Research Article Relationship between Testicular Volume...

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 145792 6 pageshttpdxdoiorg1011552013145792

Research ArticleRelationship between Testicular Volume and Conventional orNonconventional Sperm Parameters

Rosita Condorelli Aldo E Calogero and Sandro La Vignera

Section of Endocrinology Andrology and Internal Medicine Department of Medical and Pediatric Sciences University of CataniaPoliclinico ldquoG Rodolicordquo Via S Sofia 78 Building 4 Room 2C19 95123 Catania Italy

Correspondence should be addressed to Sandro La Vignera sandrolavigneraunictit

Received 8 June 2013 Accepted 2 August 2013

Academic Editor Malgorzata Kotula-Balak

Copyright copy 2013 Rosita Condorelli et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Reduced testicular volume (TV) (lt12 cm3) is associated with lower testicular function Several studies exploredthe conventional sperm parameters (concentration motility and morphology) and the endocrine function (gonadotropins andtestosterone serum concentrations) in the patients with reduction of TV No other parameters have been examinedAimThis studyaims at evaluating some biofunctional sperm parameters by flow cytometry in the semen of men with reduced TV compared withthat of subjects with normal TVMethods 78 patients without primary scrotal disease were submitted to ultrasound evaluation ofthe testisThey were divided into two groups according to testicular volume A Group including 40 patients with normal testicularvolume (TV gt 15 cm3) and B Group including 38 patients with reduced testicular volume (TV le 12 cm3) All patients underwentserumhormone concentration conventional and biofunctional (flow cytometry) spermparameters evaluationResultsWith regardto biofunctional sperm parameters all values (mitochondrial membrane potential phosphatidylserine externalization chromatincompactness and DNA fragmentation) were strongly negatively correlated with testicular volume (119875 lt 00001) Conclusions Thisstudy for the first time in the literature states that the biofunctional sperm parameters worsen and with near linear correlation withdecreasing testicular volume

1 Introduction

The testis is an oval-shaped organ that in adult men weighsabout 20 g which has an average volume (TV) of 186 plusmn48 cm3 and dimensions ranging from 36 to 55 cm in lengthand from 21 to 35 cm in width Testis consists of twostructurally distinct compartments the interstitial compart-ment and the seminiferous tubules containing germ andSertoli cells These two compartments are responsible for thetwo main testicular functions production of androgens andspermatozoa respectively [1] About 400ndash600 seminiferoustubules are found within the testis they are 70ndash80 cm longhave a diameter of 012-013mm and represent about 80ndash90of the total TV

In clinical practice the TV can be evaluated by physicalexamination using the Praderrsquos orchidometer but with agreater precision by testicular ultrasound which overcomesconfounding factors such as scrotal effusions varicose veinsintrascrotal masses testicular pain and distinction from

adjacent anatomical structures [2] It is well known that asignificant TV decrease is associated with a worst testicularperformance of both endocrine (androgen decline) andreproductive activities (abnormal sperm parameters) [3ndash6]A number of studies have explored the relationship betweenconventional sperm parameters (concentration motility andmorphology) or the endocrine function (testosterone andgonadotropin serum concentrations) and TV [7ndash9] No otherparameters have been taken into account Therefore the aimof this study was to evaluate nonconventional sperm parame-ters (mitochondrial function apoptosis and chromatinDNAintegrity) in men with reduced TV These parameters werecompared with those evaluated in men with normal TV

2 Materials and Methods

21 Patient Selection We retrospectively reviewed theclinical and laboratory (hormonal conventional and

2 International Journal of Endocrinology

nonconventional flow cytometry sperm parameters) chartsof men who requested didymo-epididymal ultrasound scanfor any reason from December 2011 to November 2012Patients having a condition known to interfere with spermparameters were excluded from analysis These included thefollowing

(i) primary scrotal disease identified by ultrasoundevaluation varicocele hydrocele testicular focalinjuryinjuries microlithiasis inhomogeneous tes-ticular echotexture and abnormal testicular arterialhemodynamic (systolic peak velocity and resistanceindex)

(ii) history of cryptorchidism varicocelectomy eversionof tunica vaginalis head injury endocrine abnor-malities (hypogonadism hyperprolactinemia andincreased estrogen concentration) systemic diseases(kidney disease liver disease [10] and diabetes mel-litus [11]) cigarette smoking [12] alcohol use [13]concomitant use of other drugs during the pre-vious 6 months leukocytospermia male accessorygland inflammatory disease [14] and positive seminalandor urine culture andor urethral swab

