|Year : 2016 | Volume
| Issue : 3 | Page : 110-114
Elevated levels of diabetes-associated peptide hormones in the follicular fluid and serum of obese polycystic ovary syndrome patients are associated with suboptimal ovarian response
Julie M Sroga-Rios1, Alan M Martinez2, David R Cool3, Krystene B DiPaola1, Steven R Lindheim4
1 Division of Reproductive Endocrinology and Infertility, Department of OB/GYN, University of Cincinnati Academic Health Center, Cincinnati, OH, USA
2 Reproductive Science Center of New Jersey, Lawrence Township, NJ, USA
3 Department of Obstetrics and Gynecology, Pharmacology and Toxicology, Wright State University, Boonshoft School of Medicine, Dayton, OH, USA
4 Department of Obstetrics and Gynecology, Wright State University, Boonshoft School of Medicine, Dayton, OH, USA
|Date of Web Publication||21-Apr-2017|
Julie M Sroga-Rios
7675 Wellness Way, Suite 315, West Chester, OH 45069
Source of Support: None, Conflict of Interest: None
Objective: To compare diabetes-associated peptide hormones (DAPHs) and inflammatory markers in the follicular fluid (FF) and serum among polycystic ovary syndrome (PCOS) patients as compared to controls undergoing in vitro fertilization (IVF). Study Design: Levels of DAPHs and inflammatory markers in FF and serum were measured and correlated to IVF cycle outcomes and clinical pregnancy rate in three IVF groups: obese PCOS (n = 8), lean PCOS (n = 12), and controls (n = 11). Methods: Follicular fluid and serum were prosectively collected from obese and lean pcos and control females undergoing IVF. BioFlex ® array was used to analyze all samples from subjects. IVF cycle outcomes were collected on all subjects. Results: Ten DAPHs were assessed. Obese PCOS had higher levels of C-peptide (194.4 ± 163.5 pg/mL, P< 0.03), insulin (172.6 ± 113.6 pg/mL, P< 0.01), and leptin (10,046.1 ± 4920.2 pg/mL, P< 0.05) in FF as compared to lean PCOS and controls. Obese PCOS patients had higher levels of serum leptin (5575.5 ± 1650.2 pg/mL), with a difference in leptin concentrations noted between obese PCOS and controls (P < 0.01) and between obese and lean PCOS patients (P < 0.01). Higher levels of C-peptide (P < 0.04) and leptin (P < 0.01) in the FF were associated with increased total gonadotropin drug usage. There was a trend toward fewer oocytes retrieved (P < 0.06) and significantly lower number of normally fertilized zygotes (P < 0.04), with higher C-peptide levels. A trend toward increased clinical pregnancy rates was noted with lower serum levels of leptin (P < 0.08). Conclusions: DAPHs may play a role in the suboptimal ovarian response seen in obese IVF patients with PCOS as compared to lean PCOS patients and controls. Further studies are needed to understand if the evaluations in DAPH are secondary to obesity itself or are specific to this subset of PCOS patients.
Keywords: Diabetes-associated peptide hormones, follicular fluid, in vitro fertilization, obesity, polycystic ovary syndrome
|How to cite this article:|
Sroga-Rios JM, Martinez AM, Cool DR, DiPaola KB, Lindheim SR. Elevated levels of diabetes-associated peptide hormones in the follicular fluid and serum of obese polycystic ovary syndrome patients are associated with suboptimal ovarian response. IVF Lite 2016;3:110-4
|How to cite this URL:|
Sroga-Rios JM, Martinez AM, Cool DR, DiPaola KB, Lindheim SR. Elevated levels of diabetes-associated peptide hormones in the follicular fluid and serum of obese polycystic ovary syndrome patients are associated with suboptimal ovarian response. IVF Lite [serial online] 2016 [cited 2023 Nov 28];3:110-4. Available from: http://www.ivflite.org/text.asp?2016/3/3/110/204672
| Introduction|| |
Polycystic ovary syndrome (PCOS) is the most common ovulatory disorder affecting reproductive age women, with an estimated prevalence of 7%–10%. PCOS is characterized by anovulation, hyperandrogenism, and polycystic ovaries. Metabolic derangements and chronic, low-grade inflammation play a key role the pathogenesis and sequelae of PCOS.,,,, The altered metabolic milieu has been implicated in the abnormal folliculogenesis seen in PCOS patients, and improvements in these parameters are associated with amelioration of menstrual irregularities., Metabolic derangements not only impact follicular development but also impact the competence of the resulting oocyte., Oocytes from diabetic, insulin-resistant, and obese mice show delayed maturation, smaller size, and increased granulosa cell apoptosis.,,,,,
The abnormal metabolic milieu in PCOS patients can be attributed to alterations in diabetes-associated peptide hormones (DAPHs). DAPH functions as intercellular communication molecules that exert endocrine control over target tissues. Certain DAPHs, such as leptin and insulin, correlate with body mass index (BMI) in overweight and obese women with PCOS., In addition, leptin levels are higher in PCOS patients compared to controls, irrespective of BMI or insulin resistance. Insulin and leptin are found in follicular fluid (FF) and have been associated with decreased pregnancy rates after in vitro fertilization (IVF).
