night of fasting before ovarian stimulation. FF was collected on first entry from follicles ≥ 18 mm, and an oocyte was retrieved. Flushing was not performed. Samples contaminated with blood were excluded. Blood and FF samples of the 160 patients were centrifuged for 15 min at 3000 rpm on the day of follicle puncture and stored at − 80 °C for 5 months for subsequent analysis. Each sample was masked to allow for blinded analysis.

Sfrp‑5 assay in serum and FF

Sfrp-5 levels in serum and FF were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Catalog No: SEC842Hu; Cloud-Clone Corp., Wuhan, China). The ELISA assay was performed according to the manufacturer’s protocol and guideline. Intra-assay and interassay CVs were < 10% and < 12%, respectively. The minimum detectable dose of Sfrp-5 was typically less than 0.62 ng/ml, and the assay detection range was 1.56–100 ng/ml.

Outcome measures

The primary outcome measures were serum and FF Sfrp-5 levels. The secondary outcome measures included the number of retrieved and metaphase-II (MII) oocytes, fertilization rate, embryo quality, clinical pregnancy rate, and live birth rate.

Statistical analysis

SPSS 15 (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA) software was used for the statistical analyses. The Shapiro–Wilk test was used to test for normal or abnormal distributions of the continuous variables. An independent samples T-test was used for the betweengroup comparisons of the continuous variables with normal distributions. The data are expressed as the mean ± SD. The Mann–Whitney U test was applied for variables with a non-normal distribution. The data are expressed as median and inter-quartile ranges. Categorical data was analysed by Pearson’s Chi square test, and Fisher’s exact test was applied if the expected frequency was less than 5 in > 20% of all cells. Whether the potential risk factors have statistically significant affect on clinical pregnancy or not was evaluated by univariate logistic regression analyses. Odds ratios and 95% confidence intervals for each independent variable were also calculated. The Pearson or Spearman correlation analysis, where appropriate, was used to study the correlations between measurements. Statistical significance was considered with a probability of 0.05.

Results

Nine of 169 patients who were not eligible for the study were excluded from the study. The remaining 160 patients were classified into two groups: a PCOS group (n = 80) and a control group (n = 80). All patients were observed from the start of the study until the time of detection of clinical pregnancy. A hundred sixty cycles (80 in the PCOS group and 80 in the control group) were analyzed (Fig. 1).
Data on age, BMI, baseline FSH, LH, E2, thyroid stimulating hormone (TSH), prolactin levels, duration of stimulation, total gonadotropin dose required, E 2 and progesterone levels on the day of hCG administration, and numbers of retrieved oocytes and MII oocytes, in addition to fertilization rates, embryo quality, leukocyte and neutrophil counts, hs-CRP, and serum-FF Sfrp-5 levels, were normally distributed and were expressed as means ± SD. Durations of infertility, stimulation day, and endometrial thickness were not normally distributed and were expressed as median and 25–75th percentiles.
The demographic, clinical, and laboratory parameters of the participants are shown in Table 1. There were no significant between-group differences in age, BMI, baseline FSH, LH, E 2, TSH, prolactin levels, duration of infertility, progesterone levels on the day of hCG administration, endometrial thickness, fertilization rates, embryo quality, and leukocyte counts (p > 0.05). There was a statistically significant between-group difference in the stimulation day (10 [9–11] vs. 9 9–10])]; total gonadotropin dose required (2000.63 ± 1051.87 vs. 1134.69 ± 286.45); E2 level on the day of hCG administration (1931.33 ± 1.010.69 vs. 2377.00 ± 733.23); numbers of retrieved oocytes (8.60 ± 2.06 vs. 11.05 ± 4.39) and MII oocytes (6.751.99 vs. 8.55 ± 3.13); neutrophil counts (56.26 ± 6.93 vs. 60.39 ± 4.35); and hs-CRP (7.86 ± 1.52 vs. 10.45 ± 2.40), serum Sfrp-5 levels (8.87 ± 1.85 vs. 11.40 ± 2.88), and FF Sfrp-5 levels (9.71 ± 2.15 vs. 11.06 ± 2.30) (p < 0.05). The perinatal outcomes of the groups are given in Table 2. There was no between-group difference in clinical pregnancy rates, miscarriages, live births, vaginal births, female sex rates, gestational ages, and birthweights.
The results of a univariate logistic regression analysis revealed that PCOS, age, BMI, day 3 FSH, LH and E 2 levels, endometrial thickness, fertilization rate, embryo quality, and serum and FF Sfrp-5 levels were not associated with clinical pregnancy rates (p > 0.05) (Table 3).
Correlations between serum Sfrp-5, FF Sfrp-5, and the levels of other measured parameters are shown in Table 4. The fasting insulin level (r = 0.355, p = 0.001; r = 0.314, p = 0.014), triglycerides level (r = 0.316, p = 0.004; r = 0.250, p = 0.025), dehydroepiandrosterone sulfate (DHEA-S) level (r = 0.380, p = 0.001; r = 0.287, p = 0.045), total gonadotropin dose required (r = − 0.347, p = 0.002; r = − 0.258, p = 0.016), hs-CRP level (r = − 0.260, p = 0.020; r = − 0.258, p = 0.021), and neutrophil counts (r = − 0.239, p = 0.033; r = − 0.294, p = 0.014) were correlated with the levels of Sfrp-5.