Applying these exclusion criteria 78 patients wereincluded in this studyThey had amean age of 250plusmn 50 years(range 23ndash45 years) and an average BMI of 250 plusmn 30 kgm2(range 20ndash30 kgm2) The testicular size was considerednormal when the mean TV ranged between 15mL and 25mL[15 16] whereas it was considered low when TV was lt12mLPatients with a mean TV in a grey zone (12ndash149mL) werearbitrarily excluded from computation [6 17 18]

22 Scrotal Ultrasound Evaluation Scrotal ultrasound scanswere carried out in two phases the first with the patient insupine position (with penis resting on suprapubic region) andthe second in upright position to evaluate blood reflux alongthe pampiniform plexus testicular malposition or extentof any fluid collections Examination was conducted witha GX Megas Esaote (Esaote SpA Genoa Italy) instrumentequipped with linear high-resolution and high-frequency(75ndash14MHz) probe dedicated to the study of soft parts andwith color Doppler for detecting slow flows and scanningsurface of at least 5 cm

The TV was calculated automatically by the ultrasonog-raphy software by applying the ellipsoid formula (length timeswidth times thickness times 052) Parenchymal echostructure wasconsidered normal in presence of thin densely packed andhomogeneously deployed echoes Presence of finely inhomo-geneous echopattern weakly hypo- or hyperechogenic areaswas considered expression of primary testicular disease

During Doppler evaluation flow was detected at thelevel of spermatic artery testicular artery and its branchesVelocitogram was considered normal in presence of lowresistance prolonged systolic phase flow maintained duringthe entire diastole and low resistance index (IR 062)

23HormonalMeasurements Hormonal evaluationwas per-formed by electrochemiluminescence with Hitachi-Rocheequipment (Cobas 6000 Roche Diagnostics IndianapolisIN USA) Blood sampling was performed at 800 am after atleast 8 hours of sleep Determination of serum LH and pro-lactin was repeated at a distance of 30minutes Normal valueswere LH = 16ndash90mIUmLminus1 FSH = 20ndash120mIUmLminus1total testosterone = 28ndash80 ngmLminus1 17120573-estradiol = 80ndash430 pgmLminus1 and prolactin = 40ndash150 ngmLminus1

24 Sperm Analysis Semen samples were collected by mas-turbation in a sterile container after 2ndash7 days of sexualabstinence and were transported to the laboratory within 30minutes from ejaculation Sperm analysis was made accord-ing to the WHO criteria [19] The following parameters wereevaluated color volume liquefaction time pH density totalcount progressive motility morphology and leukocytes

25 Evaluation of Nonconventional Sperm Parameters by FlowCytometry Analysis was performed using an EPICS XL FlowCytometer (Coulter Electronics IL Italy) equipped with anargon laser at 488 nm and three detectors of fluorescencegreen (FL-1 at 525 nm) orange (FL-2 to 575 nm) and red (FL-3 at 620 nm) For each sample 100000 events were countedat a low flow velocity and analysed by the software SystemII Version 30The following nonconventional sperm param-eters were evaluated (a) sperm mitochondrial membranepotential (MMP) (b) phosphatidylserine externalization (PS)(early apoptosis index) following stainingwith annexinV andPI (c) degree of chromatin compactness after staining withpropidium iodide (PI) and (d) spermDNA fragmentation byTUNEL assay

251 Mitochondrial Membrane Potential Evaluation Analiquot containing 1 times 106 spermatozoa was incubated withJC-1 (551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazolyl-carbocyanine iodide) (Space Import Export Milan Italy) inthe dark for 10minutes at a temperature of 37∘C At the end ofthe incubation period cells werewashed in PBS and analysed

252 Phosphatidylserine Externalization Staining withannexin V and PI was performed using Annexin V-FITC kit(Sigma Chemical Perth Australia) An aliquot containing05 times 106 spermatozoa was suspended in 05mL of buffercontaining 10 120583L of annexin V-FITC plus 20120583L of PI andincubated for 10min in the dark After incubation thesamples were immediately analysed Signals were detectedby FL-1 (FITC) and FL-3 (PI) detectors Cell populationswere identified in a plot forward (FSC) versus side scatter(SSC) Different staining patterns enabled the identificationof different cell populations FITC negative and PI negativeindicated vital cells FITC positive and PI negative indicatedapoptotic cells and FITC positive and PI positive indicatedcells in late apoptosis phase or necrosis

