Despite the fact that the major portion of the semen comes from the seminal cysts and prostate ( Gonzales 1989 ) , we have long had the feeling that low semen volume ( & lt ; 2.5 milliliter ) seems associated with an increased opportunity for obtaining testicular sperm. We found it of import to analyze whether this can be documented, and whether it is possible to find a utile threshold value.
In testicular biopsies from azoospermic work forces with a history of cryptorchidy we have frequently observed an nonuniform histological form with ripening to to the full maturate sperm in islands in tissue demoing Sertoli cell only-pattern. Therefore, we have found it of import to look into whether this histological form is more frequently seen in azoospermic work forces with a history of cryptorchidy than in other azoospermic work forces, In add-on we wanted to measure whether testicular sperm from work forces with a history of cryptorchidy set up gestation every bit efficient as do other testicular sperm.
In order to foreground these of import inquiries we have performed a historical prospective survey with the primary purposes of measuring whether:
1. ejaculate volume predicts the opportunity for obtaining testicular sperm and
2. the testicular histological form in patients with a history of cryptorchidy predicts the opportunity for obtaining testicular sperm,
Our secondary purposes were to measure whether:
1. presence or absence of vasa deferentia in azoospermic work forces transporting a Cystic Fibrosis Transmembrane conductance Regulator ( CFTR ) mutant predicts the opportunity for obtaining testicular sperm,
2. sperm from azoospermic work forces with a history of cryptorchidy are merely every bit utile as testicular sperm from other azoospermic work forces.
If the consequences should be utile in clinical pattern, it is of import to look at an unselected population of work forces referred to birthrate clinics without predating choice.
Materials and methods:
The diagnosing of azoospermia was verified by scrutiny of at least two semens, which were evaluated untreated and after centrifugation. In this survey all non-vasectomized azoospermic work forces referred to our birthrate clinic from December 1997 to December 2009 were included and examined as described below. Diagnosing “ azoospermic twosomes ” is in pattern a running procedure, which is performed in a systematic manner but might be individualized harmonizing to peculiar wants from each twosome. However, in order to analyse the prognostic value of individual factors for farther result, we chose to split the procedure into four stairss:
1. History, clinical and seminal scrutiny, and finding of serum FSH,
2. Familial scrutiny such as karyotype, Y microdeletions, and CFTR mutants,
3. Testicular biopsy for presence of motile sperm and testicular biopsy, and
4. Pregnancy ( biochemical and/or clinical ) following IntraCytoplasmic Sperm Injection
( ICSI ) with testicular or epididymal sperm.
Patients: During the survey period 277 work forces were referred to our clinic for scrutiny due to azoospermia or aspermia. Thirteen couples decided to hold intrauterine insemination utilizing donor seeds before their scrutiny programme was started, and an extra two work forces were excluded from the survey as sperm utile for intervention were in one instance found in a new semen, and in the other instance sperm were found in postejaculatory piss ( as the adult male suffered from retrograde interjection ) . In entire, 262 work forces were examined by clinical scrutiny and hormonal and familial analysis as described above. Of these, nevertheless, merely 203 had a testicular biopsy taken. From six of the staying 59 work forces sperm were observed in new semens before testicular biopsy, and 20 ( out of 22 ) patients with 47, XXY karyotype, 2 ( out of 2 ) with 46, XX karyotype, 1 ( out of 1 ) with a 47, XYY karyotype and 2 ( out of 3 ) instances with translocations chose non to hold a biopsy taken due to the modest opportunities of happening any sperm. In one instance an utricular cyst detected by rectal echography was resected ; one twosome had cryopreserved seeds, which they chose to utilize before sing testicular biopsy ; in two instances with hypogonadotrophic hypogonadism endocrine intervention was started prior to testis biopsy ; and in one instance the testicles were non available for biopsy in malice of several efforts on orchiopexia antecedently in life. In the staying 23 ( of the 59 ) instances, non-medical state of affairss were preponderantly the causes for non-participation, e.g. divorce of the twosome during the comparative short clip between blood samples and planned testicular biopsy.
Twenty work forces showed ejaculatory disfunction ( anejaculation or retrograde interjection ) due to tetraplegia, paraplegia, amyotrophic sidelong induration ( ALS ) or diabetes mellitus of many old ages, and of these, 14 were wholly unable to blurt out ( aspermic ) .