Discussion

This is the first study to investigate serum and FF levels of Sfrp-5 in PCOS patients who underwent IVF–ICSI treatment, together with the perinatal outcomes of these patients. The results revealed a significant difference in serum and FF Sfrp-5 levels of the PCOS group and control group. Serum and FF Sfrp-5 levels were correlated with insulin, free testosterone levels, and inflammatory markers, although E 2 levels on the day of hCG administration, numbers of oocytes retrieved, and MII oocytes were higher in the PCOS group. There were no between-group differences in fertilization rates and perinatal outcomes. In the PCOS group, neutrophil counts and hs-CRP levels were high because of the inflammatory nature of PCOS. We observed a significant relationship between Sfrp-5 levels, NCs, and hs-CRP levels, in agreement with the results of previous studies [12, 18].
The etiopathogenesis of PCOS is not fully understood, but genetic and environmental factors are thought to play a role [2, 3]. In the majority of PCOS cases (30–95%), IR is thought to play a role [4, 5]. More than 50% of women with PCOS have central obesity, developed IR, polycysticlike ovaries, clinical disorders of the menstrual cycle, and hirsutism, in addition to impaired glucose intolerance, type II DM, lipid metabolism disorders, and even cardiovascular diseases associated with metabolic syndrome [2–4, 10, 11, 19]. Previous research reported that IR, as evaluated by the HOMA-IR method was present in 30% of lean PCOS cases, whereas it was present in up to 95% of obese PCOS cases [3, 5]. In the current study, IR was present in 52.5% of the PCOS group, in accordance with the literature. Previous studies reported an increased to hip ratio in obese PCOS patients as compared with that in lean PCOS and non-PCOS patients [3–5]. The findings of the present study were in accordance with those in the literature.
Previous studies showed that the numbers of retrieved and MII oocytes were higher in PCOS patients who underwent IVF–ICSI treatment than patients with non-PCOS but that there was no difference between PCOS and non-PCOS patients in fertilization rates, embryo quality, and clinical pregnancy rates [2, 10, 20, 21]. In the present study, the fertilization rates, clinical pregnancy rates, and embryo quality were similar to those reported in the literature.
Visceral adipose tissue secretes a number of cytokines, such as tumor necrosis factor-α and IL-6, which increase inflammation by paracrine and endocrine pathways [7, 8, 10]. Although some studies in the literature reported increased levels of inflammatory markers, such as NC, white blood cell counts, and hs-CRP, in PCOS patients who underwent IVF–ICSI treatment [3, 10, 11, 20], other studies found no such increase [22, 23]. In the present study, white blood cell counts of patients with PCOS who underwent IVF–ICSI were unchanged as compared with those in the control group. However, NCs and hs-CRP increased in the PCOS group following IVF–ICSI.
Previous research showed that Wnt expression was correlated with BMI, hyperinsulinemia, and IR in patients with PCOS [12]. A number of studies suggested that the Wnt signaling pathway played a role in regulating pancreatic development and pancreatic beta cell functions in hyperinsulinemia [24, 25]. Research also showed that Wnt increased insulin secretion, resulting in glucose stimulation and increased Wnt signaling in the pancreas of type II DM patients. According to various studies, Wnt may increase the insulin response in patients with PCOS, resulting in increased insulin resistance, leading to hyperinsulinemia and the development of glucose intolerance in PCOS patients [12, 13, 26].
In a study of nonobese PCOS and weight-matched women that examined the association of Wnt with BMI and insulin resistance [12]. The study showed that Wnt was correlated with adiposity but not IR [12].The authors concluded that Wnt did not cause IR but that it did cause hyperinsulinemic side effects in PCOS patients. Furthermore, they concluded that Wnt had an antiandrogenic effect in women with PCOS.