253 Evaluation of Chromatin Compactness PI staining wasperformed after cell membrane permeabilisation to allowfluorochrome penetration at the nuclear level Briefly 1 times 106

International Journal of Endocrinology 3

spermatozoa were incubated with LPR DNA-Prep Reagentcontaining 01 cyanide potassium 01 NaN

3 nonionic

detergents salts and stabilizers (Beckman Coulter ILMilanItaly) in the dark at room temperature for 10 minutes andthen incubated with Stein DNA-Prep Reagent containing50120583gmL of PI (lt05) A RNAse (4KUnitzmL) lt01NaN3(Beckman Coulter IL Milan Italy) in the dark at room

temperature Flow cytometric analysiswas performed after 30minutes In this analysis FL3 detector was the only used

254 DNA Fragmentation Analysis Sperm DNA fragmen-tation was evaluated by TUNEL assay using a commerciallyavailable kit (Mebstain Apoptosis Beckman Coulter ILMilan Italy) To obtain the negative control TdTwas omittedfrom the mixing reaction Positive control was obtained bypretreating spermatozoa with 1mgmL of I deoxyribonu-clease not containing A RNAse at 37∘C for 60min beforestaining Debris was removed using the same procedureLight scattering and fluorescence datawere obtained at a fixedsetting in logarithmic scale FITC-labeled spermatozoa weremeasured using the flow cytometer FL-1 detector

The protocol was approved by the Institutional ReviewBoard and an informed written consent was obtained fromeach patient and controls

26 Statistical Analysis Results were reported as mean plusmnSEM The data were analysed by Student 119905-test The Pearsoncorrelation coefficient was used to assess the possible covari-ance linearity between TV and the laboratory parametersevaluated Statistical analysis was performed using SPSS 90for Windows (SPSS Inc Chicago IL USA) A 119875 value lowerthan 005 was accepted as statistically significant

3 Results

Altogether the patients had amean TV of 152 plusmn 17mL Fortypatients (513) had a normal TV whereas the remaining 38(487) had low TV The conventional sperm parameters ofthe patients enrolled in this study are shown in Table 1 ThepH of the seminal fluid did not vary significantly in patientswith low TV compared to those with normal TV Thesepatients with low TV had a statistically significant lowerseminal fluid volume sperm concentration and percentageof progressive motility and spermatozoa with normal formcompared with men with normal TV (119875 lt 005 Studentrsquos119905-test) Finally the percentage of immature germ cells wassignificantly higher in patients with low TV compared topatients with normal TV (119875 lt 005 Studentrsquos 119905-test) Thehormonal serum concentrations are reported in Table 2 LHFSH and prolactin levels were similar between patients withnormal or low TV These latter had significantly higher 17120573-oestradiol levels and lower total testosterone concentrationcompared to patients with normal TV (Studentrsquos 119905-test)

Nonconventional sperm parameters are reported in Fig-ure 1 The percentage of spermatozoa with low MMPabnormal chromatin compactness PS externalization andfragmented DNA was significantly higher in patients with

Table 1 Conventional sperm parameters of men with normal (119899 =40) or low (119899 = 38) testicular volume

ParameterTesticular volume

Normal(15ndash25mL)

Low(lt12mL)

Volume (mL) 30 plusmn 05 23 plusmn 04lowast

pH 76 plusmn 12 77 plusmn 13

Sperm concentration (millionmL) 423 plusmn 67 287 plusmn 47lowast

Progressive motility () 368 plusmn 58 269 plusmn 44lowast

Normal forms () 130 plusmn 21 54 plusmn 09lowast

Immature germ cells () 35 plusmn 06 96 plusmn 16lowast

lowast119875 lt 005 (Studentrsquos 119905-test)

0

5

10

15

20

25

30

Altered MMP Altered PS ext

Altered chrom compac

Altered DNA frag

Normal testicular volumeLow testicular volume

lowastlowast

lowast

lowast

lowastP lt 005 versus normal testicular volume

Sper

mat

ozoa

()

Figure 1 Percentage of spermatozoa with low mitochondrialmembrane potential (MMP) phosphatidylserine externalization(PS ext) degree of chromatin compactness (chrom compac) orDNA fragmentation (DNA frag) in patients with normal (119899 = 40)or low (119899 = 38) testicular volume

low TV compared to patients with normal TV (119875 lt 005Studentrsquos test)

