since 2009 , several studies with different methodologies and designs ( for example , stopwatch vs. questionnaire ) have investigated the relationship between genetic polymorphisms and premature ejaculation ( pe ) . in a stopwatch study , janssen et al .   found that in men with lifelong pe , the 5-ht - transporter - gene - linked promoter region ( 5-httlpr ) polymorphism is associated with statistically significant effects on the latency to ejaculate .
men with the ll genotype ejaculate 100% faster than do men with the ss genotype .
in addition , men with lifelong pe and with the cc genotype of the c(1019)g polymorphism of the 5-ht1a receptor also ejaculate statistically significantly faster than do men with the gc and gg genotypes .
.   also showed that men with lifelong pe and with the cys / cys genotype of the cys23ser polymorphism of the 5-ht2c receptor ejaculate statistically significantly faster than do men with the ser / ser genotype .
it is a well - established clinical fact that daily use of selective serotonin reuptake inhibitors ( ssris ) in men with lifelong pe clinically relevantly delays ejaculation .
compared with other ssris , daily treatment with 20 mg paroxetine exerts the strongest ejaculation delay . however ,
this is not always the case . in some men , daily use of ssris only moderately delays ejaculation , whereas in others there is no ejaculation delay at all .
animal studies have shown that pharmacodynamic factors , such as the amount of serotonin neurotransmission , 5-ht1a receptor sensitivity , and 5-ht2c receptor sensitivity are associated with the duration of the ejaculation latency time . in the current study in men with lifelong pe who responded to daily paroxetine 20-mg treatment , we investigated the role of the 5-httlpr polymorphism in paroxetine - induced ejaculation delay .
included were men who were actively seeking drug treatment for lifelong pe at the outpatient department of neurosexology .
none of them were using or had ever used drugs , such as ssris or clomipramine , for the treatment of lifelong pe .
the intravaginal ejaculation latency time ( ielt ) was defined as the time between the start of vaginal penetration and the start of intravaginal ejaculation .
lifelong pe was operationally defined according to the international society for sexual medicine definition as the lifelong presence of an ielt of 1 minute or less after vaginal penetration occurring on more than 90% of occasions of sexual intercourse with every sexual partner together with complaints of inability to delay ejaculation and feelings of frustration about it .
so as not to exclude men with particular psychological difficulties related to pe , a stable relationship with a female partner was not required .
however , it was required that during the ielt assesments in a 1-month baseline period and 10 weeks of paroxetine treatment , intercourse should have taken place with the same woman .
patients were not permitted the use of condoms , topical local anesthetic creams or sprays , or excessive consumption of alcohol within 5 hours before intercourse .
exclusion criteria included erectile dysfunction , alcohol or substance abuse , mental disorders , physical illnesses affecting ejaculatory functioning , concomitant medications , a history of sexual abuse reported by the patient or his partner , serious relationship problems , pregnancy of the partner , or the desire to become pregnant in the near future .
patients attended the outpatient department of neurosexology approximately 1 month before the start of daily ssri treatment ( first baseline assessment ) , on the day before treatment ( second baseline assessment ) , and at the end of two consecutive series of 5 weeks of daily ssri treatment . at the first visit ,
patients were interviewed by the last author and were asked to independently estimate their ielt .
the female partners measured the ielt and handled the stopwatch at home at every intercourse over the following 4 weeks .
patients were instructed not to have interrupted intromission or to change their usual way or frequency of intercourse . if intercourse took place more than once at the time of ielt measurement , only the first occurrence was included .
all laboratory testing , including blood sampling and genetic testing , was conducted by the first author .
informed consent was obtained from all patients after the purpose of the study was explained to them .
the study was approved by the hospital medical ethical committee and was conducted in accordance with the helsinki declaration of 1975 , as revised in 1983 .
the control group consisted of 92 physically and mentally healthy male individuals recruited in another study conducted by the department of psychiatry of the utrecht medical center , utrecht , the netherlands .
in addition , all male controls had at least 3 grandparents who were born in the netherlands .
