Feasibility of repeat microdissection testicular sperm extraction within 6 months for nonobstructive azoospermia

1. INTRODUCTION

With the evident trend of semen quality deterioration over time, increasing infertility between couples has become a worldwide problem, particularly in industrialized countries.1,2 Intrauterine insemination (IUI) may be offered for most couples with male partners who have exhibited suboptimal semen quality and who have failed to conceive naturally, with a reported 15% to 20% pregnancy rate per cycle.3 However, in men diagnosed with nonobstructive azoospermia (NOA), the most severe form of male infertility, testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is the sole effective treatment available for infertility.4 The strategy of using freshly retrieved or frozen-thawed sperm for the first ICSI cycle in patients with NOA varies between in vitro fertilization (IVF) centers; however, given that a comparable fertility outcome, in terms of the pregnancy success rate, can be achieved using either fresh or frozen-thawed testicular sperm,5 the excess spermatozoa are typically cryopreserved following fresh ICSI for use in future ICSI cycles in most centers.6

Viable testicular sperm cryopreservation is a valuable addition to therapies, as it could reduce the number of procedures required in the male partner to ensure pregnancy success and to reduce the need for repeated TESE attempts. Nonetheless, the post-thaw recovery of viable sperm is not always guaranteed, especially in patients with NOA who have very limited numbers of viable sperm. Previous reports have indicated that the rates of sperm loss range from 6% to 20% after freezing and thawing procedures;7,8 therefore, a substantial number of men with NOA may require repeat TESE procedures. It is important to note that a resting period of at least 6 months between TESE procedures has been recommended by some experts to ensure better sperm retrieval rates and fewer complications, such as hematoma or inflammation.9

Currently, microdissection TESE (mTESE) has been adopted by most reproductive andrologists as an optimal sperm retrieval technique, as it has yielded a superior sperm retrieval rate compared with that of conventional TESE.4 Testicular spermatozoa could be collected via mTESE in 82% of repeat attempts in men with NOA who underwent a prior successful mTESE, and seemingly lower levels of follicle-stimulating hormone (FSH), along with a larger testicular volume serve as predictive indicators of subsequent successful sperm retrieval.6 Despite the fact that some have documented encouraging sperm retrieval rates for repeat mTESE procedures, no studies have investigated the sperm retrieval rate for a repeat mTESE performed within 6 months of the original procedure, and there seems to be no scientific evidence to indicate how long a patient should wait before undergoing a second mTESE for sperm retrieval. Considering that the aneuploidy rate increases with ovarian aging, which may ultimately result in decreased fecundity, for a female partner of an infertile couple with diminished ovarian reserve or advanced maternal age, a 6-month interval after a previous unsuccessful ICSI cycle could theoretically result in decreased embryo competence.10 Therefore, the purpose of the present study was to examine whether undergoing a repeat mTESE within 6 months is a feasible option for men with NOA and to determine whether such procedures are associated with good success rates.

2. METHODS 2.1. Study design and patients

After the study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital (2020-04-004BC), data on patients with NOA who had undergone repeat mTESE procedures between May 2012 and September 2019 were retrospectively collected. Informed consent was waived by Institutional Review Board of Taipei Veterans General Hospital. All methods were carried out in accordance with relevant guidelines and regulations. Each patient underwent a detailed examination to identify the etiology of the azoospermia, and the diagnosis of azoospermia was confirmed in all patients via analysis of at least two semen samples, as required by the World Health Organization guidelines.11 Preoperative clinical characteristics were recorded for each patient, including age, hormone profile, and testicular volume (measured using an orchidometer). Comprehensive endocrinological profile analysis at the time of evaluation included quantification of FSH, luteinizing hormone (LH), testosterone, prolactin, and estradiol levels. Classical cytogenetic studies using standard karyotype analysis and additional Y chromosome microdeletion testing were performed for all subjects.

These patients were advised to undergo mTESE to retrieve sperm for the following assisted reproductive techniques to allow them to have children of their own genetic makeup. The advantages and disadvantages of ICSI with fresh sperm or cryo-banked sperm were carefully discussed with each couple. If the couple would not consider donor sperm and wished to use fresh sperm for future ICSI, then a “diagnostic” mTESE was performed to avoid unnecessary ovarian stimulation alongside oocyte retrieval.

