Endocrine Tests and/or Testicular Volume Are Not Predictive of Successful Sperm Retrieval by Multiple Testicular Sperm Extraction in NonObstructive Azoospermia

Objectives: This study aimed to determine the role of pre-surgical markers in the prediction of sperm retrieval in infertile Vietnamese men with azoospermia. Patients and Methods:Retrospective descriptive analysis of 136 infertile men with azoospermia, examined from August 2014 to July 2018. Patients underwent stepwise surgical sperm retrieval via percutaneous epididymal sperm aspiration, testicular sperm aspiration then multiple testicular sperm extraction in up to 3 locations until sperm were detected. Factorswere analyzed to determine the prediction of sperm retrieval. Results: The overall success rate of sperm retrieval was 49.3% including 88.3% and 18.4% in the OA and NOA group, respectively. The results of sperm retrieval were signicantly associated only with the OA and NOA group, not with endocrine test or testicular volume. We found no signicant difference in the endocrine test and testicular volume’s result between successful and unsuccessful sperm retrieval in either group. Conclusions: Neither an endocrine test nor testicular volume should be used for predicting the results of surgical sperm retrieval in infertile Vietnamese males with azoospermia.


Introduction
Azoospermia is one of the major reproductive disorders. It is reported in approximately 1% of males and 10-15% of infertile males,]. [1,2] It includes non-obstructive azoospermia (NOA) and obstructive azoospermia (OA). [3] OA is less common than NOA and occurs in 15 to 20% of men with azoospermia.
[4] NOA indicates impaired sperm production of the entire testis, although it has been observed that focal normal spermatogenesis can occur in 50-60% of men with NOA. [5] In fact, abnormal spermatogenesis may be caused by any factor related to the hypothalamic-pituitarytesticle axis, including the effects of follicle stimulating hormone (FSH), luteinizing hormone (LH), or testosterone on the function of the testis.
[6] Due to the pathophysiology, NOA and OA are managed differently. While sperm retrieval directly from the epididymis or testis had a dramatically high success rate in OA patients, NOA patients may have fewer chances to be father via surgery which may achieve successful sperm extraction. [7] With the achievement of assisted reproductive technologies (ART), almost every infertile man with azoospermia is a candidate for surgical sperm retrieval through percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), or multiple testicular sperm extraction (TESE) to extract sperm for treatment by the intracytoplasmic sperm injection (ICSI). Unfortunately, mature sperm are not found in certain cases and these invasive procedures may be futile. Therefore, it is important to have certain noninvasive testing that is able to predict spermatogenesis. Currently, an accurate means of predicting spermatogenesis and sperm retrieval success do not exist. Testicular function can be assessed by clinical examination, hormonal tests, semen analysis, scrotal ultrasound and biopsy of the testis.
[8] Based on the activities on the hypothalamic-pituitary-testicle axis, serum FSH and LH have been reported as important preoperative markers re ecting testicular function.
[6] Serum FSH and LH concentration is inversely related to the sperm retrieval rate. [9] In a recent meta-analysis reported by Qi Yang et al. FSH had a moderate value in independently predicating successful sperm retrieval rates in men with NOA (area under curve >0.7). Therefore, they suggested that more detailed diagnostic testing should be examined in the future in order to con rm the diagnostic value of other noninvasive parameters. [10] Scrotal ultrasound has become a more common method in the assessment of the testicle. The sensitivity and speci city of varicocele detection approaches 100% with color Doppler ultrasonography. [11] Some studies have shown that measuring the testicular volume by ultrasonography is more accurate than by the Prader orchidometer and the formula L x H x W x 0.71 is the most accurate for calculation of the testicular volume. [12] Testicular volume has been noted to have a direct correlation with semen pro les [13,14] as well as the production ability of sperm in males. [15] It is reported that the recovery of spermatozoa is successful in approximately 50% of cases and therefore the prediction of the success rate of sperm retrieval is necessary before attempted surgery.
[16] By using current popular method in sperm retrieval, micro TESE, no single clinical nding or test that is able to predict successful sperm retrieval precisely has been found. Furthermore, a variety of optimal cut-off values for testes volume and endocrine pro les for infertile males have been reported. [9,[17][18][19][20][21][22] This is possibly due to the effects of the study population and ethnicity. [23] This study aimed to determine the role of pre-surgical markers in the prediction of sperm retrieval in infertile men with azoospermia in a Vietnamese population.

