Research Article Association between Follicular Fluid...

Research ArticleAssociation between Follicular Fluid Leptin andSerum Insulin Levels in Nonoverweight Women withPolycystic Ovary Syndrome

G Garruti1 R de Palo2 M T Rotelli1 S Nocera2 I Totaro2

C Nardelli2 M A Panzarino2 M Vacca2 L E Selvaggi2 and F Giorgino1

1 Section of Internal Medicine Endocrinology Andrology and Metabolic DiseasesDepartment of Emergency and Organ Transplantation University of Bari Aldo Moro Piazza G Cesare 11 70124 Bari Italy

2 Centre of Pathophysiology of Human Reproduction and Gametes Cryopreservation Department of GynaecologyObstetrics and Neonatology Gynaecology and Obstetrics Unit A University of Bari Aldo Moro Piazza G Cesare 11 70124 Bari Italy

Correspondence should be addressed to G Garruti ggarrutiendounibait

Received 4 February 2014 Revised 12 April 2014 Accepted 14 April 2014 Published 4 May 2014

Academic Editor Irma Virant-Klun

Copyright copy 2014 G Garruti et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aims We evaluated the links between leptin and visfatin levels and fertilization rates in nonoverweight (NOW) women with PCOS(NOW-PCOS) from Apulia undergoing in vitro fertilizationembryo transfer (IVF) Materials and Methodology We recruited16 NOW women with PCOS (NOW-PCOS) and 10 normally ovulating NOW women (control-NOW) All women underwentIVF Androgens 17-120573-estradiol (17120573-E

2) and insulin levels were measured in plasma andor serum and leptin and visfatin levels

were assayed in both serum and follicular fluid (FF-leptin FF-visfatin) Results In NOW-PCOS both serum and FF-leptin weresignificantly lower than in control-NOW In NOW-PCOS significant correlations were found between BMI and serum leptin andinsulinemia and FF-leptin By contrast in control-NOW FF-leptin levels were not correlated with insulinemia Serum visfatinlevels were not significantly different in NOW-PCOS and control-NOW but FF-visfatin levels were 16-fold higher although notsignificantly in NOW-PCOS than in control-NOW Conclusions Both serum leptin levels and FF-leptin are BMI- and insulin-related in Southern Italian NOW-PCOS from Apulia In line with other reports showing that FF-leptin levels are predictive offertilization rates lower than normal FF-leptin levels in NOW-PCOS may explain their lower fertilization rate and this may berelated to the level of insulin andor insulin resistance

1 Background

Polycystic ovary syndrome (PCOS) is commonly definedaccording to the Amsterdam ESHREASRM PCOS Consen-susWorkshopGroup [1] as a disorder including anovulationmenstrual disturbances and hyperandrogenism and is oftenassociated with clinical features of the metabolic syndromewith an increased risk of type 2 diabetes PCOS is the majorcause of infertility in women in the fertile age Studies ondifferent ethnic groups support a familial and genetic pre-disposition to PCOS and oligogenicpolygenic mechanismsmight be involved in its etiology [2 3] Both hyperinsuline-mia andor insulin resistance and hyperandrogenism play an

important role in its pathogenesis [4] In addition obesity isoften but not always a fellow traveller of PCOS

In obob mice displaying a phenotype similar to that ofhuman obesity a point mutation of the ob gene encodingfor leptin accounts for hyperphagia morbid obesity andsterility [5 6] Interestingly humans that are homozygousfor a point mutation of the ob gene also display a phenotypewhose main features are morbid obesity and sterility [7 8]Impaired function andor reduced levels of leptin might thuscause infertility in women with PCOS Consistent with thisconcept circulating leptin levels and leptinmRNAexpressionin adipocytes are reduced in women with PCOS [9 10]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 980429 7 pageshttpdxdoiorg1011552014980429

2 BioMed Research International

Several studies have investigated leptin levels in over-weight andor obese women with PCOS clearly demonstrat-ing that they appear to be correlated with BMI [11] How-ever granulosa cells are able to synthesize and store leptinfromnewborn until adult life in amanner that is independentof BMI [12] Relevant to this concept few studies have inves-tigated leptin levels in normal-weight women with PCOSin specific ethnic groups in the absence of excess BMIIn an Indian study circulating leptin levels were found tobe 5-fold higher in normal-weight women with PCOS ascompared with controls [13] In a Chinese study PCOSwomen were characterized by higher leptin levels both inserum and follicular fluid than controls although the BMIof the PCOS women included in this study ranged fromnormal-weight to overweight and obesity [14] The above-mentioned data are not in line with other results in Caucasianwomen demonstrating that the concentration of leptin inthe follicular fluid (FF-leptin) seems to be predictive of thefertilization rate [15] Fertilization rate is usually lower inPCOS women as compared with normally ovulating womenThe paper by De Placido et al [15] is the only one tofind a direct correlation between the concentration of leptinin the follicular fluid (FF-leptin) and the fertilization rateInterestingly the population studied by De Placido et al wascoming from the south of Italy and certainly showed differentlifestyle and diet as compared with Indians and Chinesewomen

