Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.
Approximately 15-20% of couples in industrialized countries fail to conceive after one year [Boivin et al. 2007], and male factor infertility, characterized by semen parameters that fall below the World Health Organization (WHO) [Cooper et al. 2010] cut-offs for normozoospermia, is thought to underlie nearly half of these cases [Brugh and Lipshultz 2004]. The most severe form of male infertility, non-obstructive azoospermia (NOA), is defined by the lack of sperm in the ejaculate and very little to no sperm production within the semi- niferous tubules [Kumar 2013]. There are many poten- tial causes for NOA, including genetic and congenital abnormalities, post-infectious issues, exposure to gona- dotoxins, medications, varicocele, trauma, endocrine dis- orders, and idiopathic causes [Wosnitzer et al. 2014]. NOA accounts for approximately 10% of male infertility cases and is seen in around 1% of men in the general population [Krausz 2011]. With the development of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), many otherwise infertile men have been able to successfully overcome barriers to concep- tion, including NOA [Wang and Sauer 2006].
Because the sperm are in such low numbers in NOA patients (if any are present at all), identification and collection of these cells requires specifically-designed techniques in both the operating room and in the laboratory. The prevailing method used to collect testi- cular tissue for potential sperm recovery from NOA patients is by microdissection testicular sperm extrac- tion (microTESE). MicroTESE involves an open testi- cular biopsy under an operating microscope to selectively remove tubules that appear to have potential spermatogenesis, followed by tissue processing mea- sures to isolate sperm and store sperm for subsequent use by ICSI [Schlegel 1999]. While the advent of microTESE substantially improved sperm recovery compared to standard biopsy or other techniques [Deruyver et al. 2014], the procedure remains ineffi- cient, costly, and very time consuming.
The field of microfluidics provides interesting opportunities and has utility for many needs in cell biology, but is particularly powerful when considering applications in single cell or low cell number analyses [Sackmann et al. 2014]. “Microfluidics is the science and technology of systems that process or manipulate small volumes (10−9 and 10−18 liters) of fluids, using channels with dimensions of tens to hundreds of micrometers” [Whitesides 2006, p. 368]. This unique technology has facilitated many important innovations, including some already found in the assisted reproduc- tive technology (ART) field [Swain et al. 2013]. Because of the great need for improved efficiency and accuracy in processing the low cell counts involved in
microTESE samples, the field of microfluidics may provide unique solutions to the varied problems cur- rently encountered with the processing of microTESE samples.
While a number of microfluidic technologies have been previously developed for motile sperm separation, none have been implemented for microTESE proce- dures. Currently, sperm detection and separation in samples with low sperm concentrations (microTESE, oligozoospermic samples, etc.) is a time and labor- intensive process. As a result, there is a great need for innovative approaches to improve the speed and effi- ciency of the process. Specifically, there are two key areas that may benefit from microfluidic technology, namely the identification of sperm in heterogeneous tissue preparations derived from TESE and microTESE procedures, and the identification of ‘high quality’ or ‘genetically fit’ sperm in heterogeneous semen samples.
This review summarizes and discusses the advan- tages and disadvantages of current microTESE prac- tices, focusing on potential novel laboratory sperm identification and isolation techniques using microflui- dic technology. We examine and review microfluidic techniques that have demonstrated utility in current ART, and discuss novel microfluidic applications that may be applied to microTESE to provide improvement in the efficiency and sensitivity of sperm recovery [Lenshof and Laurell 2010].
Sperm recovery via microTESE
With the development of ICSI [Palermo et al. 1992], the importance of testicular-derived sperm in treating obstructive azoospermia (OA) has rapidly become obvious [Craft et al. 1993; Silber et al. 1995; Schoysman et al. 1993]. To obtain testicular sperm, the use of a surgical testicular sperm extraction (TESE) method was employed, in which parenchymal tissue is harvested using an open testicular biopsy [Schlegel and Su 1997]. While the simple TESE techni- que has proved to be an effective means to retrieve sperm from patients with OA [Dohle et al. 1998], the technique was less successful in obtaining sperm from patients with NOA. Because spermatogenesis is often sporadic and isolated to rare seminiferous tubules in NOA patients, a non-selective tissue biopsy approach usually missed sites of sperm production, leading to poor sperm recovery [Schlegel and Su 1997].
These shortcomings led to a modification of the TESE procedure in 1996 in order to specifically suit cases of NOA. With the assistance of a high-powered operative microscope, it became possible to distinguish
between seminiferous tubules without any germ cells, which appear smaller in diameter, and seminiferous tubules with focal spermatogenesis, which appear larger and more opaque. The resulting procedure, deemed microdissection testicular sperm extraction (microTESE), allows for selective biopsy of tissues most likely to contain sperm, thus decreasing the amount of tissue removed and increasing the chance of sperm recovery [Schlegel 1999]. This method was found to be significantly more successful in retrieving sperm from NOA patients than simple TESE [Dabaja and Schlegel 2013; Deruyver et al. 2014; Tsujimura 2007]. Representative sperm retrieval rates increased from 16-45% using simple TESE to 42-63% using microTESE [Deruyver et al. 2014]. Therefore, microTESE surgery under 15-20x magnification is cur- rently considered to be the leading option for obtaining sperm from NOA patients (Figure 1, box 1) [Dabaja and Schlegel 2013].