the control group was randomly sampled and is considered representative of the general dutch population .
neither occurrence of complaints of pe nor stopwatch assessments of ielt has been investigated in the control group .
a responder was operationally defined as an individual who with daily paroxetine 20-mg treatment had a fold increase of the geometric mean ielt of 2 or more , e.g. , a more than 100% increase of the baseline ielt value .
a non - responder was defined as an individual who had a fold increase of the geometric mean ielt of less than 2 .
the cutoff of 2 was based on the outcome data of a meta - analysis of daily ssri treatment for pe , in which placebo response was consistently lower than a twofold increase of the geometric mean ielt compared with baseline values .
genomic dna was extracted from 10 ml of edta - anticoagulated whole blood by use of a standard salting - out protocol .
the 44-bp insertion / deletion polymorphism within the promoter region of the sert ( slc6a4 ) gene was amplified by polymerase chain reaction ( pcr ) .
the insertion / deletion in the sert gene - linked polymorphic region ( 5-httlpr ) was amplified by using the following oligonucleotide primers : forward , 5'-ggcgttgccgctctgaatc-3 ' , and reverse , 5'-gag ggactgagctggacaaccac-3 ' . corresponding to the nucleotide positions ranging from -1416 to -1397 and from -910 to -889 of the 5-htt gene regulatory region , a
reagents and conditions for the pcr were as follows : 1 ml of 10 polymerase buffer , 0.2 mmol
/ l mgcl2 , 0.4 mmmol / l of each primer ( biolegio bv , nijmegen , the netherlands ) , 0.5 u accuprime pfx dna polymerase ( invitrogen life technologies , strathclyde , uk ) , and 50 ng of genomic dna in a total reaction volume of 10 ml .
the pcr program on a thermal cycler ( geneamp type 9700 ; perkin elmer , waltham , ma , usa ) was as follows : reactions were cycled with initial denaturation at 94 for 4 minutes , followed by 33 pcr cycles of 94 for 30 seconds , 61 for 60 seconds , 68 for 60 seconds , and a final extension step of 4 minutes at 72. the amplification products were electrophoresed on 2%-agarose gels at 100 v for 120 minutes . the gel and running buffers were 1 tbe ( 0.89 m tris - base , 0.89 m boric acid , 20 mm na2edta )
the mean , median , and geometric mean ielt were calculated for stopwatch - determined ielts .
hardy - weinberg equilibrium was determined to check the laboratory efficacy of pcr analysis in the control group and the patient group by using a chi - square test .
allele and genotype frequencies between patients and controls were compared by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
analysis of variance ( anova ) was performed to determine an association between the genotype in the patient group and the fold increases in the ielt .
included were men who were actively seeking drug treatment for lifelong pe at the outpatient department of neurosexology .
none of them were using or had ever used drugs , such as ssris or clomipramine , for the treatment of lifelong pe .
the intravaginal ejaculation latency time ( ielt ) was defined as the time between the start of vaginal penetration and the start of intravaginal ejaculation .
lifelong pe was operationally defined according to the international society for sexual medicine definition as the lifelong presence of an ielt of 1 minute or less after vaginal penetration occurring on more than 90% of occasions of sexual intercourse with every sexual partner together with complaints of inability to delay ejaculation and feelings of frustration about it .
so as not to exclude men with particular psychological difficulties related to pe , a stable relationship with a female partner was not required .
however , it was required that during the ielt assesments in a 1-month baseline period and 10 weeks of paroxetine treatment , intercourse should have taken place with the same woman .
patients were not permitted the use of condoms , topical local anesthetic creams or sprays , or excessive consumption of alcohol within 5 hours before intercourse .
exclusion criteria included erectile dysfunction , alcohol or substance abuse , mental disorders , physical illnesses affecting ejaculatory functioning , concomitant medications , a history of sexual abuse reported by the patient or his partner , serious relationship problems , pregnancy of the partner , or the desire to become pregnant in the near future .