In other recent studies elevated circulating levels of leptinandor increased leptinadiponectin ratios were reported inoverweight PCOSwomen but no information on leptin levelsin lean women with PCOS was provided [16 17]

Visfatin acts as a cytokine but also as an enzyme and isinvolved in metabolic and immunological diseases VisfatinmRNA was recently detected in human follicles and visfatinadministration was shown to improve ovarian response inconditions of superovulation in animal models [18 19]

In this study we havemeasured circulating leptin and vis-fatin levels and FF-leptin and FF-visfatin in a selected groupof Caucasian Southern Italian nonoverweight women withPCOS (NOW-PCOS) undergoing in vitro fertilizationembryo transfer (IVF) in order to better understand the inter-relationships if any between both cytokines and fertilizationrate

Some other papers analyzed leptin levels in both serumand follicular fluid in women having PCOS however veryfew papers included only Caucasian nonoverweight women(BMI le 249 Kgm2) with PCOS as we did Our pilot studyis also new in the fact that all women studied were comingfrom Apulia a region from the south of Italy which is verywell known for Mediterranean diet

2 Materials and Methods

In this observational pilot study we selected 16 consecutiveNOW-PCOS and 10 consecutive age- and BMI-matchednormal-ovulating nonhirsute premenopausal infertile Cau-casian women (control-NOW) requesting a procedure ofassisted reproduction at the Centre of Pathophysiology of

Human Reproduction and Gametes Cryopreservation of theUniversity of Bari All women were from Apulia in SouthernItaly and did not show any sign of metabolic syndrome Inthe control-NOW group infertility was due either to maleinfertility or tubal factors The diagnosis of PCOS was basedon the criteria indicated in the Amsterdam ESHREASRM-Sponsored 3rd PCOS Consensus Workshop Group [1 20]All women with PCOS had hirsutism oligomenorrhea andclear diagnosis of PCOS by ultrasounds Both NOW-PCOSand control-NOW were euthyroid and were undergoing IVFfor the first time All women underwent IVF according to apreviously reported protocol [21] Infertile women with adiagnosis of diabetes mellitus were excluded from the studyto rule out potential confounding effects of hyperglycemiaon leptin and fertilization rates Indeed all subjects had fast-ing glucose levels lt126mgdL (7mmolL) and underwent anoral glucose tolerance test according to the American Dia-betes Association guidelines [22] displaying plasma glucoselevels lt140mgdL (78mmolL) after 2 h All women were inthe premenopausal stage as confirmed by circulating FSHand LH levels measured between day 3 and day 5 of themenstrual cycle All women underwent IVF according toa previously reported protocol [21] Briefly daily GnRH-aadministration (Decapeptyl 01mgmL-Ipsen SpA sc) wasstarted during the middle-luteal phase of the cycle beforeovarian hyperstimulation until the day of human chorionicgonadotropin (hCG) administration Ovarian hyperstimu-lation was initiated once the serum estradiol (E

2) level

was lt30 pgmL with 150ndash225 IU of rFSH (alpha follitropin-Gonal-F Merck Serono SpA) Dosing was individualizeddepending on the patientrsquos response When at least 3 folliclesof 18mm in diameter and a serum E

2level of gt200 pgmL

per follicle were noted the patient was given 250 120583gof choriongonadotropin alpha hormone (Ovitrelle MerckSerono SpA) Monitoring was accomplished with serialvaginal ultrasound examinations and serum E

2level deter-

minations Transvaginal oocyte retrieval was performed 35hours after hCG injection After retrieval cumulus cells(COCs) were removed with a 30-second exposure to HEPES-buffed medium containing 40 IUmL hyaluronidase (SAGEUSA) and denuded oocytes were then assessed for nuclearstatus those that were observed to have released the firstpolar body were considered mature (MII) and used for ICSIprocedure [23]

Fertilization was assessed 18 hours after ICSI Normalfertilization was confirmed when 2 clearly distinct pronucleiwere present Fertilization rate was defined as the percentageof pronuleate oocytes to the number of oocytes injectedEmbryo quality was evaluated under inverted microscope onthe second day of development according to Veeck [24] Amaximum of three embryos were transferred on day 2 or3 of embryo culture under ultrasound guidance For lutealphase support all patients received a daily dose of 400mg ofvaginal micronized progesterone (Progeffik Effik Italia)Serum 120573-hCG concentration was measured 14 days afteroocyte retrieval to verify pregnancy Clinical pregnancy wasjudged by observation of the gestational sac on vaginalultrasonography after 6-7 weeks of gestation

BioMed Research International 3

Table 1 Anthropometric and metabolic characteristics and hormone levels in the experimental subjects

