Download - Clinical Study Reduced Seminal Concentration of CD45pos ...Clinical Study Reduced Seminal Concentration of CD45pos Cells after Follicle-Stimulating Hormone Treatment in Selected Patients

Clinical StudyReduced Seminal Concentration of CD45pos Cells afterFollicle-Stimulating Hormone Treatment in Selected Patientswith Idiopathic Oligoasthenoteratozoospermia

Rosita A Condorelli1 Aldo E Calogero1 Enzo Vicari1 Laura Mongioirsquo1 Giovanni Burgio1

Rossella Cannarella1 Filippo Giacone1 Linda Iacoviello1

Giuseppe Morgia2 Vincenzo Favilla2 Sebastiano Cimino2 and Sandro La Vignera1

1 Department of Medical and Pediatric Sciences Section of Endocrinology Andrology and Internal Medicine University of CataniaPoliclinico ldquoG Rodolicordquo Building 4 Rm 2C18 Via S Sofia 78 95123 Catania Italy

2 Department of Urology University of Catania Catania Italy

Correspondence should be addressed to Sandro La Vignera sandrolavigneraunictit

Received 11 September 2013 Revised 20 November 2013 Accepted 20 November 2013 Published 14 January 2014

Academic Editor Muhammad Shahab

Copyright copy 2014 Rosita A 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

The present study evaluated the conventional sperm parameters and the seminal concentration of CD45pos cells (pan-leukocytemarker) of infertile patients with idiopathic oligoasthenoteratozoospermia (OAT) The patients were arbitrarily divided into threegroups treated with recombinant follicle-stimulating hormone FSH 120572 (Group A = 20 patients) recombinant FSH-120573 (Group B = 20patients) and highly purified human FSH (Group C = 14 patients) All treated groups achieved a similar improvement of the mainsperm parameters (density progressive motility and morphology) but only the increase in the percentage of spermatozoa withnormal morphology was significant compared to the baseline in all three examined groups Moreover all groups had a significantreduction of the seminal concentration of CD45pos cells and of the percentage of immature germ cells Before and after thetreatment the concentration of CD45pos cells showed a positive linear correlation with the percentage of immature germ cellsand a negative correlation with the percentage of spermatozoa with regular morphology These results demonstrate that treatmentwith FSH is effective in patients with idiopathic OAT and that there are no significant differences between the different preparationsThe novelty of this study is in the significant reduction of the concentration of CD45pos cells observed after the treatment

1 Introduction

Follicle-stimulating hormone has an important role in testic-ular function In particular its main biological actions (medi-ated through a receptor systempresent on the Sertoli cells) arethe following cellular sperm differentiation modulation ofspermatid morphogenesis and the maturation of epididymalspermatozoa [1ndash10]

The biological actions of FSH are more relevant in theclinical condition of hypogonadotropic hypogonadism [511ndash16] and are more controversial in functional hypogo-nadism andor in patients with idiopathic oligoasthenoter-atozoospermia (OAT) [17ndash27] Functional hypogonadism isdefined as a clinical condition in which the gonadotropin

levels are in the low-normal range but the testosteronelevel is inadequate (or often in the low-normal range) [28]Idiopathic OATmeans that no etiological factor can be foundby common clinical instrumental or laboratory methods[29]

The demonstrated biological effects of FSH treatment inmale infertile patients are increased spermatogonial popu-lation and sperm count [17ndash21] increased rate of fertiliza-tion and increased pregnancy rate in programs of assistedfertilization [22ndash24] However several studies show a lack ofeffects of FSH on sperm parameters [25ndash27]

The Italian Medicines Agency authorizes the use ofpreparations of FSH for the treatment of male infertility inmales with hypogonadism and in infertile males with low or

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 372060 8 pageshttpdxdoiorg1011552014372060

2 International Journal of Endocrinology

normal levels of FSH but not more than 8mIUmL in anycase

There are however lesser known aspects relating to thepossible usefulness of FSH therapy with FSH on uncon-ventional as well as conventional sperm parameters (den-sity morphology and motility) For example FSH therapycould induce modifications that could restore the qualityof the inflammatory response of the semen Under partic-ular conditions the leukocyte concentration in semen isdirectly correlated with immature germ cell concentrationsand the percentage of abnormal forms [30] The defects ofspermatogenesis represent a potential target of hormonaltreatment as well as a potential cause of increased inflam-matory response in particular the response of leukocytesthe main mediators of this phenomenon Together withtheir classic antimicrobial action leukocytes also removeimmature germ cells andor sperm with maturational defects[31ndash33] suggesting they have a protective role in semen atconcentrations of lt1 millionmL [34] However the completecharacterization of the leukocytes requires the identificationof the specific populations (polymorphonuclear granulo-cytes macrophages and lymphocytes) [35 36] that reflectmore accurately the possible chronicization of the inflamma-tory process (in particular the increase in lymphocytes) Thefailure to characterize specific populations of leukocytes mayexplain the frequent underestimation of the inflammatoryprocess in the semen of patients with idiopathicOAT becauseonly polymorphonuclear granulocytes are reported in rou-tine practice [37] Finally in clinical practice leukospermiaand bacteriospermia frequently are not associated [33] andoften the clinical andrologist must reduce the negative effectson spermatozoa caused by the mere presence of leukocyteswhich are not improved only with anti-inflammatory treat-ment [38] From the technical point of view flow cytometry isable to discriminate (with the use of monoclonal antibodies)the different subpopulations of leukocytes [39 40]

In this context the aims of this study were the following

(a) To evaluate the quality of the conventional spermparameters (density morphology and progressivemotility) in infertile patients with idiopathic OATafter treatment with three different preparations offollicle-stimulating hormone recombinant FSH-120572and -120573 (Gonal F Puregon) and highly purified humanFSH (Fostimon)

(b) To evaluate in these patients the semen concentrationof CD45pos cells (a pan-leukocytemarker) beforeand after hormonal treatment with one of the threedifferent hormonal preparations

2 Materials and Methods

We retrospectively reviewed patient clinical instrumentaland laboratory data obtained from June 2012 to June 2013All patients enrolled in this study were referred to theDepartment of Andrology and Endocrinology of CataniaUniversity Italy for the diagnosis and treatment of maleinfertility Overall the data of 316 patients were analyzedThemen were 18ndash40 years old with a mean age of 260 plusmn 80

years and a bodymass index (BMI) ranging between 180 and270 kgm2 (mean BMI 220 plusmn 30 kgm2)