2.2. Hormone manipulation before mTESE

Hypogonadal patients (defined as those with total testosterone < 300 ng/dL) with a normal testosterone to estradiol (T/E2) ratio and an LH level in the normal range (1.24–7.8 IU/L) were administered clomiphene (50 mg/day), whereas men with low testosterone and low T/E ratios were given anastrozole (1 mg/day) initially for a period of 3 weeks, followed by measurement of serum levels of testosterone. If optimal levels of testosterone (>300 ng/dL) could not be achieved with the above treatment, a twice-weekly human chorionic gonadotropin (hCG) injection at a dose of 1,500 IU was recommended. These treatments were continued for at least 2–3 months before the mTESE.

2.3. Surgical sperm retrieval technique and reproductive outcome follow-up

The mTESE technique has been previously described in detail by Schlegel.12 Our procedure for diagnostic mTESE was performed in a similar fashion, although the tissue sampling amount was relatively limited and sufficient only for the embryologist to confirm the presence of sperm. In brief, the scrotum was incised from the scrotal median raphe, the tunica albuginea was opened, and the testicular tissues were examined under an operative microscope. Seminiferous tubules with a large diameter (≥300 μm) were sampled. The locations of the sperm-positive areas were carefully recorded. For patients with positive results (successful sperm retrieval) after the diagnostic mTESE who chose not to use cryo-banked sperm, a “therapeutic” mTESE was performed at a later date after their wives were well-prepared for oocyte retrieval. For therapeutic mTESE, sperm were retrieved directly from the area where the positive results were confirmed by the diagnostic mTESE procedure. All surgeries were performed by a single urologist. Patients were grouped based on the interval between the diagnostic mTESE and the therapeutic mTESE.

The fertility outcomes, including sperm retrieval rates, fertilization rates, embryo transfer rates, implantation rates, clinical pregnancy rates, and live birth rates were compared among the three groups that were stratified based on the time interval between the two mTESE procedures (less than 3 months, 3–6 months, and more than 6 months). Implantation was considered to be successful when a gestational sac was observed via ultrasonography after embryo transfer, and clinical pregnancy was defined as the identification of a gestational sac with a fetal heartbeat via ultrasonography. Patients with clinical pregnancies were contacted to confirm whether they had live births, either at our institute or at other clinics.

2.4. Statistical analysis

Statistical analyses were performed using Microsoft Excel and IBM Statistical Package for the Social Sciences (SPSS) Statistics version 19. Categorical variables were compared using chi-squared analyses, and continuous variables were compared using one-way analysis of variance. A p < 0.05 was considered statistically significant.

3. RESULTS 3.1. Profiles of patients with NOA and their outcomes following repeat mTESE

Data from a total of 146 patients who underwent at least two mTESE procedures were analyzed. The mean age of the patients was 37.2 (range, 26–60) years, and the mean duration of infertility of the participants in our study was 2.9 (range, 0.5–11) years. The mean interval between the two operations was 177 days, with the shortest interval being 31 days and the longest being 1,025 days. Spermatozoa were successfully retrieved in 132 (90.4%) patients at their second mTESE, resulting in 54 successful clinical pregnancies and 52 live births (Table 1). For one patient who underwent repeated mTESE at 31 days interval, sperm was successfully retrieved on the following therapeutic mTESE by examining thick seminiferous tubules from the middle part of the testis. Of note, no hematoma formation was noted during the second mTESE procedure. The timing for therapeutic mTESE is typically decided through shared decision-making by physicians and patients, according to the fertility status of the male and female partners.