Examination of semen: Ejaculate volumes were registered, and seeds samples were examined for the presence of sperm as described above. The volume of the first delivered semen was used for farther analysis.
History and clinical scrutiny: For each participant a elaborate history was obtained, and patients were examined by common nonsubjective scrutiny ( organic structure proportions, hair distribution and scrotal scrutiny, including tactual exploration for presence of scrotal parts of vessel deferentia ( VD ) ) and echography of the scrotal content. In order to obtain a precise and unvarying history and to minimise fluctuation in nonsubjective scrutiny all work forces were examined by the same clinician ( JF ) .
Hormone analysis: Degrees of FSH, Luteinizing Hormone ( LH ) , testosterone and lactogenic hormone were determined utilizing standard techniques. The degree of FSH was determined utilizing a commercial electrochemiluminescence immunoassay using two different monoclonal antibodies ( Cobas, Roche Diagnostics, Mannheim, Germany ) .
Familial analyses: Karyotyping and scrutiny for Y microdeletions and mutants in the CFTR cistron mutants was performed as described by Cruger et Al. ( 2003 ) . At least two specific sequence-tagged sites ( STSs ) for sensing of omissions in each of the three AZFa, AZFb, and AZFc parts were used. Two STSs on Yp and Yq ( term ) were used as controls. We analyzed for at least four CFTR mutants, which make up more than 90 % of the CFTR mutants in cultural Danes: I”F508 ( exon 10 ) , 394delTT ( exon 3 ) , R117H ( exon 4 ) and IVS8-5T ( intron 8 ) . In peculiar instances, including non-ethnical Danes, scrutiny for up to 33 mutants, including IVS8-5T, were performed.
Ultrasonography: Ultrasound scrutiny was used for mensurating testicular volumes and scrutiny of echogenicity of the testicles. Additionally, non-vasectomized work forces assumed to endure from clogging azoospermia, in many instances had a rectal echography in order to observe utricular cysts and dilation or absence of the seminal cysts ( Fedder et al. 2004 ) .
Testicular biopsy: Following application of funicular encirclement with 20 milliliters Lidokain ( Lidocaine, SAD ) , 20 mg/mL injected extremely into the scrotum on both sides of the funiculus, three Tru-Cut biopsies ( = Testicular sperm extraction = TESE ) were taken from different parts of one of the testiss ( 5 ) . The tissue obtained was used for immediate scrutiny in our IVF-lab, for histological scrutiny and, if the adult male had given his written consent, frequently for scientific intents. If motile sperm were found in the first testis accompaniment with the other testis being evaluated as normal ( without microcalcifications or other marks of Carcinoma in situ testicle ) , in most instances testicular biopsies were merely taken on one side. Otherwise testicular biopsies were ever taken bilaterally provided that the adult male had two testiss. Testicular biopsies were cylindrical, 18 millimeter long ( unless the testis was smaller ) and 1.3 millimeter in diameter ( ~24 mm3 ) , each incorporating more than 100 tubules for rating.
Histological scrutiny categorized testicular tissue to one of four groups: 1. normal testicle tissue, 2. unvarying tissue demoing ripening halt or atrophia, 3. Sertoli cell only-syndrome or 4. islands ( frequently less than 10 % ) of normal ( or about normal ) testicle tissue in tissue demoing Sertoli cell only-syndrome. Histological rating was performed independently of clinical history.
Statisticss: Normally distributed informations are presented as meansA±SD ( scope ) and compared utilizing Student ‘s t-test. A two-tailed I‡2-test with Yates rectification was used to compare testicular histological form and nidation and unrecorded birth rates in relation to history of old cryptorchidy.
As shown in Table 1, the distibution of work forces with chromosomal abnormalcies, e.g. 47, XXY karyotype ( Mau-Holzmann 2005 ) , CFTR mutants ( von Eckardstein et Al. 2000 ; Lissens et Al. 1996 ) , and Y omissions ( Simoni et al. 2007 ) is really similar to antecedently published information ( Fedder et al. 2004 ) , proposing that the population examined in this survey was representative for other populations. As no Y microdeletions were detected in the two work forces of 46, XX karyotype, these work forces may have on Y chromosome stuff.
