Testicular Sperm Extraction: History
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Subjects: Andrology
Contributor:
Kaan Aydos

Retrieving spermatozoa from the testicles has been a great hope for patients with non-obstructive azoospermia (NOA), but relevant methods have not yet been developed to the level necessary to provide resolutions for all cases of NOA. Although performing testicular sperm extraction under microscopic magnification has increased sperm retrieval rates, in vitro selection and processing of quality sperm plays an essential role in the success of in vitro fertilization. Moreover, sperm cryopreservation is widely used in assisted reproductive technologies, whether for therapeutic purposes or for future fertility preservation. In recent years, there have been new developments using advanced technologies to freeze and preserve even very small numbers of sperm for which conventional techniques are inadequate.

  • testicular azoospermia
  • non-obstructive azoospermia
  • sperm selection
  • sperm
  • cryopreservation
  • in vitro maturation

1. Introduction

To date, although testicular spermatozoa from patients with non-obstructive azoospermia (NOA) have been used widely for intracytoplasmic sperm injection (ICSI), this method’s effectiveness still has potential for further improvement. NOA is characterized by the absence of any spermatozoa, whether dead or alive, in the ejaculate due to reduced or nonexistent sperm production in the testicle [1]. Testicular fine needle aspiration (FNA or testicular sperm aspiration—TESA) is an effective and non-invasive method used to obtain sperm, especially from patients with obstructive azoospermia [2]. Although its simpler and less traumatic features have made FNA the preferred method, testicular sperm extraction (TESE) is the treatment of choice for patients with NOA, with a satisfactory number of successful spermatozoa retrieved in approximately half of the patients. In the conventional TESE procedure, the testis is exposed through a small incision in the tunica albuginea, and multiple biopsies are taken randomly [3]. However, microTESE carried out at high magnification under an operating microscope allows visualization of whitish, larger, and more opaque seminiferous tubules likely to contain mature germ cells [4]. Although not randomized, most studies have reported that the sperm recovery rate from microTESE is superior to that from conventional TESE [5][6][7]. In fact, a recent controlled, randomized study verified the efficacy of microTESE compared with that of TESE in retrieving spermatozoa from patients with NOA [8]. In addition to the surgical technique, however, in vitro extraction of sperm from surgically excised testicular tissue or tubules is also important for obtaining spermatozoa of sufficient quality and quantity for use in ICSI.