The correlation analysis showed a significant directcorrelation between TV and seminal volume sperm con-centration progressive motility and percentage of normalforms (Table 3) Statistically significant negative correla-tions were observed between TV and the percentage ofimmature germ cells and gonadotropin or 17120573-oestradiolserum concentrations (Table 3) No correlation was foundbetween TV and seminal pH total testosterone or prolactinserum concentration (Table 3) A statistically significantnegative correlation was found between TV and low MMPphosphatidylserine externalization chromatin compactnessor DNA fragmentation (Figure 2)

4 Discussion

This study explored the relationship between TV and con-ventional sperm parameters serum hormonal concentra-tions and using flow cytometry nonconventional spermparameters To better understand the impact of TV onthese parameters the patients were selected excluding allcases of scrotal disease detected by ultrasound and excluding


0

1

2

3

4

5

6

7

8

9

10

5 7 9 11 13 15 17 19 21 23 25

Testicular volume (mL)

Mito

chon

dria

l mem

bran

epo

tent

ial (

r = minus055 P lt 0001

)

(a)

Phos

phat

idyl

serin

eex

tern

aliz

atio

n ()


5 7 9 11 13 15 17 19 21 23 25


14

12

10

8

6

4

2

0

(b)

024681012141618202224

r = ndash076 P lt 0001

5 7 9 11 13 15 17 19 21 23 25


Chro

mat

in co

mpa

ctne

ss (

)

(c)

5 7 9 11 13 15 17 19 21 23 25


0

2

4

6

8

10

12

14

16


DN

A fr

agm

enta

tion

()

(d)

Figure 2 Correlation between mean testicular volume and percentage of spermatozoa with low mitochondrial membrane potential (a)phosphatidylserine externalization (b) degree of chromatin compactness (c) or DNA fragmentation (d) (119899 = 78)

Table 2 Serum hormone concentrations in men with normal (119899 =40) or low (119899 = 38) testicular volume


Normal(150ndash250mL)

Low(lt120mL)

LH (mUImLminus1) 48 plusmn 08 50 plusmn 08

FSH (mUImLminus1) 44 plusmn 07 51 plusmn 08

Total testosterone (ngmLminus1) 61 plusmn 10 52 plusmn 08lowast

17120573-oestradiol (pgmLminus1) 256 plusmn 41 358 plusmn 58lowast

Prolactin (ngmLminus1) 169 plusmn 27 175 plusmn 28


patients in whom the clinical history brought out factorsthat directly (cryptorchidism previous surgical trauma inthe scrotal region male accessory glands inflammatorydisease leukocytospermia positive seminal andor urineculture andor urethral swab) andor indirectly (head traumasystemic diseases drugs cigarette smoking and alcohol use)affect testicular function andor laboratory signs (endocrinediseases) that reflect testicular functional alterations Wefound that patients with reduced TV have lower seminalfluid volume sperm concentration percentage of progressivemotility and percentage of spermatozoa with normal formcompared to men with normal TV Conversely these patientshave an increased percentage of immature germ cells Thesedata suggest that patients with reduced TV (lt12 cm3) showin agreement with other observations [5 6 20] poorer

Table 3 Correlation between testicular volume and conventionalsperm parameters or serum hormone levels (119899 = 78)

Parameter 119903 119875 valueConventional sperm parameters

Seminal fluid volume (mL) 119903 = 047 lt005Seminal fluid pH 119903 = minus011 NSSperm concentration (millionmL) 119903 = minus042 lt005Spermatozoa with progressive motility () 119903 = 043 lt005Spermatozoa with normal morphology () 119903 = 082 lt005Immature germ cells () 119903 = minus064 lt005

Serum hormone concentrationsSerum LH (mUImLminus1) 119903 = minus031 NSSerum FSH (mUImLminus1) 119903 = minus037 NSSerum total testosterone (ngmLminus1) 119903 = 028 lt005Serum oestradiol (pgmLminus1) 119903 = minus035 lt005Serum prolactin (ngmLminus1) 119903 = 002 NS

NS not significant

sperm quality It is noteworthy that these patients showedsigns of tubular stress pattern as suggested by the increasedpercentage of immature germ elements in their ejaculate