patients attended the outpatient department of neurosexology approximately 1 month before the start of daily ssri treatment ( first baseline assessment ) , on the day before treatment ( second baseline assessment ) , and at the end of two consecutive series of 5 weeks of daily ssri treatment . at the first visit ,
patients were interviewed by the last author and were asked to independently estimate their ielt .
the female partners measured the ielt and handled the stopwatch at home at every intercourse over the following 4 weeks .
patients were instructed not to have interrupted intromission or to change their usual way or frequency of intercourse . if intercourse took place more than once at the time of ielt measurement , only the first occurrence was included .
all laboratory testing , including blood sampling and genetic testing , was conducted by the first author .
informed consent was obtained from all patients after the purpose of the study was explained to them .
the study was approved by the hospital medical ethical committee and was conducted in accordance with the helsinki declaration of 1975 , as revised in 1983 .
the control group consisted of 92 physically and mentally healthy male individuals recruited in another study conducted by the department of psychiatry of the utrecht medical center , utrecht , the netherlands .
in addition , all male controls had at least 3 grandparents who were born in the netherlands .
the control group was randomly sampled and is considered representative of the general dutch population .
neither occurrence of complaints of pe nor stopwatch assessments of ielt has been investigated in the control group .
a responder was operationally defined as an individual who with daily paroxetine 20-mg treatment had a fold increase of the geometric mean ielt of 2 or more , e.g. , a more than 100% increase of the baseline ielt value .
a non - responder was defined as an individual who had a fold increase of the geometric mean ielt of less than 2 .
the cutoff of 2 was based on the outcome data of a meta - analysis of daily ssri treatment for pe , in which placebo response was consistently lower than a twofold increase of the geometric mean ielt compared with baseline values .
genomic dna was extracted from 10 ml of edta - anticoagulated whole blood by use of a standard salting - out protocol .
the 44-bp insertion / deletion polymorphism within the promoter region of the sert ( slc6a4 ) gene was amplified by polymerase chain reaction ( pcr ) . the insertion / deletion in the sert gene - linked polymorphic region ( 5-httlpr ) was amplified by using the following oligonucleotide primers : forward , 5'-ggcgttgccgctctgaatc-3 ' , and reverse , 5'-gag ggactgagctggacaaccac-3 ' .
corresponding to the nucleotide positions ranging from -1416 to -1397 and from -910 to -889 of the 5-htt gene regulatory region , a 484-bp or a 528-bp fragment was generated .
reagents and conditions for the pcr were as follows : 1 ml of 10 polymerase buffer , 0.2 mmol / l deoxyribonucleotide triphosphates , 2.0 mmol / l mgcl2 , 0.4 mmmol / l of each primer ( biolegio bv , nijmegen , the netherlands ) , 0.5 u accuprime pfx dna polymerase ( invitrogen life technologies , strathclyde , uk ) , and 50 ng of genomic dna in a total reaction volume of 10 ml . the pcr program on a thermal cycler ( geneamp type 9700 ; perkin elmer , waltham , ma , usa ) was as follows : reactions were cycled with initial denaturation at 94 for 4 minutes , followed by 33 pcr cycles of 94 for 30 seconds , 61 for 60 seconds , 68 for 60 seconds , and a final extension step of 4 minutes at 72. the amplification products were electrophoresed on 2%-agarose gels at 100 v for 120 minutes .
the gel and running buffers were 1 tbe ( 0.89 m tris - base , 0.89 m boric acid , 20 mm na2edta ) .
genomic dna was extracted from 10 ml of edta - anticoagulated whole blood by use of a standard salting - out protocol .
the 44-bp insertion / deletion polymorphism within the promoter region of the sert ( slc6a4 ) gene was amplified by polymerase chain reaction ( pcr ) .
the insertion / deletion in the sert gene - linked polymorphic region ( 5-httlpr ) was amplified by using the following oligonucleotide primers : forward , 5'-ggcgttgccgctctgaatc-3 ' , and reverse , 5'-gag ggactgagctggacaaccac-3 ' .
