Research Article Etiopathogenesis of Sheehan s...

Research ArticleEtiopathogenesis of Sheehanrsquos Syndrome Roles ofCoagulation Factors and TNF-Alpha

Halit Diri1 Elif Funda Sener2 Fahri Bayram1 Nazife Tascioglu3

Yasin Simsek1 and Munis Dundar3

1 Department of Endocrinology Erciyes University Medical School 38039 Kayseri Turkey2Department of Medical Biology Erciyes University Medical School 38039 Kayseri Turkey3 Department of Medical Genetics Erciyes University Medical School 38039 Kayseri Turkey

Correspondence should be addressed to Halit Diri halitdiriyahoocom

Received 2 January 2014 Revised 10 March 2014 Accepted 26 March 2014 Published 10 April 2014

Academic Editor Kevin Sinchak

Copyright copy 2014 Halit Diri et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sheehanrsquos Syndrome (SS) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a resultof massive postpartum uterine hemorrhage Herein we aimed to investigate the roles of Factor II (G20210A) Factor V (G1691A)MTHFR (C677T and A1298C) PAI-1 4G5G and TNF-120572 (minus308 G gt A) gene polymorphisms in the etiopathogenesis of SS Venousblood samples were obtained from 53 cases with SS and 43 healthy women Standard methods were used to extract the genomicDNAs Factor II (G20210A) Factor V (G1691A) and MTHFR (C677T and A1298C) polymorphisms were identified by real-timePCR PAI-1 4G5G and TNF-120572 (minus308 G gt A) gene polymorphisms were detected with polymerase chain reaction (PCR) andrestriction fragment length polymorphism (RFLP) methods According to statistical analysis none of the polymorphisms werefound to be significantly higher in the SS group compared to the control group Hence we suggest that genetic factors other thanFactor II Factor V MTHFR PAI-1 and TNF-120572 gene polymorphisms should be researched in the etiopathogenesis of SS

1 Introduction

Sheehanrsquos Syndrome (SS) is defined as pituitary hormonedeficiency due to ischemic infarction of the pituitary glandas a result of massive postpartum uterine hemorrhage [1]Although the onset of SS can involve acute severe panhy-popituitarism in some patients themajority of SS patients arediagnosed with a clinically subtle partial pituitary deficiencyand therefore their diagnoses and treatments are delayedfor many years [2] The prevalence of SS is not clearlyknown presumably due to the great number of nondiagnosedpatients It is a rarely encountered disorder in developedcountries due to good obstetric care [3 4] However itsprevalence is estimated to be still high in developing countriesin which many deliveries take place at home [5] Zargar et alestimated the prevalence of SS to be 31 among women inIndia and about two-thirds of those women had given birthat home [6]

As the etiopathogenesis of SS is not clear disorders ofcoagulation have been investigated in some studies It hasbeen reported that disseminated intravascular coagulation(DIC) can cause postpartum hypopituitarism [7 8] Inaddition Cakir et al found protein S deficiency in 2 outof 12 patients with SS [9] Importantly in another studyGokalp et al investigated inherited hypercoagulation as arisk factor of SS They found that frequency of MTHFR(methylenetetrahydrofolate reductase) C677T and MTHFRA1298C polymorphisms was significantly higher among their38 patients with SS compared to the healthy control group[10] In additionFactor II (G20210A)FactorV (G1691A) andPAI-1 4G5G mutations were also more common among theSS patients but no significant differences were observed

Our aim in this study was to investigate gene poly-morphisms of MTHFR C677T and A1298C Factor II(G20210A) Factor V (G1691A) and PAI-1 (plasminogenactivator inhibitor-1) 4G5G that are associated with inherited

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 514891 6 pageshttpdxdoiorg1011552014514891

2 International Journal of Endocrinology

hypercoagulation and TNF-120572 (tumor necrosis factor-alpha)308 G gt A that is associated with apoptosis of pituitarycells due to autoimmunity Knowing the frequencies ofthese polymorphisms among SS patients will be helpful inunderstanding their roles in the etiopathogenesis of SS

2 Patients and Methods

21 Study Design Fifty-three patients who were previouslydiagnosed with SS and 43 healthy women were enrolled inthis study which was conducted between 2011 and 2013 Theblood samples of patients with SS who were followed up bythe EndocrinologyDepartment of ErciyesUniversityMedicalSchool were collected following 12 hours of fasting Thehealthy female volunteers in the control group were chosenfrom hospital staff and their relatives Written informedconsent was obtained from all participants before registeringthem for the study