Patient selection and evaluation
This was a retrospective cohort design which included 136 infertile males with azoospermia on semen analysis based on the World Health Organization (WHO) 2010 standard criteria (i.e. no sperm after centrifugation of total ejaculate in at least two semen analyses). The men presented at the Hue Center for Reproductive Endocrinology and Infertility (HUECREI), Hue University Hospital from August 2014 to July 2018. Azoospermia is diagnosed by the complete absence of spermatozoa in the semen after two different centrifuged samples (3000 x g for 15 minutes). [3] Azoospermia patients with FSH ≥ 7.6 mIU/ml with testicular long axis ≤ 46 mm were categorized into the NOA group. The remaining patients belonged to the OA group. [24] Patients with acute systemic diseases, acute urinary tract infection, hepatic function disorders, malignant diseases, retrograde ejaculation, and hypogonadotropic hypogonadism were excluded. All patients' general characteristics including age, geography, education, occupation, clinical history, physical examination, infertility type, infertility duration, history of mumps, history of surgery on reproductive urinary tract and testicular density were recorded.

Scrotal ultrasound technique
Testicular ultrasound was performed to evaluate testicular echogenicity and homogeneity in 2D Ultrasound and the presence of varicocele was assessed in color Doppler ultrasound (CDUS). Ultrasounds were performed in a warm room with the patient in a supine position and the penis resting on the lower abdomen. The testes were examined in at least two planes, along the long and transverse axes and each testicle was measured in three dimensions (length, width, height). The volume was then calculated by the Lambert formula V=0.71xLxWxH. We used a high frequency linear probe (7.5 MHz) for both power and spectral CDUS with Samsung Medison R5, Korea. Assessment of the varicocele was performed by measuring the largest diameter and re ux in the vessels before and after the Valsalva maneuver. Color Doppler parameters were optimized for the evaluation of slow ow (wall lter at minimum levels, gain at the maximum level permitted by the presence of artifacts, and elective focus on the region of interest) and standardized using a simpli ed version of the Sarteschi classi cation. [25] All examinations were performed by the same ultrasonographer.

Hormone assays
Venous blood samples were collected on the day of recruitment after the second semen analysis con rmed azoospermia. LH, FSH, and total testosterone levels were assessed by electrochemiluminescence (ECLIA) using Elecsys and Cobas E immunoassay analyzers (Cobas 4000/6000, Roche Diagnostics, Indianapolis, USA). The FSH's inter-assay coe cient of variation (interassay CV) is 3.95%, LH's is 2.1%, prolactin's is 3.8% and testosterone's is 3.35%. All measurements were performed at the Hue University Hospital laboratory as per manufacturer instructions.

Surgical sperm retrieval procedures
All patients underwent surgical sperm retrieval via three techniques: percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), and then multiple testicular sperm extraction (TESE) consecutively, up to three locations until sperm was found. Each procedure was carried out on the right and then the left testis in turn. The scrotum was cleaned with antiseptic and then washed with saline to eliminate any residual antiseptic. In the PESA procedure, the head of the epididymis was palpated and then stabilized between the thumb and fore nger. It was then punctured, directly through the scrotal skin. The needle was gently advanced into the epididymis. The aspirated uid was checked for sperm. TESA was performed with a 20G butter y needle in 3 different positions on the testis while applying suction with a 1 ml Becton-Dickinson syringe. If both the TESA and PESA failed to retrieve sperm, a TESE was then performed by making an incision (3 cm), just above each side of the testis, resecting up to three pieces of testicular tissue and examining it under a microscope at x300 magni cation to detect spermatozoa. The surgical procedure was stopped at any step once the lab staff found sperm successfully or after unsuccessful TESE on both sides, that is, if absolutely no sperm was found.

Statistical analysis
Statistical analysis was performed by Stata version 15.0. Patients with successful versus unsuccessful sperm recovery were compared with a Chi-square test, and an independent sample T-test. If the data were not normally distributed, then the Wilcoxon rank sum test (Mann-Whitney U test) was used. The normality distribution of the data was tested by the Shapiro-Wilk normality test. The threshold for statistical signi cance was con rmed as P<0.05.

Results
A total of 136 men with azoospermia, including 60 OA and 76 NOA patients, were divided into two groups according to the results of surgery. Surgical sperm retrieval was successful in 67 of 136 patients (49.3%). The age range of the successful sperm retrieval group was 26-70 years (mean of 37.4 ± 10.1 years) whereas the age range was 24-50 years (mean 33.8 ± 5.7 years) for the patients in the unsuccessful sperm retrieval group.
The general and clinical characteristics of the OA and NOA groups are presented in Table 1. There was no signi cant difference between the 2 groups regarding the patient's age, geography, education, occupation, infertility type, infertility duration, history of surgery and/or mumps. There were signi cant differences in testicular density between the 2 groups (P<0.001). Table 2 describes the baseline demographics of successful and unsuccessful semen retrieval in the OA and NOA groups. In terms of the OA group, there was no signi cant difference in the clinical characteristics between the two groups. The primary infertile patient's rate of unsuccessful sperm retrieval group in the NOA group was much higher than that of the successful sperm retrieval group (98.4% vs. 78.6%, P=0.018). Other characteristics were not signi cant between the two groups.
The comparisons of endocrine tests and testicular volume between two groups according to the sperm retrieval outcomes are shown in Table 3. In both the NOA and OA groups, men with successful sperm retrieval had a greater mean testicular volume than the unsuccessful sperm retrieval men, however the difference was not statistically signi cant (NOA group: 5.68±2.37 vs. 4.46±2.83, P=0.138; OA group: 14.90±7.47 vs. 11.14±5.94, P=0.208). The differences in the endocrine tests between the two groups was also not signi cant in terms of LH, FSH and testosterone (P>0.05).