21 Exclusion Criteria

(1) Primary Scrotal Disease Varicocele hydrocele testi-cular focal injuryinjuries testicular microlithiasis inhomo-geneous testicular echotexture (ultrasound examination)and abnormal hemodynamic parameters of the testis (alteredvalues of systolic peak velocity and resistance index) viaultrasound examination

(2) Other Criteria Genetic disorders (altered karyotypeandor the presence of Y chromosome microdeletions)clinical history of cryptorchidism varicocelectomy eversionof the tunica vaginalis head trauma endocrine abnormalities(increased gonadotropins reduced serum total testosterone(lt28 ngmLminus1) hyperprolactinemia and increased estro-gen concentrations) systemic diseases (kidney disease liverdisease and diabetes mellitus) cigarette smoking alcoholuse concomitant use of other drugs during the previous6 months leukocytospermia (leukocyte concentration insemen gt1millionmL (detected with the peroxidase test))male accessory gland inflammatory disease positive seminalurine andor urethral swab cultures and ultrasound signs ofepididymal obstruction

Applying these exclusion criteria we analyzed data from54 patients with an age range of 18ndash33 years (mean 240 plusmn 60years) and a BMI of 190ndash260 kgm2 (mean 230plusmn40 kgm2)

The protocol was approved by the internal InstitutionalReview Board and informed written consent was obtainedfrom each participant

22 Scrotal Ultrasound Evaluation The scrotal ultrasoundevaluation was performed in two phases the first with thepatient in a supine position (with the penis resting on thesuprapubic region) and the second in an upright position toevaluate reflux along the pampiniform plexus testicular paintesticular malposition and the extent of any fluid collectionThe examination was performed with a GX Megas Esaote(Esaote SpAmdashGenova (Italy)) device equipped with linearhigh-resolution and high-frequency (75 to 14MHz) probesdedicated to the study of soft body areas with color Dopplerfor detecting slow flow and a scanning surface of at least5 cm Testicular volume was calculated automatically by theultrasound machine using the ellipsoid formula (length timeswidth times thickness times 052) The testis was considered normalin size when it had a volume between 15 and 25 cm3 low-normal when it had a volume between 10 and 12 cm3 andhypotrophic when it had a volume of less than 10 cm3 [41 42]The parenchymal echostructure was considered normal inthe presence of thin densely packed and homogeneouslydeployed echoes The presence of a finely inhomogeneousechopattern and weakly hypo- or hyperechogenic areas wasconsidered indicative of primary testicular disease Duringthe Doppler evaluation flow was detected at the level ofthe spermatic artery and the testicular artery and theirbranches The velocity analysis was considered normal in

International Journal of Endocrinology 3

the presence of low resistance a prolonged systolic phaseflow maintenance during diastole and a low resistance index(IR 062) Systolic flow speed along the centripetal arterieswas considered normal if it was lower than 15 cmsec andorbetween 4 and 12 cmsec [41ndash43]

23 Hormone Measurements Hormonal evaluations wereperformedby electrochemiluminescencewithHitachi-Rocheequipment (Cobas 6000 Roche Diagnostics IndianapolisIN USA) The reference intervals were as follows LH = 16ndash90mIUmLminus1 FSH = 20ndash120mIUmLminus1 17120573-estradiol =80ndash430 pgmLminus1 total testosterone = 28ndash80 ngmLminus1 andprolactin = 40ndash150 ngmLminus1 Blood sampling was performedat 800 am after at least 8 hours of sleep The determinationof serum LH and prolactin was repeated after an interval of30 minutes

24 Sperm Analysis Semen samples were collected by mas-turbation into a sterile container after 2ndash7 days of sexualabstinence and were transported to the laboratory within30 minutes after ejaculation According to the 2010 WHOguidelines each sample was evaluated for seminal volumepH sperm count progressive motility morphology andround cell concentration [37] For all patients the spermanalysis was repeated after 4 months at the end of thehormonal treatment

25 Conventional Measurement of Seminal Leukocytes andImmature Germ Cells

251 Seminal Leukocytes (Peroxidase Test) The protocolused was adapted from that of Endtz [44] The working solu-tion used for the test was obtained by adding 1 120583L of H

2O2to

20120583L of a 009 331015840-diaminobenzidine tetrahydrochloridestock solution (DAB ISOPAC Sigma Milan Italy) in 40ethanol In each assay 20120583L of semen was incubated with20120583L of working solution in an Eppendorf tube for 5minutesat room temperature Before setting up the slide 40 120583L ofPBS was added Peroxidase-positive cells were marked byyellow-brown-red staining while peroxidase-negative cellsremained colorless At least 100 round cells were countedusing an optical microscope at 400x magnification and thepercentages of peroxidase-positive and -negative cells wereevaluated The total leukocyte count is expressed in millionsper milliliter of semen

252 Immature Germ Cell Evaluation in Semen Spermatidswere differentiated from leukocytes by a seminal fluid smearstained using the Papanicolaou technique [45] Spermatidswere identified on the basis of the following parame-ters coloration size core shape and size (approximately5 120583M) absence of intracellular peroxidase and the absenceof leukocyte-specific antigen (see Section 26) Morpholog-ically multinucleated spermatids were distinguished frompolymorphonuclear leukocytes by the presence of a pinkcolor in contrast to the bluish color of polymorphonuclearleukocytes

26 Cytofluorimetric Assessment Theanalysis was conductedwith an EPICS XL Flow Cytometer (Coulter Electronics ILUSA) equipped with an argon laser at 488 nm and threefluorescence detectors green (FL-1 at 525 nm) orange (FL-2 to 575 nm) and red (FL-3 at 620 nm) For each sample100000 events were measured at a low flow velocity andanalyzed using System II version 30