Sfrp-5, which is thought to be related to the anti-inflammatory response, is released in order to compensate for increased Wnt expression caused by obesity. It is secreted from white adipose tissue in both lean and obese patients [16]. Although some studies reported that Sfrp-5 levels were higher in obese patients than in lean individuals [16, 27], other studies found that they were lower in obese patients [13, 15, 16]. Conflicting results on Sfrp-5 levels in patients with PCOS have also been reported in the literature, with some studies reporting that Sfrp-5 levels were lower in PCOS patients [12, 15] but others finding that women with non-PCOS had lower Sfrp-5 levels [13, 15, 16]. The discord in the literature can be explained by the fact that the patients in the reference control group were not obese (overweight), whereas the women with PCOS were obese. In our study, there was no difference in the BMI of the two groups. However, the serum and FF Sfrp-5 levels of the patients with PCOS were significantly higher than those of the controls.
Previous research showed that Sfrp-5 levels were reduced in type II DM and gestational DM [13, 15], although one study reported increased levels [28]. Type II DM and gestational DM are considered chronic inflammatory disorders. In both disorders, hyperinsulinemia and IR are thought to increase inflammatory cytokines in adipose tissue [13]. According to one study, Sfrp-5 may be an important indicator of insulin sensitivity, and it may have a positive effect on glucose metabolism by increasing insulin sensitivity [15]. Experimental studies demonstrated that low Sfrp-5 levels induced pancreatic beta cell differentiation [27, 28]. In our study, the patients did not have an oral glucose tolerance test. Consistent with the results of the present study, other studies found a positive correlation between serum Sfrp-5 and fasting glucose and insulin levels [15, 27]. We observed no correlation between IR and Sfrp-5 levels in the present study, in common with the findings of Almario et al. [12]. Previous research reported that testosterone facilitated the Wnt signaling pathway in adipose tissue [29]. The same study found that elevated free testosterone levels in PCOS appeared to be associated with increased Sfrp-5 levels. In the present study, free testosterone and DHEA-S were correlated with Sfrp-5 levels. Triglycerides were also correlated with Sfrp-5 levels, as reported in a previous study [12]. The results suggest that Sfrp-5 may provide protection against cardiovascular diseases by affecting lipoprotein metabolism in the liver.
The strengths of our study are the synchronous measurements of serum and FF (reflecting the oocyte microenvironment) Sfrp-5 levels, analyses of the correlation of Sfrp-5 with other parameters, and assessments of perinatal outcomes in a group of patients who had not received any medical treatment that could have affected the results. The study also has some limitations. First, Sfrp-5 levels were determined in only a limited number of patients. Second, while there is a relationship between Sfrp-5 and inflammatory markers, the study is inability to correlate with IR which is thought to play an important role in the etiopathogenesis of PCOS. Third, we did not assess glucose intolerance, which is commonly seen in PCOS. Finally, we did not measure endometrial tissue Sfrp-5 levels.
In conclusion, the present study revealed elevated serum levels and FF Sfrp-5 levels in a selected population of nonobese, nonhyperandrogenic PCOS patients who underwent IVF–ICSI treatment. These results suggest that the Wnt signaling pathway and Sfrp-5 may play a significant role the etiopathogenesis of PCOS. Further studies in a larger series are warranted to determine serum and FF Sfrp-5 levels in PCOS patients with different disease severity who undergo IVF–ICSI treatment.

