Many patients with NOA do have mature spermatozoa within their testes that can be extracted with open biopsies with potential risk of vascular injury and lack of testosterone production [4], all the more since cTESE often removes unnecessary tissue without dilated tubules that are more likely to contain sperm. mTESE as described by Schlegel [15] optimizes the chance of finding spermatozoa with less testicular damage.
Our technique
The technique we described is a step-by-step macro-mTESE where × 6 loupes are used instead of × 25 operating microscope. Surprisingly, there is poor data on this modified technique in the current literature [16]. Thin and linear tubules are more likely to contain Sertoli-Cell only (SCO) wheras dilated and whitish tubules gathered together with no hyaline connective tissue in between are more likely to contain viable sperm.
As part of the procedure, we perform immediate intraoperative assessment of the retrieved tubules as described by many authors [7, 17]. This helped us to decide if the procedure must go on or terminate [18, 19], so at any step of the procedure, when motile sperm are found, we decide together with the embryologist if sperm retrieval must go on, taking into account the age, the ovarian reserve of the femal partner and the number of oocytes to be retrieved if ICSI is done in a synchronous fashion.
The overall SRR for × 6 loupe-assisted TESE was 51.8% and sperm were found in the first testis in 61% of the patients, leading to unnecessary controlateral procedures. Among these patients, 128 (12.1%) had sperm retrieved far from the superficial mid part of the testis where cTESE is usually done and we assume that without the assistance of × 6 loupes, no sperm would probably have been found in them. Even when dissection demonstrates diffuse SCO, foci of dilated tubules may be found deep in the testis (Fig. 5).
We had 8% of late SRR on the very last biopsies, which demonstrates the wide heterogenecity of the testis in NOA patients. Due to this, we assume that diagnostic biopsy and fine needle aspiration are not good predictive tools in men with NOA [3].
The usual number of testicular biopsies was 15 to 20 from each side. In our institution, the samples are dissected by the embryology team before being examined and we are now planning to pass the dissected samples through a 24G angio catheter and use digestion of the sedimented testicular tissue with collagenase before further analyses if no spermatozoa were seen and we believe that this procedure will enhance our SRR as decribed in the literature [20].
In the two last years, despite the fact that we improved our technique with deeper parenchyma dissection and spent less time to identify dilated tubules, we failed to enhance our SRR. This was due to a recruitement bias as many patients referred to us had normal FSH levels and uniform histological pattern of MA with low SRR (Fig. 6) [21]. Incidentally, obstructive azoospermia only accounted 15% of our patients these two last years versus 35% in the early years.
Since we performed × 6 loupe assisted TESE, we have had no testicular atrophy wheras we have had 3 cases with cTESE. Intra testicular haematomas were often diagnosed on ultrasound and they disappeared within 6 months [22, 23]. None had to be evacuated in our series. Deeper dissection was also thought to be more painful until we used local anesthesia.
Since half of our patients came from abroad, we could not assess post operative testosterone levels but this has been evaluated in the literature and testosterone is more likely to reach normal values within one year [24].
In our private clinics in Tunisia, having loupe assistance does not imply any overcost as long as we have a convenient operating time. If we used an operating microscope, the patient would be charged for the extra time that the procedure would demand.
Predictive factors and correlation with histology
In the literature, SRR with mTESE is correlated with the most advanced pattern of spermatogenesis rather than the predominant pattern of spermatogenesis on the results of the intraoperative testicular assesment [25].
Usually, the sample reffered to histopathology is a random single biopsy of 5x5x5 mm [26] but, in our experience, due to the frequent heterogenocity of the testis, we believe that such a tiny sample cannot always predict the most advanced histological pattern. In addition, when very superficial biopsies allowed to retrieve enough sperm, disection stopped and one could not assess precisely the different histological patterns that are present deeper within the testis. That is why in some cases, we failed to correlate histology to SRR. We are wondering if biopsies from different areas instead of a single one may help to achieve correct histopathology assesment.
We had no data on SRR related to histology but in the literature, patients with early MA have lower SRR than those with late MA. Also, focal MA was associated to higher SRR vs diffuse MA (100% of tubules showing MA) as it has been published [26]. In every histopathological subgroup, patients with heterogeneous tubules seem to have a higher SRR than patients with homogeneous tubules [27].
Many attemps have been made to define predictive models or formulas using non invasive parameters [28,29,30,31] but none of them was able to predict unnecessary surgery. Ziaee et al. [32] published that the positive predictive value for a combination of FSH and inhibin B was 100%, this conclusion is made upon a few number of cases (85 patients with 21,2% SRR). FSH, Inhibin levels, testicular volume and negative previous testicular histopathology have failed to predict the presence of spermatozoa within the testes [29, 33,34,35].