Discussion
This study investigated the predictive value of ultrasound parameters and hormone levels for successful sperm retrieval in azoospermia. The sperm retrieval rate in this study was 49.3% which was similar to the results of Salehi  showed that the sperm retrieval rate was 42% when they performed micro-TESE for NOA patients. [27] The various results between these studies could be partially explained by the various characteristics of the patients (OA, NOA) and the sperm recovery methods (TESA, PESA, TESE or Micro-TESE).
FSH is a glycoprotein that stimulates Sertoli function and some studies considered that it is associated with androgen production, which is necessary for fertility. Azoospermia has been a consistent nding in men with loss of function mutations in FSHβ. [28,29] Serum FSH could predict the existence of sperm which could be retrieved by conventional TESE. [27] Elevated plasma levels of FSH above 19.4 mIU/mL can suggest no spermatogenesis and hence an unsuccessful sperm retrieval. [19] However, the predictive value of FSH remains contentious, with certain studies showing that FSH has a low predictive value for sperm retrieval. Although serum FSH is useful marker for the evaluation of the presence of sperm in NOA patients, it is dependent on the pathologic etiology of azoospermia and may not always be a reliable predictive factor. [22,23] The various demographic characteristics in each study may explain the differences in these results. Li et al. considered FSH to only re ect the global spermatogenesis function but stated that FSH cannot determine the function of an isolated area in a testis. Micro-TESE is able to retrieve sperm even if the global spermatogenesis function of the testis is very low. This could be the reason why FSH could not precisely predict the sperm retrieval rate of micro-TESE. [23] In our study, we found that the FSH levels did indeed differ greatly in patients with whom sperm was retrieved successfully vs patients where sperm was not retrieved. However, the difference was not signi cant (see Table 3). In the NOA group, the median FSH level at which sperm was retrieved was found to be 23.17 IU/L versus 32.78 IU/L in men where sperm was not retrieved (P=0.062). Besides, 5.82 and 5.93 were the values of FSH in successful and unsuccessful semen retrieval in the OA patients, respectively.
LH is not commonly used as a predictor of sperm retrieval outcome. In males, LH stimulates Leydig cells to make and release testosterone into the testes and the blood. In contrast to FSH, LH appears to have little role in spermatogenesis outside of inducing gonadal testosterone production. Cissen et al. found that LH was also predictive for successful sperm retrieval. [9] Enatsu et al. demonstrated that older age and non-idiopathic etiology were signi cantly associated with the probability of successful sperm retrieval. However, they found no signi cant effects on testicular volume, FSH, LH, or testosterone on sperm retrieval. [20] Our data also found no signi cance difference of LH value between the two groups in OA and NOA patients (P>0.05) In our study, varicocele was found in 16 cases (11.8%). Varicoceles were present in 26% of Chinese male patients with infertility. [30] The diagnosis of varicocele by physical examination has a speci city of approximately 70%. In contrast, spectral Doppler analysis has a sensitivity of approximately 97% and a speci city of 94%. [31] Levinger U et al. found that the prevalence of varicocele increases with age. The prevalence of a varicocele increases 10% for each decade of life with the prevalence reaching 75% in the eight decade. [32] Although the relationship between varicocele and male infertility has been demonstrated in many studies, the exact mechanism of the effect of a varicocele on spermatogenesis remains unknown. [30] In our study, there was no signi cant difference between varicoceles and sperm retrieval outcomes. Scrotal ultrasound has a role in the diagnosis of the etiologies of male infertility. Abdulwahed et al. divided 268 infertile males into an OA versus NOA groups based on histopathology. The authors found, using ultrasonography, that decreased testicular volume and varicoceles (intra-and extra-testicular) were the most common abnormalities in NOA patients. In contrast, epididymitis, spermatocele and duct ectasia were the most common in OA patients with a sensitivity 87% but only 30% speci city. Therefore, scrotal ultrasound is used to exclude these diagnoses. [17] Sharath et al. (2013) found that a mean testicular volume is 15 ml (right testis 7.62±4.056 ml, left testis 6.99±3.60 ml) in males with an abnormal semen analysis. [14] There is a distinction between the selection of sperm recovery methods in OA patients (TESA, Percutaneous Biopsy, PESA, MESA) vs NOA patients (TESE, micro TESE). In particular, we found that the median testicular volume was 14.46 ml (right testicular volume 14.32 ml and left testicular volume 