References

1. Ehrmann DA (2005) Polycystic ovary syndrome. N Eng J Med 352:1223–1236
2. Inal HA, Yilmaz N, Gorkem U, Oruc AS, Timur H (2016) The impact of follicular fluid adiponectin and ghrelin levels based on BMI on IVF outcomes in PCOS. J Endocrinol Invest 4:431–437
3. Inal ZO, Erdem S, Gederet Y, Duran C, Kucukaydin Z, Kurku H et al (2018) The impact of serum adropin and ischemia modified albumin levels based on BMI in PCOS. Endokrynol Pol 69:135–141
4. Diamanti-Kandarakis E, Dunaif A (2012) Insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications. Endocr Rev 33:981–1030
5. Alebic MS, Bulum T, Stojanović N, Duvnjak L (2014) Definition of insulin resistance using the homeostasis model assessment (HOMAIR) in IVF patients diagnosed with polycystic ovary syndrome (PCOS) according to the Rotterdam criteria. Endocrine 47:625–630
6. Lovren F, Pan Y, Quan A, Singh KK, Shukla PC, Gupta M et al (2010) Adropin is a novel regulator of endothelial function. Circulation 122:185–192
7. Escobar-Morreale HF, Luque-Ramírez M, González F (2011) Circulating inflammatory markers in polycystic ovary syndrome: a systematic review and metaanalysis. Fertil Steril 95:1048–1058
8. Duleba AJ, Dokras A (2012) Is PCOS an inflammatory process? Fertil Steril 97:7–12
9. Timur H, Yimaz N, Kahyaoglu I, Inal HA, Erkaya S (2015) The effect of serum and follicular fluid amyloid-associated protein levels on in vitro fertilization outcome in patients with polycystic ovary syndrome. J Assist Reprod Genet 11:1637–1642
10. Winger EE, Reed JL, Ashoush S, El-Toukhy T, Taranissi M (2012) Die-off ratio correlates with increased TNF-α:IL-10 ratio and decreased IVF success rates correctable with H umira. Am J Reprod Immunol 68:428–437
11. Levin I, Gamzu R, Mashiach R, Lessing JB, Amit A, Almog B (2007) Higher C-reactive protein levels during IVF stimulation are associated with ART failure. J Reprod Immunol 75:141–144
12. Almario RU, Karakas SE (2015) Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation. Horm Metab Res 47:152–157
13 . Hu Z, Deng H, Qu H (2013) Plasma SFRP5 levels are decreased in Chinese subjects with obesity and type 2 diabetes and negatively correlated with parameters of insulin resistance. Diabetes Res Clin Pract 99:391–395
14. Hu W, Li L, Yang M, Luo X, Ran W, Liu D et al (2013) Circulating WAY-316606 Sfrp5 is a signature of obesity-related metabolic disorders and is regulated by glucose and liraglutide in humans. J Clin Endocrinol Metab 98:290–298
15. Carstensen M, Herder C, Kempf K, Erlund I, Martin S, Koenig W et al (2013) Sfrp5 correlates with insulin resistance and oxidative stress. Eur J Clin Invest 43:350–357
16 . Schulte DM, Muller N, Neumann K, Oberhauser F, Faust M, Gudelhofer H et al (2012) Pro-inflammatory wnt5a and antiinflammatory sFRP5 are differentially regulated by nutritional factors in obese human subjects. PLoS ONE 7:e32437
17. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 81:19–25
18. Ouchi N, Higuchi A, Ohashi K, Oshima Y, Gokce N, Shibata R et al (2010) Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity. Science 329:454–457
19. Kahyaoglu I, Yılmaz N, Timur H, Inal HA, Erkaya S (2015) Granulocyte colony-stimulating factor: a relation between serum and follicular fluid levels and in-vitro fertilization outcome in patients with polycystic ovary syndrome. Cytokine 74:113–116
20. Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Tesquier A, Serkine AM et al (2003) Oocyte and embryo quality in polycystic ovary syndrome. Gynecol Obstet Fertil 31:350–354
21. Sahu B, Ozturk O, Ranierri M, Serhal P (2008) Comparison of oocyte quality and intracytoplasmic sperm injection outcome in women with isolated polycystic ovaries or polycystic ovarian syndrome. Arch Gynecol Obstet 277:239–244
22. Wunder DM, Kretschmer R, Bersinger NA (2005) Concentrations of leptin and C-reactive protein in serum and follicular fluid during assisted reproductive cycles. Hum Reprod 20:1266–1271
23. Robinson S, Pemberton P, Laing I, Nardo LG (2008) Low grade inflammation, as evidenced by basal high sensitivity CRP, is not correlated to outcome measures in IVF. J Assist Reprod Genet 25:383–388
24. Schinner S, Ulgen F, Papewalis C, Schott M, Woelk A, Vidal-Puig A, Scherbaum WA (2008) Regulation of insulin secretion, glucokinase gene transcription and beta cell proliferation by adipocyte-derived Wnt signalling molecules. Diabetologia 51:147–154
25. Mori H, Prestwich TC, Reid MA, Longo KA, Gerin I, Cawthorn WP et al (2012) Secreted frizzled-related protein 5 suppresses adipocyte mitochondrial metabolism through WNT inhibition. J Clin Invest 122:2405–2416
26. Flehmig G, Fasshauer M, Klöting N, Schön MR, Stumvoll M, Blüher M (2012) Sfrp5 serum concentration is associated with measures of glucose homeostasis and insulin sensitivity in human obesity (Abstract). Diabetologie Stoffwechs 7(S1):S35
27. Papadopoulou S, Edlund H (2005) Attenuated Wnt signaling perturbs pancreatic growth but not pancreatic function. Diabetes 54:2844–2851
28. Rebuffat SA, Oliveira JM, Altirriba J, Palau N, Garcia A, Esteban Y et al (2013) Down regulation of Sfrp5 promotes beta cell proliferation during obesity in the rat. Diabetologia 56:2446–2455
29. Singh R, Artaza JN, Taylor WE, Braga M, Yuan X, GonzalezCadavid NF et al (2006) Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with betacatenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors. Endocrinology 147:141–154