Azoospermic males comprise 1% of the general population and up to 10–20% of those presenting to infertility clinics.1 Azoospermia is defined as the absence of spermatozoa in two different ejaculated semen samples after centrifugation (1000×g for 15 min),2,3 and the etiology can be classified into three principle categories: pre-testicular, testicular, and post-testicular.
An accurate diagnosis of the etiology of azoospermia is crucial and mandatory for these patients, as the treatment approach differs according to the cause of azoospermia. Male hypogonadotropic hypogonadism, the most common cause of pre-testicular azoospermia, is treated with gonadotropin replacement therapy, whereas restoration of fertility may be possible for patients with post-testicular azoospermia caused by reproductive tract obstruction after recanalization. For patients with testicular azoospermia caused by congenital (genetic abnormalities), acquired (testicular torsion, trauma, mumps, surgery and varicocele) or idiopathic disorders, microdissection testicular sperm extraction (TESE) has been proven to be a preferable surgical technique in terms of higher sperm retrieval rate and low complication rate compared with conventional TESE.4–6
In clinical practice, azoospermia can be categorized as obstructive or non-obstructive. Obstructive azoospermia (OA) is less common than non-obstructive azoospermia (NOA), and accounts for 15–20% of all men with azoospermia.7 Testicular biopsy plays an important role in the diagnosis of OA, however hormone profiles and testicular size have also been used to predict the cause of azoospermia by using a cutoff value of 7.6 mIU/ml for follicular stimulating hormone (FSH) and a testicular long axis of 4.6 cm.8 However compared to Caucasians, Asian males tend to have higher FSH and smaller testes,9,10 and as a result it is essential to establish reference points for Asians.
The aim of this study was to determine the optimal cut-off values for testes size and endocrine profile for Asian males to predict the cause of azoospermia. Azoospermia related to hypogonadotropic hypogonadism is classified as being NOA and it can be identified by hormone profile, therefore these patients were not included in this study.
From April 2008 to July 2016, a total of 217 azoospermic patients with no history of cryptorchidism or testicular-related surgery were enrolled, including 166 with NOA and 51 with OA. Ten hypogonadotropic hypogonadism patients were excluded, and the remaining 156 patients with NOA were analyzed. Every patient underwent a detailed examination to identify the etiology of azoospermia, including a detailed history, physical examination, two consecutive semen analyses, hormone profile (FSH, Leutinizing hormone (LH), testosterone, prolactin, and estradiol), chromosome karyotyping, and Y chromosome microdeletion. Bilateral testicular size was measured by a single andrologist with the assistance of an orchidometer. To differentiate between OA and NOA, bilateral testes biopsies or microdissection TESE were performed for a definitive diagnosis. The study was performed according to the Taipei Veterans General Hospital approved Institutional Review Board protocol (IRB number: 2017–07–013CC).
2.2. Statistical analysis
The Student's t test was used to compare hormone profiles and testes size between the OA and NOA groups. Results were expressed as mean ± standard deviation and analyzed using SAS ver.9.0, and a P value less than 0.05 was considered to be statistically significant. Receiver operating characteristic (ROC) curves were used to determine the appropriate cut-off values for hormone profile and testes size to discriminate NOA from OA. Accuracy was assessed by the area under the ROC curve (AUC), and results were considered to be excellent for AUC values between 0.9 and 1, good for AUC values between 0.8 and 0.9, fair for 0.7–0.8 AUC values, poor for 0.6–0.7 AUC values, and failed for AUC values between 0.5–0.6.11
The demographic data and characteristics of the patients are listed in Table 1. The mean levels of testosterone (4.5 vs. 3.4 ng/ml) and E2 (26.3 vs. 19.2 pg/ml) were significantly higher, and the levels of FSH (5.6 vs. 25.4 mIU/ml) and LH (3.7 vs. 11.6) were significantly lower in the OA group. The NOA group had a significantly smaller bilateral testes size compared to the OA group (p < 0.0001). There were no differences between the two groups in age or prolactin level. In order to determine the cutoff values to discriminate between the two groups, we performed ROC curve analysis.
Fig. 1 depicts the ROC curve for FSH, LH, testosterone, E2, right testis and left testis size. Overall, FSH had the best response in terms of AUC (with a 95% CI for the area being between 0.9253 and 0.9897), followed by right testis (with a 95% CI for the area being between 0.8680 and 0.9552), left testis (with a 95% CI for the area being between 0.8587 and 0.9489), LH, E2 and testosterone. A cutoff value of 9.2 mIU/ml for FSH could discriminate the etiology of azoospermia with a sensitivity of 89.7% and a specificity of 90.2%, and using a cutoff value of right testis size of 15 ml, had a sensitivity of 76.3% and a specificity 92.2% (Fig. 2). Using a combination of FSH >9.2 mIU/ml and right testis size <15 ml (Table 2), the positive predictive value for NOA was 99.2% and 81.8% for OA.