Ejaculate volume: Of 203 work forces undergoing TESE ( Tru-Cut biopsies ) , motile sperm were found in 95 work forces ( 47 % ) and nonmotile sperm in extra 21 work forces ( 10 % ) . As expected, lower FSH degrees ( 11.3 IU/L versus 21.2 IU/L ) and larger testicular volumes ( 25.0 milliliter versus 14.1 milliliter ) were found in the group of work forces in whom motile sperm were found. In add-on hereto, a significantly lower semen volumes ( 2.3 milliliter versus 3.6 milliliter ) were detected in the group with motile sperm, even after exclusion of the work forces transporting CFTR mutants ( 2.8 milliliter versus 3.4 milliliter ) ( Table 2 ) . Significant associations between low semen volume and low FSH degree ( Figure 1 ) or low semen volume and high testicular volume ( informations non shown ) could non be demonstrated.
Ejaculate volumes were by and large lower for the 33 ( 33 out of 34 were able to blurt out ) work forces transporting at least one CFTR mutant ( 1.9 milliliter A± 1.7 milliliter ; scope: 0.2 mL-7.5 milliliter ) compared to 214 azoospermic work forces without a CFTR mutant ( 3.0 milliliter A± 1.7 milliliter ; scope: 0.1 mL-7.5 milliliter ) ( p & lt ; 0.01, Student ‘s t-test. For farther inside informations see Table 3 ) . Fourteen work forces unable to blurt out were non included in this computation. Furthermore, for the 13 CFTR-men holding Congenital Bilateral Absence of Vasa Deferentia ( CBAVD ) , the semen volumes were even lower ( 0.9 milliliter A± 0.6 milliliter ; scope: 0.3 mL-2.0 milliliter ) than for the 18 CFTR-men with seemingly normal presence of VD ( 2.7 milliliter A± 1.9 milliliter ; scope: 0.2 mL-7.5 milliliter ) ( p & lt ; 0.01, Student ‘s t-test ) . The 13 work forces with CBAVD showed low FSH values, and it was in all these instances possible to happen motile sperm for TESA intervention. In testicles of the work forces with CFTR mutant and present VD, it was merely possible to happen sperm in 44 % ( 8 of 18 ) of the instances. The work forces from whom sperm were found, most frequently showed low semen volumes and low FSH values. In the instance of the two work forces with a I”F508 mutant and VD presence, ejaculate volumes were 0.2 milliliters and 0.5 milliliter and FSH 4.7 IE/L and 4.0 IE/L. In the instance of the two work forces with Congenital Unilateral Absence of Vasa Deferentia ( CUAVD ) and uncomplete VD, the semen volumes were in between.
As ejaculate volumes were associated to the opportunity of happening sperm utile for birthrate intervention ( Table 2 ) , specifying a threshold value for practical clinical work laid nigh at manus. Therefore, the stuff was divided harmonizing to whether it was possible to happen motile sperm, merely nonmotile sperm, or no sperm at all in testicular biopsies, and informations on work forces and semen volume were arranged in 0.5 milliliter intervals ( Table 4 ) . On the footing of these informations we consider 2.5 milliliter as a utile threshold value. Sing ejaculate volume & lt ; 2.5 milliliter as forecaster for the presence of motile testicular sperm, the sensitivenesss were 57 % and 44 % , severally, and the specificities severally 75 % and 74 % after inclusion and exclusion of work forces transporting CFTR mutants. Positive prognostic values of 63 % ( CFTR mutants included ) and 50 % ( CFTR mutants excluded ) and negative prognostic values of 70 % ( CFTR mutants included ) and 69 % ( CFTR mutants excluded ) were found.
History of cryptorchidy: As stated in Table 5, an nonuniform form with spermatogenesis and ripening of sperm in stray islands in an ocean of seminiferous tubules with merely Sertoli cells was peculiarly characteristic for work forces with a old history of cryptorchidy ( p & lt ; 0.0001, Figure 2 ) . However, for at least 25 of the 59 work forces with a history of cryptorchidy, the status had been bilateral, and for at least 35 instances the patients have had an orchiopexia. Reliable information about the precise localisation of the testicles in childhood was non possible to obtain, and it was non possible to separate between true undescended and ectopic testicles ( Fedder and Boesen 1998 ) .
