Predictive factors for sperm retrieval from males with azoospermia who are eligible for testicular sperm extraction (TESE)

ABSTRACT

The aim of this study was to evaluate the predictive value of factors in infertile male patients to retrieve sperm from their testicles before they undergo testicular sperm extraction (TESE). In total, 64 males were enrolled in this study. Infertility was identified as obstructive azoospermia (OA); non-obstructive azoospermia (NOA); Klinefelter syndrome (KS); and cryptozoospermia (Crypt). Age, body mass index and concomitant conditions were noted. Testicular volumes, serum levels of Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), testosterone (T) and prolactin were investigated. Sperm retrieval after TESE was noted as positive or negative. Average age of the cases was 36.8 years. TESE success rate of testicular sperm retrieval rate was 50.0%. Majority of men (83.9%) had a 46, XY karyotype, minority had genetic anomalies, and 5 of were diagnosed with KS – all were TESE-negative. There was no statistical difference in age, testicular volume, presence of varicocele and microcalcifications or hormonal levels in the TESE-positive and negative groups. Smoking as an independent variable was the only risk factor statistically showing (OR = 0.269; p = 0.045) prediction of negative sperm retrieval after the TESE procedure. None of the parameters investigated herein predicted successful TESE outcomes. However, in cases with negative TESE only smoking was identified as a predictive factor for negative sperm retrieval and was established as a risk factor.

Abbreviations: AZF: azoospermia factor; BMI: body mass index; Crypt: cryptozoospermia; FSH: Follicle-Stimulating Hormone; ICSI: intracytoplasmic sperm injection; IU: international unit; KS: Klinefelter syndrome; LH: Luteinizing Hormone; mL: milliliter; NOA: non-obstructive azoospermia; OA: obstructive azoospermia; T: testosterone; TESA: testicular sperm aspiration; TESE: testicular sperm extraction

KEYWORDS:

• Azoospermia
• risk factors
• sperm retrieval
• testicular sperm extraction

Introduction

Azoospermia is defined as a lack of sperm in the ejaculated semen and is found in approximately 10–15% of infertile men (Irvine 1998). The most common cause of infertility in 60% of the cases is due to non-obstructive azoospermia (NOA), a condition associated with impaired production of sperm (Willott 1982), followed by obstructive azoospermia (OA), a lack of the azoospermia factor (AZF) at the c region of the Y chromosome and Klinefelter syndrome (KS). Despite there might be an impression of the absence of spermatozoa in the ejaculate, sperm from NOA, may still be retrieved due to active spermatogenesis (Eken and Gulec 2018). In 1993 for the first time, and in several later studies testicular sperm extraction (TESE)-intracytoplasmic sperm injection (ICSI) was described as a procedure to significantly increase the possibility of pregnancy (Craft et al. 1993; Schoysman et al. 1993; Vernaeve et al. 2003; Kyono et al. 2007). During the time, TESE as a surgical technique improved and variants developed; multibiopsy/conventional TESE, microdissection TESE and testicular sperm aspiration (TESA) (Craft et al. 1993; Bourne et al. 1995; Schlegel and Li 1998).

Although a higher sperm retrieval rate was described for men with NOA after microdissection TESE, this is not true for all men and depends on the type and cause of azoospermia (Deruyver et al. 2014). Successful sperm retrieval has been reported in up to 63% of men undergoing micro-TESE (Schlegel 1999; Tsujimura 2007). Although effective, the surgical procedure is not without complications; hematoma, devascularization, inflammation and a decrease in testosterone level (Eliveld et al. 2018). The procedure is also very expensive; thus, it is important to identify the males from which sperm could be retrieved (Eken and Gulec 2018). Blood hormone levels, follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, testosterone (T), testicular volume, Y chromosome microdeletions, and chromosome analysis have been used as predictors of sperm retrieval (Cetinkaya et al. 2015; Xu et al. 2016; Guler et al. 2016). In this aspect, a question arose, can we predict the successful retrieval of sperm from male patients avoiding execution of TESE? Therefore, the aim of this study was to evaluate the predictive value of factors in infertile male patients for positive sperm retrieval from their testicles before they undergo TESE.

Results and discussion

In the past, men who were diagnosed with azoospermia and desired to start a family were advised to try donor insemination or adoption. After the introduction of TESE, couples had the option of having their own biological children with intracytoplasmic sperm injection (ICSI). TESE is a routine procedure with a success rate of retrieval of spermatozoa of ~50% for men with NOA and KS, and up to 100% for men with OA (Chan and Schlegel 2000; Cissen et al. 2016; Corona et al. 2017). However, the procedure is costly and men could encounter postoperative complications, such as hematoma, devascularisation and inflammation, eventually leading to scars and calcification (Donoso et al. 2007; Eliveld et al. 2018). Therefore, the aim was to evaluate the predictive value of factors in infertile male patients for positive sperm retrieval from their testicles before they undergo TESE.