Given the metabolic abnormalities seen in PCOS, and their independent associations with obesity and decreased pregnancy rates after IVF, it is interesting to consider what differences may exist between obese PCOS patients and lean PCOS patients with a normal BMI with respect to the follicular environment and pregnancy outcomes after IVF. In this study, we investigate the relationship between DAPHs and inflammatory markers in the serum and FF of obese and normal BMI PCOS patients and correlate these findings to IVF cycle parameters and pregnancy outcomes as compared to women without PCOS.
| Materials and Methods|| |
In this Institutional Review Board-approved study, consecutive patients undergoing IVF at a single center were recruited between January 2012 and December 2013. Participants were approached if they were <35 years old, met Rotterdam criteria for PCOS, or were fertile oocyte donors or women with tubal factor infertility to serve as controls. Informed consent was obtained and participants were assigned to one of three groups: (1) Obese PCOS (BMI >30 kg/m 2; n = 8), (2) lean PCOS (BMI <25 kg/m 2; n = 12), and (3) donor or tubal factor controls (n = 11).
Ovarian stimulation and in vitro fertilization
Recombinant follicle stimulating hormone (Follistim; Organon USA) with or without urinary human menopausal gonadotropin (Menopur; Ferring Pharmaceuticals, NJ USA) was used for controlled ovarian hyperstimulation and adjusted per patient response. GnRH antagonist (Ganirelix; Merck & Co., NJ, USA) was initiated when the leading follicle was 12–14 mm in diameter. When two or more follicles had attained a minimum mean diameter of 18–20 mm, follicular maturation was achieved with recombinant human chorionic gonadotropin (hCG; Ovidrel; Serono, MA, USA). Oocyte retrieval was performed 35 h later.
Follicular fluid and serum samples
At the time of oocyte retrieval, fluid from the first and largest punctured follicle was collected. FF was centrifuged for 5 min at 1500 ×g. The supernatant was removed and stored at −80°C. Serum was collected before IVF cycle initiation and samples were centrifuged, red blood cells were discarded, and serum was similarly stored at −80°C until processing.
In vitro fertilization-intracytoplasmic sperm injection-embryo culture and transfer
Fertilization was performed 3–4 h after oocyte retrieval, using either conventional IVF or intracytoplasmic sperm injection. The following day, fertilization was confirmed by the presence of two pronuclei. All normally fertilized embryos were cultured in center well organ dishes in 1 mL Global ® medium (Life-Global ®, CT, USA) at 6% CO2 and atmosphere O2 and transferred on either day 3 or day 5 when at least four good quality cleavage stage embryos were present to continue culture to the blastocyst stage using ultrasound guidance. All patients received luteal phase support in 90 mg bioadhesive vaginal gel (8% Crinone; Watson Pharmaceuticals, NJ, USA).
Pregnancy was confirmed by serial serum β-hCG levels 7–10 days, following embryo transfer. Clinical pregnancies were confirmed with transvaginal ultrasonography by a gestational sac at 6–7 weeks. Hormonal supplementation was continued until 10 weeks gestation.