When analyzing the prognostic value of a history of cryptorchidy for the opportunity to obtain motile sperm, besides concentrating on the birthrate potency of motile sperm used for ICSI was obvious for us, although it has non been the chief purpose of this survey. Nineteen of 81 twosomes with motile sperm were, after andrological scrutiny in our centre, treated in a local birthrate centre. Of the present stuff, to day of the month 96 stirred microinsemination intervention rhythms with multiple testicular biopsies in 50 twosomes have been finished in our birthrate clinic. In farther 7 twosomes where the female spouse was stimulated for oocyte aspiration, it was non possible to refind testicular sperm ; of those 4 had a history of cryptorchidy ( Table 6 ) . The extra 5 azoospermic work forces with motile testicular sperm either had no female spouse, a female spouse with excessively high organic structure mass index ( BMI & gt ; 30 ) , or were merely non ready for intervention for other grounds.
Except for a about indistinguishable fertilisation rate for the work forces with and the work forces without a history of cryptorchidy, history of cryptorchidy seemed to give somewhat reduced successes in refinding motile testicular sperm, cleavage, nidation, clinical gestation and unrecorded birth rates ( Table 6 ) . Patients treated with epididymal ( normally vasectomised work forces ) or frozen-thawed sperm were non included in this analysis.
As ripening of sperm in islands might name for multiple biopsies compared to a unvarying histology, it is of import to analyze the prognostic value of a old history of cryptorchidy while sing the opportunity of demoing the nonuniform histological form of spermatogenesis in islands. Associating cryptorchidy and histological forms of spermatogenesis in islands, a sensitiveness of 81 % and a specificity of 83 % were observed, while the positive and negative prognostic values were 53 % and 95 % , severally ( Table 5 ) . Among the 31 work forces with a history of old cryptorchidy and spermatogenesis with mature sperm in islands, motile sperm were obtained in 15 ( 48 % ) .
As mentioned, the distribution of familial diagnosings in this study-population is in conformity with other surveies reflecting that the population is unselected ( Table 1 ) . A frequence of 11.1 % unnatural karyotypes is consistent with the 13.1 % found by Mau-Holzman ( 2005 ) , and a frequence of Y omissions of 5.3 % is compatible with an estimated frequence of around 8 % in work forces with non-obstructive azoospermia ( Simoni et al. 2007 ) .
Ejaculate volume: The prognostic value of testicular volume and FSH for the opportunities of happening motile sperm utile for intervention is good documented ( de Kretser et Al. 1974 ) , and the usage of these parametric quantities is widespread. In add-on, several documents have focused on the possible linear prognostic value of inhibin B and AMH ( Goulis et al. 2009 ) . This paper focuses on the prognostic value of simple historic information and seed volume for the opportunities of happening motile sperm in testicular biopsies from azoospermic work forces. A significantly lower average semen volume was found for the group of work forces with motile sperm compared to those without motile sperm. Low semen volumes did non demo any clear correlativity to FSH degrees ( Figure 1 ) .
Sing the 34 work forces shown to transport a CFTR mutant, significantly smaller semen volumes were observed. An account to this phenomenon might be that the seminal cysts are losing or hypoplastic, instead than obstructor of the vessel deferentes. As merely a minor portion of the work forces transporting CFTR mutants had rectal echography performed, it was non possible to tie in seminal volumes and specific CFTR mutants to the size of the seminal cysts. However, even after exclusion of the work forces transporting CFTR mutants, the average semen volume was significantly lower for work forces with noticeable motile sperm ( Table 2 ) .
This survey indicates that CBAVD and low semen volumes increase the opportunity of happening motile testicular sperm, as motile sperm were found in all 13 CFTR bearers with CBAVD. The IVS8-5T mutant, located to the noncoding DNA of the CFTR cistron, may hold a less intensive negative influence on the male venereal piece of land, as merely one of 8 work forces transporting merely this mutant showed CBAVD. Motile sperm were found in merely 4 of the 7 work forces with evident normal VD, proposing another etiology being the cause of the azoospermia. This well illustrates some of the troubles in spliting into clogging and non-obstructive azoospermia ( see subsequently ) .