In addition to the demographic information the medicalhistory and drug use of the participants were examined indetail The most important inclusion criterion for the SSgroup was having the exact diagnosis of SS Therefore it wasemphasized that all of the following criteria for diagnosiswere met (a) at least one pituitary hormone deficiency foundin basal levels or via dynamic tests if required (b) massivepostpartum uterine hemorrhage history at last delivery (c)agalactia and amenorrhea after the last delivery (d) exclusionof all other causes of pituitary deficiency (e) observationof partial or complete empty sella on magnetic resonanceimaging (MRI) Exclusion criteria for SS patients were havingcomorbidity or being on other treatments than glucocor-ticoid and thyroid hormone replacement therapies whichwere adequately performed In order to avoid the effects ofreplacement therapies of growth hormone (GH) and gonadalsteroids on genotyping studies such treatments were stopped3 months before the blood collection for the genetic analysesIn addition women in the control group did not have anyhistories of disease and were not on any drug therapies

22 Hormonal Analyses Basal levels of hormones wereanalyzed to determine hypopituitarism in all patients Inaddition the insulin tolerance test or glucagon stimulationtest was performed to identify growth hormone (GH) defi-ciency and secondary adrenal failure Basal hormone levelsincluding free T4 (normal 088ndash172 ngdL) thyroid stimu-lating hormone (TSH normal 057ndash56mIUmL) adreno-corticotropic hormone (ACTH normal 0ndash46 pgmL) corti-sol (normal 9ndash23 120583gdL) prolactin (PRL normal for post-menopausal women 24ndash298 and premenopausal women33ndash298 ngmL) follicle stimulating hormone (FSH normalfor postmenopausal women 239ndash1191 and premenopausalwomen 20ndash98mIUmL) luteinizing hormone (LH normalfor postmenopausal women 163ndash548 and premenopausalwomen 07ndash173mIUmL) estradiol (E2 normal for post-menopausal women 144ndash445 and premenopausal women189ndash2467 pgmL) and insulin-like growth factor-1 (IGF-1 reference intervals varied by age) were measured in thehormone laboratories of Erciyes University Medical School

Methods of assays and commercial kits were as followsGH immunoradiometric assay (IRMA) Immunotech SASFrance IGF-1 IRMA Immunotech SAS France ACTHIRMA Cisbio Bioassays France cortisol radioimmunoassay(RIA) Immunotech sro Czech Republic PRL TSH fT4FSH LH estradiol Immunoassay Siemens Advia centaurXP-USA

23 Genotyping Studies 2mL venous blood samples werecollected in EDTA-containing tubes for DNA analyses Totalgenomic DNA was extracted by standard methods and DNAsamples were stored at minus20∘C until the analyses of poly-morphisms Genotyping studies were conducted in ErciyesUniversity Genome and Stem Cell Center (GENKOK) Atotal of 20120583L PCR mixture with 5 120583L sample DNA wasanalyzed by using a LightCycler FastStart DNA MasterHybProbe to perform the genotypings of MTHFR C677Tand A1298C Factor II (prothrombin) G20210A and FactorV G1691A according to manufacturerrsquos instructions (RocheDiagnostics Germany) In addition Roche LightCycler 480Software was used for detecting different genotypes of thesepolymorphisms

Genotypes of the TNF-120572-308 GA and PAI-1 4G5Gpolymorphisms were detected by analyses of polymerasechain reaction (PCR) and restriction fragment length poly-morphism (RFLP) results PAI-1 4G5G polymorphisms wereanalyzed by forward primer 51015840-CACAGAGAGAGTCTG-GCCACGT-31015840 and 51015840-CCAACAGAGGACTCTTGGTCT-31015840reverse primer The genomic region of interest was amplifiedby using PCR for 30 cycles with a denaturation temperatureof 94∘C for 3min 94∘C for 30 sec 60∘C for 30 sec and 72∘Cfor 30 sec The final extension step at 72∘C was extended for1min Amplified 98 base pair (bp) products were digestedovernight with Bsl I at 55∘C and subjected to 4 agarose gelelectrophoresis [11]

The PCR procedure was performed in a total volume of50 120583L containing 5120583L genomic DNA 10x PCR buffer dNTPs(25mM) MgCl

2(15mM) Taq DNA polymerase (1 UmL)