2. Processing Immature Germ Cells

Despite extensive searching, mature spermatozoa can be obtained with TESE in only approximately 40% to 60% of cases of NOA for use in ICSI [9][10]. In the remaining cases, round spermatids were attempted as a last resort, although until recently, the results were not satisfactory enough to encourage routine practice [11]. However, in 2015, Tanaka et al. reported 14 healthy babies born with round spermatid injection (ROSI) in oocytes previously activated by electric current; 3 years later, from the 2-year follow-up results of 90 ROSI babies, it was determined that round spermatids enabled patients with NOA to have their own genetic offspring [12][13]. Subsequently, Papuccu et al. reported their results of 472 couples who underwent 904 cycles using elongating (Sb2) spermatids for the ROSI technique and achieved a 9.6% ongoing pregnancy rate [14]. Since transformation of immature germ cells into spermatozoa with fully developed flagella has had limited success in in vitro experiments, culturing samples to achieve at least haploid round spermatids may have wider clinical application [15].
If testicular tissue samples from TESE do not contain spermatozoa, various approaches have been described for in vitro maturation of early stage germ cells. For in vitro maturation, either (1) testicular tissue is used in whole pieces while preserving its three-dimensional (3D) structure or (2) after isolation and purification, different cell types are exposed to culture conditions in which the spermatogenic process is recreated. In TESE-negative cases, it makes sense to try culturing small fragments of testicular tissue or intact pieces of tubules first. However, since the limited diffusion rates of the tissue do not allow tissue viability to be maintained over a long period, it is challenging to culture the tissue as a whole. After long-standing efforts, in 2011, Sato et al. reported healthy offspring from haploid cells developed from testicular fragments cultured on agarose gel in modified Minimum Essential Medium (α-MEM) supplemented with knockout serum replacement [16]. Others have also shown culturing of frozen/thawed testicular tissue on agarose gel to restore spermatogenesis up to haploid spermatids, leading to offspring [17]. Furthermore, from seminiferous tubule segments cultured in chitosan hydrogel bioreactors, the development of spermatids and spermatozoa were achieved on days 34 and 55, respectively [18]. In another organotypic culture system described for human immature testicular tissue, germ cells were shown to differentiate up to round spermatids within 16 days [19]. However, the inability of organ culture systems to restore spermatogenesis in cryopreserved human testicular specimens has also been reported [20]. These contradictions in results may be due to the fact that culture methods are not yet fully optimized or that the nature of subcellular defects is different [21]. Later, developed microfluidic technology further improved organotypic culture systems, allowing ex vivo sustainability of the structure and viability of germ cells in testicular tissue for producing mature sperm [22]. As an option for in vitro maturation of isolated germ cells, 2D culture systems have been developed to support enzymatically digested testicular cell suspensions. In 2D cultures, isolated SSCs are maintained either on feeder cells or on mixed cell populations co-cultured with somatic cells [23]. Different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells and laminin-coated plate were used to support spermatogenesis [24]. Apart from feeders, different culture systems, such as human amnion mesenchymal stem cells [25], in vitro reprogramming of fibroblasts to human induced Sertoli-like cells [26], and isolated cell culture with growth factor supplementation [27], have been defined to support in vitro spermatogenesis. Nevertheless, 2D culture systems provided in vitro restoration of spermatogenesis and supported the development of haploid spermatids with fertilization potential [28]. Experiences gained from previous studies ultimately led to the development of artificially constructed 3D structures [29]. By creating structures that mimic the composition of the main testicular components, 3D cultures allow immature germ cells to be reconstructed similarly to their original tissue architecture, thereby allowing further maturation [30][31][32][33]. Although this system has the ability to direct the differentiation of germ cells, microenvironmental conditions favorable for complete maturation must also be achieved.
In addition to attempting complex and intricate methods for in vitro maturation of immature germ cells from testicular tissue, there is a need for simple methods that can be used more easily in clinical practice. When Aslam et al. compared suspensions of mixed cell populations and isolated homogeneous populations of spermatogenic cells prepared from testicular tissue, they showed that most of the isolated round spermatids developed tails and remained intact and viable for 72 h in modified Eagle’s minimum essential medium with no hormonal supplementation [34]. However, since they used a mixture of obstructive and non-obstructive tissue samples, the contribution of in vitro culturing to the development of flagella in immature germ cells in cases of NOA is not clear. Similarly, it has been shown that human round spermatids can mature up to spermatozoa when cocultured on Vero cell monolayers [35]. Other researchers have also verified the maturation of primary spermatocytes into haploid spermatids through in vitro coculture with Vero cells [36]. Subsequently, round spermatids generated from human SSCs were shown to fertilize mouse oocytes [27]. However, even without