Table 1 - Demographic data of men with NOA and hypospermatogenesis Characteristics N (%) or mean (range) Number of patients 146 Mean interval between repeat mTESE (days) 177 ± 160 (31–1,025) Age (years) 37.2 ± 5.0 (26–60) BMI (kg/m2) 26.7 ± 4.3 (17.3–43.4) Infertility duration (years) 2.9 ± 2.3 (0.5–11) Right testis volume (mL) 9.5 ± 5.6 (1–25) Left testis volume (mL) 9.3 ± 5.4 (1–25) Varicocele 58 (39.7%) Previous testis surgery 14 (9.6%) Karyotyping 46 XY 121 (82.9%) 47 XXY 19 (13.0%) Others* 6 (4.1%) AZFc microdeletion 24 (16.4%) FSH (mIU/mL) 19.4 ± 11.7 (0.03–60.1) LH (mIU/mL) 8.5 ± 5.8 (0.01–32.1) Testosterone (ng/mL) 3.7 ± 2.1 (0.2–18.0) Prolactin (ng/mL) 12.0 ± 8.0 (3.4–61.7) Estradiol (pg/mL) 21.0 ± 11.4 (5.0–86.1) Sperm retrieval 132 (90.4) Biochemical pregnancy 59 (40.4) Clinical pregnancy 54 (37.0) Live birth 52 (35.6)

*Others: mos 47, XY, +15[2]/46, XY [18]; mos 46 XY,inv(1)(p31q42); mos 46,X,del(Y)(q12)[28]/47,X,del(Y)(q12),+mar[2]; mos 47,XXY[40]/46,XY[10]; mos46,XY,t(13;14)(p13;q24.1).

NOA = nonobstructive azoospermia, mTESE = microdissection testicular sperm extraction, BMI = body mass index, FSH = follicle-stimulating hormone, LH = luteinizing hormone.

The characteristics and preoperative hormone profiles of all patients are shown in Table 1. These patients were categorized into three groups according to the time interval between the two mTESE procedures. Forty-four patients who underwent a repeat mTESE within 3 months were classified into group I, whereas 60 patients and 42 patients who received a second mTESE 3 to 6 months and more than 6 months after their first mTESE procedure were classified into groups II and III, respectively. Eight patients in group I, six patients in group II, and five patients in group III had nonmosaic Klinefelter Syndrome. AZFc microdeletion was also observed in eight, seven, and nine cases in groups I, II, and III, respectively. Baseline gonadotropin levels were higher than the upper limit of the normal range in all three groups, although 88 (60.3%) men reached eugonadal status after hormone optimization treatment before mTESE. Retrieval of sperm was unsuccessful in 14 patients (three in group I, six in group II, and five in group III). The testicular histology were as follows: late maturation arrest in 5 patients, early maturation arrest in 4, and Sertoli-cell only in 5. Overall sperm retrieval rates did not differ among the three groups (93.2%, 90.0%, and 88.1% in groups I, II, and III, respectively [p = 0.719]) (Table 2). With regard to the chance of obtaining sperm during repeat mTESE, no clinical or biological parameters allowed us to predict the outcome (Table 3).

Table 2 - Demographic data of men with NOA and hypospermatogenesis who underwent repeat mTESE procedures at various intervals Characteristics <3 months (I)(n = 44) 3-6 months (II) (n = 60) >6 months (III) (n = 42) p Mean interval (days) 64.3 ± 17.4 130.2 ± 25.9 363.0 ± 191.7 Age (years) 37.5 ± 6.4 36.1 ± 4.2 38.4 ± 4.1 0.060 BMI (kg/m2) 26.9 ± 4.3 26.6 ± 4.3 26.8 ± 4.5 0.930 Infertility duration (years) 2.8 ± 2.4 2.7 ± 1.9 3.4 ± 2.7 0.259 Right testis volume (mL) 8.4 ± 5.1 9.5 ± 5.1 10.8 ± 6.6 0.135 Left testis volume (mL) 8.1 ± 5.0 9.0 ± 5.1 10.8 ± 6.6 0.205 Varicocele 17 (38.6%) 20 (33.3%) 21 (50.0%) 0.235 Previous testis surgery 3 (6.8%) 7 (11.7%) 4 (9.5%) 0.709 Karyotyping 46 XY 36 (81.8%) 51 (85.0%) 34 (81.0%) 0.369 47 XXY 8 (18.2%) 6 (10.0%) 5 (11.9%) Others* 0 3 (5.0%) 3 (7.1%) AZFc microdeletion 8 (18.2%) 7 (11.7%) 9 (21.4%) 0.396 FSH (mIU/mL) 18.4 ± 9.7 22.1 ± 12.9 16.6 ± 11.3 0.054 LH (mIU/mL) 8.0 ± 5.1 9.4 ± 6.3 7.8 ± 5.8 0.301 Testosterone (ng/mL) 3.7 ± 2.7 3.6 ± 1.7 3.6 ± 2.1 0.907 Prolactin (ng/mL) 13.7 ± 10.2 12.4 ± 8.1 9.8 ± 4.1 0.073 Estradiol (pg/mL) 22.0 ± 12.2 19.2 ± 9.3 19.0 ± 13.9 0.414 Sperm retrieval (%) 41 (93.2%) 54 (90.0%) 37 (88.1%) 0.719