51015840AGGCAATAGGTTTTGAGGGCCAT-31015840 forward primersand 51015840TCCTCCCTGCTCCGATTCCG-31015840 reverse primersfor TNF-120572 [12] The cycling conditions consisted of denatu-ration at 95∘C for 5min followed by 30 consecutive cyclesof 95∘C for 1min 60∘C for 1min and 72∘C for 1min with afinal elongation at 72∘C for 5min The 107 bp PCR productswere separated by electrophoresis on a 2 agarose gel andvisualized with UV illumination and ethidium bromidestaining After amplification the PCR products were digestedovernight at 37∘CwithNco I restriction enzyme and analyzedby 3 agarose gel electrophoresis

24 Statistical Analyses Statistical analyses were performedby using SPSS 150 (SPSS Inc Chicago IL) Demographicdata were presented as mean plusmn SD Binomial variables wereanalyzed using Pearsonrsquos chi-square test or Fisherrsquos exact testMoreover the odds ratios (OR) were calculated with 95confidence intervals (CI) using logistic regression analysisComparisons of genotype frequencies were made between

International Journal of Endocrinology 3

Table 1 Distribution of deficient hormones in patients with SS

Deficient hormonesNumber andpercentage of

patientsFSH-LH + GH + TSH + ACTH + PRL 38 (717)FSH-LH + GH + TSH + PRL 4 (76)FSH-LH + GH + ACTH + PRL 2 (38)FSH-LH + GH + ACTH + TSH 3 (57)FSH-LH + GH + PRL 1 (19)FSH-LH + GH + TSH 4 (76)FSH-LH + GH 1 (19)Total 53 (100)

patients and controls and 119875 values less than 005 wereconsidered as statistically significant

3 Results

Panhypopituitarism was detected in 38 (717) patients withSS while partial hypopituitarism was detected in 15 (283)patients Importantly all patients had gonadotropin and GHdeficiencies but deficiencies of ACTH TSH and prolactinwere not found in all of the patients (Table 1)

When comparing groups it was revealed that there wasno significant difference between the mean age of the 53cases with SS and the 43 healthy controls 632 plusmn 125 and603 plusmn 93 years respectively In addition no differencewas detected in terms of mean body mass indexes (BMIs)The mean BMI was 289 plusmn 28 kgm2 in the SS group and292 plusmn 33 kgm2 in healthy women Moreover the SS andhealthy control groups did not significantly differ accordingto polymorphism ratesof Factor II (G20210A) Factor V(G1691A)MTHFR (C677T andA1298C) PAI-1 (4G5G) andTNF-120572 (minus308 G gt A) genes except for PAI-1 (4G5G) ThePAI-1 (4G5G) mutation was detected in 26 (605) of the 43control cases and in 20 (377) of the 53 SS cases Frequenciesand comparisons of polymorphisms are demonstrated inTable 2

4 Discussion

The physiological enlargement of the pituitary gland duringpregnancy plays a significant role in onset of SS becausesevere bleeding does not lead to pituitary deficiency inwomen unless they are pregnant Even though the patho-genesis of SS has not yet been fully clarified the basis ofits pathology has been identified as infarction and ischemicnecrosis that develops due to the interruption of arterialblood flow in the anterior pituitary gland [13] Howeverthe cause of the interruption in the blood flow is not clearConsidered potential mechanisms are arterial thrombosissimilar to that seen in stroke development of arterial spasmas a result of severe hypotension that is due to massiveuterine bleeding or compression of pituitary vessels due to

relatively small sella turcica volume associated with enlarge-ment of the pituitary during pregnancy [14] Furthermoreautoantibodies detected inmany patients against the pituitarygland have been suggested as a contributing factor in theetiopathogenesis of SS [15]

In the etiopathogenesis of SS our study findings didnot yield a significant difference between the control and SSpatient groups in terms of mutations of blood compositionanomalies that lead to inherited hypercoagulation In otherwords in the SS group none of the genes had higherpolymorphism rates than those in the control group Thereare numerous other acquired (prolonged immobilizationpregnancy oral contraceptive pills advanced age obesitycigarette use hypertension etc) or genetic (protein C orS deficiency antithrombin-III deficiency platelet GPIIbIIIaHPA-Ibmutation elevated levels of Factors VII VIII IX andXII VonWillebrand disease fibrinogen etc) factors that areknown to cause hypercoagulation An important limitationof our study is that not all of the parameters associated withhypercoagulation were investigated Therefore it is hard toconclude based only on our study findings that inheritedhypercoagulation is not involved in SS