Smaller testicular volume and high FSH levels are not correlated to a poor SRR for small foci of advanced spermatogenesis may be present in these conditions (Fig. 7) [9, 36]. Patients with testis volume > 10 cc and FSH < 10 are correlated to a poorer SRR [26].
In our series, older age does not adversely affect SRR, which seems to support the idea that elderly patients are more likely to have acquired azoospermia with a higher frequency of hypospermatogenesis than congenital NOA [37].
Cryptochidism
In our series, the overall SRR was 57.8%. Successfull × 6 Loupe-TESE was correlated to early age of orchiopexy and scrotal location of the testis at the time of biopsy. When the testis was located in a low inguinal position and remained still palpable, we had a poor SRR of 10% which jumped to 20% if the testis could be descended to a scrotal position 6 months prior to biopsy. We never found sperm in patients with unpalpable testes. Compared to other NOA patients, a man with a history of cryptorchidism tends to have a slightly higher chance of sperm retrieval, and even if he had a history of bilateral cryptorchidism, mTESE will lead to find sperm in up to 62% [10, 38].SRR is also correlated to a subnormal testicular volume but independant from FSH [39].
AZF microdeletions
It is very well documented that men with complete AZFa microdeletion will demonstrate diffuse SCO pattern whereas those with complete AZFb have diffuse MA [40]. None of them will have sperm in their testes and thus, testicular biopsy is contraindicated.
The most frequent microdeletion is AZFc [41]. In this condition, patients may have oligozoospermia, typically < 1 M/ml and freezing, if possible, is mandatory since these patients may continue to impair their spermatogenesis until they become azoospermic [40, 42].
The usual SRR in these AZFc patients is around 55% [42] but in our series, we found sperm in only 21.7% of these patients. All patients with sperm at biopsy had SCO with small foci of spermatogenesis rather than HS. We initially thought that this poor SRR might be due to an older age in this group compared to standard patients but there was no significnt difference. This may only be related to the small number of AZFc patients in this cohort.
Klinefelter
In klinefelter patients, SRR is strongly correlated to the age of the patient and his testoterone level at the time of surgery [34] although in older patients, SRR is still high if sperm is retrieved with mTESE [43,44,45].
We had very few cases of Klinefelter in the early years because they were labelled to be infertile. We operated 72 patients and always performed bilateral biopsies since they may continue to impair their spermatogenesis, which would lead to poorer retrieval rates if further biopsies are needed. We had a very poor SRR of 18% and we believe that this is not related to our technique.
This seems to be more likely due to the fact that our patients were not diagnosed as Klinefelters and even had testesterone therapy for a long period, usually for erectile dysfunction, before they were referred to us.
Also, in our country, there is a lack of information on the possibility of sperm retrieval in them [44, 46, 47]. Thus, when they are eventually referred to our centre, they are often over 35 years old, which is correlated to a poor SRR [11]. In our series, the mean age of Klinefelter patients is 37.8 +/− 4.9. We had only one Klinefelter patient that we operated at the age of 23 and we could freeze 3 pellets of viable sperm. ICSI was performed 6 years later, as he got married, which led to one take home baby.
On the other hand, we performed synchronous × 6 loupe TESE in a 41 year-old patient. Surprisingly, not only sperm were found, but the couple could have taken home twins at the first ICSI attempt.
Due to this, we always offer biopsies in Klinefelter patients unless 4 conditions are present: more than 40 years old, previous failed testicular biopsy, testicular volume less than 1,5 ml and low testorene level (< 3 ng/ml) with no response to hormonal stimulation. We usually begin with one tablet every second day of clomifen citrate 50 mg, and if testosterone doen’t increase within one month, we recommand one daily tablet. After a 3 month-period, if teststerone is not beyond 3 ng/ml, we may add 5000 IU of HCG twice a week.
Collaboration between urologists and endocrinologists should be made to discuss wether sperm retrieval should be done by the age of 20, before these young men undergo testosterone replacement therapy, which seems to be a promising option [44].
Testicular tumors
In 11 patients with NOA, ultrasound revealed unexpected infra-clinic testicular tumors. × 6 loupes were useful to identify the tumor from the surrounding tissue and perform tumorectomy with safe margins, together with TESE. Our SRR in such patients is 72.7%.
In benign cases, we performed conservative surgery and in case of leydigomas, 3 of the 6 patients demonstrated sperm in their ejaculate from 3 to 6 months after surgery. In them, freezing was mandatory because they may have recurrence with de novo NOA.
In a patient with unilateral tumor, when no sperm were found in the ipsilateral testis, we never performed contralateral TESE in the same session.
Conservative treatment may be offered in very few selected patients with testis cancer if 5 conditions are present: tumor size< 20 mm, safety distance from the rete testis, no intraepithelial neoplasia in the remaining parenchyma, normal preoperative testosterone levels, close follow-up [48].