
 sheehan 's syndrome ( ss ) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage .
although the onset of ss can involve acute severe panhypopituitarism in some patients , the majority of ss patients are diagnosed with a clinically subtle partial pituitary deficiency and therefore their diagnoses and treatments are delayed for many years .
the prevalence of ss is not clearly known , presumably due to the great number of nondiagnosed patients .
it is a rarely encountered disorder in developed countries due to good obstetric care [ 3 , 4 ] .
however , its prevalence is estimated to be still high in developing countries in which many deliveries take place at home .
zargar et al . estimated the prevalence of ss to be 3.1% among women in india , and about two - thirds of those women had given birth at home .
as the etiopathogenesis of ss is not clear , disorders of coagulation have been investigated in some studies .
it has been reported that disseminated intravascular coagulation ( dic ) can cause postpartum hypopituitarism [ 7 , 8 ] .
in addition , cakir et al . found protein s deficiency in 2 out of 12 patients with ss .
importantly , in another study , gokalp et al . investigated inherited hypercoagulation as a risk factor of ss .
they found that frequency of mthfr ( methylenetetrahydrofolate reductase ) c677 t and mthfr a1298c polymorphisms was significantly higher among their 38 patients with ss compared to the healthy control group .
in addition , factor ii ( g20210a ) , factor v ( g1691a ) , and pai-1 4g/5 g mutations were also more common among the ss patients , but no significant differences were observed . our aim in this study was to investigate gene polymorphisms of mthfr c677 t and a1298c , factor ii ( g20210a ) , factor v ( g1691a ) , and pai-1 ( plasminogen activator inhibitor-1 ) 4g/5 g that are associated with inherited hypercoagulation and tnf- ( tumor necrosis factor - alpha ) 308 g > a that is associated with apoptosis of pituitary cells due to autoimmunity .
knowing the frequencies of these polymorphisms among ss patients will be helpful in understanding their roles in the etiopathogenesis of ss .
fifty - three patients who were previously diagnosed with ss and 43 healthy women were enrolled in this study which was conducted between 2011 and 2013 .
the blood samples of patients with ss who were followed up by the endocrinology department of erciyes university medical school were collected following 12 hours of fasting .
the healthy female volunteers in the control group were chosen from hospital staff and their relatives .
in addition to the demographic information , the medical history and drug use of the participants were examined in detail . the most important inclusion criterion for the ss group was having the exact diagnosis of ss .
therefore , it was emphasized that all of the following criteria for diagnosis were met : ( a ) at least one pituitary hormone deficiency found in basal levels or via dynamic tests if required ; ( b ) massive postpartum uterine hemorrhage history at last delivery ; ( c ) agalactia and amenorrhea after the last delivery ; ( d ) exclusion of all other causes of pituitary deficiency ; ( e ) observation of partial or complete empty sella on magnetic resonance imaging ( mri ) .
exclusion criteria for ss patients were having comorbidity or being on other treatments than glucocorticoid and thyroid hormone replacement therapies which were adequately performed . in order to avoid the effects of replacement therapies of growth hormone ( gh ) and gonadal steroids on genotyping studies , such treatments were stopped 3 months before the blood collection for the genetic analyses .
in addition , women in the control group did not have any histories of disease and were not on any drug therapies .
in addition , the insulin tolerance test or glucagon stimulation test was performed to identify growth hormone ( gh ) deficiency and secondary adrenal failure .