NOW-PCOS Control-NOW 119901 valueAge (years) 3209 plusmn 360 3435 plusmn 313 004BMI (Kgm2) 2255 plusmn 261 2255 plusmn 255 099Insulinemia (120583UmL) 1030 plusmn 744 939 plusmn 927 073HOMA-IR 257 plusmn 206 237 plusmn 272 076FSH (mlUmL) 571 plusmn 202 696 plusmn 234 007LH (mlUmL) 812 plusmn 4 477 plusmn 161 000117120573-E2 (basal) (pgmL) 326 plusmn 1340 9556 plusmn 13163 007FF-leptin (ngmL) 712 plusmn 112 1444 plusmn 467 0037Serum leptin (ngmL) 531 plusmn 113 1149 plusmn 421 0045FF-visfatin (ngmL) 455 plusmn 059 288 plusmn 088 0075Serum visfatin (ngmL) 866 plusmn 081 724 plusmn 148 074NOW nonoverweight control-NOW controls BMI body mass index E2 estradiol FF follicular fluid FSH follicle stimulating hormone LH luteinizinghormone Data are expressed as mean plusmn SEM Differences between groups were considered significant for 119901 le 005

Follicular fluid (FF) samples were obtained during IVFtreatment at the time of oocyte aspiration Bloodless aspiratesfrom different follicles containing mature oocytes of onepatient were pooled and centrifuged (1200 rpm for 5min)(for 10min at 2000 g) The supernatants were divided inaliquots and stored in sterile tubes and stored at minus80∘Crdquo

Blood samples for metabolic and hormonal evaluationswere collected before the IVF stimulation protocol Totalcholesterol HDL-cholesterol LDL-cholesterol and triglyc-erides circulating levels were measured with specific Dimen-sion clinical chemistry systems (Siemens Healthcare Diag-nostics Ltd) Androgens (free and total testosterone Δ4-androstenedione SHBG DHT DHEA and DHEAS) weremeasured between day 3 and day 5 of the menstrual cycle17120573-E

2and insulin levels were measured in plasma andor

serum and leptin and visfatin levels in both serum andfollicular fluid (FF) (DIAsource Leptin-EASIA Kit Visfatin-EASIA Kit)

The homeostasis model assessment of insulin resistance(HOMA-IR) indexwas calculated as previously reported [2526]

The fertilization rates were calculated and correlated withthe anthropometric and hormonal parameters

All results are expressed as mean plusmn SEM Comparisonof continuous variables among groups was performed withpaired Studentrsquos 119905-test All statistical calculations were per-formedwithNCSS2004 software (Kaysville UTUSA) Statis-tical tests were conducted as an alpha level of 005 [27 28] Astepwise regression analysis was performed to identify signif-icant links between anthropometric and metabolic variablesand between the above mentioned variables and fertilizationrates

3 Results

In this study we have investigatedNOW-PCOS who request-ed a procedure of assisted reproduction with no signs ofmetabolic syndrome Both NOW-PCOS and control-NOWhad total cholesterol HDL-cholesterol LDL-cholesterol and

triglycerides levels as well as arterial blood pressure levelsin the normal range and showed a normal glucose tolerance(data not shown)

NOW-PCOS and control-NOW had comparable fastingglucose and insulin levels and HOMA-IR was also notsignificantly different in the two groups (Table 1) As far as cir-culating insulin levels are concerned they were significantlycorrelated with BMI in both NOW-PCOS and control-NOW(Figures 1(a) and 1(b))

In both NOW-PCOS and control-NOW serum leptinlevels positively correlated with FF-leptin levels (Figures 2(a)and 2(b)) A positive linear correlation between FF-leptin andcirculating insulin levels was found inNOW-PCOS but not incontrol-NOW (Figures 3(a) and 3(b))

In our cohort of NOW-PCOS a significant positivecorrelation was found between BMI and serum leptin (1198772 =0 3838 119903 06195 119901 = 00181) and a positive although non-significant correlation was also observed between BMI andFF-leptin (1198772 = 0 2379 119901 = 0 0553)

As far as visfatin is concerned serum visfatin levels werenot different in NOW-PCOS and control-NOW but FF-visfatin levels were 16-fold higher even though not signifi-cantly in NOW-PCOS as compared with control-NOW (119901 =0075 Table 1) No correlation was found between FF-visfatinand circulating insulin HOMA-IR and FF-leptin in eitherNOW-PCOS or control-NOW

Basal nonstimulated FSH levels were not significantlydifferent between the two groups but basal nonstimulated LHlevels were significantly higher in NOW-PCOS than control-NOW (119901 = 0001 Table 1) and basal 17120573-E

2levels were

29-fold lower in NOW-PCOS as compared to control-NOWeven though this difference was not statistically significant(119901 = 007) (Table 1) In regard to the IVF stimulation pro-cedure the number of total oocytes was significantly higher(119901 = 0001) but in-vitro-matured metaphase II oocytes werelower although not significantly (119901 = 007) in NOW-PCOSthan in control-NOW (Table 2) Even if fertilization rate hasbeen known to be lower in PCOS women as compared withnormally ovulating women due to IVF the fertilization rate


300

225

150

75

00

180 200 220 240 260

Insu

lin (120583

Um

L)

BMI (kgm2)

(a)