*Others: mos 47, XY, +15[2]/46, XY [18]; mos 46 XY,inv(1)(p31q42); mos 46,X,del(Y)(q12)[28]/47,X,del(Y)(q12),+mar[2]; mos 47,XXY[40]/46,XY[10]; mos46,XY,t(13;14)(p13;q24.1)

NOA = nonobstructive azoospermia; mTESE = microdissection testicular sperm extraction; BMI = body mass index; FSH = follicle-stimulating hormone; LH = luteinizing hormone.


Table 3 - Comparison of demographic data between those with successful and unsuccessful sperm retrieval Sperm retrieval Characteristics Yes (n = 132) No (n = 14) p Mean interval (days) 176.2 ± 164.8 188.1 ± 105.6 0.792 Age (years) 37.2 ± 5.1 37.5 ± 3.1 0.811 BMI (kg/m2) 26.6 ± 4.3 28.2 ± 4.3 0.176 Infertility duration (years) 2.9 ± 2.2 3.5 ± 3.1 0.469 Right testis volume (mL) 9.4 ± 5.4 10.9 ± 7.4 0.268 Left testis volume (mL) 8.9 ± 5.4 10.6 ± 7.2 0.205 Varicocele 51 (38.6%) 7 (50.0%) 0.409 Previous testis surgery 14 (10.6%) 0 (0.0%) 0.200 Karyotyping 46 XY 111 (84.1%) 10 (71.4%) 0.152 47 XXY 15 (11.4%) 4 (28.6%) Others* 6 (4.5%) 0 (0.0%) AZFc microdeletion 22 (16.7%) 2 (14.3%) 0.819 FSH (mIU/mL) 19.3 ± 11.4 20.6 ± 14.6 0.697 LH (mIU/mL) 8.4 ± 5.6 9.8 ± 8.1 0.411 Testosterone (ng/mL) 3.7 ± 2.2 3.3 ± 2.2 0.583 Prolactin (ng/mL) 11.9 ± 7.9 12.9 ± 9.2 0.649 Estradiol (pg/mL) 20.0 ± 12.2 19.5 ± 5.9 0.874

*Others: mos 47, XY, +15[2]/46, XY [18]; mos 46 XY,inv(1)(p31q42); mos 46,X,del(Y)(q12)[28]/47,X,del(Y)(q12),+mar[2]; mos 47,XXY[40]/46,XY[10]; mos46,XY,t(13;14)(p13;q24.1)

BMI = body mass index; FSH = follicle-stimulating hormone; LH = luteinizing hormone.


3.2. Reproductive outcomes

Assisted reproductive technology involving mTESE-ICSI was performed on 132 couples, with at least one mature oocyte injected with fresh testicular sperm. No group differences were observed in terms of the biochemical pregnancy rates (43.9%, 53.7%, and 32.4% in groups I, II, and III, respectively; p=0.133), clinical pregnancy rates (41.5%, 46.3%, and 32.4% in groups I, II, and III, respectively; p = 0.416), or live birth rates (38.6%, 41.7%, and 23.8% in groups I, II, and III, respectively; p = 0.158). Regarding other fertility outcomes, in terms of fertilization and implantation rates, comparable results were also recorded (Table 4, Fig. 1).

