Spermatozoa retrieved from the testis of azoospermic patients are found to be as effective as ejaculated spermatozoa in intracytoplasmic sperm injection (ICSI) cycles [1]. Performing ICSI involves treatments for both partners: the male undergoes surgery for testicular sperm recovery and the female undergoes the ovarian stimulation protocol for oocyte pick-up. For this reason, an unsuccessful sperm recovery procedure could have important emotional and financial implications for the infertile couple.

In this context, sperm retrieval (SR) rates for men with obstructive azoospermia (OA) are excellent (96–100 percent), whereas the reported overall SR rates for patients with nonobstructive azoospermia (NOA) are less optimal and range from 30 to 60 percent [2]. Therefore, it is important to evaluate all clinical parameters that may predict the possibility of SR, because they would be crucial for assisting surgeons and clinicians in the correct counseling of the azoospermic patient [3].

In fact, physical examination, detailed medical history, hormonal analysis, and genetic studies will help to determine the type of azoospermia [4].

However, testicular histopathology has been found to be the most reliable predictive factor for successful SR in NOA patients since the late 1990s [5]. As reported by previous authors, the histological evaluation of testicular samples is performed after staining the tissue and cell sections with hematoxylin and eosin (H&E), and examining at least 100 different sections of seminiferous tubules. The counting is performed with a 10× objective lens, exposing several tubuli in one field of vision. In cases of doubt, the presence of spermatozoa is checked using a higher magnification, and counting is performed with the 25× objective lens. Tubules having their majority portion in this field are scored, and the slide is then moved sideways to bring the adjacent area into the field. When coming to the edge of the biopsy, the slide is moved up to bring the structure at the bottom edge to the top edge and scoring continued. Based on the principal histopathological pattern, testicular histology is categorized as follows: normal spermatogenesis (NS), as shown in Figure 3.1, comprising cells from all stages of spermatogenesis and an adequate number of elongated spermatids/spermatozoa; hypospermatogenesis (HYPO), in which there is a reduction in the number of spermatogenetic cells but all stages are present, as shown in Figure 3.2; maturation arrest (MA), characterized by the absence of the later stages of spermatogenesis (Figure 3.3); Sertoli-cell-only syndrome, in which all tubules lack germ cells and are lined with Sertoli cells in the seminiferous tubules (Figure 3.4); and tubular atrophy. In addition to the registration of spermatogenesis, the number of Leydig cells is judged and recorded [6].

Figure 3.1 Normal spermatogenesis.

Figure 3.2 Hypospermatogenesis.

Figure 3.3 Maturation arrest.

Figure 3.4 Sertoli-cell-only syndrome.

Follicle-stimulating hormone (FSH) concentrations and testicular volume seem to be less accurate than testicular histopathology for prediction of SR success [7]. This is supported by the fact that many patients with MA are found to have normal plasma FSH levels and testicular volume [8]. Indeed, variations in FSH concentrations can occur for reasons unrelated to spermatogenesis – FSH secretion and release are controlled by too many endocrine and paracrine factors [9]. Moreover, although the smaller testicular volume is associated with the worse possibility of SR, there is no minimum limit of testicular volume that predicts the presence of spermatozoa [10]. Previous studies also reported that higher serum FSH concentrations were inversely related to the probability of SR in patients with NOA [11]. Indeed, FSH concentrations were inversely related to the total number of testicular germ cells; however, FSH values have poor predictive value for SR in NOA [6]. Along the same lines, the presence of associated male pathologies cannot be used as predictive factors of success [12].

The role of testicular histology as a predictor of SR has been suggested by previous several studies. Caroppo et al. showed that including the pattern of testicular histology in a model for predicting SR rates in patients with NOA improves its diagnostic accuracy [7]. Nevertheless, its application in clinical practice has been debated due to some criticism. The major and unquestionable aspect is that testicular histology may be obtained only after surgery, since a diagnostic testicular biopsy might not be a cost-effective procedure.

Based on the fact that testicular histopathology is the most accurate predictor of successful SR, some studies have proposed performing a diagnostic biopsy before assisted reproductive technologies (ARTs) [4]. In fact, the presence of elongated spermatids or spermatozoa in testicular biopsy is associated with increased SR rates [13].

However, a diagnostic biopsy itself is an invasive procedure that may have complications similar to the testicular sperm extraction (TESE) procedure, including infection, bleeding, hematoma, and tubular sclerosis [14]. This means patients undergo surgery and anesthesia twice, which increases the cost and the risk of complications. Moreover, because testicular tubules of patients with NOA are usually heterogeneous, the absence of sperm in a single biopsy does not offer assurance of a similar pattern in the entire testis. Indeed, a testicular biopsy specimen may not reflect the entire testicular tissue, bilaterally, as showed by a study that documented a 28 percent intra-individual difference in testicular histology in bilateral testicular biopsy specimens [15].

In this regard, in addition to the standard histological biopsy, previous authors have described the use of the remnants of TESE specimens after sperm has been isolated through embryological processing. In fact, only the supernatant with free sperm cells is cryopreserved or utilized for ICSI. This testicular solid tissue, defined as a “testicular pool,” is waste material consisting of the residual seminiferous tubules after stretching, centrifugation, and extraction of the spermatozoa. The testicular pool has proved to be an easy-to-analyze tissue, and to be practical, manageable, and more enlightening than a single testicular biopsy for the prediction of SR and the presence of germ cell neoplasia in situ (GCNIS), being a tissue more representative of the entire testicular parenchyma [6].

Furthermore, when testicular histopathology shows NS, it will accurately diagnose cases of OA that were ignored clinically. However, up to 15 percent of patients with OA have an intra-testicular obstruction that is not clinically revealed [16]. It should be noted, however, that patients with OA may have some damage in spermatogenesis. It should also be noted that although patients with NOA have a serious injury of spermatogenesis, it may be possible to have areas in the testes showing foci of active NS [17]. This occurs because spermatogenesis can be very unequally distributed in a testis, which may result in a negative finding using a randomly taken testicular open biopsy. This is due to irregular distribution of spermatogenesis in testicular parenchyma, which can explain the presence of focal areas of intact spermatogenesis in patients with severe testicular atrophy. In these cases, in fact, the distribution of a minute quantity of spermatogenesis must be diffusely multi-focal. A small diagnostic testicle biopsy specimen represents perhaps 1/1000 of the total testicular volume and possibly predicts the presence of a minute patch of spermatogenesis, according to the potential heterogeneity of testicular histopathology.

Despite these observations, another important advantage of histopathology is the revealing of GCNIS, which occurs in 1–3 percent of patients with severe male factor infertility, and which progresses to testicular cancer. Both carcinoma in situ and seminoma of the testis have been reported in the literature for patients undergoing TESE [18]. Banz-Jansen et al. found a tumor frequency of 1.8 percent by standard testicular biopsy in a TESE population [19].

In conclusion, testicular histopathology offers important information for counseling patients about the chances of SR success. Moreover, the results can guide clinical management in cases of failed SR, and confirm the presence of active areas or spermatogenesis on histopathology specimens. The optimal timing for the biopsy is during TESE, aiming to avoid the need for a second operation. As the pathologist analyzes the testis sample only after the surgical procedure, the histopathological pattern could guide the clinician in choosing a more suitable therapeutic option for the azoospermic patient.