分步法小切口改良睾丸显微取精术在隐睾下降固定术后非梗阻性无精子症的临床应用研究

Modified stepwise mini-incision microdissection testicular sperm extraction: a useful technique for patients with a history of orchidopexy affected by non-obstructive azoospermia 分步法小切口改良睾丸显微取精术在隐睾下降固定术后非梗阻性无精子症的临床应用研究 1 2 first-author 1 1 3 1 1 4 1 1 1 1 1 1 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:lizhengboshi@sjtu.edu.cn †E-mail:lizhengboshi@sjtu.edu.cn lizhengboshi@sjtu.edu.cn Department of Andrology, Center for Men’s Health, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China Department of Andrology, Center for Men’s Health, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China Department of Urology, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China Department of Urology, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China Reproductive Medicine Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China Reproductive Medicine Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China Department of Urology, Zhoushan Hospital, Zhoushan 316021, China Department of Urology, Zhoushan Hospital, Zhoushan 316021, China Non-obstructive azoospermia (NOA), which is defined as the absence of spermatozoa in the ejaculate secondary to impaired spermatogenesis within the testis, may be caused by a variety of etiologies, including varicocele-induced testicular damage, cryptorchidism, prior testicular torsion, post-pubertal mumps orchitis, gonadotoxic effects from medications, genetic abnormalities, chemotherapy/radiation, and other unknown causes currently classified as idiopathic (Cocuzza et al., 2013 2013 ). The microdissection testicular sperm extraction (micro-TESE) technique involves a meticulous microsurgical exploration of the testicular parenchyma to identify and selectively extract larger seminiferous tubules that carry a higher probability of complete spermatogenesis (Schlegel, 1999 1999 ). The Cornell group evaluated the efficacy of micro-TESE in 152 NOA patients with an associated history of cryptorchidism. In their series, spermatozoa were successfully retrieved in 116/181 attempts (64%), and the resulting pregnancy rate was 50% with a delivery rate of 38% (Dabaja and Schlegel, 2013 2013 ). Franco et al. ( 2016 2016 ) described a stepwise micro-TESE approach in NOA patients, which was considered to reduce the cost, time, and effort associated with the surgery. Alrabeeah et al. ( 2016 2016 ) further reported that a mini-incision micro-TESE, carried through a 1-cm equatorial testicular incision, can be useful for micro-TESE candidates, particularly in patients with cryptozoospermia. We conducted a retrospective study of 20 consecutive NOA patients with a history of orchidopexy from May 2015 to March 2017. 目的 2 探讨分步法小切口改良睾丸显微取精术在隐睾下降固定术后非梗阻性无精子症的有效性. 创新点 2 既往有隐睾下降固定术手术史的非梗阻性无精子症的患者睾丸局部粘连严重,所以睾丸显微取精术时睾丸暴露困难,因此手术难度明显增大.术者将常规睾丸显微取精术改良为分步法小切口睾丸显微取精术,术中无需完全游离暴露睾丸,仅通过小切口睾丸显微取精术就可使一半的患者成功获取精子.因此分步法小切口改良睾丸显微取精术可在不降低睾丸取精成功率的前提下缩短手术时间和减少手术创伤. 方法 2 收集2015年5月至2017年3月于上海交通大学附属第一人民医院泌尿中心男科接受睾丸显微取精术的20例既往有隐睾下降固定术手术史的非梗阻性无精子症,采用分步法小切口改良睾丸显微取精术获取睾丸精子.分步法小切口改良睾丸显微取精术具体步骤为:第一步,小切口睾丸取精术;第二步,将小切口延长为标准切口,行标准睾丸显微取精术;第三步,对侧睾丸标准睾丸显微取精术. 结论 2 小切口睾丸显微取精术可快速有效获取睾丸内表浅位置饱满的生精小管,标准的睾丸显微取精术可作为小切口睾丸显微取精术失败时的补救措施.分步法小切口睾丸显微取精术可降低睾丸损伤前提下,快速有效地获取睾丸精子.