In terms of hormonal concentrations we found thatpatients with low TV have increased 17120573-estradiol serumlevels and lower concentrations of total testosterone Nostatistically significant differences were detected in the con-centrations of gonadotropins and prolactin between patients


with normal and low TV These findings emphasize thata TV reduction may be a result of spermatogenic or hor-monal impairment [21 22] and not only on the quality ofsemen [5 6 20] It is known that the endocrine testicularfunction has important implications on metabolism [23]cardiovascular profile [24] and bone metabolism [25] Inpresence of low androgen concentrations peripheral insulinsensitivity is reduced [26] blood vessels aremore predisposedto atherosclerosis [27] and finally osteoporosis is more com-mon in patients with impaired testicular function Recentfindings have shown that the Leydig cell plays an importantrole in vitamin D metabolism [28]

At last to our knowledge for the first time this studyinvestigated the relationship between TV and nonconven-tional sperm parametersWe found that patients with low TVhave a lower number of spermatozoa with lowmitochondrialmembrane potential early signs of sperm apoptosis (PSexternalization) and an abnormal sperm chromatinDNAintegrity

Only one similar study was carried out by Zorn andcolleagues in particular the authors evaluated phospholipidasymmetry mitochondrial membrane potential and DNAdenaturation in 142 males of infertile couples showing thatDNA denaturation correlated negatively with TV normalmitochondrialmembrane potential correlated positively withTV and finally normal viable sperm correlated positivelywith TV [29] However the study does not examine theultrasound appearance of the testis and does not identify aclear thresholds for TV estimated by ultrasound

The dilemma on the causes responsible for TV reductionapparently unjustified on a clinical ground remains openbecause of the absence of testicular disease identified byscrotal ultrasonography In this regard it is worthmentioningthat some alterations of testicular morphostructural charac-teristicsmay be undetectable by physical andor instrumentalexamination In particular chronic orchitis is difficult to clin-ically diagnose because of its asymptomatic course howeverthe histological examination ofmany infertile patients detectslymphocytic peritubular infiltrate of inflammatory meaningin absence of relevant scrotal pathology associated with TVand sperm count reduction [30 31]

In conclusion the results of this study indicate thatpatients with a reduced TV (lt12mL) in the absence oftesticular disease show poorer conventional and noncon-ventional sperm parameters The patients enrolled in thisstudy did not show any frank endocrine alteration but totaltestosterone serum concentration was lower in men withreduced TV This suggests that men with low TV needto be monitored overtime for possible onset of an overtendocrine dysfunction Finally further studies are needed toclarify the relationship between TV reduction volume andultrastructural alterations of the testicular parenchyma inpatients with low TV and without evidence of disease at theclinical and ultrasound examinations

References

[1] B P Setchell and D E Brooks ldquoAnatomy vasculature innerva-tion and fluids of themale reproductive tractrdquo inThePhysiology

of Reproduction E Knobil and J D Neill Eds pp 753ndash836Raven Press New York NY USA 1988

[2] B Mirochnik P Bhargava M K Dighe and N Kanth ldquoUltra-sound evaluation of scrotal pathologyrdquo Radiologic Clinics ofNorth America vol 50 no 2 pp 317ndash332 2012

[3] H Takihara M J Cosentino J Sakatoku and A T K CockettldquoSignificance of testicular size measurement in andrology IICorrelation of testicular size with testicular functionrdquo Journalof Urology vol 137 no 3 pp 416ndash419 1987

[4] L Bujan R Mieusset A Mansat J P Moatti C Mondinatand F Pontonnier ldquoTesticular size in infertile men relationshipto semen characteristics and hormonal blood levelsrdquo BritishJournal of Urology vol 64 no 6 pp 632ndash637 1989

[5] H Sakamoto T YajimaM Nagata T Okumura K Suzuki andY Ogawa ldquoRelationship between testicular size by ultrasonog-raphy and testicular functionmeasurement of testicular lengthwidth and depth in patients with infertilityrdquo InternationalJournal of Urology vol 15 no 6 pp 529ndash533 2008

[6] H Sakamoto Y Ogawa andH Yoshida ldquoRelationship betweentesticular volume and testicular function comparison of thePrader orchidometric and ultrasonographic measurements inpatients with infertilityrdquo Asian Journal of Andrology vol 10 no2 pp 319ndash324 2008

[7] A Aribarg W Kenkeerati V Vorapaiboonsak et al ldquoTesticularvolume semen profile and serum hormone levels in fertileThaimalesrdquo International Journal of Andrology vol 9 no 3 pp 170ndash180 1986

[8] M Sigman and J P Jarow ldquoIpsilateral testicular hypotrophy isassociated with decreased sperm counts in infertile men withvaricocelesrdquo Journal of Urology vol 158 no 2 pp 605ndash607 1997