Aiming to specify an semen volume threshold value utile in clinical pattern, patients were arranged after size of the semen volumes ( Table 4 ) . In our sentiment, the present informations support 2.5 milliliter as a utile threshold value when semen volume is used as a prognostic parametric quantity for the opportunity to obtain testicular sperm.
Histology: For most work forces the about 2 centimeter long and comparatively big testicular biopsies ought to be representative for the histological image throughout the whole testicle. This claim is supported by a clear feeling of merely a negligible difference between biopsies taken from the same patient at the same juncture.
An association between cryptorchidy and reduced sperm concentration is good documented in the literature ( Wohlfahrt-Veje et Al. 2009 ) . The consequences presented in this survey show that a histological form with little islands of normal testicular tissue in tissue demoing Sertoli cell merely ( SCO ) -syndrome is peculiarly characteristic for work forces with a history of cryptorchidy. Naturally, even for the work forces with a history of cryptorchidy and SCO syndrome, presence of little islands with normal spermatogenesis, non caught by the biopsy acerate leaf, can non be excluded. For azoospermic work forces with a history of cryptorchidy it therefore might be peculiarly relevant to see multiple TESEs ( Seo and Ko 2001 ) and microsurgery ( Tsujimura 2007 ) for obtaining sperm from the frequently stray islands with normal testicle tissue. Although a positive prognostic value of happening an nonuniform form with ripening of sperm in islands of merely 51 % were found, a negative prognostic value of 95 % shows that the opportunity of happening an nonuniform histological form in an azoospermic adult male is really low unless the adult male has a history of cryptorchidy. This suggests that if the adult male has no history of cryptorchidy, one testicular biopsy most likely is representative, and the adult male could be spared for multiple TESEs.
In this survey it was non possible to show any important associations between a history of cryptorchidy and ability to subsequently ( months ) refind testicular sperm ( following diagnostic TESE ) , fertilisation rate, cleavage rate, nidation rate, clinical gestation rate or unrecorded birth rate. This is in conformity with other surveies ( Haimov-Kochman et al. 2010 ; Vernaeve V et Al. 2004 ; Negri et Al. 2003 ) . Unless taking footing in a cohorte of cryptorchid male childs chiseled through childhood ( Fedder and Boesen 1998 ) it is really hard to acquire exact information about the old places of the testiss and inside informations about old therapy, including day of the month for intervention ( Raman and Schlegel 2003 ) . Such information would assist in specifying subgroups of work forces with a history of cryptorchidy, who should be treated otherwise from the chief group of azoospermic work forces.
General treatment: Specifying one individual parametric quantity which can foretell whether a adult male ( with at least one testicle ) produces sperm at this point seems impossible. FSH concentration and testicular volume are wellknown utile parametric quantities. This survey shows that in add-on, low seeds volumes increase the opportunity of happening motile testicular sperm. Low semen volumes in combination with CBAVD suggest that the adult male carriers a CFTR mutant, and that the opportunities of happening motile testicular sperm are good.
The survey besides pinpoints the importance of paying peculiar attending to work forces with a history of cryptorchidy in trying to recover motile sperm from testicular biopsy as normal sperm production is frequently found in little islands located in testicular tissue demoing Sertoli cell only-syndrome. Therefore, multiple TESEs may be considered for these work forces, while work forces without a history of cryptorchidy could be spared for such extended intervention.
In the literature azoospermia is frequently divided into clogging ( OA ) and non-obstructive azoospermia ( NOA ) . This is done on the footing of testicular histology, history, clinical scrutiny ( e.g. deficiency of the scrotal parts of the vessel deferentia ) , and research lab analyses ( e.g. presence of circular source cells in seeds ) ( Fedder et al. 2004 ) . However, it might be hard to separate between OA and NOA, and long-run obstructor of the seminal piece of land may do impaired spermatogenesis, as seen after vasectomy ( Thomas 1987 ) . Therefore, it was chosen non to utilize the terms/definitions of OA and NOA in this survey.
Future prospective surveies may farther clear up the prognostic value of ejaculate volume and history of cryptorchidy for the ability to happen testicular sperm and for intervention result.