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Despite the fact that the major part of the ejaculate comes from the seminal vesicles and prostate (Gonzales 1989), we have long had the impression that low ejaculate volume (<2.5 mL) seems associated with an increased chance for obtaining testicular sperm. We found it important to examine whether this can be documented, and whether it is possible to determine a useful threshold value.
In testicular biopsies from azoospermic men with a history of cryptorchidism we have often observed an inhomogeneous histological pattern with maturation to fully mature sperm in islands in tissue showing Sertoli cell only-pattern. Therefore, we have found it important to investigate whether this histological pattern is more often seen in azoospermic men with a history of cryptorchidism than in other azoospermic men, In addition we wanted to evaluate whether testicular sperm from men with a history of cryptorchidism establish pregnancy equally efficient as do other testicular sperm.
In order to highlight these important questions we have performed a historical prospective study with the primary aims of evaluating whether:
1. ejaculate volume predicts the chance for obtaining testicular sperm and
2. the testicular histological pattern in patients with a history of cryptorchidism predicts the chance for obtaining testicular sperm,
Our secondary aims were to evaluate whether:
1. presence or absence of vasa deferentia in azoospermic men carrying a Cystic Fibrosis Transmembrane conductance Regulator (CFTR) mutation predicts the chance for obtaining testicular sperm,
2. sperm from azoospermic men with a history of cryptorchidism are just as useful as testicular sperm from other azoospermic men.
If the results should be useful in clinical practice, it is important to look at an unselected population of men referred to fertility clinics without preceding selection.
Materials and methods:
The diagnosis of azoospermia was verified by examination of at least two ejaculates, which were evaluated untreated and after centrifugation. In this study all non-vasectomized azoospermic men referred to our fertility clinic from December 1997 to December 2009 were included and examined as described below. Diagnosing "azoospermic couples" is in practice a running process, which is performed in a systematic way but might be individualized according to particular wishes from each couple. However, in order to analyze the predictive value of single factors for further outcome, we chose to divide the process into four steps:
1. History, clinical and seminal examination, and determination of serum FSH,
2. Genetic examination such as karyotype, Y microdeletions, and CFTR mutations,
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 study period 277 men were referred to our clinic for examination due to azoospermia or aspermia. Thirteen couples decided to have intrauterine insemination using donor semen before their examination programme was started, and an additional two men were excluded from the study as sperm useful for treatment were in one case found in a new ejaculate, and in the other case sperm were found in postejaculatory urine (as the man suffered from retrograde ejaculation). In total, 262 men were examined by clinical examination and hormonal and genetic analysis as described above. Of these, however, only 203 had a testicular biopsy taken. From six of the remaining 59 men sperm were observed in new ejaculates 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) cases with translocations chose not to have a biopsy taken due to the modest chances of finding any sperm. In one case an utricular cyst detected by rectal ultrasonography was resected; one couple had cryopreserved semen, which they chose to use before considering testicular biopsy; in two cases with hypogonadotrophic hypogonadism hormone treatment was started prior to testis biopsy; and in one case the testes were not available for biopsy in spite of several attempts on orchiopexia previously in life. In the remaining 23 (of the 59) cases, non-medical situations were predominantly the causes for non-participation, e.g. divorce of the couple during the relative short time between blood samples and planned testicular biopsy.
Twenty men showed ejaculatory dysfunction (anejaculation or retrograde ejaculation) due to tetraplegia, paraplegia, amyotrophic lateral sclerosis (ALS) or diabetes mellitus of many years, and of these, 14 were completely unable to ejaculate (aspermic).
Examination of ejaculate: Ejaculate volumes were registered, and semen samples were examined for the presence of sperm as described above. The volume of the first delivered ejaculate was used for further analysis.
History and clinical examination: For each participant a detailed history was obtained, and patients were examined by common objective examination (body proportions, hair distribution and scrotal examination, including palpation for presence of scrotal parts of vasa deferentia (VD)) and ultrasonography of the scrotal content. In order to obtain a precise and uniform history and to minimize variation in objective examination all men were examined by the same clinician (JF).
Hormone analysis: Levels of FSH, Luteinizing Hormone (LH), testosterone and prolactin were determined using standard techniques. The level of FSH was determined using a commercial electrochemiluminescence immunoassay employing two different monoclonal antibodies (Cobas, Roche Diagnostics, Mannheim, Germany).