Diabetes-associated peptide hormones and inflammatory markers
Using the BioPlex ® flow-based suspension array system, BioPlex ® Pro Human Diabetes 10-plex or BioPlex ® Pro Human Cytokine 27-plex assay kits were used for analyses. All samples were analyzed in duplicate. The protein concentration of FF and serum was determined by Bradford analysis for normalization. Multiplexed analyte standard curve wells were analyzed at the same time to provide quantification for each sample. Following incubation and wash steps according to the manufacturer's protocols, data were acquired by the BioPlex ® 200 plate reader at the low photomultiplier tube (PMT) setting. Bead count was set to 50 and bead map set to 100, with a 60 s time limit. Following another round of wash and incubation steps, data were acquired at a high PMT setting, to ensure complete analysis of all analytes. The data were grouped across each analyte according to the study group and normalized to protein concentration before statistical analysis.
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 18; SPSS, Inc., Chicago, IL, USA). Results were analyzed with Chi-square, one-way ANOVA, and Tukey post hoc analysis, or Pearson correlation as appropriate. Significance was defined as P< 0.05.
| Results|| |
Clinical parameters are listed in [Table 1]. Obese PCOS patients had a significantly higher total serum testosterone than control patients (obese PCOS 73.4 ng/dL ± 56.5, control 34.1 ng/dL ± 12.3, P< 0.05). Lean PCOS patients had a significantly higher AMH compared to controls (lean PCOS 6.9 ng/mL ± 3.8, control 4.0 ng/mL ± 3.5; P< 0.05).
|Table 1: Clinical parameters of control and polycystic ovary syndrome patients|
Click here to view
Of the 10 DAPH assessed, significant differences in C-peptide, insulin, and leptin were noted between groups [Figure 1],[Figure 2],[Figure 3]. Obese PCOS patients had higher levels of C-peptide (194.4 ± 163.5 pg/mL), insulin (172.6 ± 113.6 pg/mL), and leptin (10,046.1 ± 4920.2 pg/mL) in FF as compared to lean PCOS patients and controls. A difference in follicular C-peptide (P < 0.03), insulin (P < 0.01), and leptin (P < 0.05) was noted between obese and lean PCOS patients [Figure 1],[Figure 2],[Figure 3]. A difference in follicular C-peptide (P < 0.03) and insulin (P < 0.01) was noted between obese PCOS and control patients. Obese PCOS patients had higher levels of serum leptin (55765.5 ± 1650.2 pg/mL), with a difference in leptin concentrations noted between obese PCOS and controls (P < 0.01) and between obese and lean PCOS patients (P < 0.01) [Figure 1],[Figure 2],[Figure 3].
|Figure 1: Follicular fluid and serum concentrations of c-peptide. *P < 0.05 Obese PCOS vs. Lean PCOS, +P < 0.05 Obese PCOS vs. Control|
Click here to view
|Figure 2: Follicular fluid and serum concentrations of insulin. *P < 0.05 Obese PCOS vs. Lean PCOS, +P < 0.05 Obese PCOS vs. Control|
Click here to view
|Figure 3: Follicular fl uid and serum concentrations of leptin. *P < 0.05 Obese PCOS vs. Lean PCOS, +P < 0.05 Obese PCOS vs. Control|
Click here to view
Higher levels of C-peptide (P < 0.04) and leptin (P < 0.01) in the FF were associated with increased total gonadotropin drug usage [Table 2]. There was a trend toward fewer oocytes retrieved (P < 0.06) and significantly lower number of normally fertilized zygotes (P < 0.04) with higher C-peptide levels [Table 2]. A trend toward increased clinical pregnancy rates was noted with lower serum levels of leptin (P < 0.08) [Table 3].
|Table 3: Correlation of C-peptide, insulin, and leptin with IVF outcomes|
Click here to view
There was no significant difference in inflammatory marker concentration or pregnancy outcomes between the groups.
| Discussion|| |
To the best of our knowledge, this is the first study to correlate FF and serum levels of DAPHs in obese and lean PCOS patients and correlate these values with IVF cycle parameters. Insulin is a pancreatic peptide hormone that plays a major role in the regulation of carbohydrate, fat, and protein metabolism. The ovary is an important target tissue for insulin., Insulin has been demonstrated to suppress apoptosis in ovarian follicles., Our findings indicate that obese patients with PCOS undergoing IVF have higher levels of DAPHs than lean PCOS patients or patients without PCOS. In particular, C-peptide and insulin levels were higher in the FF of obese PCOS patients.