261 Flow-Cytometric Analysis of CD45pos Cells To performthe absolute lymphocyte count 100 120583L of each liquefiedsemen sample was incubated with a mixture containingSyto-16 green fluorescent nucleic acid stain to identifythe spermatozoa and exclude debris (final concentration200 nM) (Molecular Probes Eugene Oregon USA) 7-amino-actinomycin D (7-AAD Via-Probe BD PharmingenSan Diego CA USA) to assess viability anti-CD45-APC(pan-leukocyte antigen) to recognize white blood cells andanti-CD16-PE to PMN recognize The addition of 100 120583Lof Flow-Count Fluorospheres (Beckmann-Coulter FullertonCA USA) at 1034 beadsmL allowed us to determine theabsolute lymphocyte count by flow cytometry After incuba-tion in the dark for 20 minutes at room temperature 1mLof PBS was added and the sample was analyzed by flowcytometry (FACSCalibur Becton Dickinson San Jose CAUSA) For each test 100000 events were acquired The totalnumber of leukocytes permilliliterwas calculated by applyingthe following formula [39]

monoclonal antibody minus (positive cells times no of spermatozoamL)100

(1)

27 Different Hormonal Treatments Analyzed Sperm param-eters of three different groups (arbitrarily assigned) beforeand after the treatment with different preparations of follicle-stimulating hormone were evaluated

(i) Group A (20 patients) treated with recombinantFSH-120572 (Gonal F Merck Serono Europe)

(ii) Group B (119899 = 20 patients) treated with recombinantFSH-120573 (Puregon Organon)

(iii) Group C (14 patients) treated with highly purifiedhuman FSH (Fostimon IBSA)

All patients were treated for 4 months (according to theperiod of treatment admitted from the current ministerialnote (note 74)) with the following scheme of therapy150 units 3 times a week (Monday Wednesday and Fri-day) always injected subcutaneously No patient receivedcombination therapy with chorionic gonadotropin becauseno patient had a low concentration of total testosterone(exclusion criteria)

28 Statistical Analysis The results are reported as themean plusmn SEM and percentage The data were analyzed using aone-way analysis of variance (ANOVA) followed by Duncanrsquosmultiple range test A correlation analysis was conductedby evaluating the Pearson correlation coefficient to assesspossible covariance linearity between CD45pos cells and


the other examined parameters Statistical analyses wereperformed using SPSS 90 for Windows (SPSS Inc ChicagoIL USA) A119875 value less than 005 was accepted as statisticallysignificant

3 Result

The three examined groups did not show statistically sig-nificant difference at baseline in the evaluated physicalparameters (age body mass index and testicular volume) orhormonal parameters (FSH LH total testosterone estradioland prolactin) (Table 1)

The correlation analysis conducted on all patients beforeand after the treatment showed the presence of a positivelinear correlation between the concentration of CD45poscells in the semen and the percentage of spermatids and a neg-ative correlation between the concentration of CD45pos cellsand the percentage of spermatozoa with normal morphology(Table 2)

The three examined groups did not show statistically sig-nificant differences at baseline in the evaluated sperm param-eters (density morphology progressive motility percentageof spermatids leukocyte concentration and CD45pos cellconcentrations) (Table 3)

After hormonal treatment we did not detect a statisticallysignificant difference between the examined groups in anyof the investigated sperm parameters However within eachgroup the following sperm parameters showed statisticallysignificant differences (119875 lt 005) compared to the baselinepercentage of spermatozoa with normal morphology per-centage of spermatids and concentration of CD45pos cells(Table 3)

Table 4 shows the percentage distribution of patientsaccording to the different concentrations of CD45pos cellsbefore and after therapy

Finally the mean testicular volume in the three examinedgroups did not change significantly from before to aftertherapy (Group A = 160 plusmn 70 versus 162 plusmn 74 cm3 GroupB = 155 plusmn 85 versus 158 plusmn 80 cm3 Group C = 162 plusmn 66versus 163 plusmn 82 cm3)

4 Discussion

The results of this study show that a group of selected patientswith idiopathic OAT had a significant improvement of thepercentage of spermatozoa with regular morphology afterpharmacological treatment with FSH for 4 months Thepatients treated with different formulations of FSH (recom-binant FSH-120572 and -120573 and recombinant purified human FSH)after therapy showed similar improvements of this parameterIn the three examined groups we observed a significantreduction of the seminal concentration of CD45pos cells andof the percentage of immature germ cells (spermatids) afterthe pharmacological treatmentThe seminal concentration ofCD45pos cells before and after treatment showed a positivelinear correlation with the percentage of spermatids and anegative correlation with the percentage of spermatozoa withnormal morphology On the basis of these results we think

Table 1 Physical and hormonal characteristics at baseline of thethree examined groups

ParametersGroup A(119899 = 20patients)

Group B(119899 = 20patients)

Group C(119899 = 14patients)

Physical parametersAge (years) 250 plusmn 40 230 plusmn 30 235 plusmn 20BMI (kgm2) 235 plusmn 30 244 plusmn 25 236 plusmn 20Testicular volume (cm3) 160 plusmn 70 155 plusmn 85 162 plusmn 66

Hormonal parametersFSH (mIUmLminus1) 26 plusmn 11 28 plusmn 18 31 plusmn 46LH (mIUmLminus1) 45 plusmn 33 52 plusmn 27 41 plusmn 22Testosterone (ngmLminus1) 61 plusmn 51 64 plusmn 47 59 plusmn 42Estradiol (pgmLminus1) 146 plusmn 120 184 plusmn 137 169 plusmn 78Prolactin (ngmLminus1) 46 plusmn 47 89 plusmn 57 67 plusmn 42

further studies should examine the frequency of increasedconcentration of seminal CD45pos cells in infertile patientswith idiopathic OAT because this parameter is not routinelyassessed in practice [37]

Other studies have examined the quality of conventionalsperm parameters after pharmacological treatment with FSHin patients with idiopathic infertility with discordant results[46]

In the placebo-controlled study of Kamischke and col-leagues [26] 34 patients were treated with 150 IU rh-FSH onalternate days for 3 months and 33 patients received placebo(30mg saccharose) After the treatment they observed asignificant increase of testicular volume in patients receivingFSH but they did not find any significant modification ofconventional spermparameters Caroppo and colleagues [20]confirmed the increase of testicular volume in patients treatedwith FSH Other studies in accordance with Kamischke andcolleagues observed an absence of significant effects of thistreatment on the conventional sperm parameters [22 27 47ndash50]