Genetic analyses: Karyotyping and examination for Y microdeletions and mutations in the CFTR gene mutations was performed as described by Crüger et al. (2003). At least two specific sequence-tagged sites (STSs) for detection of deletions in each of the three AZFa, AZFb, and AZFc regions were used. Two STSs on Yp and Yq(term) were used as controls. We analyzed for at least four CFTR mutations, which make up more than 90% of the CFTR mutations in ethnical Danes: Î”F508 (exon 10), 394delTT (exon 3), R117H (exon 4) and IVS8-5T (intron 8). In particular cases, including non-ethnical Danes, examination for up to 33 mutations, including IVS8-5T, were performed.
Ultrasonography: Ultrasound examination was used for measuring testicular volumes and examination of echogenicity of the testes. Additionally, non-vasectomized men assumed to suffer from obstructive azoospermia, in many cases had a rectal ultrasonography in order to detect utricular cysts and dilation or absence of the seminal vesicles (Fedder et al. 2004).
Testicular biopsy: Following application of funicular blockade with 20 mL Lidokain (lidocaine, SAD), 20 mg/mL injected highly into the scrotum on both sides of the funicle, three Tru-Cut biopsies (= Testicular sperm extraction = TESE) were taken from different parts of one of the testicles (5). The tissue obtained was used for immediate examination in our IVF-lab, for histological examination and, if the man had given his written consent, often for scientific purposes. If motile sperm were found in the first testicle concomitant with the other testicle being evaluated as normal (without microcalcifications or other signs of Carcinoma in situ testis), in most cases testicular biopsies were only taken on one side. Otherwise testicular biopsies were always taken bilaterally provided that the man had two testicles. Testicular biopsies were cylindrical, 18 mm long (unless the testicle was smaller) and 1.3 mm in diameter (~24 mm3), each containing more than 100 tubules for evaluation.
Histological examination categorized testicular tissue to one of four groups: 1. normal testis tissue, 2. uniform tissue showing maturation stop or atrophia, 3. Sertoli cell only-syndrome or 4. islands (often less than 10%) of normal (or nearly normal) testis tissue in tissue showing Sertoli cell only-syndrome. Histological evaluation was performed independently of clinical history.
Statistics: Normally distributed data are presented as meansÂ±SD (range) and compared using Student's t-test. A two-tailed Ï‡2-test with Yates correction was used to compare testicular histological pattern and implantation and live birth rates in relation to history of previous cryptorchidism.
As shown in Table 1, the distibution of men with chromosomal abnormalities, e.g. 47,XXY karyotype (Mau-Holzmann 2005), CFTR mutations (von Eckardstein et al. 2000; Lissens et al. 1996), and Y deletions (Simoni et al. 2007) is very similar to previously published data (Fedder et al. 2004), suggesting that the population examined in this study was representative for other populations. As no Y microdeletions were detected in the two men of 46,XX karyotype, these men may wear Y chromosome material.
Ejaculate volume: Of 203 men undergoing TESE (Tru-Cut biopsies), motile sperm were found in 95 men (47%) and immotile sperm in additional 21 men (10%). As expected, lower FSH levels (11.3 IU/L versus 21.2 IU/L) and larger testicular volumes (25.0 mL versus 14.1 mL) were found in the group of men in whom motile sperm were found. In addition hereto, a significantly lower ejaculate volumes (2.3 mL versus 3.6 mL) were detected in the group with motile sperm, even after exclusion of the men carrying CFTR mutations (2.8 mL versus 3.4 mL) (Table 2). Significant associations between low ejaculate volume and low FSH level (Figure 1) or low ejaculate volume and high testicular volume (data not shown) could not be demonstrated.
Ejaculate volumes were generally lower for the 33 (33 out of 34 were able to ejaculate) men carrying at least one CFTR mutation (1.9 mL Â± 1.7 mL; range: 0.2 mL-7.5 mL) compared to 214 azoospermic men without a CFTR mutation (3.0 mL Â± 1.7 mL; range: 0.1 mL-7.5 mL)(p<0.01, Student's t-test. For further details see Table 3). Fourteen men unable to ejaculate were not included in this calculation. Furthermore, for the 13 CFTR-men having Congenital Bilateral Absence of Vasa Deferentia (CBAVD), the ejaculate volumes were even lower (0.9 mL Â± 0.6 mL; range: 0.3 mL-2.0 mL) than for the 18 CFTR-men with apparently normal presence of VD (2.7 mL Â± 1.9 mL; range: 0.2 mL-7.5 mL)(p<0.01, Student's t-test). The 13 men with CBAVD showed low FSH values, and it was in all these cases possible to find motile sperm for TESA treatment. In testes of the men with CFTR mutation and present VD, it was only possible to find sperm in 44% (8 of 18) of the cases. The men from whom sperm were found, most often showed low ejaculate volumes and low FSH values. In the case of the two men with a Î”F508 mutation and VD presence, ejaculate volumes were 0.2 mL and 0.5 mL and FSH 4.7 IE/L and 4.0 IE/L. In the case of the two men with Congenital Unilateral Absence of Vasa Deferentia (CUAVD) and incomplete VD, the ejaculate volumes were in between.