Insu

lin (120583

Um

L)

BMI (kgm2)

160

125

90

55

20

190 203 215 228 240

(b)

Figure 1 Correlation between circulating insulin levels and BMI in NOW-PCOS and control-NOW (a) Positive linear correlation betweencirculating insulin levels and BMI in NOW-PCOS 1198772 = 03385 119903 = 05818 119901 = 00181 (b) Positive linear correlation between circulatinginsulin levels and BMI in control NOW 1198772 = 07169 119903 = 08467 119901 = 00040

160

120

80

40

00

00 50 100 150 200

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(a)

350

263

175

88

00

00 125 250 375 500

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(b)

Figure 2 Correlation between leptin levels in the follicular fluid and circulating leptin levels in NOW-PCOS and control-NOW (a) Positivelinear correlation between leptin levels in the follicular fluid and circulating leptin levels in NOW-PCOS 1198772 = 08517 119903 = 09229 119901 =000001 (b) Positive linear correlation between leptin levels in the follicular fluid and circulating leptin levels in control-NOW 1198772 = 09376119903 = 09683 119901 = 00001

resulted to be not different in the two groups At the end of theIVF protocol estrogen levels were threefold higher in NOW-PCOS than in control-NOW (119901 = 0001)

4 Discussion

PCOS is highly prevalent (about 4ndash7) among women inthe reproductive age [25 26] and recognizes in the impairedhypothalamus-pituitary-ovarian axis dysfunctions andhyperinsulinemia major causes driving the infertile pheno-type Nonetheless overweight and obesity are often but notalways associated with PCOS and adipokines including

leptin and visfatin might also be involved in the pathogenesisof this disease [18ndash20 29ndash34]

Some previous reports suggest that adipokines might beinvolved in the pathogenesis of PCOS [18 31] Since in bothanimal and human models of leptin deficiency infertilityis present together with the obesity phenotype [5ndash8] it isto be expected that altered levels andor function of leptinmight be involved in causing infertility in overweight womenwith PCOS too Leptin levels usually correlate with body fatmass and since PCOS is often associated with overweightthe latter might represent a confounding factor in the effortto understand the role of leptin in the pathogenesis of PCOSIn this study we investigated strictly nonoverweight womenrequesting a procedure of assisted reproduction and excluded


200

150

100

50

00

00 75 150 225 300

Insulin (pgmL)

FF-le

ptin

(pg

mL)

(a)

Insulin (pgmL)

500

375

250

125

00

20 55 90 125 160

FF-le

ptin

(pg

mL)

(b)

Figure 3 Correlation between leptin levels in the follicular fluid and circulating insulin levels in NOW-PCOS and control-NOW (a) Positivelinear correlation between leptin levels in the follicular fluid (FF) and circulating insulin levels in NOW-PCOS 1198772 = 03173 119903 = 05633119901 = 00231 (b) Absence of correlation between leptin levels in FF and circulating insulin levels in control-NOW 1198772 = 00919 119903 = minus03032119901 = 03944

both overweight and obese women to rule out potential con-founding effects of overweight and obesity on leptin andorvisfatin levels and fertilization ratesWe studied a small groupof homogeneous Italian-Caucasian women from Apulia Wefound significantly lower serum and FF-leptin levels inNOW-PCOS compared to BMI-matched infertile womenand a positive correlation between insulinemia and FF-leptinlevels in NOW-PCOS only

In studies performed in non-Caucasian ethnic groups(Chinese Indian) higher levels of leptin in both serum andFF were reported in PCOS women than in controls howeverin these studies the experimental groups included normal-weight overweight and obese PCOS women (range 1981ndash3062Kgm2) [13 14] In another recent study on Caucasianwomen with PCOS elevated circulating levels of leptinandor increased leptinadiponectin ratios were found inoverweight PCOS women but leptin levels in lean womenwith PCOS were not investigated [16]

Nonetheless results on interethnic differences in body fatdistribution and phenotype make it reasonable to find differ-ent adipokine levels in different ethnic groups particularlywhen different cutoff values for waist circumferences have tobe considered [17]

It is interesting to note that in another study involvingCaucasian PCOS women from Southern Italy FF-leptinconcentrations were found to be predictive of the fertilizationrates [15] In PCOS women the fertilization rate is known tobe lower than in normally ovulating women and therefore itmay seem reasonable to find reduced FF-leptin levels in thesesubjects

In our study both NOW-PCOS and control-NOW hadnormal glucose tolerance Fasting insulin levels and HOMA-IR index were not significantly different in the two groupsHowever circulating insulin levels correlated with FF-leptinin NOW-PCOS only even if in both NOW-PCOS andcontrol-NOW they correlated with BMI

It has been already demonstrated that hyperleptinemiaandor high FF-leptinmay result in impaired steroidogenesisreproductive function and fertility [15 33 34] However itis also true that follicle maturation occurs in the presence ofphysiologic levels of leptin through the activation of the STATsignal transduction pathway [13 15] The possibility existsthat too low levels of FF-leptin decrease fertility in NOW-PCOS women as well as too high FF-leptin levels might favorinfertility in overweightobese PCOS women In overweightPCOS women insulin resistance is certainly associated withincreased steroidogenesis even if the adipose cell lineage isnot intrinsically insulin-resistant [35 36]