Tissue obtained by microTESE requires careful pro- cessing in the laboratory in order to separate and iden- tify sperm within the biopsied tissue. First, mechanical tissue-mincing methods are utilized in order to release spermatocytes from seminiferous tubules and even- tually to achieve a tissue suspension. Various mechan- ical techniques are employed, including passing tissue through angiocaths/syringe needles, tissue shredding with fine needles or glass slides, tubule squeezing, or cell straining [Esteves and Varghese 2012; Popal and Nagy 2013]. Next, and most time-consuming, is the tissue-processing step which involves manually search- ing through the testicular tissue specimens for sperm. Testicular sperm are generally non-motile, making the search for sperm more difficult [Bettegowda and Wilkinson 2010]. Each microscopic field examined under 200-400x magnification contains a combination of red blood cells, white blood cells, sertoli cells, sperm precursor cells, and debris that must be distinguished from the spermatocytes [Esteves and Varghese 2012; Popal and Nagy 2013]. Depending on the level of spermatogenesis and the number of sperm cells pre- sent, this step may take as little as one hour to find a sufficient number of sperm, or as long as 12-14 hours with multiple personnel examining tissue specimens to find just a handful of sperm [Ramasamy et al. 2011a] (Figure 1, box 2). Some clinics use an erythrocyte lysing buffer to reduce the number of red blood cells within the cell suspension, or pentoxyfylline to chemically induce motility in testicular sperm; however, these additives, like collagenase, are controversial for their effects on sperm viability [Popal and Nagy 2013].
Not only is manual microscopic testicular specimen examination time-consuming and tedious, it is also
greatly dependent on personnel skill-level [Ramasamy et al. 2011b]. Sperm can easily be overlooked due to a number of variables including incomplete cell dissocia- tion, elevated levels of other cell types, inexperience, exhaustion, or human-error. Furthermore, the longer a single person searches for sperm cells, the more error- prone they become [Ramasamy et al. 2011b]. For patients that produce a small number of sperm to begin with, failing to identify even a couple of sperm could mean the difference between infertility and a successful pregnancy. For these reasons, the sperm identification process is a crucial step of microTESE. By removing the human-factor and moving towards automated and efficient cell separation, not only would it be possible for sperm to be identified faster and more accurately, but sperm could be separated from somatic cells and debris simultaneously, facilitat- ing downstream preparation for ICSI.
Predicting successful microTESE sperm retrieval for any given patient is difficult, with the chances of iden- tifying sperm at about 50% [Dabaja and Schlegel 2013]. Because the only route to fertilization using testicular derived sperm is via ICSI, sperm retrieval by microTESE represents only half of the equation to achieve pregnancy for NOA patients. In preparation for ICSI, the female partner must undergo precisely timed hormonal triggers for weeks prior to oocyte retrieval (Figure 1). Thus, coordinating fresh oocytes simultaneously with microTESE sperm retrieval has a major drawback; if no sperm are recovered from the male, the couple will have unnecessarily spent a great deal of time, effort, and money on ovarian stimulation and oocyte retrieval. Therefore, many clinics opt to cryopreserve sperm retrieved from microTESE before retrieving eggs from the female partner [Ben-Yosef et al. 1999; Gangrade 2013; Tavukcuoglu et al. 2013; Oates et al. 1997]. This course of action, however, requires additional microTESE processing steps. The ability to efficiently aliquot and cryopreserve small quantities of spermatozoa (minimum volume vitrifica- tion) sufficient for a single assisted reproduction cycle would benefit patients with limited quantities of sperm. Some laboratories use density gradient centrifugation to purge some of the unwanted somatic cells and to collect a more viable sperm population, yet this practice comes with the major drawback that sperm may be lost to the density gradient (Figure 1, box 4) [Oates et al. 1997]. Accordingly, the entire cryopreservation and thawing process could be optimized specifically for microTESE cases in order to improve sperm recovery rates for ICSI. Development of a micro-scale cryopre- servation system could allow sperm to be separated from somatic cells upon their identification, and then thawed without additional cell-processing steps.
In summary, goals for improving microTESE success in the near future should include increasing tissue processing consistency/efficiency, decreasing tissue processing time/cost, decreasing technician variability, and increasing cryopreservation consistency/efficiency. It is likely that such improvements can be made by the development of appropriate automated technologies that incorporate microfluidic systems for processing microTESE samples.
Read Full Article: http://www.tandfonline.com/doi/pdf/10.3109/19396368.2016.1159748