As ejaculate volumes were associated to the chance of finding sperm useful for fertility treatment (Table 2), defining a threshold value for practical clinical work laid near at hand. Therefore, the material was divided according to whether it was possible to find motile sperm, only immotile sperm, or no sperm at all in testicular biopsies, and data on men and ejaculate volume were arranged in 0.5 mL intervals (Table 4). On the basis of these data we consider 2.5 mL as a useful threshold value. Considering ejaculate volume < 2.5 mL as predictor for the presence of motile testicular sperm, the sensitivities were 57% and 44%, respectively, and the specificities respectively 75% and 74% after inclusion and exclusion of men carrying CFTR mutations. Positive predictive values of 63% (CFTR mutations included) and 50% (CFTR mutations excluded) and negative predictive values of 70% (CFTR mutations included) and 69% (CFTR mutations excluded) were found.
History of cryptorchidism: As stated in Table 5, an inhomogeneous pattern with spermatogenesis and maturation of sperm in isolated islands in an ocean of seminiferous tubules with only Sertoli cells was particularly characteristic for men with a previous history of cryptorchidism (p<0.0001, Figure 2). However, for at least 25 of the 59 men with a history of cryptorchidism, the condition had been bilateral, and for at least 35 cases the patients have had an orchiopexia. Reliable information about the precise localization of the testes in childhood was not possible to obtain, and it was not possible to distinguish between true undescended and ectopic testes (Fedder and Boesen 1998).
When examining the predictive value of a history of cryptorchidism for the chance to obtain motile sperm, also focusing on the fertility potential of motile sperm used for ICSI was obvious for us, although it has not been the main aim of this study. Nineteen of 81 couples with motile sperm were, after andrological examination in our center, treated in a local fertility center. Of the present material, to date 96 stimulated microinsemination treatment cycles with multiple testicular biopsies in 50 couples have been finished in our fertility clinic. In further 7 couples where the female partner was stimulated for oocyte aspiration, it was not possible to refind testicular sperm; of those 4 had a history of cryptorchidism (Table 6). The excess 5 azoospermic men with motile testicular sperm either had no female partner, a female partner with too high body mass index (BMI>30), or were just not ready for treatment for other reasons.
Except for a nearly identical fertilization rate for the men with and the men without a history of cryptorchidism, history of cryptorchidism seemed to give slightly reduced successes in refinding motile testicular sperm, cleavage, implantation, clinical pregnancy and live birth rates (Table 6). Patients treated with epididymal (usually vasectomised men) or frozen-thawed sperm were not included in this analysis.
As maturation of sperm in islands might call for multiple biopsies compared to a uniform histology, it is important to analyse the predictive value of a previous history of cryptorchidism while considering the chance of showing the inhomogeneous histological pattern of spermatogenesis in islands. Associating cryptorchidism and histological patterns of spermatogenesis in islands, a sensitivity of 81% and a specificity of 83% were observed, while the positive and negative predictive values were 53% and 95%, respectively (Table 5). Among the 31 men with a history of previous cryptorchidism and spermatogenesis with mature sperm in islands, motile sperm were obtained in 15 (48%).
As mentioned, the distribution of genetic diagnoses in this study-population is in accordance with other studies reflecting that the population is unselected (Table 1). A frequency of 11.1% abnormal karyotypes is consistent with the 13.1% found by Mau-Holzman (2005), and a frequency of Y deletions of 5.3% is compatible with an estimated frequency of around 8% in men with non-obstructive azoospermia (Simoni et al. 2007).
Ejaculate volume: The predictive value of testicular volume and FSH for the chances of finding motile sperm useful for treatment is well documented (de Kretser et al. 1974), and the use of these parameters is widespread. In addition, several papers have focused on the possible additive predictive value of inhibin B and AMH (Goulis et al. 2009). This paper focuses on the predictive value of simple historic information and semen volume for the chances of finding motile sperm in testicular biopsies from azoospermic men. A significantly lower mean ejaculate volume was found for the group of men with motile sperm compared to those without motile sperm. Low ejaculate volumes did not show any clear correlation to FSH levels (Figure 1).