这种手术方式特别适用于睾丸内存在表浅饱满生精小管的非梗阻性无精子症,如隐睾下降固定术后的非梗阻性无精子症. Non-obstructive azoospermia Microdissection testicular sperm extraction Orchidopexy Cryptorchidism 非梗阻性无精子症 隐睾 睾丸显微取精术 睾丸下降固定术 精子获取 Non-obstructive azoospermia (NOA), which is defined as the absence of spermatozoa in the ejaculate secondary to impaired spermatogenesis within the testis, may be caused by a variety of etiologies, including varicocele-induced testicular damage, cryptorchidism, prior testicular torsion, post-pubertal mumps orchitis, gonadotoxic effects from medications, genetic abnormalities, chemotherapy/radiation, and other unknown causes currently classified as idiopathic (Cocuzza et al., 2013 2013 ). The microdissection testicular sperm extraction (micro-TESE) technique involves a meticulous microsurgical exploration of the testicular parenchyma to identify and selectively extract larger seminiferous tubules that carry a higher probability of complete spermatogenesis (Schlegel, 1999 1999 ). The Cornell group evaluated the efficacy of micro-TESE in 152 NOA patients with an associated history of cryptorchidism. In their series, spermatozoa were successfully retrieved in 116/181 attempts (64%), and the resulting pregnancy rate was 50% with a delivery rate of 38% (Dabaja and Schlegel, 2013 2013 ). Franco et al. ( 2016 2016 ) described a stepwise micro-TESE approach in NOA patients, which was considered to reduce the cost, time, and effort associated with the surgery. Alrabeeah et al. ( 2016 2016 ) further reported that a mini-incision micro-TESE, carried through a 1-cm equatorial testicular incision, can be useful for micro-TESE candidates, particularly in patients with cryptozoospermia. We conducted a retrospective study of 20 consecutive NOA patients with a history of orchidopexy from May 2015 to March 2017. We employed a modified stepwise mini-incision micro-TESE consisting of three steps: (1) mini-incision micro-TESE; (2) standard micro-TESE of the ipsilateral testis; (3) contralateral micro-TESE. The operating time was evaluated, and it revealed statistically significant differences between the mini-incision micro-TESE group ((52±23) min), the standard micro-TESE group ((87±10) min) and the bilateral-standard micro-TESE group ((121±9) min) (mini-incision micro-TESE group vs. standard micro-TESE group, P < 0.05; mini-incision micro-TESE group vs. bilateral-standard micro-TESE group, P < 0.001). The overall sperm retrieval rate (SRR) was 80% (16/20). In 50% of patients (10/20) mini-incision micro-TESE alone was required because of the rapid identification of spermatozoa harvested from the superficial testicular tissue. The remaining 10 patients underwent a standard micro-TESE, and 4 patients progressed to a bilateral micro-TESE. The sperm were cryopreserved for future intracytoplasmic sperm injection (ICSI), and, to date, 10 of 16 couples have attempted ICSI. Clinical pregnancies were achieved in 5 of the 10 couples, whereas ongoing pregnancy or deliveries occurred in 4 of the 10. Mini-incision micro-TESE can prove sufficient, reserving standard micro-TESE as a salvage method, for patients affected by NOA with a history of orchidopexy. The modified stepwise mini-incision micro-TESE, which gradually increases surgical invasiveness, may represent an optimal approach for sperm retrieval for patients affected by NOA with a history of orchidopexy. Cryptorchidism is the most common genital anomaly in boys. The diagnosis is rendered when one or both testes fail to descend to a normal scrotal position during development (Wood and Elder, 2009 2009 ; Serrano et al., 2013 2013 ). The incidence of NOA in men with unilateral cryptorchidism is 13% regardless of whether the patient was treated or not; in the most severe cases, approximately 88% of patients with untreated bilateral cryptorchidism will develop into azoospermia (Hadziselimovic and Herzog, 2001 2001 ). Fortunately, surgical orchidopexy is associated with a success rate exceeding 80% (Docimo, 1995 1995 ). Despite best efforts, 10% of infertile men still have a history of cryptorchidism and orchidopexy (Grasso et