corresponding to the nucleotide positions ranging from -1416 to -1397 and from -910 to -889 of the 5-htt gene regulatory region , a 484-bp or a 528-bp fragment was generated .
reagents and conditions for the pcr were as follows : 1 ml of 10 polymerase buffer , 0.2 mmol / l deoxyribonucleotide triphosphates , 2.0 mmol / l mgcl2 , 0.4 mmmol / l of each primer ( biolegio bv , nijmegen , the netherlands ) , 0.5 u accuprime pfx dna polymerase ( invitrogen life technologies , strathclyde , uk ) , and 50 ng of genomic dna in a total reaction volume of 10 ml .
the pcr program on a thermal cycler ( geneamp type 9700 ; perkin elmer , waltham , ma , usa ) was as follows : reactions were cycled with initial denaturation at 94 for 4 minutes , followed by 33 pcr cycles of 94 for 30 seconds , 61 for 60 seconds , 68 for 60 seconds , and a final extension step of 4 minutes at 72. the amplification products were electrophoresed on 2%-agarose gels at 100 v for 120 minutes .
the gel and running buffers were 1 tbe ( 0.89 m tris - base , 0.89 m boric acid , 20 mm na2edta ) .
the mean , median , and geometric mean ielt were calculated for stopwatch - determined ielts .
hardy - weinberg equilibrium was determined to check the laboratory efficacy of pcr analysis in the control group and the patient group by using a chi - square test .
allele and genotype frequencies between patients and controls were compared by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
analysis of variance ( anova ) was performed to determine an association between the genotype in the patient group and the fold increases in the ielt .
the mean age of the controls was significantly higher than that of the patients . however ,
because lifelong pe affects all age categories , this difference did not affect the purpose of the study .
the paroxetine - induced ejaculation delay , expressed as percentage fold increase of the geometric mean ielt compared with baseline , is shown in fig .
notably , paroxetine was used only by the patients with lifelong pe and not by the controls . of
the 54 patients , 43 ( 79.6% ) responded to paroxetine treatment with an ejaculation delay , whereas 11 patients ( 20.4% ) did not respond to paroxetine treatment ; 50% , 12% , and 18% had a fold increase of 2 - 10 , 10 - 20 , or more than 20 , respectively .
a photograph of dna fragments on a gel under ultraviolet light is shown in fig .
hardy - weinberg equilibrium was not rejected for genotype distributions of the polymorphisms investigated in patients ( p=0.83 ) and controls ( p=0.59 ) .
of the 54 men with lifelong pe , 14 ( 25.9% ) had the ll genotype , 29 ( 53.7% ) had the sl genotype , and 11 ( 20.4% ) had the ss genotype .
of the 92 controls , the ll , sl , and ss genotypes were present in 27 ( 29.3% ) , 41 ( 44.6% ) , and 24 ( 26.1% ) , respectively .
treated with 20 mg paroxetine , including the 80% of men with ejaculation delay and the 20% who did not respond with an ejaculation delay , anova of the natural logarithm of the fold increase of the ielt showed no statistically significant difference in men with the ll , sl , and ss genotypes ( p=0.83 ) ( table 3 ) .
owing to the lack of genome - wide studies in men with lifelong pe , we selectively chose to investigate the 5-httlpr polymorphism because the results of in vivo animal and clinical human research with ssris indiate that the serotonin content in the neuronal synapse is involved in ejaculation delay . moreover , daily use of 20 mg paroxetine , which inhibits the reuptake of 5-ht by inhibiting the activity of the 5-ht transporter , exerts the strongest ejaculation delay of the ssris .
the difference in age between the patients and controls in the current study did not influence the comparison of men with lifelong pe and controls , because lifelong pe is present " lifelong , " that is , both at a young age and in older aged men .
why the number of married men was higher in the control group is unclear but , speculatively , may be explained by the difference in age , as younger men may be more inclined to live together with a female partner instead of being married . in the current study of a cohort of 54 men with lifelong pe it was shown that with daily treatment with 20 mg paroxetine , 80% of men responded with ejaculation delay expressed in a fold increase of the ielt of more than 2 .