Significant increases in sperm concentration motilityand morphology were reported in oligoasthenozoospermicpatients receiving high p-FSH (150 IU p-FSH every day for3 months) but not low p-FSH (150 IU p-FSH on alternatedays for 3 months) in another study conducted by Iaconoand colleagues [17] Similarly Paradisi et al [51] showedthat oligoasthenozoospermic patients benefitted from theadministration of a high dosage of rh-FSH (300 IU onalternate days for 4 months) Their results agreed with otherstudies reporting the ability of this hormone to quantitativelyimprove spermatogenesis in mammals [52 53]

Foresta and colleagues found after 3months of treatmentwith hp-FSH (75 IU on alternate days) increased spermato-gonial cell population The authors subdivided the cohort ofpatients into responders (normal serum FSH and isolatedhypospermatogenesis) and nonresponders (hypospermato-genesis associated with abnormal spermatogenetic matu-ration) [19 54] Two recent studies confirmed that theadministration of either rh-FSH (150 IU on alternate days for3 months) or hp-FSH (150 IU on alternate days for 3 months)


Table 2 Correlation analysis between semen concentrations of CD45pos cells and other sperm and hormonal parameters before and aftertreatment (119899 = 54 patients)

Parameter CD45pos cells (before treatment) CD45pos cells (after treatment)Semen volume (mL) 119903 = 045 119875 = NS 119903 = 040 119875 = NSSemen pH 119903 = 054 119875 = NS 119903 = 050 119875 = NSTotal sperm count (million) 119903 = minus058 119875 = NS 119903 = minus054 119875 = NSSperm density (millionmL) 119903 = minus047 119875 = NS 119903 = minus042 119875 = NSSpermatozoa with normal morphology () 119903 = minus075 119875 lt 005 119903 = minus073 119875 lt 005

Spermatozoa with progressive motility () 119903 = minus063 119875 = NS 119903 = minus060 119875 = NSImmature germ elements () 119903 = 088 119875 lt 005 119903 = 085 119875 lt 005

Peroxidase-positive leukocytes (millionmL) 119903 = 045 119875 = NS 119903 = 043 119875 = NSSerum FSH (mIUmLminus1) 119903 = 052 119875 = NS 119903 = 051 119875 = NSSerum LH (mIUmLminus1) 119903 = 047 119875 = NS 119903 = 044 119875 = NSSerum total testosterone (ngmLminus1) 119903 = minus063 119875 = NS 119903 = minus060 119875 = NSSerum estradiol (pgmLminus1) 119903 = 059 119875 = NS 119903 = 055 119875 = NSSerum prolactin (ngmLminus1) 119903 = 040 119875 = NS 119903 = 030 119875 = NS

Table 3 Sperm parameters of the three examined groups before andafter the hormonal treatment




Sperm parameters before treatmentDensity (milmL) 66 plusmn 33 84 plusmn 57 77 plusmn 42Normal forms () 22 plusmn 23 26 plusmn 13 19 plusmn 12Progressive motility () 110 plusmn 60 140 plusmn 40 100 plusmn 70Spermatids () 120 plusmn 60 140 plusmn 40 110 plusmn 40Leukocytes (milmL) 04 plusmn 03 06 plusmn 03 03 plusmn 02CD45pos cells (milmL) 18 plusmn 12 15 plusmn 14 17 plusmn 13

Sperm parameters after treatmentDensity (milmL) 88 plusmn 42 108 plusmn 46 90 plusmn 30Normal forms () 42 plusmn 28lowast 50 plusmn 17lowast 36 plusmn 16lowast

Progressive motility () 140 plusmn 80 190 plusmn 30 150 plusmn 60Spermatids () 40 plusmn 20lowast 50 plusmn 30lowast 30 plusmn 20lowast

Leukocytes (milmL) 02 plusmn 02 04 plusmn 02 02 plusmn 03CD45pos cells (milmL) 05 plusmn 02lowast 04 plusmn 03lowast 04 plusmn 02lowastlowast119875 lt 005 versus baseline

significantly improved conventional sperm parameters [5556]

To our knowledge this is the first study that hasdemonstrated a significant reduction in the concentrationof CD45pos cells in the semen of infertile patients withidiopathic OAT How should this result be interpreted

Leukocytes are detectable in the male reproductive tractand in the seminal fluid [57] under physiological condi-tions they represent approximately 5 of the round cellsin these locations [58] The three subtypes of leukocytes arepresent in different quantities in the ejaculate Particularlypolymorphonuclear granulocytes represent 50ndash60 of theleukocytes macrophages represent 20ndash30 and T lympho-cytes represent approximatively 5 [35 59] According to

Table 4 The percentage distribution of patients according to thedifferent concentrations of CD45pos cells before and after therapy

CD45pos cellsGroup A(119899 = 20patients)



Before treatment01ndash05milmL 2 (10) 2 (10) 1 (7)05ndash10milmL 3 (15) 3 (15) 2 (14)10ndash15milmL 3 (15) 5 (25) 2 (14)15ndash20milmL 8 (40)lowast 7 (35)lowast 7 (50)lowast

20ndash25milmL 2 (10) 2 (10) 1 (7)25ndash30milmL 2 (10) 1 (5) 1 (7)

After treatment01ndash05milmL 14 (70)lowast 15 (75)lowast 9 (64)lowast

05ndash10milmL 2 (10) 1 (5) 1 (7)10ndash15milmL 1 (5) 1 (5) 1 (7)15ndash20milmL 1 (5) 1 (5) 1 (7)20ndash25milmL 1 (5) 1 (5) 1 (7)25ndash30milmL 1 (5) 1 (5) 1 (7)lowast119875 lt 005 versus other concentrations

the WHO a concentration of leukocytes in the ejaculateexceeding 106mL as assessed by the peroxidase test definesa condition known as leukocytospermia

The clinical significance of this condition in the patho-genesis of male infertility is still controversial [30 60 61]In fact leukocytospermia is frequently not associated witha microbiologically provable genitourinary infection [33]In these cases it has been suggested that white blood cellsoriginate from the epididymis and appear to have favor-able effects on semen quality with an important role inimmunosurveillance and in phagocytosis of morphologicallyabnormal spermatozoa [31 62 63] or apoptosis [39]