Although the mutation rates of MTHFR C677T andA1298C genes show social differences homozygous muta-tions are estimated to be 10 and heterozygous mutationsare about 40 [16 17] Approximately 15 of the populationis reported to carry both heterozygous genetic mutationstogether due to their close relations [17] Although the ratesof MTHFR gene polymorphisms were not higher in the SSgroup than in the control group in our study more importantissues are whether the SS patients had vitamin B6 vitaminB12 andor folate deficiency and thus a disorder in MTHFRgene expression during their last pregnancies

The heterozygous mutations in Factor II (prothrombin)G20210A are encountered at a 2-3 rate among Caucasians[18] Plasma Factor-II levels (prothrombin) increase as aresult of mutations In that case risk of venous thrombosisis higher than arterial thrombosis risk Venous thrombosisrisk among individuals carrying this mutation is increasedby 2-3 times One study reported that coronary artery riskis increased by 131 times in individuals with prothrombinG20210A mutation [19]

While varying across different populations the incidencerate of Factor V Leiden heterozygous mutation is approxi-mately 5 but its homozygous mutation is rare [20] As aresult of the mutation in the Factor V gene (G1691A) acti-vated protein-C cannot inhibit Factor V molecules This inturn results in a deterioration of the bleeding and coagulationbalance in favor of coagulation This mutation is reportedto increase thrombosis risk fivefold when heterozygousand by 10ndash80-fold when homozygous [21 22] Factor VLeiden has also been shown to increase complications suchas miscarriage preeclampsia and abruptio placentae by aminimum of 2-3 times [20]

Plasminogen activator inhibitor-1 (PAI-1) inhibits tissueplasminogen activator (tPA) and urokinasewhich are proteinsleading to plasminogen activation and fibrinolysisThe resultis inhibition of fibrinolysisThe estimated frequences of PAI-14G4G 4G5G and 5G5G are respectively about 35 50


Table 2 Distribution of thrombophilic and cytokine genes among Sheehanrsquos Syndrome cases and controls

Sheehanrsquos Syndrome patients (n 53) Controls (n 43) OR (95 CI) P valueDaggern () n ()

MTHFR C677TGenotype

CC (normal) 31 585 25 581 1 (Ref) mdashCT (heterozy) 19 358 13 302 1179 (0489ndash2843) 0714TT (homozy) 3 57 5 116 0484 (0105ndash2224) 0351

P valuedagger 0538MTHFR A1298C

GenotypeAA (normal) 17 321 15 349 1 (Ref) mdashAC (heterozy) 28 528 19 442 13 (0525ndash3219) 0570CC (homozy) 8 151 9 209 0784 (0241ndash2549) 0686

P valuedagger 0646Factor II G20210A

GenotypeGG (normal) 50 943 42 977 1 (Ref) mdashGA (heterozy) 3 57 1 23 2520 (0253ndash25136) 0431AA (homozy) mdash mdash mdash mdash mdash mdash

P valuedagger 0416Factor V G1691A

GenotypeGG (normal) 47 887 37 86 1 (Ref) mdashGA (heterozy) 5 94 4 93 0984 (0247ndash3925) 0982AA (homozy) 1 19 2 47 0394 (0034ndash4511) 0454

P valuedagger 0741PAI-1 4G5G

Genotype5G5G (normal) 21 396 7 163 1 (Ref) mdash4G5G (heterozy) 20 377 26 605 0256 (0091ndash0722) 0014G4G (homozy) 12 226 10 233 04 (0121ndash1326) 0134

P valuedagger 003TNF-120572 minus308 GgtA

GenotypeGG (normal) 43 811 34 791 1 (Ref) mdashGA (heterozy) 9 17 9 209 0791 (0283ndash2210) 0654AA (homozy) 1 19 mdash mdash mdash mdash

P valuedagger 06Notes heterozy heterozygous homozy homozygousdaggerSignificance of 1205942 values which were obtained from a Chi-Square testDaggerSignificance of odds ratios which were obtained from a logistic regression model

and 15 [23 24] Polymorphisms of PAI-1 4G4G and 4G5Gare reported to cause an increase in PAI-1 levels and thusrisk of arterial thrombosis rather than of venous thrombosis[25 26] Pregnant women who carry the homozygous PAI-14G4G mutation are also known to have an increased risk ofmiscarriage [27] However there are other studies reportingthat patients with ischemic stroke and coronary artery diseasedo not have increased PAI-1 4G5G polymorphism [28 29]