[9] T Arai S Kitahara S Horiuchi S Sumi and K YoshidaldquoRelationship of testicular volume to semen profiles and serumhormone concentrations in infertile Japanese malesrdquo Interna-tional Journal of Fertility and Womenrsquos Medicine vol 43 no 1pp 40ndash47 1998

[10] S La Vignera R A Condorelli E Vicari R DrsquoAgata and A ECalogero ldquoSpermDNA damage in patients with chronic viral Chepatitisrdquo European Journal of Internal Medicine vol 23 no 1pp e19ndashe24 2012

[11] S La Vignera R Condorelli E Vicari R DrsquoAgata and A ECalogero ldquoDiabetes mellitus and minireview sperm parame-tersrdquo Journal of Andrology vol 33 no 2 pp 145ndash153 2012

[12] A Calogero R Polosa A Perdichizzi et al ldquoCigarette smokeextract immobilizes human spermatozoa and induces spermapoptosisrdquo Reproductive BioMedicine Online vol 19 no 4 pp564ndash571 2009

[13] J Pajarinen P J Karhunen V Savolainen K Lalu A Penttilaand P Laippala ldquoModerate alcohol consumption and disordersof human spermatogenesisrdquo Alcoholism vol 20 no 2 pp 332ndash337 1996

[14] S La Vignera E Vicari R A Condorelli R DrsquoAgata andA E Calogero ldquoMale accessory gland infection and spermparameters (review)rdquo International Journal of Andrology vol34 no 5 part 2 pp 330ndash347 2011

[15] A Pilatz A Rusz F Wagenlehner W Weidner and BAltinkilic ldquoReference values for testicular volume epididymalhead size and peak systolic velocity of the testicular artery inadult males measured by ultrasonographyrdquo Ultraschall in derMedizin vol 34 no 4 pp 349ndash354 2012

[16] C Foresta AGarolla A C Frigo et al ldquoAnthropometric penileand testis measures in post-pubertal Italian malesrdquo Journal ofEndocrinological Investigation vol 36 no 5 pp 287ndash292 2012


[17] V S Dogra R H Gottlieb M Oka and D J RubensldquoSonography of the scrotumrdquo Radiology vol 227 no 1 pp 18ndash36 2003

[18] I Mihmanli and F Kantarci ldquoSonography of scrotal abnor-malities in adults an updaterdquo Diagnostic and InterventionalRadiology vol 15 no 1 pp 64ndash73 2009

[19] World Health Organization WHO Laboratory Manual Forthe Examination and Processing of Human Semen CambridgeUniversity Press Cambridge UK 5th edition 2010

[20] M Schurich F Aigner F Frauscher and L Pallwein ldquoThe roleof ultrasound in assessment of male fertilityrdquo European Journalof Obstetrics amp Gynecology and Reproductive Biology vol 144supplement 1 pp S192ndashS198 2009

[21] M Grossmann ldquoLow testosterone in men with type 2 diabetessignificance and treatmentrdquo Journal of Clinical Endocrinologyand Metabolism vol 96 no 8 pp 2341ndash2353 2011

[22] A M Traish F Saad R J Feeley and A Guay ldquoThe dark sideof testosterone deficiency III Cardiovascular diseaserdquo Journalof Andrology vol 30 no 5 pp 477ndash494 2009

[23] R F Spark ldquoTestosterone diabetes mellitus and the metabolicsyndromerdquo Current Urology Reports vol 8 no 6 pp 467ndash4712007

[24] G Corona G Rastrelli L Vignozzi E Mannucci and MMaggi ldquoTestosterone cardiovascular disease and the metabolicsyndromerdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 25 no 2 pp 337ndash353 2011

[25] S P Tuck andRM Francis ldquoTestosterone bone and osteoporo-sisrdquo Frontiers of Hormone Research vol 37 pp 123ndash132 2009

[26] M Zitzmann ldquoTestosterone deficiency insulin resistance andthe metabolic syndromerdquoNature Reviews Endocrinology vol 5no 12 pp 673ndash681 2009

[27] M D Schwarcz and W H Frishman ldquoTestosterone and coro-nary artery diseaserdquoCardiology in Review vol 18 no 5 pp 251ndash257 2010

[28] C Foresta R Selice A Di Mambro and G StrapazzonldquoTesticulopathy and vitamin D insufficiencyrdquo The Lancet vol377 no 9769 p 904 2011