As far as visfatin is concerned it belongs to the familyof cytokines and has been implicated in several metabolicconditions such as central obesity metabolic syndrome andtype 2 diabetes In an animal model (mouse) administrationof visfatin during superovulation improves developmentalcompetency of oocytes and fertility potential despite theage These and other data support a role for visfatin inreproduction [18 19] In our experimental cohort serumvisfatin levels were not different in NOW-PCOS and control-NOW but FF-visfatin levels were 16-fold higher even thoughnot significantly in NOW-PCOS as compared with control-NOW

In regard to the IVF stimulation procedure even if fer-tilization rate has been known to be lower in PCOS womenas compared with normally ovulating women due to IVFthe fertilization rate resulted to be not different in the twogroups of women analysed in our study Since during the IVFprotocol NOW-PCOS and control-NOW followed a lifestylechange program including a Mediterranean low-glycemicindex isocaloric diet and daily moderate exercise accordingto standard care protocols we can argue that this lifestylechange might have resulted in improved fertilization rates inwomen with PCOS even if estrogen levels were still higher inNOW-PCOS than in control-NOW


Table 2 Outcomes at the stimulation end in the experimental women

NOW-PCOS Control-NOW 119901 valueFollicles lt18mm 814 plusmn 307 258 plusmn 248 211Follicles gt18mm 624 plusmn 202 605 plusmn 255 0817120573-E2 (pgmL) 318426 plusmn 147709 168967 plusmn 107579 0001Total oocytes 933 plusmn 328 595 plusmn 286 0001M II 376 plusmn 137 489 plusmn 226 007M IItotal oocytes () 4538 plusmn 2278 8123 plusmn 2373 212Fertilization rate () 6016 plusmn 3522 6282 plusmn 3377 081Embryos (I∘) 174 plusmn 152 205 plusmn 147 052Pregnancy rate () 30 35NOW nonoverweight control-NOW controls BMI bodymass index E2 estradiolM II in-vitro-maturedmetaphase II oocytes Data are expressed asmean plusmnSEM Differences between groups were considered significant for 119901 lt 005

A possible take-homemessage to get from our pilot studyis that NOW-PCOS might represent a completely differentphenotype as compared with those found in overweight orobese women with PCOS FF-leptin levels lower and FF-visfatin levels higher than those found in control-NOWmight be markers of this different phenotype and low fertil-ization rate

Considering the correlation between insulin circulatinglevels and FF-leptin in NOW-PCOS future studies shouldmore closely focus on direct measurements of insulin sensi-tivity and insulin secretion inNOW-PCOS to better elucidatethe relationship between changes in insulin secretion andoraction and changes in cytokines relevant to the fertility statusin this population

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

G Garruti R de Palo M T Rotelli S Nocera I Totaro CNardelli M A Panzarino M Vacca L E Selvaggi and FGiorgino equally contributed to the paper

References

[1] Amsterdam ESHREASRM-Sponsored 3rd PCOS ConsensusWorkshop Group ldquoConsensus on womenrsquos health aspects ofpolycystic ovary syndrome (PCOS)rdquoHuman Reproduction vol27 no 1 pp 14ndash24 2012

[2] R S Legro D Driscoll J F Strauss III J Fox and A DunaifldquoEvidence for a genetic basis for hyperandrogenemia in poly-cystic ovary syndromerdquo Proceedings of the National Academyof Sciences of the United States of America vol 95 no 25 pp14956ndash14960 1998

[3] K G Ewens M R Jones W Ankener et al ldquoFTO and MC4Rgene variants are associated with obesity in polycystic ovarysyndromerdquo PLoS ONE vol 6 no 1 Article ID e16390 2011

[4] ADunaif XWuA Lee andEDiamanti-Kandarakis ldquoDefectsin insulin receptor signaling in vivo in the polycystic ovary syn-drome (PCOS)rdquo American Journal of Physiology Endocrinologyand Metabolism vol 281 no 2 pp E392ndashE399 2001

[5] Y Zhang R Proenca M Maffei M Barone L Leopold and JM Friedman ldquoPositional cloning of the mouse obese gene andits human homologuerdquo Nature vol 372 no 6505 pp 425ndash4321994

[6] G Fr 120592hbeck ldquoIntracellular signalling pathways activated byleptinrdquo Biochemical Journal vol 393 part 1 pp 7ndash20 2006

[7] C T Montague I S Farooqi J P Whitehead et al ldquoCongenitalleptin deficiency is associated with severe early-onset obesity inhumansrdquo Nature vol 387 no 6636 pp 903ndash908 1997

[8] A Strobel T Issad L Camoin M Ozata and A D StrosbergldquoA leptinmissensemutation associated with hypogonadism andmorbid obesityrdquoNatureGenetics vol 18 no 3 pp 213ndash215 1998