Between February 2014 and February 2018, 64 men with infertility conditions (NOA, OA, KS and Crypt) underwent TESE at the Urology Department of our clinic. Due to different pathophysiology of OA, NOA and Crypt, one patient with OA and one patient with Crypt were excluded from the analysis. Basic characteristics of the patients are presented in Table 1. Average age was 36.8 ± 4.9 years with body mass index (BMI) 26.9 ± 4.2.

Table 1. Clinical results of infertile male patients included in the study and comparison of factors between positive sperm retrieval and negative sperm retrieval after TESE.

	Total N of males (N = 62)	Sperm presence (TESE positive) n = 31	Sperm absence (TESE negative) n = 31	p-value
Age [years]	36.8 ± 4.9	37.2 ± 5.4	36.4 ± 4.4	0.519
BMI	26.9 ± 4.2	27.4 ± 5.2	26.4 ± 3.0	0.426
Smoking	15 (24.2%)	4 (12.9%)	11 (35.5%)	0.038
T [nmol/L]	17.4 ± 35.3	22.0 ± 50.3	13.2 ± 6.5	0.335
LH [IU/L]	7.4 ± 5.5	6.4 ± 5.5	8.3 ± 5.5	0.210
FSH [IU/L]	18.6 ± 12.7	16.7 ± 12.4	20.7 ± 12.9	0.241
Prolactin [mIU/L]	260.6 ± 259.5	239.4 ± 291.0	282.9 ± 227.5	0.607
Testis volume right [mL]	11.9 ± 9.8	10.8 ± 7.9	12.7 ± 11.1	0.536
Testis volume left [mL]	8.7 ± 7.1	7.7 ± 3.8	9.7 ± 9.5	0.505
Testical biopsy – right	62 (100%)	31 (100%)	31 (100%)	/
Testical biopsy – left	58 (93.5%)	29 (93.5%)	29 (93.5%)	1.000
Presence of varicocele	18 (29.0%)	7 (22.6%)	11 (35.5%)	0.263
Surgery†	11 (17.7%)	3 (9.7%)	8 (25.8%)	0.084
Presence of microcalcifications	6 (9.7%)	3 (9.7%)	3 (9.7%)	0.965
Mutations of Y chromosome	1 (1.6%)	0	1 (3.2%)	0.322
Mutation of CFTR F 508	2 (3.2%)	2 (6.5%)	0	0.143
TESE complications‡	4 (6.5%)	4 (12.9%)	0	0.039
Chromosomal profile				0.051
46,XY	52 (83.9%)	29 (93.5%)	23 (74.2%)
46,XY,inv(9)	2 (3.2%)	0	2 (6.5%)
46,XY,reciprocal translocation of 11th & 15th ch	1 (1.6%)	0	1 (3.2%)
46,XY,H63D polymorphism of HFE	1 (1.6%)	1 (3.2%)	0
46,XY,16qh+	1 (1.6%)	0	1 (3.2%)
46,XXY	5 (8.1%)	0	5 (16.1%)

BMI – body mass index; T – total testosterone; FSH – follicle stimulating hormone; LH – luteinizing hormone; ch – chromosome

†surgery included operations of varicocele testis lat. sin.; orchidectomia testis l.s. seminoma; orchidopexiam bill; appendectomiam; unilateral orchiopexy; inguinal hernia. ‡3 cases of dehiscences, 1 case of hematoma.

Overall TESE-was successful in 50% of the patients with unilateral and bilateral testicular biopsies. Sub-analysis of the respective biopsy showed 50% successful sperm retrieval in unilateral (2 out of 4 patients) and 50% of sperm retrieval in bilateral biopsies (30 out of 60 patients). There are some reports showing much higher extraction with unilateral testicular biopsies (92.6%) than compared to patients who underwent bilateral testicular biopsies (14.2%) (Güneri et al. 2016). This important difference demonstrates that the patients who had previously undergone bilateral TESE are showing a lower rate of sperm retrieval. A similar proportion was observed in the study by Eken and Gulec (2018) which described sperm retrieval rates in 65.5%. Moreover, they even managed to retrieve sperm from 4 out of 7 patients with KS. Furthermore, Okada et al. (2005) reported successful sperm retrieval in 26 (51%) of 51 KS patients in their study. In our study, we encountered 5 cases of KS, but with no success in sperm retrieval after TESE. According to the study by Okuyama et al. (2017) the testicular sperm recovery rate was 57.8% overall: 89.6% in the Crypt, 97.1% in the OA, 28.9% in the NOA, and 42.2% in the KS groups. Unfortunately, we have not analyzed separately infertility conditions among the patients, but overall retrieval coincided with the reports in the literature of about half of the patients (Schlegel 1999). The majority of men in our study (83.9%) had a normal karyotype with 46,XY chromosomal profile, a minority had genetic anomalies, and five of them were diagnosed with 46,XXY knows as KS.