al., 1991 1991 ), probably due to the loss of Ad spermatogonia (Hadziselimovic and Herzog, 2001 2001 ). All patient characteristics are listed in Table 1 1 . No genetic abnormalities were present. Average age (mean±standard deviation (SD)) was (30.7±5.5) years (range from 23 to 47 years). The average age at orchidopexy (mean±SD) was (12.8±9.3) years (range from 3 to 35 years). In the 20 men, 13 suffered from bilateral cryptorchidism whereas the remaining 7 had unilateral cryptorchidism. The average right testicular volume (mean±SD) was (7.1±2.1) mL, and the left was (7.6±2.3) mL. Notably, one patient had a history of radiotherapy/chemotherapy for left testicular carcinoma. Among the study subjects, 10 required mini-incision micro-TESE alone, 6 underwent mini-incision and ipsilateral standard micro-TESE, and the remainder had standard bilateral micro-TESE procedures. All surgical procedures were carried out under general anesthesia. Generally, the larger testicle, determined by physical examination and/or prior scrotal ultrasound, was explored first. A longitudinal 3-cm scrotal incision was made over the right or left testicle (a contralateral incision was made if a bilateral procedure proved necessary) (Fig. 1a 1a ), not the median raphe incision (Fig. 1b 1b , the dotted line). The subcutaneous tissue was then gently dissected to expose the testis, which was fixed outside of the dartos muscle given the prior history of orchidopexy. The testicle was delivered, which at times was difficult due to scarring. A planned transverse, equatorial, approximately 1-cm incision (“mini-incision”) was then carried down through the tunica albuginea to expose the testicular parenchyma under the operating microscope (Fig. 1d 1d ). The available testicular tissue beneath the mini-incision was examined under the operating microscope at 15×to 24×magnification to locate and collect dilated seminiferous tubules (Fig. 1e 1e ). A specimen was only collected if dilated tubules were identified, which was immediately evaluated by an embryologist available in the operating room. If spermatozoa were present, the motility, morphology, and quantification were provided. Usually, the surgeon would examine the slides with the embryologist to confirm the result. If no spermatozoa were found in this superficial exposure, the incision was then extended to perform the standard micro-TESE (Fig. 1f 1f ). Failure resulted in a full contralateral micro-TESE. When sperm was found, it was cryopreserved for future ICSI. The procedure was terminated when several spermatozoa were observed or after complete and thorough examination of the entire testicular parenchyma. At the completion of the procedure, the tunica albuginea and skin were closed with 5–0 suture (Ethicon, Puerto Rico, USA). If sperm were present, they were cryopreserved using either the LSL straw (Cryo BioSystem, Paris, France) (Liu et al., 2017 2017 ) or the cryopiece system (Sun et al., 2017 2017 ), according to the quantity and quality of the spermatozoa available for ICSI. (a, b) Photograph showing the pair of longitudinal scrotal incisions made just over the right or left testicle (solid line), and the median raphe incision (dotted line); (c, d) Photograph showing the planned equatorial testicular mini-incision; (e) Photograph of the mini-incision micro-TESE; (f) Photograph showing the mini-incision extended to the standard micro-TESE incision NOA: non-obstructive azoospermia; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; ICSI: intracytoplasmic sperm injection; Mini: mini-incision micro-TESE; Stan: standard micro-TESE; Bi-Stan: bilateral standard micro-TESE; Y: yes; N: no; P: pregnancy. History of radiotherapy/chemotherapy for a left testicular carcinoma; Data are expressed as mean±standard deviation; Oocyte frozen, immotile sperm post-thawing; Ongoing pregnancy No. Age (year) Age atorchidopexy (year) Cryptorchidism FSH (IU/L) LH (IU/mL) T (ng/mL) Surgical procedure Operation time (min) Sperm ICSI Clinical pregnancy Live birth  1 35 10 Unilateral 15.05 5.77 3.27 Mini 65 Positive Y P d d  2 26 19 Unilateral 20.12 11.79 2.86 Mini 30 Positive Y N  3 30 3 Unilateral 6.60 10.18 11.46 Mini 45 Positive Y N  4 29 6 Unilateral 1.78 2.62 7.61 Mini 50 Positive Y P Abortion  5 31 6 Bilateral 11.85 8.32 2.02 Mini 80 Positive Y N  6 35 6 Bilateral 5.68 5.97 8.07 Mini 105 Positive  7 47 Unilateral 8.17 1.30 3.83 Mini 40 Positive  8 31 11 Bilateral 33.56 7.20 3.94 Mini 45 Positive Y P Y (girl)  9 26 23 Bilateral 6.43 3.62 6.71 Mini 30 Positive  10 30 5 Unilateral 1.34 3.00 15.63 Mini 30 Positive Y P Y (girl)  11 23 22 Bilateral 7.98 3.62 5.06 Bi-Stan 120 Positive  12 25 10 Bilateral 28.94 6.73 2.82 Stan 80 Positive Y c c  13 34 6 Bilateral 25.73 10.85 2.41 Stan 90 Positive Y c c  14 a a 31 8 Bilateral 26.20 12.21 4.32 Stan 100 Positive Y P Y (boy)  15 28 Bilateral 84.59 4.39 4.39 Stan 80 Positive Y N  16 39 35 Bilateral 51.10 34.06 7.73 Stan 75 Positive Y N  17 27 5 Bilateral 22.63 7.11 1.96 Bi-Stan 110 Negative  18 27 15 Bilateral 9.12 4.87 2.33 Bi-Stan 135 Negative  19 35 32 Bilateral 68.60 37.54 1.12 Bi-Stan 120 Negative  20 25 8 Unilateral 24.53 61.92 5.92 Stan 100 Negative Average b b 30.7±5.5 12.8±9.3 19.4±18.9 12.2±14.7 5.1±3.3 76.5±32.5 The operating time among the mini-incision micro-TESE group ((52±23) min), the standard micro-TESE group ((87±10) min) (mini-incision micro-TESE group vs. standard micro-TESE group, P < 0.05) and the bilateral-standard micro-TESE group ((121±9) min) (mini-incision micro-TESE group vs. bilateral-standard micro-TESE group, P < 0.001) was evaluated (Fig. 2 2 ), and it indicated that the stepwise mini-incision micro-TESE could reduce operating times markedly. SRR has been documented to be between 51.9% and 73.0% (Lin et al., 2001 2001 ; Negri et al., 2003 2003 ; Vernaeve et al., 2004 2004 ; Haimov-Kochman et al., 2010 2010 ). Raman and Schlegel ( 2003 2003 ) reported that spermatozoa were successfully retrieved in 35 of 47 TESE attempts (74%), including 5 of 8 (63%) attempts made by the multiple testicular biopsy technique, and 30 of 39 (77%) attempts by the micro-TESE method. The Cornell group reported an SRR of 64% (116/181) in 152 patients with NOA associated with cryptorchidism. Their resulting pregnancy and delivery rates were 50% and 38%, respectively (Dabaja and Schlegel, 2013 2013 ). Findings suggest that NOA patients, after correction of maldescended testes, tend to have a significantly higher SRR (80%) than men with a primary fine needle biopsy (FNB) diagnosis of spermatogenic arrest (SRR 40%–45%) and a clearly higher SSR than men with Sertoli cell only (SRR 30%–35%) (Bernie et al., 2013 2013 ). Due to rapid identification of spermatozoa harvested from the superficial testicular tissue, 50% (10/20) required only a mini-incision micro-TESE. However, of men who required the full bilateral micro-TESE approach, spermatozoa were successfully retrieved in only one of four. The SRR was 85.7% (6/7) among patients with a history of unilateral cryptorchidism, and 76.9% (10/13) in those with a history of bilateral cryptorchidism. Data are expressed as mean±standard deviation (SD). * P < 0.05, *** P < 0.001 Sperm were successfully cryopreserved for future ICSI in all cases of successful micro-TESE. In some centers, micro-TESE is usually performed on the day