Interestingly our results showed that the 4G5G polymor-phism rate was 605 among healthy women while it was377 among SS patients This finding may suggest that the4G5Gpolymorphismdoes not always lead to thrombophiliaunless accompanied by some acquired factors

TNF-120572 (tumor necrosis factor-alpha) is a cytokine thatplays an important role in the regulations of immune func-tions cell proliferation and differentiation apoptosis due


to autoimmunity coagulation adipocyte lipid and glucosemetabolism The 308 GA polymorphism of TNF-120572 whichleads to elevated TNF-120572 levels is known to have an impor-tant impact on the development of autoimmunity diseasesmetabolic syndromes cancers and psychiatric disorders [3031] Additionally an association between high TNF-120572 levelsand hypercoagulation has also been detected [32]

Autoimmunity is a potential risk factor in the devel-opment of SS and 35 of patients have been shown tohave anti-pituitary autoantibodies [33] These antibodies arepresumably generated in reaction to the necrotic pituitarygland tissue found after ischemic infarction and seem to beresponsible for the chronic but progressive course of SS [15]On another note increased estrogen levels during pregnancymake adenohypophysis cells particularly the lactotroph cellsmore vulnerable to apoptosis due to autoimmunity as a resultof elevated expression of the TNF-120572 gene in rats [34] Thiscondition may also be associated with the vulnerability ofan enlarged pituitary to ischemic necrosis at the end ofpregnancy Estrogen levels gradually increase during preg-nancy and peak close to completion of the full term In thisregard we hypothesized that autoimmunity caused by TNF-120572 308 GA mutation can be a contributing factor in theetiopathogenesis of SS However no increase in the rate ofTNF-120572 308 GA polymorphism was observed among the SSpatients when compared to the control group

5 Conclusion

As demonstrated in our study the disorders suggested forSS etiopathogenesis and the associations among them arehighly complicated Thrombophilia and autoimmunity in SSare alsomultifaceted and complicatedmechanismsHoweverthe results of our study showed no increase in Factor IIFactor V MTHFR PAI-1 and TNF-120572 gene polymorphismrates among SS patients compared to the control groupHence genetic factors other than these gene polymorphismsshould be researched in the etiopathogenesis of SS ClarifyingSS etiopathogenesis via further studies will be particularlybeneficial in identifying women susceptible to SS prior todisease development

Conflict of Interests

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

Authorsrsquo Contribution

Halit Diri and Elif Funda Sener contributed equally to thiswork and should be considered cofirst authors

Acknowledgment

This work was funded by Erciyes University The authorswould like to thank Gokmen Zararsiz for statistical analysis

References

[1] H L Sheehan ldquoPostpartum necrosis of the anterior pituitaryrdquoJournal of Pathology amp Bacteriology vol 45 no 1 pp 189ndash2141937

[2] F Kelestimur ldquoSheehanrsquos syndromerdquo Pituitary vol 6 no 4 pp181ndash188 2003

[3] E C Feinberg M E Molitch L K Endres and A MPeaceman ldquoThe incidence of Sheehanrsquos syndrome after obstetrichemorrhagerdquo Fertility and Sterility vol 84 no 4 pp 975ndash9792005

[4] M Regal C Paramo J M Sierra and R V Garci-MayorldquoPrevalence and incidence of hypopituitarism in an adult Cau-casian population in northwestern Spainrdquo Clinical Endocrinol-ogy vol 55 no 6 pp 735ndash740 2001

[5] O O Famuyiwa A F Bella and A O Akanji ldquoSheehanrsquossyndrome in a developing country Nigeria a rare disease orproblem of diagnosisrdquo East AfricanMedical Journal vol 69 no1 pp 40ndash43 1992

[6] A H Zargar B Singh B A Laway S R Masoodi A IWani and M I Bashir ldquoEpidemiologic aspects of postpartumpituitary hypofunction (Sheehanrsquos syndrome)rdquo Fertility andSterility vol 84 no 2 pp 523ndash528 2005

[7] T Yamauchi N Yoshio T Mizuguchi E Negoro N Kamitaniand T Ueda ldquoAcute fatty liver of pregnancy complicated withanterior pituitary insufficiencyrdquo Internal Medicine vol 40 no12 pp 1227ndash1231 2001

[8] J J Piech P Thieblot and J P Haberer ldquoTwin pregnancy withacute hepatic steatosis followed by hypopituitarism anddiabetesinsipidusrdquo Presse Medicale vol 14 no 26 pp 1421ndash1423 1985