[29] B Zorn B Golob A Ihan A Kopitar andM Kolbezen ldquoApop-totic sperm biomarkers and their correlation with conventionalsperm parameters and male fertility potentialrdquo Journal ofAssisted Reproduction and Genetics vol 29 no 4 pp 357ndash3642012

[30] H C Schuppe A Meinhardt J P Allam M Bergmann WWeidner and G Haidl ldquoChronic orchitis a neglected cause ofmale infertilityrdquo Andrologia vol 40 no 2 pp 84ndash91 2008

[31] J Suominen and K O Soderstrom ldquoLymphocyte infiltration inhuman testicular biopsiesrdquo International Journal of Andrologyvol 5 no 5 pp 461ndash466 1982

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


nonconventional flow cytometry sperm parameters) chartsof men who requested didymo-epididymal ultrasound scanfor any reason from December 2011 to November 2012Patients having a condition known to interfere with spermparameters were excluded from analysis These included thefollowing

(i) primary scrotal disease identified by ultrasoundevaluation varicocele hydrocele testicular focalinjuryinjuries microlithiasis inhomogeneous tes-ticular echotexture and abnormal testicular arterialhemodynamic (systolic peak velocity and resistanceindex)

(ii) history of cryptorchidism varicocelectomy eversionof tunica vaginalis head injury endocrine abnor-malities (hypogonadism hyperprolactinemia andincreased estrogen concentration) systemic diseases(kidney disease liver disease [10] and diabetes mel-litus [11]) cigarette smoking [12] alcohol use [13]concomitant use of other drugs during the pre-vious 6 months leukocytospermia male accessorygland inflammatory disease [14] and positive seminalandor urine culture andor urethral swab

Applying these exclusion criteria 78 patients wereincluded in this studyThey had amean age of 250plusmn 50 years(range 23ndash45 years) and an average BMI of 250 plusmn 30 kgm2(range 20ndash30 kgm2) The testicular size was considerednormal when the mean TV ranged between 15mL and 25mL[15 16] whereas it was considered low when TV was lt12mLPatients with a mean TV in a grey zone (12ndash149mL) werearbitrarily excluded from computation [6 17 18]

22 Scrotal Ultrasound Evaluation Scrotal ultrasound scanswere carried out in two phases the first with the patient insupine position (with penis resting on suprapubic region) andthe second in upright position to evaluate blood reflux alongthe pampiniform plexus testicular malposition or extentof any fluid collections Examination was conducted witha GX Megas Esaote (Esaote SpA Genoa Italy) instrumentequipped with linear high-resolution and high-frequency(75ndash14MHz) probe dedicated to the study of soft parts andwith color Doppler for detecting slow flows and scanningsurface of at least 5 cm

The TV was calculated automatically by the ultrasonog-raphy software by applying the ellipsoid formula (length timeswidth times thickness times 052) Parenchymal echostructure wasconsidered normal in presence of thin densely packed andhomogeneously deployed echoes Presence of finely inhomo-geneous echopattern weakly hypo- or hyperechogenic areaswas considered expression of primary testicular disease

During Doppler evaluation flow was detected at thelevel of spermatic artery testicular artery and its branchesVelocitogram was considered normal in presence of lowresistance prolonged systolic phase flow maintained duringthe entire diastole and low resistance index (IR 062)

23HormonalMeasurements Hormonal evaluationwas per-formed by electrochemiluminescence with Hitachi-Rocheequipment (Cobas 6000 Roche Diagnostics IndianapolisIN USA) Blood sampling was performed at 800 am after atleast 8 hours of sleep Determination of serum LH and pro-lactin was repeated at a distance of 30minutes Normal valueswere LH = 16ndash90mIUmLminus1 FSH = 20ndash120mIUmLminus1total testosterone = 28ndash80 ngmLminus1 17120573-estradiol = 80ndash430 pgmLminus1 and prolactin = 40ndash150 ngmLminus1

24 Sperm Analysis Semen samples were collected by mas-turbation in a sterile container after 2ndash7 days of sexualabstinence and were transported to the laboratory within 30minutes from ejaculation Sperm analysis was made accord-ing to the WHO criteria [19] The following parameters wereevaluated color volume liquefaction time pH density totalcount progressive motility morphology and leukocytes