Normal FSH levels range from 1.3 to 13.58 IU/L, while the mean FSH level of the patients in our study group was relatively higher (18.6 ± 12.7 IU/L). Right, and left mean testicular volumes were 11.9 ± 9.8 mL, and 8.7 ± 7.1 mL which were lower than normal values (18–20 mL). As expected, levels of serum FSH, LH, and prolactin were high in patients with negative TESE. These patients had lower serum concentration of T. A decrease in serum testosterone levels in men after TESE procedures have also been described (Donoso et al. 2007; Shin and Turek 2013; Eliveld et al. 2018), but were not measured in our study. Decreased testosterone levels can lead to hypogonadism and were most profound in men with KS. Recovery to baseline serum levels occurred after 18–26 months (Guler et al. 2016). However, the differences in hormonal levels were not statistically significant between the groups. Although studies have included various parameters such as testicular pathology, levels of FSH, inhibin, testosterone, testicular volumes, genetic variables, and diagnostic testicular biopsy, only testicular biopsy, FSH, and testicular volumes have been identified as predictive factors for sperm retrieval. Increased FSH levels and small testicular volumes have been associated with testicular sperm failure (Tournaye et al. 1997; Su et al. 1999; Ishikawa et al. 2004). Even though this inverse proportion is well-known, in one third of the patients with this spermatogenetic defect, normal FSH levels were detected. Thus, defective spermatogenesis can be observed in patients with normal FSH levels and in patients with high FSH levels. According to our results, TESE-negative patients had higher levels of FSH, but the difference was not statistically significant; thus, FSH was not evaluated as a predictive factor. Testicle volumes, were measured for both testes (left and right) where TESE-positive patients had smaller volume in comparison to TESE-negative patients (not statistically significant). This does not coincide with the previous reports, but it has been reported in the literature that there is no statistically significant difference in testicular volume between NOA patients in whom sperm could be retrieved and NOA patients in whom sperm could not be retrieved (Nicopoullos et al. 2004; Okuyama et al. 2017).

FSH, LH and prolactin levels were higher in TESE-negative men in our study. However, the T levels were lower in these men. Comparisons were not statistically significant. In general, low T levels were reported in men with NOA and OA (Sussman et al. 2008). The same results were observed by Okuyama et al. (2017). The associations between hormones FSH, LH, prolactin, T and the presence of spermatogenesis are not straightforward. Serum levels of all hormones have poor predictive values for TESE. However, the result that FSH and LH were higher in the TESE-negative cases simply reflects that they are usually higher in a NOA cohort, compared to others such as OA, KS or Crypt cases, but as mentioned in our study patients were not divided according to infertility condition. The reports investigating levels of FSH, LH and T for sperm retrieval show that high FSH and LH levels have the possibility that sperm retrieval will fail (Xu et al. 2016; Guler et al. 2016). Moreover, LH levels can change in patients with NOA. Based on the data by Güneri et al. (2016) average LH levels of NOA patients were within normal limits, LH levels in the TESE-negative group were found to be significantly higher versus TESE-positive group and a positive correlation was found between levels of LH and FSH.