about 18% of these men had a clinically very relevant ejaculation delay , as mirrored by a fold increase of 10 - 20 .
another 18% of these men had a clinically very strong ejaculation delay ( fold increase>20 ) . however
the patient and control groups were in hardy - weinberg equilibrium with regard to the 5-httlpr genotype polymorphism .
in addition , with regard to the 5-httlpr polymorphism , the patient and control groups did not differ in their genotype frequencies .
importantly , although most men responded with a clinically relevant ejaculation delay , in the current study it was found that the 5-httlpr genotype polymorphism was not associated with the paroxetine - induced ejaculation delay .
interestingly , in a previous study in 89 men with lifelong pe ( who at the moment of investigation did not use medication ) , it was found that the 5-httlpr polymorphism is associated with the duration of the ielt .
men with the ll genotype had a 100% shorter ielt than that in men with the ss or sl genotype .
in the current study , which included some of the previous group of men , the paroxetine - induced ejaculation delay was not associated with the 5-httlpr polymorphism .
it might be argued that this negative outcome was due to the low number of participants of the study .
however , note that the data of the current study did not show any tendency toward a paroxetine - induced ejaculation delay that was associated with the 5-httlpr polymorphism , as mirrored by a p - value of 0.83 . for a definite answer , a stopwatch study in a much larger cohort of men with lifelong pe and treated by daily 20-mg paroxetine is required .
notably , because various genetic polymorphisms may interact , future research of paroxetine - induced ejaculation delay in men with lifelong pe should also focus on the interaction of various genetic polymorphisms .
in the current study of 54 men with lifelong pe , 80% of men reported a clinically relevant ejaculation delay and 20% did not report any paroxetine - induced ejaculation delay .
in addition , the 5-httlpr polymorphism in these men was not associated with daily 20-mg paroxetine - induced ejaculation delay .
 purposeto investigate the association between the 5-ht - transporter - gene - linked promoter region ( 5-httlpr ) polymorphism and 20-mg paroxetine - induced ejaculation delay in men with lifelong premature ejaculation ( lpe).materials and methodsthis was a prospective study of 10 weeks of paroxetine treatment in 54 men with lpe . 
 intravaginal ejaculation latency time ( ielt ) was measured by stopwatch . 
 controls consisted of 92 caucasian men . 
 all men with lpe were genotyped for the 5-httlpr polymorphism . 
 allele frequencies and genotypes of short ( s ) and long ( l ) variants of the polymorphism were compared between patients and controls . 
 associations between the ll , sl , and ss genotypes and fold increase of mean ielt were investigated.resultsof the 54 patients , 43 ( 79.6% ) responded to 20-mg paroxetine treatment with an ejaculation delay , whereas 11 patients ( 20.4% ) did not respond ; 44% , 18% , and 18% of the patients showed a fold increase in mean ielt of 2 - 10 , 10 - 20 , and more than 20 , respectively . 
 of the 54 men , 14 ( 25.9% ) had the ll genotype , 29 ( 53.7% ) had the sl genotype , and 11 ( 20.4% ) had the ss genotype . 
 in the 92 controls , the ll , sl , and ss genotypes were present in 27 ( 29.3% ) , 41 ( 44.6% ) , and 24 ( 26.1% ) , respectively . 
 no statistically significant differences were found in 5-httlpr allelic variations or in 5-httlpr gene variations . in all men treated with 20 mg paroxetine , analysis of variance of the natural logarithm of fold increase in the ielt showed no statistically significant difference according to genotype ( p=0.83).conclusionsthe 5-httlpr polymorphism is not associated with daily 20-mg paroxetine treatment - induced ejaculation delay in men with lpe . 
INTRODUCTION
MATERIALS AND METHODS
1. Patients and assessments
2. Genotyping
1) DNA isolation
2) Polymerase chain reaction analysis
3. Statistics
RESULTS
DISCUSSION
CONCLUSIONS