25 Evaluation of Nonconventional Sperm Parameters by FlowCytometry Analysis was performed using an EPICS XL FlowCytometer (Coulter Electronics IL Italy) equipped with anargon laser at 488 nm and three detectors of fluorescencegreen (FL-1 at 525 nm) orange (FL-2 to 575 nm) and red (FL-3 at 620 nm) For each sample 100000 events were countedat a low flow velocity and analysed by the software SystemII Version 30The following nonconventional sperm param-eters were evaluated (a) sperm mitochondrial membranepotential (MMP) (b) phosphatidylserine externalization (PS)(early apoptosis index) following stainingwith annexinV andPI (c) degree of chromatin compactness after staining withpropidium iodide (PI) and (d) spermDNA fragmentation byTUNEL assay

251 Mitochondrial Membrane Potential Evaluation Analiquot containing 1 times 106 spermatozoa was incubated withJC-1 (551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazolyl-carbocyanine iodide) (Space Import Export Milan Italy) inthe dark for 10minutes at a temperature of 37∘C At the end ofthe incubation period cells werewashed in PBS and analysed

252 Phosphatidylserine Externalization Staining withannexin V and PI was performed using Annexin V-FITC kit(Sigma Chemical Perth Australia) An aliquot containing05 times 106 spermatozoa was suspended in 05mL of buffercontaining 10 120583L of annexin V-FITC plus 20120583L of PI andincubated for 10min in the dark After incubation thesamples were immediately analysed Signals were detectedby FL-1 (FITC) and FL-3 (PI) detectors Cell populationswere identified in a plot forward (FSC) versus side scatter(SSC) Different staining patterns enabled the identificationof different cell populations FITC negative and PI negativeindicated vital cells FITC positive and PI negative indicatedapoptotic cells and FITC positive and PI positive indicatedcells in late apoptosis phase or necrosis

253 Evaluation of Chromatin Compactness PI staining wasperformed after cell membrane permeabilisation to allowfluorochrome penetration at the nuclear level Briefly 1 times 106



3 nonionic






3 Results





Normal(15ndash25mL)

Low(lt12mL)








0

5

10

15

20

25

30



Altered DNA frag


lowastlowast

lowast

lowast


Sper

mat

ozoa

()




4 Discussion



0

1

2

3

4

5

6

7

8

9

10

5 7 9 11 13 15 17 19 21 23 25


Mito

chon

dria

l mem

bran

epo

tent

ial (


)

(a)

Phos

phat

idyl

serin

eex

tern

aliz

atio

n ()


5 7 9 11 13 15 17 19 21 23 25


14

12

10

8

6

4

2

0

(b)

024681012141618202224


5 7 9 11 13 15 17 19 21 23 25


Chro

mat

in co

mpa

ctne

ss (

)

(c)

5 7 9 11 13 15 17 19 21 23 25


0

2

4

6

8

10

12

14

16


DN

A fr

agm

enta

tion

()

(d)





Low(lt120mL)












NS not significant









References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 nonionic






3 Results





Normal(15ndash25mL)

Low(lt12mL)








0

5

10

15

20

25

30



Altered DNA frag


lowastlowast

lowast

lowast


Sper

mat

ozoa

()




4 Discussion



0

1

2

3

4

5

6

7

8

9

10

5 7 9 11 13 15 17 19 21 23 25


Mito

chon

dria

l mem

bran

epo

tent

ial (


)

(a)

Phos

phat

idyl

serin

eex

tern

aliz

atio

n ()


5 7 9 11 13 15 17 19 21 23 25


14

12

10

8

6

4

2

0

(b)

024681012141618202224


5 7 9 11 13 15 17 19 21 23 25


Chro

mat

in co

mpa

ctne

ss (

)

(c)

5 7 9 11 13 15 17 19 21 23 25


0

2

4

6

8

10

12

14

16


DN

A fr

agm

enta

tion

()

(d)





Low(lt120mL)












NS not significant









References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

8

9

10

5 7 9 11 13 15 17 19 21 23 25


Mito

chon

dria

l mem

bran

epo

tent

ial (


)

(a)

Phos

phat

idyl

serin

eex

tern

aliz

atio

n ()


5 7 9 11 13 15 17 19 21 23 25


14

12

10

8

6

4

2

0

(b)

024681012141618202224


5 7 9 11 13 15 17 19 21 23 25


Chro

mat

in co

mpa

ctne

ss (

)

(c)

5 7 9 11 13 15 17 19 21 23 25


0

2

4

6

8

10

12

14

16


DN

A fr

agm

enta

tion

()

(d)





Low(lt120mL)












NS not significant









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 Relationship between Testicular Volume...

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