In the present study the patients were carefully selectedIn particular the exclusion criteria included the absence of


leukocytospermia the absence of MAGI and the absence ofurogenital infection detected through bacterial culture Thesame patients after hormonal treatment showed a significantreduction in the seminal concentration of CD45pos cells anda significant improvement of conventional spermparametersMoreover before the treatment the seminal concentrationof CD45pos cells was correlated with the percentage ofspermatids and negatively correlated with the percentage ofspermatozoa with normal morphology

Most likely the maintenance of a high concentrationof CD45pos cells (there are poor data in the literatureconcerning the threshold value but Politch and colleaguesin 1993 suggested a threshold value of 2 times 106WBCmL[40]) is initially protective for spermatozoa due to theirsuggested action of immunosurveillance for thematurationaldefects of the spermatozoa However no clinical studieshave demonstrated whether the chronicization of this clinicalmodel maintains these characteristics or is associated withthe worsening of the sperm parameters The results of thisstudy suggest the latter hypothesis but these data shouldbe confirmed by further studies that evaluate other mark-ers of inflammation andor oxidative stress in the semenof infertile patients with high concentrations of CD45poscells

Theoretically according to the data provided by trans-mission electron microscopy the proportions of the mainconstituents of the nonsperm cellular components are thefollowing germinal elements (84) (anucleate bodies =43 spermatids = 222 cellular masses with anucleateorganelles and spermatocytes = 18) leukocytes (13)(neutrophils = 12 macrophages = 09 lymphocytes =01) epithelial cells (23) and Sertoli cells (07)[59]

The prevalence of leukocytospermia in semen of infertilepatients is between 10 and 20 [33 35] while the presenceof lymphocytes is detectable up to 20 [57] Frequentlythe concentration of leukocytes detected in the ejaculateis significantly higher with the immunocytological methodthan the traditional technique [40]

In most cases chlamydia infections represent the mainstimulus for the persistence of the inflammatory processin the urogenital tract [35] However bacteriospermia andleukocytospermia are not statistically associated with eachother and the presence of bacteriospermia alone is associatedwith sperm damage but the presence of only leukocytesis also associated with deterioration of sperm parameters[33]

Leukocytes in addition to their antimicrobial role par-ticipate in the response to defects of sperm maturation Inparticular the concentration of CD45pos cells is reducedin semen samples that have defects in the morphology ofthe head of gt50 of spermatozoa [31 58] Additionallyfragments of spermatozoa have been detected inside of cellswith phagocytic activity [31] Finally semen samples withhigh concentrations of white blood cells contain a higherfrequency of sperm with ideal morphology compared withsamples with a high number of immature germ cells and alow concentration of leukocytes [63]

5 Limitations of the Study

This study has limitations that may explain some seem-ingly unfavorable results such as the nonsignificant increaseof sperm density andor testicular volume after therapyThese limitations include the lack of data concerning thepolymorphism of the FSH receptor a factor known to beassociated with different individual responses to therapythe first evaluation of sperm parameters immediately afterthe end of therapy and the lack of a control group Futurestudies should examine these factors with a larger number ofpatients

In conclusion the results of this study suggest thattreatment with FSH has significant positive effects on spermmorphology in patients with idiopathic OAT The threepreparations seem to have similar effects on spermmorphol-ogyThemost novel finding is the reduction of CD45pos cellsin the semen of infertile males Future studies should clarifythe importance of this diagnostic parameter in patients withidiopathic OAT and the significance of this result in partic-ular the relationships between CD45pos cells and spermatidsand spermmorphology before and after hormonal treatment

Conflict of Interests

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

References

[1] E Steinberg ldquoHormonal control of mammalian spermiogene-sisrdquo Physiological Reviews vol 5 pp 1ndash72 1971

[2] J L Courtens and M Courot ldquoAcrosomal and nuclear mor-phogenesis in ram spermatids an experimental study ofhypophysectomized and testosterone-supplemented animalsrdquoAnatomical Record vol 197 no 2 pp 143ndash152 1980

[3] A M Matsumoto A E Karpas C A Paulsen and W JBremner ldquoReinitiation of sperm production in gonadotropin-suppressed normal men by administration of follicle-stimu-lating hormonerdquo Journal of Clinical Investigation vol 72 no 3pp 1005ndash1015 1983

[4] A M Matsumoto C A Paulsen and W J Bremner ldquoStim-ulation of sperm production by human luteinizing hormonein gonadotropin-suppressed normal menrdquo Journal of ClinicalEndocrinology and Metabolism vol 59 no 5 pp 882ndash887 1984

[5] A M Matsumoto A E Karpas and W J Bremner ldquoChronichuman chorionic gonadotropin administration in normal menevidence that follicle-stimulating hormone is necessary for themaintenance of quantitatively normal spermatogenesis inmanrdquoJournal of Clinical Endocrinology andMetabolism vol 62 no 6pp 1184ndash1192 1986

[6] M M A van Alphen H J G van de Kant and D G de RooijldquoFollicle-stimulating hormone stimulates spermatogenesis inthe adult monkeyrdquo Endocrinology vol 123 no 3 pp 1449ndash14551988

[7] R M Sharpe ldquoFollicle-stimulating hormone and spermatogen-esis in the adult malerdquo Journal of Endocrinology vol 121 no 3pp 405ndash407 1989

[8] J S Tapanainen K Aittomaki J Min T Vaskivuo and I THuhtaniemi ldquoMen homozygous for an inactivating mutation of


the Follicle-Stimulating Hormone (FSH) receptor gene presentvariable suppression of spermatogenesis and fertilityrdquo NatureGenetics vol 15 no 2 pp 205ndash206 1997

[9] E Nieschlag M Simoni J Gromoll and G FWeinbauer ldquoRoleof FSH in the regulation of spermatogenesis clinical aspectsrdquoClinical Endocrinology vol 51 no 2 pp 139ndash146 1999

[10] M Simoni G FWeinbauer J Gromoll and E Nieschlag ldquoRoleof FSH inmale gonadal functionrdquoAnnales drsquoEndocrinologie vol60 no 2 pp 102ndash106 1999