a co-culture, in vitro hormonal supplementation has been demonstrated to be capable of providing sufficient support to mature premeiotic germ cells [37]. Thus, culturing testicular samples from patients with NOA in medium containing recombinant FSH and testosterone for 48 h transformed FISH-proven primary spermatocytes into mature round spermatids, after which injection into the oocyte resulted in healthy offspring [38]. It has been shown that hormones added to in vitro culture medium in cases of NOA not only accelerate spermiogenesis but also improve apoptosis-related cell damage in enhancing the reproductive performance of germ cells [39]. Contrary to most studies indicating that FSH and testosterone added to testicular culture media play a role in the development of different stages of in vitro spermatogenesis, it has also been suggested that their supplementation does not induce meiotic and post-meiotic cells and therefore cannot differentiate premeiotic germ cells [40]. Differences in germ cell development in in vitro maturation studies may be due to insufficient support of established culture conditions. The maintenance of healthy spermatogenesis from SSCs can only be achieved with the support of a complicated and precise “niche” microenvironment [41]. Sertoli cells establish the most important component of the “niche,” and by producing growth factors and cytokines, they regulate proper self-renewing and differentiation of SSCs, transition to meiosis, and, finally, differentiation of round spermatids into spermatozoa [42].
Under proper culture conditions, reaggregation of Sertoli cells forms organized monolayer structures. Therefore, in most of the in vitro maturation studies performed on testicular tissue samples, a co-culture with Sertoli cells has been used effectively to provide structural and nutritional support for differentiation of germ cells [43][44][45]. In the co-culture of round spermatids and Sertoli cells, it has been shown that supplementation with recombinant FSH and testosterone contributes significantly to the differentiation of round spermatids into elongating spermatids [46]. However, other studies have also reported that no matter how much FSH stimulation in organotypic cultures of immature testicular tissue increases the percentage of premeiotic cells, it does not allow for further maturation [21]. Some similar studies have also confirmed that FSH supplementation in cultures does not support post-meiotic maturation [47]. Actually, the underlying mechanism for the contradictions in the reports may be the impaired ability of testes to respond to the endogenous hormonal milieu due to compromised androgen receptor and FSH receptor (FSHR) signaling pathways [48]. In fact, when compared to obstructive azoospermia, the FSHR expression level in isolated and purified Sertoli cell cultures was found to be 2.7 times lower in the NOA group; hence, it has been claimed that there may be an altered Sertoli cell response to in vitro FSH stimulation [49]. Alternatively, without the need for hormone supplementation, spermatogenesis from testicular SSCs to fertility-competent sperm formation could be induced in organ cultures using different techniques [50][51]. Considering this fact, before choosing a method to be used in co-culture studies with Sertoli cells, it is important to investigate the hormone/receptor interaction along with the response to FSH and testosterone.
To date, a number of studies have been conducted on the development of many different culture systems, with varying levels of success. Furthermore, with their innovations in in vitro germ cell maturation, advanced technology products created in the field of regenerative medicine using cell/tissue culture, biomaterials, and bioactive products have become promising treatment alternatives for patients with NOA [52][53]. However, before suggesting potential clinical uses of haploid male gametes exposed to in vitro manipulations, further analyses of fecundity, epigenetic consequences, and safety are essential.

3. Conclusions

The primary outcome associated with the efficiency of ARTs is successful, healthy live births. In addition to allowing only a small amount of sperm retrieval, cases of NOA require a more demanding process in the treatment of infertility due to the fact that the available sperm are also products of impaired spermatogenesis. The whole process begins with collection of the highest-quality surgical specimens possible. Following microTESE, with its verified efficacy, researchers have attempted sophisticated techniques such as Raman spectroscopy, multiphoton microscopy, and full field optical coherence tomography to identify testicular tubules with spermatogenesis. As further developments, laser-assisted sperm selection and microfluidic systems appear to be promising for extracting viable spermatozoa from surgically removed testicular samples. Moreover, there is an ongoing effort to develop optimized freezing protocols and effective technologies that will allow patients with NOA to retain their fertility by implementing cryopreservation of even a single sperm. However, the initial promising results of all of these developments must be confirmed by large studies in the context of clinical practice. The use of nanoparticles for in vitro maturation of germ cells is also another promising innovation, as it will allow previously unsuccessful patients with NOA to have children using their own biological material. Undoubtedly, the clinical consequences of all of these manipulations that could result from potential changes in the offspring’s genomes must be followed very carefully.

This entry is adapted from the peer-reviewed paper 10.3390/jcm10122687

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