basal hormone levels including free t4 ( normal : 0.881.72  ng / dl ) , thyroid stimulating hormone ( tsh ; normal : 0.575.6  miu / ml ) , adrenocorticotropic hormone ( acth ; normal : 046  pg / ml ) , cortisol ( normal : 923  g / dl ) , prolactin ( prl ; normal for postmenopausal women : 2.429.8 , and premenopausal women : 3.329.8  ng / ml ) , follicle stimulating hormone ( fsh ; normal for postmenopausal women : 23.9119.1 , and premenopausal women : 2.09.8  miu / ml ) , luteinizing hormone ( lh ; normal for postmenopausal women : 16.354.8 , and premenopausal women : 0.717.3  miu / ml ) , estradiol ( e2 ; normal for postmenopausal women : 14.444.5 , and premenopausal women : 18.9246.7  pg / ml ) , and insulin - like growth factor-1 ( igf-1 ; reference intervals varied by age ) were measured in the hormone laboratories of erciyes university medical school .
methods of assays and commercial kits were as follows : gh : immunoradiometric assay ( irma ) , immunotech sas , france ; igf-1 : irma , immunotech sas , france ; acth : irma , cisbio bioassays , france ; cortisol : radioimmunoassay ( ria ) , immunotech s.r.o , czech republic ; prl , tsh , ft4 , fsh , lh , estradiol : immunoassay , siemens advia centaur xp - usa .
2  ml venous blood samples were collected in edta - containing tubes for dna analyses .
total genomic dna was extracted by standard methods and dna samples were stored at 20c until the analyses of polymorphisms .
genotyping studies were conducted in erciyes university genome and stem cell center ( genkok ) .
a total of 20  l pcr mixture with 5  l sample dna was analyzed by using a lightcycler faststart dna master hybprobe to perform the genotypings of mthfr c677 t and a1298c , factor ii ( prothrombin ) g20210a , and factor v g1691a according to manufacturer 's instructions ( roche diagnostics , germany ) .
in addition , roche lightcycler 480 software was used for detecting different genotypes of these polymorphisms .
genotypes of the tnf--308 g / a and pai-1 4g/5 g polymorphisms were detected by analyses of polymerase chain reaction ( pcr ) and restriction fragment length polymorphism ( rflp ) results .
the genomic region of interest was amplified by using pcr for 30 cycles with a denaturation temperature of 94c for 3  min , 94c for 30  sec , 60c for 30  sec , and 72c for 30  sec .
amplified 98 base pair ( bp ) products were digested overnight with bsl i at 55c and subjected to 4% agarose gel electrophoresis .
the pcr procedure was performed in a total volume of 50  l containing 5  l genomic dna , 10x pcr buffer , dntps ( 2.5  mm ) , mgcl2 ( 1.5  mm ) , taq dna polymerase ( 1  u / ml ) , 5aggcaataggttttgagggccat-3 forward primers , and 5tcctccctgctccgattccg-3 reverse primers for tnf- . the cycling conditions consisted of denaturation at 95c for 5  min , followed by 30 consecutive cycles of 95c for 1  min , 60c for 1  min , and 72c for 1  min , with a final elongation at 72c for 5  min .
the 107  bp pcr products were separated by electrophoresis on a 2% agarose gel and visualized with uv illumination and ethidium bromide staining .
after amplification , the pcr products were digested overnight at 37c with nco i restriction enzyme and analyzed by 3% agarose gel electrophoresis .
binomial variables were analyzed using pearson 's chi - square test or fisher 's exact test .
moreover , the odds ratios ( or ) were calculated with 95% confidence intervals ( ci ) using logistic regression analysis .
comparisons of genotype frequencies were made between patients and controls , and p values less than 0.05 were considered as statistically significant .
panhypopituitarism was detected in 38 ( 71.7% ) patients with ss , while partial hypopituitarism was detected in 15 ( 28.3% ) patients .
importantly , all patients had gonadotropin and gh deficiencies , but deficiencies of acth , tsh , and prolactin were not found in all of the patients ( table 1 ) . when comparing groups , it was revealed that there was no significant difference between the mean age of the 53 cases with ss and the 43 healthy controls , 63.2  12.5 and 60.3  9.3 years , respectively .
in addition , no difference was detected in terms of mean body mass indexes ( bmis ) .
kg / m in the ss group and 29.2  3.3  kg / m in healthy women .