Considering the 34 men shown to carry a CFTR mutation, significantly smaller ejaculate volumes were observed. An explanation to this phenomenon might be that the seminal vesicles are missing or hypoplastic, rather than obstruction of the vas deferentes. As only a minor part of the men carrying CFTR mutations had rectal sonography performed, it was not possible to associate seminal volumes and specific CFTR mutations to the size of the seminal vesicles. However, even after exclusion of the men carrying CFTR mutations, the mean ejaculate volume was significantly lower for men with detectable motile sperm (Table 2).
This study indicates that CBAVD and low ejaculate volumes increase the chance of finding motile testicular sperm, as motile sperm were found in all 13 CFTR carriers with CBAVD. The IVS8-5T mutation, located to the intron of the CFTR gene, may have a less intensive negative influence on the male genital tract, as only one of 8 men carrying only this mutation showed CBAVD. Motile sperm were found in only 4 of the 7 men with apparent normal VD, suggesting another etiology being the cause of the azoospermia. This well illustrates some of the difficulties in dividing into obstructive and non-obstructive azoospermia (see later).
Aiming to define an ejaculate volume threshold value useful in clinical practice, patients were arranged after size of the ejaculate volumes (Table 4). In our opinion, the present data support 2.5 mL as a useful threshold value when ejaculate volume is used as a predictive parameter for the chance to obtain testicular sperm.
Histology: For most men the nearly 2 cm long and relatively large testicular biopsies ought to be representative for the histological picture throughout the whole testis. This claim is supported by a clear impression of only a negligible difference between biopsies taken from the same patient at the same occasion.
An association between cryptorchidism and decreased sperm concentration is well documented in the literature (Wohlfahrt-Veje et al. 2009). The results presented in this study show that a histological pattern with small islands of normal testicular tissue in tissue showing Sertoli cell only (SCO)-syndrome is particularly characteristic for men with a history of cryptorchidism. Naturally, even for the men with a history of cryptorchidism and SCO syndrome, presence of small islands with normal spermatogenesis, not caught by the biopsy needle, cannot be excluded. For azoospermic men with a history of cryptorchidism it therefore might be particularly relevant to consider multiple TESEs (Seo and Ko 2001) and microsurgery (Tsujimura 2007) for obtaining sperm from the often isolated islands with normal testis tissue. Although a positive predictive value of finding an inhomogeneous pattern with maturation of sperm in islands of only 51% were found, a negative predictive value of 95% shows that the chance of finding an inhomogeneous histological pattern in an azoospermic man is very low unless the man has a history of cryptorchidism. This suggests that if the man has no history of cryptorchidism, one testicular biopsy most probably is representative, and the man could be spared for multiple TESEs.
In this study it was not possible to demonstrate any significant associations between a history of cryptorchidism and ability to later (months) refind testicular sperm (following diagnostic TESE), fertilization rate, cleavage rate, implantation rate, clinical pregnancy rate or live birth rate. This is in accordance with other studies (Haimov-Kochman et al. 2010; Vernaeve V et al. 2004; Negri et al. 2003). Unless taking basis in a cohorte of cryptorchid boys well-defined through childhood (Fedder and Boesen 1998) it is very difficult to get exact information about the previous positions of the testicles and details about previous therapy, including date for treatment (Raman and Schlegel 2003). Such information would help in defining subgroups of men with a history of cryptorchidism, who should be treated differently from the main group of azoospermic men.
General discussion: Defining one single parameter which can predict whether a man (with at least one testis) produces sperm at this point seems impossible. FSH concentration and testicular volume are wellknown useful parameters. This study shows that in addition, low semen volumes increase the chance of finding motile testicular sperm. Low ejaculate volumes in combination with CBAVD suggest that the man carriers a CFTR mutation, and that the chances of finding motile testicular sperm are good.
The study also pinpoints the importance of paying particular attention to men with a history of cryptorchidism in attempting to retrieve motile sperm from testicular biopsy as normal sperm production is often found in small islands located in testicular tissue showing Sertoli cell only-syndrome. Therefore, multiple TESEs may be considered for these men, while men without a history of cryptorchidism could be spared for such extensive treatment.
In the literature azoospermia is often divided into obstructive (OA) and non-obstructive azoospermia (NOA). This is done on the basis of testicular histology, history, clinical examination (e.g. lack of the scrotal parts of the vasa deferentia), and laboratory analyses (e.g. presence of round germ cells in semen) (Fedder et al. 2004). However, it might be difficult to distinguish between OA and NOA, and long-term obstruction of the seminal tract may cause impaired spermatogenesis, as seen after vasectomy (Thomas 1987). Therefore, it was chosen not to use the terms/definitions of OA and NOA in this study.
Future prospective studies may further clarify the predictive value of ejaculate volume and history of cryptorchidism for the ability to find testicular sperm and for treatment outcome.