[11] A M Matsumoto ldquoHormonal therapy of male hypogonadismrdquoEndocrinology andMetabolism Clinics of North America vol 23no 4 pp 857ndash875 1994

[12] R W Whitcomb and W F Crowley Jr ldquoClinical review 4diagnosis and treatment of isolated gonadotropin-releasinghormone deficiency in menrdquo Journal of Clinical Endocrinologyand Metabolism vol 70 no 1 pp 3ndash7 1990

[13] I Mastrogiacomo R G Motta S Botteon G Bonanni and MSchiesaro ldquoAchievement of spermatogenesis and genital tractmaturation in hypogonadotropic hypogonadic subjects duringlong term treatment with gonadotropins or LHRHrdquoAndrologiavol 23 no 4 pp 285ndash289 1991

[14] European Metrodin HP Study Group ldquoEfficacy and safetyof highly purified urinary folliclestimulating hormone withchorionic gonadotropin for treating men with isolated hypog-onadotropic hypogonadismrdquo Fertility and Sterility vol 70 pp256ndash262 1998

[15] A Isidori M Latini and F Romanelli ldquoTreatment of maleinfertilityrdquo Contraception vol 72 no 4 pp 314ndash318 2005

[16] D Madhukar and S Rajender ldquoHormonal treatment of maleinfertility promise and pitfallsrdquo Journal of Andrology vol 30no 2 pp 95ndash112 2009

[17] F Iacono S Barra L Montano and T Lotti ldquoContributionof high-dose pure FSH in the treatment of idiopathic maleinfertilityrdquo Journal drsquoUrologie vol 102 no 2 pp 81ndash84 1996

[18] G Arnaldi G Balercia G Barbatelli and F Mantero ldquoEffectsof long-term treatment with human pure follicle-stimulatinghormone on semen parameters and sperm-cell ultrastructure inidiopathic oligoteratoasthenozoospermiardquo Andrologia vol 32no 3 pp 155ndash161 2000

[19] C Foresta A Bettella M Merico et al ldquoFSH in the treatmentof oligozoospermiardquoMolecular and Cellular Endocrinology vol161 no 1-2 pp 89ndash97 2000

[20] E Caroppo C Niederberger G M Vizziello and G DrsquoAmatoldquoRecombinant human follicle-stimulating hormone as a pre-treatment for idiopathic oligoasthenoteratozoospermic patientsundergoing intracytoplasmic sperm injectionrdquo Fertility andSterility vol 80 no 6 pp 1398ndash1403 2003

[21] M Fernandez-Arjona J Dıaz I Cortes J Gonzalez J MRodriguez and E Alvarez ldquoRelationship between gonado-trophin secretion inhibin B and spermatogenesis in oligo-zoospermic men treated with highly purified urinary follicle-stimulating hormone (uFSH-HP) a preliminary reportrdquo Euro-pean Journal of Obstetrics Gynecology and Reproductive Biologyvol 107 no 1 pp 47ndash51 2003

[22] A A Acosta E Khalifa and S Oehninger ldquoPure human folliclestimulating hormone has a role in the treatment of severe maleinfertility by assisted reproduction norfolkrsquos total experiencerdquoHuman Reproduction vol 7 no 8 pp 1067ndash1072 1992

[23] Z Ben-Rafael J Farhi D Feldberg et al ldquoFollicle-stimulatinghormone treatment for men with idiopathic oligoteratoas-thenozoospermia before in vitro fertilization the impact on

sperm microstructure and fertilization potentialrdquo Fertility andSterility vol 73 no 1 pp 24ndash30 2000

[24] B Baccetti P Piomboni E Bruni et al ldquoEffect of follicle-stimulating hormone on sperm quality and pregnancy raterdquoAsian Journal of Andrology vol 6 no 2 pp 133ndash137 2004

[25] R Matorras C Perez B Corcostegui et al ldquoTreatment of themale with follicle-stimulating hormone in intrauterine insem-ination with husbandrsquos spermatozoa a randomized studyrdquoHuman Reproduction vol 12 no 1 pp 24ndash28 1997

[26] A Kamischke H M Behre M Bergmann M Simoni TSchafer and E Nieschlag ldquoRecombinant human follicle stim-ulating hormone for treatment of male idiopathic infertilitya randomized double-blind placebo-controlled clinical trialrdquoHuman Reproduction vol 13 no 3 pp 596ndash603 1998

[27] O Efesoy S Cayan and E Akbay ldquoThe efficacy of recombinanthuman follicle-stimulating hormone in the treatment of varioustypes of male-factor infertility at a single university hospitalrdquoJournal of Andrology vol 30 no 6 pp 679ndash684 2009

[28] A Lenzi G Balercia A Bellastella et al ldquoEpidemiologydiagnosis and treatment of male hypogonadotropic hypogo-nadismrdquo Journal of Endocrinological Investigation vol 32 no11 pp 934ndash938 2009

[29] G Cavallini ldquoMale idiopathic oligoasthenoteratozoospermiardquoAsian Journal of Andrology vol 8 no 2 pp 143ndash157 2006

[30] J G Alvarez R K Sharma M Ollero et al ldquoIncreased DNAdamage in sperm from leukocytospermic semen samples asdetermined by the sperm chromatin structure assayrdquo Fertilityand Sterility vol 78 no 2 pp 319ndash329 2002

[31] M J Tomlinson A White C L R Barratt A E Bolton andI D Cooke ldquoThe removal of morphologically abnormal spermforms by phagocytes a positive role for seminal leukocytesrdquoHuman Reproduction vol 7 no 4 pp 517ndash522 1992

[32] J Aitken C Krausz and D Buckingham ldquoRelationshipsbetween biochemical markers for residual sperm cytoplasmreactive oxygen species generation and the presence of leuko-cytes and precursor germ cells in human sperm suspensionsrdquoMolecular Reproduction and Development vol 39 no 3 pp268ndash279 1994

[33] T Domes K C Lo E D Grober J B M Mullen T Mazzulliand K Jarvi ldquoThe incidence and effect of bacteriospermia andelevated seminal leukocytes on semen parametersrdquo Fertility andSterility vol 97 no 5 pp 1050ndash1055 2012