before oocyte retrieval or the same day to benefit from the use of fresh testicular sperm. However, this strategy may cause unnecessary ovarian stimulation and oocyte retrieval when no sperm are detected during the male procedure (Schachter-Safrai et al., 2017 2017 ). Furthermore, a meta-analysis reporting on 574 cycles revealed no difference in the clinical outcomes for the use of fresh vs. frozen sperm for ICSI in men with NOA (Ohlander et al., 2014 2014 ). In our institution, uncoupled micro-TESE/oocyte retrieval is performed in NOA cases to prevent possible unnecessary morbidity to the female partner. Cryopreservation also maximizes flexibility for the couple to initiate an ICSI cycle. Twelve couples attempted to initiate ICSI, two couples resorted to oocyte cryopreservation due to the absence of motile sperm post-thaw, which is the shortcoming of sperm banking. Of the remaining 10 couples, 1 couple is currently carrying an ongoing pregnancy, and 3 deliveries have occurred in other couples. A spontaneous abortion occurred in one couple. Micro-TESE, which involves a careful dissection of the testicular tissue to minimize diminished testicular function (Schlegel, 1999 1999 ), may also cause extensive tissue damage and potential long-lasting injury to the testis. To this end, we believe that optimization of sperm retrieval technique will contribute to reduced testicular injury without compromising SSR. Furthermore, due to post-surgical scarring, men with a history of prior orchidopexy may present with a challenge as the anatomy is slightly more challenging to release the testis for microscopic evaluation. The presented stepwise mini-incision micro-TESE for patients with prior orchidopexy carries the same goals as the standard micro-TESE technique: (1) to successfully harvest spermatozoa for ICSI, (2) to reduce the operating time and effort involved in surgery, and (3) to potentially minimize tissue loss without subsequent loss of testicular function. We argue that the presented “three-step” sperm retrieval method accomplishes these goals while gradually increasing surgical invasiveness (Ma et al., 2012 2012 ). Our data demonstrate that half of the patients with NOA associated with a prior history orchidopexy had rapid identification of sperm in the superficial tissue, which is less invasive and less detrimental to testicular function (Alrabeeah et al., 2016 2016 ). Upon failure of the first two steps, sperm were found in the contralateral testis of one patient following progression to bilateral standard micro-TESE. Although we did not evaluate the post-operative testicular function in this cohort, we postulate that the modified stepwise micro-TESE will minimize the reduction of testicular function due to less disruption of testicular parenchyma. Our preliminary data showed that the stepwise mini-incision micro-TESE technique could gradually increase surgical invasiveness and reduce operating time without compromising outcomes. 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Cryptorchidism and testicular cancer: separating fact from fiction Compliance with ethics guidelines Peng LI, Chen-cheng YAO, Er-lei ZHI, Yuan XU, Zhong WAN, Ying-chuan JIANG, Yu-hua HUANG, Yue-hua GONG, Hui-xing CHEN, Ru-hui TIAN, Chao YANG, Liang-yu ZHAO, and Zheng LI declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients included in the study. 10.1631/jzus.B1900232 Project supported by the National Natural Science Foundation of China (Nos. 81701524, 81671512, and 81701428), and the Frontier Technology Project of Shanghai (No. SHDC12015122), China 2019-07-12 2019-09-04 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 21