[9] I Cakir F Tanriverdi Z Karaca et al ldquoEvaluation of coagu-lation and fibrinolytic parameters in adult onset GH deficiencyand the effects of GH replacement therapy a placebo controlledstudyrdquoGrowth Hormone and IGF Research vol 22 no 1 pp 17ndash21 2012

[10] D Gokalp A Tuzcu M Bahceci et al ldquoAnalysis of throm-bophilic geneticmutations in patientswith Sheehanrsquos syndromeis thrombophilia responsible for the pathogenesis of Sheehanrsquossyndromerdquo Pituitary vol 14 no 2 pp 168ndash173 2011

[11] E F Emirogullari C Saatci A Unal A Sahin and YOzkul ldquoProthrombin factor-V leiden and plasminogen acti-vator inhibitor type 1 gene polymorphisms in hemodialysispatients withwithout arteriovenous fistula thrombosisrdquo TheJournal of Nephro-Urology vol 2 no 2 pp 314ndash319 2010

[12] M Dundar A Kiraz B Balta et al ldquoThe role of TNF-alpha andPAI-1 gene polymorphisms in familial Mediterranean feverrdquoModern Rheumatology vol 23 no 1 pp 140ndash145 2013

[13] K Kovacs ldquoSheehan syndromerdquo The Lancet vol 361 no 9356pp 520ndash522 2003

[14] A H Tessnow and J DWilson ldquoThe changing face of Sheehanrsquossyndromerdquo American Journal of the Medical Sciences vol 340no 5 pp 402ndash406 2010

[15] R Goswami N Kochupillai P A Crock A Jaleel and NGupta ldquoPituitary autoimmunity in patients with Sheehanrsquossyndromerdquo Journal of Clinical Endocrinology and Metabolismvol 87 no 9 pp 4137ndash4141 2002

[16] B Zappacosta L Romano S Persichilli et al ldquoGenotype preva-lence and allele frequencies of 510- methylenetetrahydrofolatereductase (MTHFR) C677T and A1298C polymorphisms inItalian newbornsrdquo Laboratory Medicine vol 40 no 12 pp 732ndash736 2009


[17] I Weisberg P Tran B Christensen S Sibani and R RozenldquoA second genetic polymorphism in methylenetetrahydrofolatereductase (MTHFR) associated with decreased enzyme activ-ityrdquoMolecular Genetics and Metabolism vol 64 no 3 pp 169ndash172 1998

[18] F R Rosendaal C J M Doggen A Zivelin et al ldquoGeographicdistribution of the 20210 G to A prothrombin variantrdquo Throm-bosis and Haemostasis vol 79 no 4 pp 706ndash708 1998

[19] Z Ye E H C Liu J P T Higgins et al ldquoSeven haemostaticgene polymorphisms in coronary disease meta-analysis of 66155 cases and 91 307 controlsrdquoThe Lancet vol 367 no 9511 pp651ndash658 2006

[20] J L Kujovich ldquoFactor v Leiden thrombophiliardquo Genetics inMedicine vol 13 no 1 pp 1ndash16 2011

[21] S Middeldorp J R Meinardi M M W Koopman et alldquoA prospective study of asymptomatic carriers of the factorV Leiden mutation to determine the incidence of venousthromboembolismrdquo Annals of Internal Medicine vol 135 no 5pp 322ndash327 2001

[22] F R Rosendaal T Koster J P Vandenbroucke and P HReitsma ldquoHigh risk of thrombosis in patients homozygous forfactor V Leiden (activated protein C resistance)rdquo Blood vol 85no 6 pp 1504ndash1508 1995

[23] Y Matsubara M Murata I Isshiki et al ldquoGenotype frequencyof plasminogen activator inhibitor-1 (PAI-1) 4G5G polymor-phism in healthy Japanesemales and its relation to PAI-1 levelsrdquoInternational Journal of Hematology vol 69 no 1 pp 43ndash471999

[24] J Kvasnicka J Hajkova P Bobcıkova et al ldquoPrevalence ofthrombophilic mutations of FV Leiden prothrombin G20210Aand PAI-1 4G5G and their combinations in a group of 1450healthy middle-aged individuals in the Prague and CentralBohemian regions (results of FRET real-time PCR assay)rdquoCasopis Lekaru Ceskych vol 151 no 2 pp 76ndash82 2012