[9] C S Mantzoros A Dunaif and J S Flier ldquoLeptin concentra-tions in the polycystic ovary syndromerdquo Journal of ClinicalEndocrinology and Metabolism vol 82 no 6 pp 1687ndash16911997

[10] S B Lecke FMattei DMMorsch and PM Spritzer ldquoAbdom-inal subcutaneous fat gene expression and circulating levels ofleptin and adiponectin in polycystic ovary syndromerdquo Fertilityand Sterility vol 95 no 6 pp 2044ndash2049 2011

[11] R Pasquali and A Gambineri ldquoPolycystic ovary syndrome amultifaceted disease from adolescence to adult agerdquo Annals ofthe New York Academy of Sciences vol 1092 pp 158ndash174 2006

[12] R Abir A Ao S Jin et al ldquoLeptin and its receptors in humanfetal and adult ovariesrdquo Fertility and Sterility vol 84 no 6 pp1779ndash1782 2005

[13] M R Ram P G Sundararaman and R Malathi ldquoBody fat dis-tribution and leptin correlation in womenwith polycystic ovarysyndrome endocrine and biochemical evaluation in southIndian populationrdquo Reproductive Medicine and Biology vol 4no 1 pp 71ndash78 2005

[14] M-G LiG-LDing X-J Chen et al ldquoAssociation of serumandfollicular fluid leptin concentrations with granulosa cell phos-phorylated signal transducer and activator of transcription 3expression in fertile patients with polycystic ovarian syndromerdquoJournal of Clinical Endocrinology andMetabolism vol 92 no 12pp 4771ndash4776 2007

[15] G De Placido C Alviggi R Clarizia et al ldquoIntra-follicularleptin concentration as a predictive factor for in vitro oocyte


fertilization in assisted reproductive techniquesrdquo Journal ofEndocrinological Investigation vol 29 no 8 pp 719ndash726 2006

[16] S Savastano R Valentino C Di Somma et al ldquoSerum 25-HydroxyvitaminDLevels phosphoprotein enriched in diabetesgene product (PEDPEA-15) and leptin-to-adiponectin ratio inwomenwith PCOSrdquoNutrition andMetabolism vol 8 article 842011


[18] M ReverchonMCornuau L Cloix et al ldquoVisfatin is expressedin human granulosa cells regulation by metformin throughAMPKSIRT1 pathways and its role in steroidogenesisrdquoMolec-ular Human Reproduction vol 19 no 5 pp 313ndash326 2013

[19] K-H Choi B-S Joo S-T Sun et al ldquoAdministration of visfatinduring superovulation improves developmental competency ofoocytes and fertility potential in aged femalemicerdquo Fertility andSterility vol 97 no 5 pp 1234ndash1241 2012

[20] G Garruti R Depalo M G Vita et al ldquoAdipose tissue meta-bolic syndrome and polycystic ovary syndrome from patho-physiology to treatmentrdquo Reproductive BioMedicine Online vol19 no 4 pp 552ndash563 2009

[21] R Depalo F Lorusso M Palmisano et al ldquoFollicular growthand oocyte maturation in GnRH agonist and antagonist proto-cols for invitro fertilisation and embryo transferrdquo GynecologicalEndocrinology vol 25 no 5 pp 328ndash334 2009

[22] The Expert Committee on the Diagnosis and Classification ofDiabetesMellitus ldquoReport of the expert committee on the diag-nosis and classification of diabetes mellitusrdquo Diabetes Care vol20 no 7 pp 1183ndash1197 1997

[23] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo vol 29 no11 pp 1281ndash1287 2012

[24] L L Veeck Atlas of the Human Oocyte and Early ConceptusWilliams ampWilkins Baltimore Md USA 1986

[25] D R Matthews J P Hosker and A S Rudenski ldquoHomeostasismodel assessment insulin resistance and 120573-cell function fromfasting plasma glucose and insulin concentrations in manrdquoDiabetologia vol 28 no 7 pp 412ndash419 1985

[26] M Matsuda and R A DeFronzo ldquoInsulin sensitivity indicesobtained from oral glucose tolerance testing comparison withthe euglycemic insulin clamprdquo Diabetes Care vol 22 no 9 pp1462ndash1470 1999

[27] P Armitage and G Berry Statistical Methods in MedicalResearch Blackwell Science Oxford UK 3rd edition 1994

[28] B Dawson and R G Trapp Basic amp Clinical Biostatistics McGraw-Hill New York NY USA 3rd edition 2001

[29] R Azziz K S Woods R Reyna T J Key E S Knochenhauerand B O Yildiz ldquoThe prevalence and features of the polycysticovary syndrome in an unselected populationrdquo Journal of Clini-cal Endocrinology andMetabolism vol 89 no 6 pp 2745ndash27492004