Table 4 - Fertility outcomes in patients with NOA and hypospermatogenesis who underwent repeat mTESE at different intervals Fertility outcomes < 3 months (I) 3-6Months (II) > 6 months (III) p Fertilization rate 53.4 ± 26.1 % 54.3 ± 29.9 % 45.5 ± 24.5 % 0.587 Biochemical pregnancy/cycle 18/41 (43.9%) 29/54 (53.7%) 12/37 (32.4%) 0.133 Clinical pregnancy/cycle 17/41 (41.5%) 25/54 (46.3%) 12/37 (32.4%) 0.416 Cumulative live birth rate 17/44 (38.6%) 25/60 (41.7%) 10/42 (23.8%) 0.158
F1Fig. 1:

Fertility outcomes for men with NOA who underwent repeat mTESE procedures at different intervals after the first operation. NOA = nonobstructive azoospermia, mTESE = microdissection testicular sperm extraction.

4. DISCUSSION

The present study investigated a group of men with NOA who underwent consecutive mTESE procedures at different time intervals after the first mTESE. Repeated attempts at sperm recovery were successful in 90.4% of all men with NOA who previously had a successful sperm extraction using mTESE and, more importantly, the chance of obtaining testicular sperm was not dependent on the interval between sperm retrieval procedures, as demonstrated in patients who underwent subsequent attempts at <3 months, 3–6 months, and >6 months after the initial procedure. These findings suggest that a repeat mTESE can be performed in as little as 3 months after the first.

The recommendation of a minimum 6-month interval between sperm retrieval procedures was proposed by several previously published articles9,13 and was based on the notion that the initial operation could induce deleterious changes to the microenvironment of the testes, thereby negatively affecting spermatogenesis and the outcome of the second attempt. Circumstantial evidence supports a delay in repeated operations based on the observation that an identifiable hematoma may still be present 3 months after TESE, characterized by a hypoechoic region within the testis parenchyma, which is theoretically likely to increase intratesticular pressure and thereby cause detrimental effects on spermatogenesis by decreasing microcirculation.9 Although inflammation or hematoma formation was evident in 82% (14 out of 17) of patients after surgery, the acute inflammatory changes had typically resolved by 6 months, and in that small-scale study, the sperm retrieval rate of repeat TESE within an interval of <6 months after the initial procedure in men with NOA was 25% (1/4), whereas the rate was 80% (12/15) in patients with an interval of >6 months between procedures.9 However, equivalent sperm retrieval outcomes using TESE for repeat attempts (82.7% vs. 76.5% for <6 months and >6 months, respectively) have been demonstrated in a subsequent large-scale study involving 156 procedures.14 However, a comparison of sperm retrieval rates based on time intervals between repeat mTESE procedures, which is standard treatment for patients with NOA, has not been reported in the literature. Okada et al and Ramasamy et al have conducted comparative studies assessing postoperative testicular structural changes at one or 3 months following two retrieval approaches, either using conventional TESE or mTESE procedures.13,15 Both studies described fewer acute and chronic testicular changes in patients who underwent mTESE based on ultrasound assessments, which may have been due to the careful and limited sampling of the tissue and meticulous hemostatic control achieved with the aid of an operating microscope. Similarly, in the current study, we removed the seminiferous tubules using microforceps or microscopic fine scissors and used microbipolar forceps for hemostasis, which allowed for precise cauterization without significant current flow that can traumatize adjacent tissue. In addition, the tunica albuginea was closed with a running 6-0 Prolene suture, as this type of suture incites minimal inflammation and dissuades a foreign body response to the tunica vaginalis, preventing any subsequent increase in intratesticular pressure. Accordingly, the intricate process of spermatogenesis appeared to be secured, as demonstrated by the 90.4% (132/146) sperm retrieval rate for repeated attempts within 6 months in our cohort. Besides, even sperm was retrieved in second mTESE according to diagnostic mTESE result, there was still some patients were failed. Ramasamy et al. had reported higher testicular volume and lower FSH level were appear to have predictive value for successful sperm retrieval at second mTESE.6 Although there was no predictive factor in our cohort (Table 3), but they may be possible factors to affect sperm recovery between two procedures.