Table 2 shows the calculated predictive values of factors with accompanied odds ratios (OR) that could potentially influence positive sperm retrieval. The hormonal level was not intended to set the criteria for the positive or negative result of sperm retrieval. It is important to consider the patient’s characteristics. Male age was considered to be a predictive factor (Kyono et al. 2007; Corona et al. 2017). In the evaluation of demographic data, patients’ age was similar between TESE-negative (36.4 years) and TESE-positive (37.2 years) groups, showing a bit higher age for TESE-positive group. This is similar to Güneri et al. in 2016 (38 vs. 42 years of age). Theoretically, in older patients, remarkably lower rates of successful outcomes might be expected. Therefore, with aging concomitant diseases might emerge which adversely affect testicular perfusion, and germ cell loss. In a study of Okada et al. (2005), patient’s age was demonstrated as the factor affecting TESE outcomes. Median ages in TESE-negative TESE-positive group were significantly different (38 (28–43), and 31 (25–40) years, respectively). We observed that smoking status could indicate a potential factor for negative sperm retrieval (p = 0.038). Only 4 out of 15 smokers (26.7%) patients were TESE-positive. Kovac et al.’s (2015) review highlighted that smoking can have unfavorable effects on male semen parameters, and may reduce the success of reproduction techniques, such as in vitro fertilization (IVF) and (ICSI). Smoking was associated with a poorer semen quality, lower sperm count and an increase in the number of morphological defects of spermatozoa including head, neck and tail defects. Reasons associated with smoking might be attributed to increased oxidative stress and in-sufficient scavenging by antioxidant enzymes in the seminal fluids or contribution of zinc (Bundhun et al. 2019). Tobacco smoke contains more than 4,000 compounds including nicotine, tar, carbonic monoxide, polycyclic aromatic hydrocarbons, radioactive substances, heavy metals, etc., which are toxic and can affect sperm. Elevated oxidative stress, DNA damage, and cell apoptosis may play important roles and are reflected in sperm parameters (Dai et al. 2015). The review of Dai et al. (2015) provides additional detail. However, smoking’s effect on TESE outcome or testicular histopathology of men with NOA has never been evaluated. There was a similar retrospective study by Shrem et al. (2019) on 52 azoospermic men. They found significantly fewer motile sperm in the smokers compared to the non-smokers, but showed no effect on the pregnancy and delivery rates between the two groups. However, the authors agreed that smoking is associated with decreased IVF–ICSI success rates as reported before. The issue with both studies is that we have only analyzed 15 smokers and they have analyzed 34 smokers. In the future, we recommend conducting additional studies with higher sample sizes so that adequate power is attained.

Table 2. Predictive values of factors in infertile male patients for positive retrieval of sperm from testicles before they undergo TESE.

	B	OR	95% CI	p-value
Age [years]	0.035	1.035	0.933–1.149	0.513
BMI	0.058	1.059	0.921–1.218	0.420
Smoking	−1.312	0.269	0.075–0.971	0.045
T [nmol/L]	0.012	1.012	0.979–1.045	0.484
LH [IU/L]	−0.067	0.935	0.841–1.041	0.219
FSH [IU/L]	−0.026	0.975	0.934–1.017	0.238
Prolactin [mIU/L]	−0.001	0.999	0.997–1.002	0.601
Testis volume right [mL]	−0.021	0.980	0.919–1,044	0.528
Testis volume left [mL]	−0.043	0.958	0.845–1.085	0.495
Varicocele	−0.634	0.530	0.173–1.622	0.266
Surgery	−1.222	0.295	0.070–1.243	0.096
Microcalcifications	−0.038	0.963	0.178–5.211	0.965
Mutations of Y chromosome	−21.203	0	0	1.000
Mutation of CFTR F 508	21.312	1	0	0.999
TESE complications	21.341	1	0	0.999
Chromosomal profile	−0.690	0.502	0.248–1.016	0.055

Odds ratios (OR) were calculated with binary logistic regression, where sperm retrieval presented the dependent variable. B value was determined as the correlation coefficient between the independent and dependent variables.

There was no significant difference in other factors, such as age, testicular volume, or presence of varicocele and microcalcifications in patients who were TESE-positive or negative. Their chromosomal profiles did not differ between the groups, but fewer patients with normal karyotype 46,XY profile were found in TESE-negative group. Based on our data, karyotypic anomalies and Y-chromosome microdeletion were primarily observed in all TESE-negative cases; however, there were 2 cases of mutation of CFTR F 508 and 1 case of H63D polymorphism of HFE. When patients were evaluated based on TESE outcomes, the differences were not statistically significant, most probably due to a low number of patients included in the study. As described, sperm retrieval attempts failed in the majority of patients with karyotypic anomalies.

In 11 (17.7%) out of 62 patients, history of genital region surgery was elicited. The patients had previously undergone a series of different operations including varicocele testis lat. sin.; orchidectomia testis l.s. seminoma; orchidopexiam bill; appendectomiam; unilateral orchiopexy; inguinal hernia. Patients also had a history of concomitant disease that might affect fertility which included varicoceles in 18 cases and microcalcifications in 6 cases.

Four cases of complications were identified in males who underwent TESE (1 case of hematoma and 3 cases of dehiscences). All complications were in patients who had positive sperm findings after TESE, which was statistically different comparing to the TESE-negative group (p = 0.039).