[30] R A Wild E Carmina E Diamanti-Kandarakis et al ldquoAssess-ment of cardiovascular risk and prevention of cardiovasculardisease in women with the polycystic ovary syndrome a con-sensus statement by the androgen excess and polycystic ovarysyndrome (AE-PCOS) societyrdquo Journal of Clinical Endocrinol-ogy and Metabolism vol 95 no 5 pp 2038ndash2049 2010

[31] C J Alexander E P Tangchitnob and N E Lepor ldquoPolycysticovary syndrome a major unrecognized cardiovascular risk fac-tor in womenrdquo Reviews in Obstetrics amp Gynecology vol 2 no 4pp 232ndash239 2009

[32] P Zimmet G Alberti and J A Shaw ldquoA new IDF worldwidedefinition of the metabolic syndrome the rationale and theresultsrdquo Diabetes Voice vol 50 no 3 pp 31ndash33 2005

[33] Q Lin S L Poon J Chen L Cheng B HoYuen and P C KLeung ldquoLeptin interferes with 3101584051015840-Cyclic Adenosine Mono-phosphate (cAMP) signaling to inhibit steroidogenesis inhuman granulosa cellsrdquo Reproductive Biology and Endocrinol-ogy vol 7 article 115 2009

[34] A Cervero F Dominguez J A Horcajadas A Quinonero APellicer and C Simon ldquoThe role of the leptin in reproductionrdquoCurrent Opinion in Obstetrics and Gynecology vol 18 no 3 pp297ndash303 2006

[35] S Sam and A Dunaif ldquoPolycystic ovary syndrome syndromeXXrdquo Trends in Endocrinology and Metabolism vol 14 no 8pp 365ndash370 2003

[36] A Corbould and A Dunaif ldquoThe adipose cell lineage is notintrinsically insulin resistant in polycystic ovary syndromerdquoMetabolism Clinical and Experimental vol 56 no 5 pp 716ndash722 2007

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Evidence-Based Complementary and Alternative Medicine

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Several studies have investigated leptin levels in over-weight andor obese women with PCOS clearly demonstrat-ing that they appear to be correlated with BMI [11] How-ever granulosa cells are able to synthesize and store leptinfromnewborn until adult life in amanner that is independentof BMI [12] Relevant to this concept few studies have inves-tigated leptin levels in normal-weight women with PCOSin specific ethnic groups in the absence of excess BMIIn an Indian study circulating leptin levels were found tobe 5-fold higher in normal-weight women with PCOS ascompared with controls [13] In a Chinese study PCOSwomen were characterized by higher leptin levels both inserum and follicular fluid than controls although the BMIof the PCOS women included in this study ranged fromnormal-weight to overweight and obesity [14] The above-mentioned data are not in line with other results in Caucasianwomen demonstrating that the concentration of leptin inthe follicular fluid (FF-leptin) seems to be predictive of thefertilization rate [15] Fertilization rate is usually lower inPCOS women as compared with normally ovulating womenThe paper by De Placido et al [15] is the only one tofind a direct correlation between the concentration of leptinin the follicular fluid (FF-leptin) and the fertilization rateInterestingly the population studied by De Placido et al wascoming from the south of Italy and certainly showed differentlifestyle and diet as compared with Indians and Chinesewomen

In other recent studies elevated circulating levels of leptinandor increased leptinadiponectin ratios were reported inoverweight PCOSwomen but no information on leptin levelsin lean women with PCOS was provided [16 17]

Visfatin acts as a cytokine but also as an enzyme and isinvolved in metabolic and immunological diseases VisfatinmRNA was recently detected in human follicles and visfatinadministration was shown to improve ovarian response inconditions of superovulation in animal models [18 19]

In this study we havemeasured circulating leptin and vis-fatin levels and FF-leptin and FF-visfatin in a selected groupof Caucasian Southern Italian nonoverweight women withPCOS (NOW-PCOS) undergoing in vitro fertilizationembryo transfer (IVF) in order to better understand the inter-relationships if any between both cytokines and fertilizationrate

Some other papers analyzed leptin levels in both serumand follicular fluid in women having PCOS however veryfew papers included only Caucasian nonoverweight women(BMI le 249 Kgm2) with PCOS as we did Our pilot studyis also new in the fact that all women studied were comingfrom Apulia a region from the south of Italy which is verywell known for Mediterranean diet

2 Materials and Methods

In this observational pilot study we selected 16 consecutiveNOW-PCOS and 10 consecutive age- and BMI-matchednormal-ovulating nonhirsute premenopausal infertile Cau-casian women (control-NOW) requesting a procedure ofassisted reproduction at the Centre of Pathophysiology of