C-peptide is produced in the beta cells of the pancreas where proinsulin is cleaved to form two peptides: Insulin and C-peptide. Since C-peptide and insulin are produced at the same rate, C-peptide is a useful marker of insulin production. Therefore, the elevated C-peptide levels validate the elevated insulin levels seen in the FF of these patients.
Leptin aids in body weight regulation by binding hypothalamic receptors and decreasing food intake and also has receptors located in the ovary., Leptin levels are proportional to adipose mass, and leptin secretion is stimulated by estrogen and inhibited by androgens, and as such plays a role in inhibiting insulin-induced progesterone and estradiol production.,,, We found that leptin levels are higher in both the FF and serum for obese PCOS patients as compared to both control and lean patients. Therefore, the results support previous work that suggests its role in regulating progesterone and estradiol production.
FF elevations of the C-peptide, insulin, and leptin provide further evidence of the altered metabolic milieu seen in obese patients with PCOS. These findings correlate with increased gonadotropin dosing and decreased oocyte and zygote yield seen in obese PCOS patients, suggesting that elevations in DAPHs may contribute to the suboptimal response seen in obese PCOS patients undergoing IVF.
A limitation of our study is that direct comparison of DAPHs in obese patients with and without PCOS is needed to determine if these elevations are specific to the obese subset of PCOS patients or rather due to the obesity alone. In addition, our small sample size made it difficult to ascertain what effect hyperandrogenism had on outcomes independently from obesity.
Our data suggest that PCOS results in a modified hormonal milieu that decreases gonadotropin response in IVF cycles. Improved understanding of the metabolic disturbances seen in obese compared to lean PCOS patients may help tailor treatment options. For example, diet, exercise, and medications which improve insulin resistance may be more beneficial in obese, compared to lean, PCOS patients.
This work was completed in collaboration with the technical assistance of Mr. William C. Grunwald, Jr., in the Proteome Analysis Laboratory at Wright State University. Statistical analysis of the data was completed with the assistance of Rose Maxwell PhD.
Financial support and sponsorship
The study was supported in part by a research grant from Investigator-initiated Studies Program of Merck Sharp & Dohme Corp. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp.
Conflicts of interest
There are no confl icts of interest.
| References|| |
Franks S. Polycystic ovary syndrome. N Engl J Med 1995;333:853-61.
Rios JS. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19-25.
Dunaif A. Insulin resistance in women with polycystic ovary syndrome. Fertil Steril 2006;86 Suppl 1:S13-4.
Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 1989;38:1165-74.
Dunaif A. Insulin resistance and the polycystic ovary syndrome: Mechanism and implications for pathogenesis. Endocr Rev 1997;18:774-800.
Svendsen PF, Madsbad S, Nilas L. The insulin-resistant phenotype of polycystic ovary syndrome. Fertil Steril 2010;94:1052-8.
Repaci A, Gambineri A, Pasquali R. The role of low-grade inflammation in the polycystic ovary syndrome. Mol Cell Endocrinol 2011;335:30-41.
Mason HD, Willis DS, Beard RW, Winston RM, Margara R, Franks S. Estradiol production by granulosa cells of normal and polycystic ovaries: Relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid. J Clin Endocrinol Metab 1994;79:1355-60.
Romualdi D, De Cicco S, Tagliaferri V, Proto C, Lanzone A, Guido M. The metabolic status modulates the effect of metformin on the antimullerian hormone-androgens-insulin interplay in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 2011;96:E821-4.
Desforges-Bullet V, Gallo C, Lefebvre C, Pigny P, Dewailly D, Catteau-Jonard S. Increased anti-müllerian hormone and decreased FSH levels in follicular fluid obtained in women with polycystic ovaries at the time of follicle puncture for in vitro
fertilization. Fertil Steril 2010;94:198-204.
Franks S, Stark J, Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Hum Reprod Update 2008;14:367-78.
Colton SA, Pieper GM, Downs SM. Altered meiotic regulation in oocytes from diabetic mice. Biol Reprod 2002;67:220-31.
Diamond MP, Moley KH, Pellicer A, Vaughn WK, DeCherney AH. Effects of streptozotocin-and alloxan-induced diabetes mellitus on mouse follicular and early embryo development. J Reprod Fertil 1989;86:1-10.
Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE, Moley KH. Diet-induced obesity model: Abnormal oocytes and persistent growth abnormalities in the offspring. Endocrinology 2010;151:4039-46.
Niu Z, Lin N, Gu R, Sun Y, Feng Y. Associations between insulin resistance, free fatty acids, and oocyte quality in polycystic ovary syndrome during in vitro
fertilization. J Clin Endocrinol Metab 2014;99:E2269-76.
Rouru J, Anttila L, Koskinen P, Penttilä TA, Irjala K, Huupponen R, et al
. Serum leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997;82:1697-700.
Chakrabarti J. Serum leptin level in women with polycystic ovary syndrome: Correlation with adiposity, insulin, and circulating testosterone. Ann Med Health Sci Res 2013;3:191-6.
] [Full text]
Takikawa S, Iwase A, Goto M, Harata T, Umezu T, Nakahara T, et al
. Assessment of the predictive value of follicular fluid insulin, leptin and adiponectin in assisted reproductive cycles. Gynecol Endocrinol 2010;26:494-9.
Shuldiner AR, Berbetti F, Raben N, Scavo L, Serrano J. Insulin. In: LeRoith D, editor. Insulin-like Growth Factors: Molecular and Cellular Aspects. Boca Raton, FL: RC Press; 1998.
Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS. Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary. J Clin Endocrinol Metab 1985;61:728-34.
Poretsky L, Smith D, Seibel M, Pazianos A, Moses AC, Flier JS. Specific insulin binding sites in human ovary. J Clin Endocrinol Metab 1984;59:809-11.
Chun SY, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJ. Gonadotropin suppression of apoptosis in cultured preovulatory follicles: Mediatory role of endogenous insulin-like growth factor I. Endocrinology 1994;135:1845-53.
Hsueh AJ, Billig H, Tsafriri A. Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocr Rev 1994;15:707-24.
Spicer LJ, Francisco CC. The adipose obese gene product, leptin: Evidence of a direct inhibitory role in ovarian function. Endocrinology 1997;138:3374-9.
Flier JS, Maratos-Flier E. Obesity and the hypothalamus: Novel peptides for new pathways. Cell 1998 20;92:437-40.
Mantzoros CS, Moschos S, Avramopoulos I, Kaklamani V, Liolios A, Doulgerakis DE, et al
. Leptin concentrations in relation to body mass index and the tumor necrosis factor-alpha system in humans. J Clin Endocrinol Metab 1997;82:3408-13.
Blum WF, Englaro P, Hanitsch S, Juul A, Hertel NT, Müller J, et al
. Plasma leptin levels in healthy children and adolescents: Dependence on body mass index, body fat mass, gender, pubertal stage, and testosterone. J Clin Endocrinol Metab 1997;82:2904-10.
Saad MF, Riad-Gabriel MG, Khan A, Sharma A, Michael R, Jinagouda SD, et al
. Diurnal and ultradian rhythmicity of plasma leptin: Effects of gender and adiposity. J Clin Endocrinol Metab 1998;83:453-9.
| Authors|| |
Julie M. Sroga-Rios, MD, joined the UC Center for Reproductive Health in 2012. She is board certifi ed in Obstetrics and Gynecology and in Reproductive Endocrinology and Infertility. Dr. Sroga Rios is an Assistant Professor at the University Of Cincinnati College Of Medicine, an active member of the American Congress of Obstetricians and Gynecologists and of the American Society for Reproductive Medicine. Dr. Sroga Rios is experienced in all areas of infertility and gynecologic surgery, and her clinical and research interests focus on polycystic ovarian syndrome, endometriosis, and oncofertility.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Artificial intelligence and amniotic fluid multiomics: prediction of perinatal outcome in asymptomatic women with short cervix
| ||R. O. Bahado-Singh,J. Sonek,D. McKenna,D. Cool,B. Aydas,O. Turkoglu,T. Bjorndahl,R. Mandal,D. Wishart,P. Friedman,S. F. Graham,A. Yilmaz |
| ||Ultrasound in Obstetrics & Gynecology. 2019; 54(1): 110 |
|[Pubmed] | [DOI]|