[34] V Barraud-Lange J-C Pont A Ziyyat et al ldquoSeminal leuko-cytes are good Samaritans for spermatozoardquo Fertility andSterility vol 96 no 6 pp 1315ndash1319 2011

[35] H Wolff ldquoThe biologic significance of white blood cells insemenrdquo Fertility and Sterility vol 63 no 6 pp 1143ndash1157 1995

[36] S Seshadri B Flanagan G Vince and D J Lewis-JonesldquoDetection of subpopulations of leucocytes in different sub-groups of semen sample qualitiesrdquo Andrologia vol 44 pp 354ndash361 2012

[37] World Health Organization WHO Laboratory Manual forthe Examination and Processing of Human Semen CambridgeUniversity Press Cambridge Mass USA 5th edition 2010

[38] J E Lackner R Herwig J Schmidbauer G Schatzl C Kratzikand M Marberger ldquoCorrelation of leukocytospermia withclinical infection and the positive effect of antiinflammatorytreatment on semen qualityrdquo Fertility and Sterility vol 86 no3 pp 601ndash605 2006

[39] G Ricci G Presani S Guaschino R Simeone and S Per-ticarari ldquoLeukocyte detection in human semen using flow


cytometryrdquo Human Reproduction vol 15 no 6 pp 1329ndash13372000

[40] J A Politch H Wolff J A Hill and D J Anderson ldquoCom-parison of methods to enumerate white blood cells in semenrdquoFertility and Sterility vol 60 no 2 pp 372ndash375 1993

[41] 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

[42] 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 pp 349ndash354 2013

[43] G-M Pinggera M Mitterberger G Bartsch et al ldquoAssessmentof the intratesticular resistive index by colour Doppler ultra-sonography measurements as a predictor of spermatogenesisrdquoBJU International vol 101 no 6 pp 722ndash726 2008

[44] A W Endtz ldquoA rapid staining method for differentiatinggranulocytes from ldquogerminal cellsrdquo in papanicolaou stainedsemenrdquo Acta Cytologica vol 18 no 1 pp 2ndash7 1974

[45] E Johanisson A Campana R Luthi and A de AgostinildquoEvaluation of rsquoround cellsrsquo in semen analysis a comparativestudyrdquo Human Reproduction Update vol 6 no 4 pp 404ndash4122000

[46] D Valenti S La Vignera R A Condorelli et al ldquoFollicle-stimulating hormone treatment in normogonadotropic infertilemenrdquo Nature Reviews Urology vol 10 pp 55ndash62 2013

[47] A A Acosta S Oehninger H Ertunc and C Philput ldquoPossiblerole of pure human follicle-stimulating hormone in the treat-ment of severe male-factor infertility by assisted reproductionpreliminary reportrdquo Fertility and Sterility vol 55 no 6 pp1150ndash1156 1991

[48] B Bartoov F Eltes E Lunenfeld D Har-Even H Ledermanand B Lunenfeld ldquoSperm quality of subfertile males beforeand after treatment with human follicle-stimulating hormonerdquoFertility and Sterility vol 61 no 4 pp 727ndash734 1994

[49] B Baccetti E Strehler S Capitani et al ldquoThe effect of folliclestimulating hormone therapy on human sperm structurerdquoHuman Reproduction vol 12 no 9 pp 1955ndash1968 1997

[50] H-J Glander and J Kratzschr ldquoEffects of pure human Follicle-Stimulating Hormone (pFSH) on sperm quality correlate withthe hypophyseal response to gonadotrophin-releasing hormone(GnRH)rdquo Andrologia vol 29 no 1 pp 23ndash28 1997

[51] R Paradisi P Busacchi R Seracchioli E Porcu and S Ven-turoli ldquoEffects of high doses of recombinant human follicle-stimulating hormone in the treatment of male factor infertilityresults of a pilot studyrdquo Fertility and Sterility vol 86 no 3 pp728ndash731 2006

[52] G R Marshall D S Zorub and T M Plant ldquoFollicle-stimulating hormone amplifies the population of differenti-ated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta)rdquo Endocrinol-ogy vol 136 no 8 pp 3504ndash3511 1995

[53] P Y Liu L T Xirner D Rushford et al ldquoEfficacy and safetyof recombinant human follicle stimulating hormone (Gonal-F)with urinary human chorionic gonadotrophin for induction ofspermatogenesis and fertility in gonadotrophin-deficient menrdquoHuman Reproduction vol 14 no 6 pp 1540ndash1545 1999

[54] C Foresta A Bettella A Ferlin A Garolla and M RossatoldquoEvidence for a stimulatory role of follicle-stimulating hormone

on the spermatogonial population in adult malesrdquo Fertility andSterility vol 69 no 4 pp 636ndash642 1998

[55] S Palomba A Falbo S Espinola et al ldquoEffects of highlypurified follicle-stimulating hormone on sperm DNA damageinmenwithmale idiopathic subfertility a pilot studyrdquo Journal ofEndocrinological Investigation vol 34 no 10 pp 747ndash752 2011

[56] N Colacurci M G Monti F Fornaro et al ldquoRecombinanthuman FSH reduces sperm DNA fragmentation in men withidiopathic oligoasthenoteratozoospermiardquo Journal of Androl-ogy vol 33 pp 588ndash593 2012

[57] M I M El-Demiry H Young and R A Elton ldquoLeucocytes inthe ejaculate from fertile and infertile menrdquo British Journal ofUrology vol 58 no 6 pp 715ndash720 1986

[58] W Eggert-Kruse A Bellmann G Rohr W Tilgen and BRunnebaum ldquoDifferentiation of round cells in semen by meansof monoclonal antibodies and relationship with male fertilityrdquoFertility and Sterility vol 58 no 5 pp 1046ndash1055 1992

[59] D C Smith C L R Barratt and M A Williams ldquoThecharacterisation of non-sperm cells in the ejaculates of fertilemen using transmission electron microscopyrdquo Andrologia vol21 no 4 pp 319ndash333 1989

[60] R A Saleh A Agarwal E Kandirali et al ldquoLeukocytospermiais associated with increased reactive oxygen species productionby human spermatozoardquo Fertility and Sterility vol 78 no 6 pp1215ndash1224 2002