The study presented here had some limitations. A major limitation is the retrospective study design with a low number of patients. The study was designed as a single-center study, so we encourage other researchers to conduct larger studies including more patients, potentially from more centers. Moreover, we did not distinguish between the diagnosis of our cohort to NOA, OA, KS and Crypt cohort or respective condition on pathologic subgroups, but we excluded one patient with OA and one patient with Crypt. Among NOA patients who had undergone TESE, the success rates varied according to pathologic subgroups. According to the difference between histopathological subgroups, clinicians can evaluate the outcomes of testicular biopsy more precisely before the TESE procedure. Lastly, we did not analyze the outcome of ICSI and associated factors for pregnancy outcomes.

This study has demonstrated that the parameters examined, namely hormonal levels, patients’ characteristics or genetic anomalies, do not provide clear predictive factors for successful sperm retrieval, in patients with azoospermia. However, the findings can provide important clues. The success rate of TESE was approximately 50% and was much lower in patients who were smokers. After detailed information of the patients who had previously undergone a bilateral procedure, it is important to understand the patients’ background, such as smoking status, so that they can understand that the probability of successful TESE is much lower in such cases irrespective of the potential for complications after TESE.

Methods

Study design and patients

The study was designed as a retrospective analysis of the clinical records of 64 men with infertility conditions such as non-obstructive azoospermia (NOA), obstructive azoospermia (OA), Klinefelter syndrome (KS) and cryptozoospermia (Crypt). All included patients underwent TESE at the Urology Department of the Merkur Clinical Hospital, Zagreb, Croatia between February 2014 and February 2018.

Clinical assessment of infertile men was done. Each patient underwent a physical examination, semen analyses, and endocrinologic evaluations. Patients' semen analyses were performed at two separate occasions. The following parameters were recorded for each patient: medical history, physical and urogenital examinations, the presence or absence of a varicocele and a hormone profile. Orchidometer was used for the measurement of testicular volumes.

Laboratory analyses

Endocrinologic evaluations including serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were performed. These were done 1–2 months prior to the TESE procedure. Venous blood samples drawn from the forearm of the patients were evaluated with the immune assay method (Access 2, Beckman Coulter, CA, USA).

Chromosomal analyses of the patients were indicated with cytogenetic and Y chromosome microdeletion analyses. In cytogenetic studies, for chromosomal analysis, peripheral blood samples drawn into heparinized tubes were incubated on phytohemagglutinin (PHA) media and cultivated at 37°C for 72 h. Forty-five minutes before the study, cells cultivated in colcemid-added culture media were lysed in hypotonic solution and fixed with Camoy’s fixative. The chromosomes retrieved were analyzed using Giemsa banding technique.

TESE procedure

TESE for each patient was timed before the partner’s enrolment in the ovarian stimulation cycle. All TESE procedures were performed by the urologist with expertise in microsurgery. Procedures were performed under local anesthesia with the patient positioned on the operating table in a supine position. Any viable sperm was collected and prepared for later use in ICSI. Patients were discharged on the day of operation. Prior to discharge, patients were examined for scrotal hematoma.

Ethical approval

The study procedure was conducted according to the Declaration of Helsinki and approved by the departmental committee. Patients did not need to give the consent of participation, since the data were obtained from our database.

Statistical analyses

The data collected were analyzed with the statistical software SPSS 21.0 (IBM, New York, USA). Statistical significance for all tests was set at p-value less than 0.05. Numeric variables were analyzed by a quantitative t-test for normally distributed data or by Mann–Whitney U-test in case of abnormally distributed variables. Statistical power and probability of type II error (beta) were determined by the use of the Bonferroni correction and observed at 0.80. Therefore, type II error probability was calculated at 20%. Pearson’s chi-square test was used to find qualitative statistical significance between positive sperm retrieval as a dependent and each predictive factor as an independent variable. A binary logistic regression with odds ratios was calculated to predict positive sperm retrieval from male patients who were eligible for TESE with the help of Hosmer-Lemeshow goodness of fit, which was statistically significant with smoking as the parameter.

Authors’ contributions

Conceived and designed the experiments: DPJ; Acquisition of the data: DPJ; Analysis and interpretation of the data: DPJ, DS, TJ, AS; Drafting of the manuscript: DS, TJ; Critical revision of the manuscript for important intellectual content: DS, AS; Statistical analysis: DS, TJ; Supervision: DS.

Disclosure statement

None of the contributing authors have any conflict of interest, including specific financial interests or relationships and affiliations relevant to the subject matter or materials discussed in the manuscript.