moreover , the ss and healthy control groups did not significantly differ according to polymorphism ratesof factor ii ( g20210a ) , factor v ( g1691a ) , mthfr ( c677 t and a1298c ) , pai-1 ( 4g/5 g ) , and tnf- ( 308 g > a ) genes , except for pai-1 ( 4g/5 g ) .
the pai-1 ( 4g/5 g ) mutation was detected in 26 ( 60.5% ) of the 43 control cases and in 20 ( 37.7% ) of the 53 ss cases .
the physiological enlargement of the pituitary gland during pregnancy plays a significant role in onset of ss , because severe bleeding does not lead to pituitary deficiency in women unless they are pregnant .
even though the pathogenesis of ss has not yet been fully clarified , the basis of its pathology has been identified as infarction and ischemic necrosis that develops due to the interruption of arterial blood flow in the anterior pituitary gland . however , the cause of the interruption in the blood flow is not clear
. considered potential mechanisms are arterial thrombosis similar to that seen in stroke , development of arterial spasm as a result of severe hypotension that is due to massive uterine bleeding , or compression of pituitary vessels due to relatively small sella turcica volume associated with enlargement of the pituitary during pregnancy .
furthermore , autoantibodies detected in many patients against the pituitary gland have been suggested as a contributing factor in the etiopathogenesis of ss . in the etiopathogenesis of ss , our study findings did not yield a significant difference between the control and ss patient groups in terms of mutations of blood composition anomalies that lead to inherited hypercoagulation .
in other words , in the ss group none of the genes had higher polymorphism rates than those in the control group .
there are numerous other acquired ( prolonged immobilization , pregnancy , oral contraceptive pills , advanced age , obesity , cigarette use , hypertension , etc . ) or genetic ( protein c or s deficiency , antithrombin - iii deficiency , platelet gpiib / iiia hpa - ib mutation , elevated levels of factors vii , viii , ix , and xii , von willebrand disease , fibrinogen , etc . ) factors that are known to cause hypercoagulation .
an important limitation of our study is that not all of the parameters associated with hypercoagulation were investigated .
therefore , it is hard to conclude , based only on our study findings , that inherited hypercoagulation is not involved in ss .
although , the mutation rates of mthfr c677 t and a1298c genes show social differences , homozygous mutations are estimated to be 10% , and heterozygous mutations are about 40% [ 16 , 17 ] .
approximately , 15% of the population is reported to carry both heterozygous genetic mutations together , due to their close relations .
although the rates of mthfr gene polymorphisms were not higher in the ss group than in the control group in our study , more important issues are whether the ss patients had vitamin b6 , vitamin b12 , and/or folate deficiency and thus a disorder in mthfr gene expression during their last pregnancies .
the heterozygous mutations in factor ii ( prothrombin ) g20210a are encountered at a 2 - 3% rate among caucasians
. plasma factor - ii levels ( prothrombin ) increase as a result of mutations . in that case ,
venous thrombosis risk among individuals carrying this mutation is increased by 2 - 3 times .
one study reported that coronary artery risk is increased by 1.31 times in individuals with prothrombin g20210a mutation .
while varying across different populations , the incidence rate of factor v leiden heterozygous mutation is approximately 5% , but its homozygous mutation is rare . as a result of the mutation in the factor v gene ( g1691a ) , activated
this , in turn , results in a deterioration of the bleeding and coagulation balance in favor of coagulation .
this mutation is reported to increase thrombosis risk fivefold when heterozygous , and by 1080-fold when homozygous [ 21 , 22 ] .
factor v leiden has also been shown to increase complications such as miscarriage , preeclampsia , and abruptio placentae by a minimum of 2 - 3 times . 