[61] R K Sharma A E Pasqualotto D R Nelson A J Thomasand A Agarwal ldquoRelationship between seminal white bloodcell counts and oxidative stress in men treated at an infertilityclinicrdquo Journal of Andrology vol 22 no 4 pp 575ndash583 2001

[62] S Kaleli F Ocer T Irez E Budak and M F Aksu ldquoDoesleukocytospermia associate with poor semen parameters andsperm functions in male infertility The role of different sem-inal leukocyte concentrationsrdquo European Journal of ObstetricsGynecology and Reproductive Biology vol 89 no 2 pp 185ndash1912000

[63] AA KiesslingN Lamparelli H-Z YinMM Seibel andRCEyre ldquoSemen leukocytes friends or foesrdquo Fertility and Sterilityvol 64 no 1 pp 196ndash198 1995

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


normal levels of FSH but not more than 8mIUmL in anycase

There are however lesser known aspects relating to thepossible usefulness of FSH therapy with FSH on uncon-ventional as well as conventional sperm parameters (den-sity morphology and motility) For example FSH therapycould induce modifications that could restore the qualityof the inflammatory response of the semen Under partic-ular conditions the leukocyte concentration in semen isdirectly correlated with immature germ cell concentrationsand the percentage of abnormal forms [30] The defects ofspermatogenesis represent a potential target of hormonaltreatment as well as a potential cause of increased inflam-matory response in particular the response of leukocytesthe main mediators of this phenomenon Together withtheir classic antimicrobial action leukocytes also removeimmature germ cells andor sperm with maturational defects[31ndash33] suggesting they have a protective role in semen atconcentrations of lt1 millionmL [34] However the completecharacterization of the leukocytes requires the identificationof the specific populations (polymorphonuclear granulo-cytes macrophages and lymphocytes) [35 36] that reflectmore accurately the possible chronicization of the inflamma-tory process (in particular the increase in lymphocytes) Thefailure to characterize specific populations of leukocytes mayexplain the frequent underestimation of the inflammatoryprocess in the semen of patients with idiopathicOAT becauseonly polymorphonuclear granulocytes are reported in rou-tine practice [37] Finally in clinical practice leukospermiaand bacteriospermia frequently are not associated [33] andoften the clinical andrologist must reduce the negative effectson spermatozoa caused by the mere presence of leukocyteswhich are not improved only with anti-inflammatory treat-ment [38] From the technical point of view flow cytometry isable to discriminate (with the use of monoclonal antibodies)the different subpopulations of leukocytes [39 40]

In this context the aims of this study were the following

(a) To evaluate the quality of the conventional spermparameters (density morphology and progressivemotility) in infertile patients with idiopathic OATafter treatment with three different preparations offollicle-stimulating hormone recombinant FSH-120572and -120573 (Gonal F Puregon) and highly purified humanFSH (Fostimon)

(b) To evaluate in these patients the semen concentrationof CD45pos cells (a pan-leukocytemarker) beforeand after hormonal treatment with one of the threedifferent hormonal preparations

2 Materials and Methods

We retrospectively reviewed patient clinical instrumentaland laboratory data obtained from June 2012 to June 2013All patients enrolled in this study were referred to theDepartment of Andrology and Endocrinology of CataniaUniversity Italy for the diagnosis and treatment of maleinfertility Overall the data of 316 patients were analyzedThemen were 18ndash40 years old with a mean age of 260 plusmn 80

years and a bodymass index (BMI) ranging between 180 and270 kgm2 (mean BMI 220 plusmn 30 kgm2)

21 Exclusion Criteria

(1) Primary Scrotal Disease Varicocele hydrocele testi-cular focal injuryinjuries testicular microlithiasis inhomo-geneous testicular echotexture (ultrasound examination)and abnormal hemodynamic parameters of the testis (alteredvalues of systolic peak velocity and resistance index) viaultrasound examination

(2) Other Criteria Genetic disorders (altered karyotypeandor the presence of Y chromosome microdeletions)clinical history of cryptorchidism varicocelectomy eversionof the tunica vaginalis head trauma endocrine abnormalities(increased gonadotropins reduced serum total testosterone(lt28 ngmLminus1) hyperprolactinemia and increased estro-gen concentrations) systemic diseases (kidney disease liverdisease and diabetes mellitus) cigarette smoking alcoholuse concomitant use of other drugs during the previous6 months leukocytospermia (leukocyte concentration insemen gt1millionmL (detected with the peroxidase test))male accessory gland inflammatory disease positive seminalurine andor urethral swab cultures and ultrasound signs ofepididymal obstruction

Applying these exclusion criteria we analyzed data from54 patients with an age range of 18ndash33 years (mean 240 plusmn 60years) and a BMI of 190ndash260 kgm2 (mean 230plusmn40 kgm2)

The protocol was approved by the internal InstitutionalReview Board and informed written consent was obtainedfrom each participant

22 Scrotal Ultrasound Evaluation The scrotal ultrasoundevaluation was performed in two phases the first with thepatient in a supine position (with the penis resting on thesuprapubic region) and the second in an upright position toevaluate reflux along the pampiniform plexus testicular paintesticular malposition and the extent of any fluid collectionThe examination was performed with a GX Megas Esaote(Esaote SpAmdashGenova (Italy)) device equipped with linearhigh-resolution and high-frequency (75 to 14MHz) probesdedicated to the study of soft body areas with color Dopplerfor detecting slow flow and a scanning surface of at least5 cm Testicular volume was calculated automatically by theultrasound machine using the ellipsoid formula (length timeswidth times thickness times 052) The testis was considered normalin size when it had a volume between 15 and 25 cm3 low-normal when it had a volume between 10 and 12 cm3 andhypotrophic when it had a volume of less than 10 cm3 [41 42]The parenchymal echostructure was considered normal inthe presence of thin densely packed and homogeneouslydeployed echoes The presence of a finely inhomogeneousechopattern and weakly hypo- or hyperechogenic areas wasconsidered indicative of primary testicular disease Duringthe Doppler evaluation flow was detected at the level ofthe spermatic artery and the testicular artery and theirbranches The velocity analysis was considered normal in







2O2to






(1)









3 Result







4 Discussion




















































References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2O2to






(1)









3 Result







4 Discussion




















































References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Result







4 Discussion




















































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