Human Reproduction and Gametes Cryopreservation of theUniversity of Bari All women were from Apulia in SouthernItaly and did not show any sign of metabolic syndrome Inthe control-NOW group infertility was due either to maleinfertility or tubal factors The diagnosis of PCOS was basedon the criteria indicated in the Amsterdam ESHREASRM-Sponsored 3rd PCOS Consensus Workshop Group [1 20]All women with PCOS had hirsutism oligomenorrhea andclear diagnosis of PCOS by ultrasounds Both NOW-PCOSand control-NOW were euthyroid and were undergoing IVFfor the first time All women underwent IVF according to apreviously reported protocol [21] Infertile women with adiagnosis of diabetes mellitus were excluded from the studyto rule out potential confounding effects of hyperglycemiaon leptin and fertilization rates Indeed all subjects had fast-ing glucose levels lt126mgdL (7mmolL) and underwent anoral glucose tolerance test according to the American Dia-betes Association guidelines [22] displaying plasma glucoselevels lt140mgdL (78mmolL) after 2 h All women were inthe premenopausal stage as confirmed by circulating FSHand LH levels measured between day 3 and day 5 of themenstrual cycle All women underwent IVF according toa previously reported protocol [21] Briefly daily GnRH-aadministration (Decapeptyl 01mgmL-Ipsen SpA sc) wasstarted during the middle-luteal phase of the cycle beforeovarian hyperstimulation until the day of human chorionicgonadotropin (hCG) administration Ovarian hyperstimu-lation was initiated once the serum estradiol (E

2) level

was lt30 pgmL with 150ndash225 IU of rFSH (alpha follitropin-Gonal-F Merck Serono SpA) Dosing was individualizeddepending on the patientrsquos response When at least 3 folliclesof 18mm in diameter and a serum E

2level of gt200 pgmL

per follicle were noted the patient was given 250 120583gof choriongonadotropin alpha hormone (Ovitrelle MerckSerono SpA) Monitoring was accomplished with serialvaginal ultrasound examinations and serum E

2level deter-

minations Transvaginal oocyte retrieval was performed 35hours after hCG injection After retrieval cumulus cells(COCs) were removed with a 30-second exposure to HEPES-buffed medium containing 40 IUmL hyaluronidase (SAGEUSA) and denuded oocytes were then assessed for nuclearstatus those that were observed to have released the firstpolar body were considered mature (MII) and used for ICSIprocedure [23]

Fertilization was assessed 18 hours after ICSI Normalfertilization was confirmed when 2 clearly distinct pronucleiwere present Fertilization rate was defined as the percentageof pronuleate oocytes to the number of oocytes injectedEmbryo quality was evaluated under inverted microscope onthe second day of development according to Veeck [24] Amaximum of three embryos were transferred on day 2 or3 of embryo culture under ultrasound guidance For lutealphase support all patients received a daily dose of 400mg ofvaginal micronized progesterone (Progeffik Effik Italia)Serum 120573-hCG concentration was measured 14 days afteroocyte retrieval to verify pregnancy Clinical pregnancy wasjudged by observation of the gestational sac on vaginalultrasonography after 6-7 weeks of gestation











3 Results








2levels were



300

225

150

75

00

180 200 220 240 260

Insu

lin (120583

Um

L)

BMI (kgm2)

(a)

Insu

lin (120583

Um

L)

BMI (kgm2)

160

125

90

55

20

190 203 215 228 240

(b)


160

120

80

40

00

00 50 100 150 200

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(a)

350

263

175

88

00

00 125 250 375 500

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(b)



4 Discussion





200

150

100

50

00

00 75 150 225 300

Insulin (pgmL)

FF-le

ptin

(pg

mL)

(a)

Insulin (pgmL)

500

375

250

125

00

20 55 90 125 160

FF-le

ptin

(pg

mL)

(b)



















References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results








2levels were



300

225

150

75

00

180 200 220 240 260

Insu

lin (120583

Um

L)

BMI (kgm2)

(a)

Insu

lin (120583

Um

L)

BMI (kgm2)

160

125

90

55

20

190 203 215 228 240

(b)


160

120

80

40

00

00 50 100 150 200

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(a)

350

263

175

88

00

00 125 250 375 500

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(b)



4 Discussion





200

150

100

50

00

00 75 150 225 300

Insulin (pgmL)

FF-le

ptin

(pg

mL)

(a)

Insulin (pgmL)

500

375

250

125

00

20 55 90 125 160

FF-le

ptin

(pg

mL)

(b)



















References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















300

225

150

75

00

180 200 220 240 260

Insu

lin (120583

Um

L)

BMI (kgm2)

(a)

Insu

lin (120583

Um

L)

BMI (kgm2)

160

125

90

55

20

190 203 215 228 240

(b)


160

120

80

40

00

00 50 100 150 200

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(a)

350

263

175

88

00

00 125 250 375 500

Seru

m le

ptin

(pg

mL)

FF-leptin (pgmL)

(b)



4 Discussion





200

150

100

50

00

00 75 150 225 300

Insulin (pgmL)

FF-le

ptin

(pg

mL)

(a)

Insulin (pgmL)

500

375

250

125

00

20 55 90 125 160

FF-le

ptin

(pg

mL)

(b)



















References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200

150

100

50

00

00 75 150 225 300

Insulin (pgmL)

FF-le

ptin

(pg

mL)

(a)

Insulin (pgmL)

500

375

250

125

00

20 55 90 125 160

FF-le

ptin

(pg

mL)

(b)



















References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Association between Follicular Fluid...

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