plasminogen activator inhibitor-1 ( pai-1 ) inhibits tissue plasminogen activator ( tpa ) and urokinase which are proteins leading to plasminogen activation and fibrinolysis .
the estimated frequences of pai-1 4g/4 g , 4g/5 g and 5g/5 g are , respectively , about 35% , 50% , and 15% [ 23 , 24 ] .
polymorphisms of pai-1 4g/4 g and 4g/5 g are reported to cause an increase in pai-1 levels and thus risk of arterial thrombosis rather than of venous thrombosis [ 25 , 26 ] .
pregnant women who carry the homozygous pai-1 4g/4 g mutation are also known to have an increased risk of miscarriage . however , there are other studies reporting that patients with ischemic stroke and coronary artery disease do not have increased pai-1 4g/5 g polymorphism [ 28 , 29 ] .
interestingly , our results showed that the 4g/5 g polymorphism rate was 60.5% among healthy women , while it was 37.7% among ss patients .
this finding may suggest that the 4g/5 g polymorphism does not always lead to thrombophilia , unless accompanied by some acquired factors .
tnf- ( tumor necrosis factor - alpha ) is a cytokine that plays an important role in the regulations of immune functions , cell proliferation and differentiation , apoptosis due to autoimmunity , coagulation , adipocyte , lipid and glucose metabolism .
the 308 g / a polymorphism of tnf- which leads to elevated tnf- levels is known to have an important impact on the development of autoimmunity diseases , metabolic syndromes , cancers , and psychiatric disorders [ 30 , 31 ] .
autoimmunity is a potential risk factor in the development of ss and 35% of patients have been shown to have anti - pituitary autoantibodies .
these antibodies are presumably generated in reaction to the necrotic pituitary gland tissue found after ischemic infarction and seem to be responsible for the chronic but progressive course of ss . on another note , increased estrogen levels during pregnancy make adenohypophysis cells , particularly the lactotroph cells , more vulnerable to apoptosis due to autoimmunity as a result of elevated expression of the tnf- gene in rats .
this condition may also be associated with the vulnerability of an enlarged pituitary to ischemic necrosis at the end of pregnancy .
estrogen levels gradually increase during pregnancy and peak close to completion of the full term . in this regard
, we hypothesized that autoimmunity caused by tnf- 308 g / a mutation can be a contributing factor in the etiopathogenesis of ss .
however , no increase in the rate of tnf- 308 g / a polymorphism was observed among the ss patients when compared to the control group .
as demonstrated in our study , the disorders suggested for ss etiopathogenesis and the associations among them are highly complicated .
however , the results of our study showed no increase in factor ii , factor v , mthfr , pai-1 , and tnf- gene polymorphism rates among ss patients compared to the control group .
hence , genetic factors other than these gene polymorphisms should be researched in the etiopathogenesis of ss .
clarifying ss etiopathogenesis via further studies will be particularly beneficial in identifying women susceptible to ss prior to disease development .
 sheehan 's syndrome ( ss ) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage . herein , we aimed to investigate the roles of factor ii ( g20210a ) , factor v ( g1691a ) , mthfr ( c677 t and a1298c ) , pai-1 4g/5 g , and tnf- ( 308  g > a ) gene polymorphisms in the etiopathogenesis of ss . 
 venous blood samples were obtained from 53 cases with ss and 43 healthy women . 
 standard methods were used to extract the genomic dnas . 
 factor ii ( g20210a ) , factor v ( g1691a ) , and mthfr ( c677 t and a1298c ) polymorphisms were identified by real - time pcr . 
 pai-1 4g/5 g and tnf- ( 308  g > a ) gene polymorphisms were detected with polymerase chain reaction ( pcr ) and restriction fragment length polymorphism ( rflp ) methods . according to statistical analysis , none of the polymorphisms were found to be significantly higher in the ss group compared to the control group . 
 hence , we suggest that genetic factors other than factor ii , factor v , mthfr , pai-1 , and tnf- gene polymorphisms should be researched in the etiopathogenesis of ss . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion
5. Conclusion