[25] M-L P J van Goor E G Garcıa F Leebeek G-J Brouw-ers P Koudstaal and D Dippel ldquoThe plasminogen activatorinhibitor (PAI-1) 4G5G promoter polymorphism and PAI-1levels in ischemic stroke A case-control studyrdquoThrombosis andHaemostasis vol 93 no 1 pp 92ndash96 2005

[26] L L Gong J H Peng F F Han et al ldquoAssociation of tissueplasminogen activator and plasminogen activator inhibitorpolymorphism with myocardial infarction a meta-analysisrdquoThrombosis Research vol 130 no 3 pp e43ndashe51 2012

[27] A Dossenbach-Glaninger M Van Trotsenburg M Dossen-bach et al ldquoPlasminogen activator inhibitor I 4G5G polymor-phism and coagulation factor XIII Val34Leu polymorphismimpaired fibrinolysis and early pregnancy lossrdquo Clinical Chem-istry vol 49 no 7 pp 1081ndash1086 2003

[28] W Koch M Schrempf A Erl et al ldquo4G5G polymorphism andhaplotypes of SERPINE1 in atherosclerotic diseases of coronaryarteriesrdquoThrombosis and Haemostasis vol 103 no 6 pp 1170ndash1180 2010

[29] K Jood P Ladenvall A Tjarnlund-Wolf et al ldquoFibrinolyticgene polymorphism and ischemic strokerdquo Stroke vol 36 no 10pp 2077ndash2081 2005

[30] H T Idriss and J H Naismith ldquoTNF alpha and the TNF recep-tor superfamily structure-function relationship(s)rdquoMicroscopyResearch and Technique vol 50 no 3 pp 184ndash195 2000

[31] W P Cawthorn and J K Sethi ldquoTNF-120572 and adipocyte biologyrdquoFEBS Letters vol 582 no 1 pp 117ndash131 2008

[32] M G Macey S I Wolf C P D Wheeler-Jones and C LawsonldquoExpression of blood coagulation factors on monocytes after

exposure to TNF-treated endothelium in a novel whole bloodmodel of arterial flowrdquo Journal of Immunological Methods vol350 no 1-2 pp 133ndash141 2009

[33] A De Bellis F Kelestimur A A Sinisi et al ldquoAnti-hypothalamus and anti-pituitary antibodies may contributeto perpetuate the hypopituitarism in patients with Sheehanrsquossyndromerdquo European Journal of Endocrinology vol 158 no 2pp 147ndash152 2008

[34] V Zaldivar M L Magri S Zarate et al ldquoEstradiol increasesthe expression of TNF-120572 and TNF receptor 1 in lactotropesrdquoNeuroendocrinology vol 93 no 2 pp 106ndash113 2011

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


hypercoagulation and TNF-120572 (tumor necrosis factor-alpha)308 G gt A that is associated with apoptosis of pituitarycells due to autoimmunity Knowing the frequencies ofthese polymorphisms among SS patients will be helpful inunderstanding their roles in the etiopathogenesis of SS

2 Patients and Methods

21 Study Design Fifty-three patients who were previouslydiagnosed with SS and 43 healthy women were enrolled inthis study which was conducted between 2011 and 2013 Theblood samples of patients with SS who were followed up bythe EndocrinologyDepartment of ErciyesUniversityMedicalSchool were collected following 12 hours of fasting Thehealthy female volunteers in the control group were chosenfrom hospital staff and their relatives Written informedconsent was obtained from all participants before registeringthem for the study

In addition to the demographic information the medicalhistory and drug use of the participants were examined indetail The most important inclusion criterion for the SSgroup was having the exact diagnosis of SS Therefore it wasemphasized that all of the following criteria for diagnosiswere met (a) at least one pituitary hormone deficiency foundin basal levels or via dynamic tests if required (b) massivepostpartum uterine hemorrhage history at last delivery (c)agalactia and amenorrhea after the last delivery (d) exclusionof all other causes of pituitary deficiency (e) observationof partial or complete empty sella on magnetic resonanceimaging (MRI) Exclusion criteria for SS patients were havingcomorbidity or being on other treatments than glucocor-ticoid and thyroid hormone replacement therapies whichwere adequately performed In order to avoid the effects ofreplacement therapies of growth hormone (GH) and gonadalsteroids on genotyping studies such treatments were stopped3 months before the blood collection for the genetic analysesIn addition women in the control group did not have anyhistories of disease and were not on any drug therapies