Using fresh sperm for ICSI is associated with several disadvantages that are likely to reduce the clinical benefit of its use, and cryopreserved sperm for ICSI has been widely preferred and utilized in modern reproductive centers. These well-known drawbacks include, but are not limited to, the requirement for concurrent fresh sperm recovery on the day of oocyte retrieval and the necessity for a separate surgery for each cycle. Such disadvantages cause undue psychological distress for the couple and may potentiate devascularization of the highly dysfunctional testis.16 While the use of frozen sperm derived from the testis can circumvent up to 50% of pointless ovarian stimulation in the female partner, given that sperm recovery has reportedly been unsuccessful in 50% of men with NOA undergoing mTESE,17 the ultimate concern is whether cryopreserved testicular sperm retains its fertilizing capacity after freezing and thawing, and whether it results in comparable reproductive outcomes as that of fresh samples remains a matter of debate. Based on a growing body of emerging evidence, two recent meta-analyses concluded that there was no difference in reproductive outcomes in terms of fertilization and pregnancy rates between the use of fresh sperm or cryopreserved sperm for ICSI in men with azoospermia due to spermatogenic dysfunction; thus, there is now agreement among reproductive andrologists recognizing the equivalent efficacy of fresh and cryopreserved sperm for ICSI.18 However, a note of caution must nevertheless be added here: men with NOA who are attempting to achieve successful outcomes in ICSI using frozen-thawed testicular sperm must be carefully counseled about the fact that post-thaw identification of viable sperm is not always guaranteed. For example, some studies have estimated a 6% or 20% cellular loss in the recovery of post-thawed sperm7,8; furthermore, Schlegel et al have documented a loss in viability in up to 70% of testicular samples from men with NOA after freeze-thawing.19 Patients whose cryopreserved sperm cannot be used for ICSI should undergo a salvage mTESE. As such, many fertility specialists still prefer fresh to frozen-thawed testicular sperm.

In infertile couples, treating female partners with diminished ovarian reserve (OR) is a daunting task that should be undertaken in a timely manner. Historical studies have shown that the decreased fecundity in women begins gradually in their early 30s and accelerates rapidly after 37 years of age, as reflected by a decline in oocyte quality in combination with elevations of circulating FSH levels.20,21 Furthermore, in addition to an increasing chronological age, a low baseline level of anti-Müllerian hormone has also been identified as an independent factor associated with reduced OR, which limits the overall success of treatment with assisted reproductive technology due to its correlation with a poor ovarian response to ovarian stimulation.22 Consequently, women at risk of early depletion of ovarian follicles require immediate attention and more appropriate allocation of assisted reproductive technology to help achieve pregnancy within their shorter reproductive lifespan. Since time is of the essence for these individuals, especially those who have not experienced a successful first ICSI attempt, undergoing repeat ICSI within 6 months may be beneficial before some of their fertility potential is lost.

Several limitations of the current study need to be acknowledged. Although this is the only study conducted to date that has evaluated sperm retrieval rates in men undergoing repeat mTESE procedures within 6 months, it is limited by its retrospective nature and small sample size. Another limitation is that although the diagnostic mTESE followed by therapeutic mTESE described in this study is a typical practice in our center, it is atypical for most mTESE/IVF centers. Nevertheless, the medical expenses associated with obtaining sperm via mTESE techniques are largely covered under the government-run, single-payer National Health Insurance (NHI) system in Taiwan; thus, this unique treatment approach would not, in turn, impose a financial burden on the individuals undergoing such a procedure. Lastly, scrotal ultrasound was not performed after the first surgery; therefore, the scrotal condition was unclear, and it is impossible to say whether any inflammation or the presence of a hematoma had an impact on successful sperm retrieval. Future studies with a follow-up imaging (ultrasonography) and assessments of endocrine function are required to investigate the effects any potential testicular damage induced by mTESE may have had on the subsequent success of sperm retrieval.

In conclusion, the results of this single-institute study do not support the suggestion that sperm retrieval procedures should be performed at intervals longer than 6 months to allow for sperm recovery in men with NOA. In fact, the sperm retrieval rate exceeded 90% in repeat mTESE procedures performed within 6 months after the first procedure. There were no significant differences between the patients’ characteristics, preoperative hormone profiles, sperm retrieval rates, and fertility outcomes across the different study groups. Therefore, a repeat mTESE performed within 6 months of the first procedure does not appear to be a limitation for men with NOA, if it is required clinically.

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