The hypothalamic-pituitary-gonadal (HPG) axis is critical for the development of reproductive organs and spermatogenesis,12,13 and three different activation periods of the HPG axis have been identified. At the 8th week of gestation, the testes of the fetus secrete testosterone and anti-Müllerian hormone to induce masculinization, Wolffian duct differentiation, and regression of the Müllerian duct to prevent the formation of a uterus and fallopian tubes.14,15 The second period of testosterone surge starts at 1 week of age and peaks to a pubertal level at 1–3 months. The elevated testosterone level then gradually decreases to a pre-pubertal level by 6 months of age. The level of peripheral blood testosterone in infancy reflects the number of Leydig cells and testosterone concentration in the testis.16–18 An increase in the number of Sertoli cells and germ cells is associated with postnatal testicular activation, but not spermatogenesis due to a lack of androgen receptors in Sertoli cells during infancy.19,20 Postnatal testosterone activation, namely mini-puberty, is essential for penile growth,16,21 prostatic activity,16 and descent of the testes.22,23 Furthermore, from the neurobehavioral development point of view, a higher postnatal testosterone level measured from postnatal day 7 to an age of 6 months has been associated with male-type behavior at the age of 14 months.24
For boys with hypogonadotropic hypogonadism, gonadotropin therapy has been used to treat undescended testes and induce penile growth.25 In the final stage, during puberty, the HPG axis is reinitiated accompanies by spermatogenesis.12,13
Although Asian men are identical to Caucasian men in most aspects of reproductive endocrinology, previous studies have reported difference in testes volume and responsiveness of gonadotropin negative feedback between Asian and Caucasian men. Nevertheless, semen parameters in Asian men are comparable to Caucasian men, and the sperm concentration has been positively correlated to testis volume. The average testis size in Asians is 17 ml compared to 25 ml in Caucasians. By infusing testosterone for 48 h at different concentrations from 0, 7, 14 and 28 mg/1.7 m2, the suppression of LH secretory pulse has been reported to be significantly more pronounced in Asians during the lowest infusion dose, whereas ethnic differences in the suppression of pulsatile LH secretion disappear with increasing testosterone dosage. In contrast to the suppressive effect of LH after testosterone infusion, the responsiveness of pulsatile FSH secretion has not been reported to be significantly different, however, the relatively elevated level of FSH at baseline in Asians may indicate a possible decrease in spermatogenic reserve.9,10,26
The difference in gonadotropin negative feedback response may explain the excellent contraceptive efficacy with weekly 200 mg testosterone enanthate injections in Asians, with consistent azoospermia being achieved in 95% of Asians and 68% in non-Asians. The median time for the first semen sample to reach azoospermia has also been reported to occur sooner (91 days compared with 112 days) and the period for recovery to normal sperm concentration to be longer (126 days compared with 105 days) for Asians compared to non-Asians.26 One potential explanation for the observed lower secretion of inhibitory gonadotropin with the administration of exogenous testosterone in Caucasian men may be due is an increase in 5 alpha-reductase activity.27,28 A clinical trial also demonstrated the activity of 5 alpha-reductase in relation to the suppression of testicular spermatogenesis after the administration of supraphysiological doses of testosterone. By dividing subjects into azoospermic and oligozoospermic responders, an increase in dihydrotestosterone (DHT) concentration in seminal plasma was observed in the oligozoospermic subjects but not in those becoming azoospermic, indicating an increase in 5 alpha-reductase with the greater availability of DHT, and thereby lower spermatogenesis suppression.29
Determining the etiology of azoospermia allows physicians to identify whether the patient is amenable to treatment and to search for possible significant underlying medical disorders. An FSH level of less than 2 times greater than normal and the absence of bilateral testicular atrophy have been reported to be reliable markers to predict OA.1 In addition, Schoor et al. reported that a precise cutoff value of FSH ≤7.6 mIU/L and a testicular long axis >4.6 cm allowed for a detection rate of 96% of patients with OA, whereas using FSH ≤7.6 mIU/L or testicular long axis >4.6 cm resulted in 77% and 72% sensitivity, and 93% and 78% specificity, respectively.8 The size of the testes can be calculated by the Lambert equation (length×width×height×0.71) or with an orchidometer,30 and a long axis testicular length of 4.7 cm is equivalent to a testicular volume of 22 ml.31 Although using cutoff values of FSH ≤ 7.6 mIU/L and testis size ≥ 22 ml yielded a positive predictive value of 100% for a diagnosis of OA in the patients in the current study, it only allowed for a detection rate of 4% of patients with OA. Overall, only three of our patients had a testicular size exceeding 22 ml, including one with NOA and two with OA.
Similar to the investigation by Schoor et al., based on ROC analysis, FSH and testis size served as the best clinical indicators to discriminate between NOA and OA.8 The optimal cutoff value for FSH in the current study was 9.2 mIU/ml, which is higher compared to previous studies, which may be due to patient selection and different ethnicity. The reason why we excluded patients with hypogonadotropic hypogonadism from our NOA group is because these NOA patients can be identified by an inadequate production of gonadotropin. Therefore, excluding the 10 patients with hypogonadotropic hypogonadism may have resulted in an elevation of mean FSH level and cutoff value. Furthermore, the mean FSH levels in the NOA and OA groups were 25.4 mIU/ml and 5.6 mIU/ml, respectively, both of which are higher than those in Schoor et al.'s study (16.9 mIU/ml and 5.34 mIU/ml, respectively).8 The higher baseline FSH level in the current study is consistent with the study by Wang et al., who reported an elevated basal serum FSH concentration in Asians, and this may suggest a small relative decrease in spermatogenic reserve and/or gonadal negative feedback in Asians.9
Various methods have been used to investigate the etiology of azoospermia. Genetic testing to detect Y chromosome microdeletions or chromosome abnormalities are suggested in the initial work-up for the diagnosis of azoospermia, however these chromosome alterations only account for 15% of cases, with up to 20% of patients remaining idiopathic.32–34 In this scenario, an FSH level >9.2 mIU/ml and right testis size <15 ml will be beneficial and can avoid unnecessary testis biopsies. Our findings emphasize the necessity of adjusting cutoff values based on the ethnicity of the patient to improve the diagnostic rate.
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