22 Hormonal Analyses Basal levels of hormones wereanalyzed to determine hypopituitarism in all patients Inaddition the insulin tolerance test or glucagon stimulationtest was performed to identify growth hormone (GH) defi-ciency and secondary adrenal failure Basal hormone levelsincluding free T4 (normal 088ndash172 ngdL) thyroid stimu-lating hormone (TSH normal 057ndash56mIUmL) adreno-corticotropic hormone (ACTH normal 0ndash46 pgmL) corti-sol (normal 9ndash23 120583gdL) prolactin (PRL normal for post-menopausal women 24ndash298 and premenopausal women33ndash298 ngmL) follicle stimulating hormone (FSH normalfor postmenopausal women 239ndash1191 and premenopausalwomen 20ndash98mIUmL) luteinizing hormone (LH normalfor postmenopausal women 163ndash548 and premenopausalwomen 07ndash173mIUmL) estradiol (E2 normal for post-menopausal women 144ndash445 and premenopausal women189ndash2467 pgmL) and insulin-like growth factor-1 (IGF-1 reference intervals varied by age) were measured in thehormone laboratories of Erciyes University Medical School

Methods of assays and commercial kits were as followsGH immunoradiometric assay (IRMA) Immunotech SASFrance IGF-1 IRMA Immunotech SAS France ACTHIRMA Cisbio Bioassays France cortisol radioimmunoassay(RIA) Immunotech sro Czech Republic PRL TSH fT4FSH LH estradiol Immunoassay Siemens Advia centaurXP-USA

23 Genotyping Studies 2mL venous blood samples werecollected in EDTA-containing tubes for DNA analyses Totalgenomic DNA was extracted by standard methods and DNAsamples were stored at minus20∘C until the analyses of poly-morphisms Genotyping studies were conducted in ErciyesUniversity Genome and Stem Cell Center (GENKOK) Atotal of 20120583L PCR mixture with 5 120583L sample DNA wasanalyzed by using a LightCycler FastStart DNA MasterHybProbe to perform the genotypings of MTHFR C677Tand A1298C Factor II (prothrombin) G20210A and FactorV G1691A according to manufacturerrsquos instructions (RocheDiagnostics Germany) In addition Roche LightCycler 480Software was used for detecting different genotypes of thesepolymorphisms

Genotypes of the TNF-120572-308 GA and PAI-1 4G5Gpolymorphisms were detected by analyses of polymerasechain reaction (PCR) and restriction fragment length poly-morphism (RFLP) results PAI-1 4G5G polymorphisms wereanalyzed by forward primer 51015840-CACAGAGAGAGTCTG-GCCACGT-31015840 and 51015840-CCAACAGAGGACTCTTGGTCT-31015840reverse primer The genomic region of interest was amplifiedby using PCR for 30 cycles with a denaturation temperatureof 94∘C for 3min 94∘C for 30 sec 60∘C for 30 sec and 72∘Cfor 30 sec The final extension step at 72∘C was extended for1min Amplified 98 base pair (bp) products were digestedovernight with Bsl I at 55∘C and subjected to 4 agarose gelelectrophoresis [11]

The PCR procedure was performed in a total volume of50 120583L containing 5120583L genomic DNA 10x PCR buffer dNTPs(25mM) MgCl

2(15mM) Taq DNA polymerase (1 UmL)

51015840AGGCAATAGGTTTTGAGGGCCAT-31015840 forward primersand 51015840TCCTCCCTGCTCCGATTCCG-31015840 reverse primersfor TNF-120572 [12] The cycling conditions consisted of denatu-ration at 95∘C for 5min followed by 30 consecutive cyclesof 95∘C for 1min 60∘C for 1min and 72∘C for 1min with afinal elongation at 72∘C for 5min The 107 bp PCR productswere separated by electrophoresis on a 2 agarose gel andvisualized with UV illumination and ethidium bromidestaining After amplification the PCR products were digestedovernight at 37∘CwithNco I restriction enzyme and analyzedby 3 agarose gel electrophoresis

24 Statistical Analyses Statistical analyses were performedby using SPSS 150 (SPSS Inc Chicago IL) Demographicdata were presented as mean plusmn SD Binomial variables wereanalyzed using Pearsonrsquos chi-square test or Fisherrsquos exact testMoreover the odds ratios (OR) were calculated with 95confidence intervals (CI) using logistic regression analysisComparisons of genotype frequencies were made between






3 Results



4 Discussion











MTHFR C677TGenotype



















5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results



4 Discussion











MTHFR C677TGenotype



















5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















MTHFR C677TGenotype



















5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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