14.60 ml) in OA patients compared to 4.68 ml (Right testis 4.67 and left testis 4.69 ml) in NOA patients. According to Moon et al. testicular volume was found to be statistically signi cant between the OA and NOA patients (mean testicular volume of OA and NOA patients was 11.6 ml and 8.3 ml, respectively, P<0.05). [33] Huang et al. showed that a combination of FSH >9.2 mIU/ml and right testis size <15 ml may be used to distinguish NOA patients from OA patients. The positive predictive value for NOA patients was 99.2% and for OA patients was 81.8%. [21] In fact, many studies showed that testicular volume was not reduced in OA patients. Despite the difference in testicular volume between these studies, the majority of studies showed that the mean total testicular volume in infertile males was lower than normal. There was an insigni cant difference in total testicular volume between the successful and unsuccessful sperm retrieval groups (5.68 ml versus 4.46 ml in NOA group, P=0.138; 14.90 ml versus 11.14 ml in OA group, P=0.208). Tang WH et al. and Enatsu N et al. found that the testicular volume of the successful sperm retrieval group was higher than that of the unsuccessful sperm retrieval group, however there was no statistical signi cance between the two groups. [20,34] Salehi et al. (2017) performed TESE and micro-TESE in 170 NOA patients and found that the sperm recovery rate was 48.8% and that testicular volume was related to the surgical sperm retrieval results (OR,10.5, P<0.01).
[16] The result was similar to Boitrelle's study. [18] In our study, successful sperm retrieval in the OA and NOA groups was 88.3% and 18.4%, respectively (P<0.001). In the OA group, sperm production was totally normal, so the sperm retrieval rates via TESA or PESA was very high. Levine et al. assessed the effect of PESA and TESA in the NOA and the OA groups and found that SRR in the NOA group was 47% and OA this rate amounted to 100%. [35] In fact, it is di cult to predict the outcome of surgical sperm retrieval with only testicular volume or any hormone value as many factors can affect the process of spermatogenesis. Therefore, a combination of these values may prove to be more reliable. However, after multivariate logistic regression analysis, we found no signi cance difference between the two groups according to the results of semen retrieval. Boitrelle et al. found that there was no signi cant difference between the two groups (sperm present and no sperm) with age, testosterone, or LH. However, they found that a combination of FSH concentration, inhibin B, and total testicular volume were the best predictors of TESE outcomes (AUC=0.663).
[18] Li H et al. analyzed ve studies with a total of 1764 patients involving testicular volume and found AUC=0.6389, indicating a low predictive value. [23] Boitrelle et al. developed a formula to predict TESE outcome that included three parameters: total testicular volume, FSH and inhibin B. This formula was shown to be the best predictor of successful TESE (positive likelihood ratio:+3.01).
[18] Otherwise, certain studies showed that testicular volume did not affect the sperm retrieval rate for micro-TESE. These studies suggested that testicular volume was an in uential factor in successful sperm retrieval, as it is correlated with spermatogenesis, however topographical variations in testicular pathology can occur. Consequently testicular volume may not be a good predictive factor for successful sperm retrieval for ICSI. [22,36] Ramasamy et al. found that BMI and age had no predictive value for sperm retrieval rates [37,38] which was similar to the ndings in our study. From our research, preoperative variables, namely FSH and testicular volume could not be used as a predictive factor for the success of sperm retrieval in men with non-obstructive azoospermia. Histopathological examination has been the most reliable predictive factor of SRR to date. However, it is contraindicated to perform a testicular biopsy just to predict the SRR of microdissection TESE. [23] We believe that a trial for sperm retrieval should not be denied to any man with azoospermia based solely on the values of the preoperative variables. The patient should be informed, prior to the operation that nding mature cells may not be guaranteed (even in cases with normal FSH and testicular volume) and allowed to make an informed decision on whether to proceed.
In conclusion, in a variety of optimal cut-off values for testis volume and endocrine pro les for men with azoospermia have been reported with controversial results. This study demonstrated that testicular volume and endocrine tests should not be used as predictive factors for sperm retrieval outcomes in infertile males with azoospermia, both in the OA and NOA groups.    All participants were explained and agreed with written consent form.
Competing interests: The authors have no competing nancial or other interests to declare in relation to this manuscript.