Novel concepts in male factor infertility: clinical and laboratory perspectives

Abstract

In recent years, the management of male factor infertility has undergone important changes with the introduction of novel concepts, advanced testing, and therapeutic interventions. This review highlights some of these changes and discusses their impact to routine clinical practice. First, we discuss the recent changes in the World Health Organization (WHO) laboratory methods and reference values for the examination of human semen. Second, we examine the role of sperm chromatin integrity tests in light of increasing evidence of the detrimental effect of sperm DNA fragmentation on reproductive outcomes. Third, we summarize the main findings of varicocele-related infertility and the outcomes of microsurgical varicocele repair to different case scenarios. Lastly, we critically discuss the current management of men with nonobstructive azoospermia seeking fertility and the new opportunities that emerged to help these men achieve biological fatherhood.

Keywords: Andrology; Male infertility; Microsurgery; Nonobstructive azoospermia; Semen analysis; Sperm DNA fragmentation; Varicocele.

Fig. 1

a Comparison of sperm DNA fragmentation rates in ejaculated and testicular sperm of 81 infertile men undergoing ICSI. Use of testicular sperm for ICSI resulted in an absolute reduction of 32.6 % (relative reduction of 79.7 %) in SDF. b Sperm chromatin dispersion (SCD) test for assessing SDF in testicular sperm. A variant of the Halosperm test (Halotech DNA, Spain) that combines a dual fluorescent cocktail probe to discriminate somatic cells from spermatozoa was used. Spermatozoa and somatic cells exhibit differences in the wavelength emission associated with each fluorochrome (green for proteins and red for DNA). Spermatozoa exhibit only red fluorescence on the sperm head owing to protamine removal, while nonsperm cells fluoresce yellow as a result of the combined emission of both fluorochromes (a). Spermatozoa exhibiting red fluorescence with a green flagellum and no halo of chromatin dispersion represented those with fragmented DNA (arrow cap). In contrast, spermatozoa exhibiting red fluorescence with a green flagellum and haloes of chromatin dispersion represented those with nonfragmented DNA (arrow). A somatic cell with its typical high protein and DNA contents and a spermatozoon with its characteristic low protein remnant and high DNA content are seen in b and c, respectively, using a single channel fluorescence emission. After merging the information provided by protein and DNA selective staining, somatic cells and spermatozoa can be easily distinguished (d and d′). In addition, the sperm tail fluoresces in green, and this feature also helps to distinguish spermatozoa from other cell elements (a and d′). Adapted with permission from Esteves et al. [41]

Fig. 2

Clinical pregnancy, miscarriage, and live birth rates after sperm injections using either ejaculated sperm (EJA-ICSI; n = 91) or testicular sperm retrieved by TESE or TESA (TESTI-ICSI; n = 81) cohorts. Adapted with permission from Esteves et al. [41]

Fig. 3

Possible treatment alternatives to overcome high sperm DNA fragmentation. The figure highlights the role of SDF testing to better manage couples facing infertility. Possible treatment strategies to overcome high SDF are indicated. ART assisted reproductive technology, ICSI intracytoplasmic sperm injection, IMSI ultra-high magnification sperm injection

Fig. 4

Step-by-step approach in the clinical management of men with nonobstructive azoospermia seeking fertility (adapted with permission from Esteves [14])

Fig. 5

Human Y chromosome map depicting the AZF subregions and gene content. The AZFa region maps from approximately 12.9 to 13.7 Mb of the chromosome and contains two single copy genes, USP9Y and DDX3Y. AZFb spans from approximately 18 to 24.7 Mb of the chromosome and AZFc from approximately 23 to 26.7 Mb. Both regions contain multiple genes as depicted in the bottom of the figure. The location of the basic set of sequence-tagged sites primers to be investigated in azoospermic men with spermatogenic failure, according to the European Association of Andrology and the European Molecular Genetics Quality Network 2013 guidelines, is identified by solid vertical lines (adapted with permission from Esteves [14])

Fig. 6

Microdissection testicular sperm extraction (micro-TESE). The flowchart illustrates the consecutive steps from the microsurgical procedure to the laboratory processing of testicular specimens. The rationale of micro-TESE is to identify focal areas of sperm production within the testes, based on the size and appearance of the seminiferous tubules, with the aid of the operating microscope (A). A large incision is made in an avascular area of the tunica albuginea and the testicular parenchyma is widely exposed (B). The parenchyma is then dissected at ×16 to ×25 magnification to enable the search and isolation of seminiferous tubules exhibiting larger diameter in comparison with nonenlarged or collapsed counterparts (C). These enlarged tubules are more likely to contain germ cells and eventually normal sperm production. Microsurgical-guided biopsies are performed by carefully removing such tubules, which are sent to the laboratory for examination (D). The minimal tissue extracted facilitates laboratory processing and sperm search thus increasing the process efficiency (E). The initial laboratory step involves mechanical mincing of the seminiferous tubules and examination of specimens for sperm identification (F). The use of optical magnification also reduces the chances of vascular injury by proper identification of testicular blood supply, thus reducing the chances of hematoma formation and testicular devascularization

Fig. 7

The Cell Sleeper method for low count sperm freezing. The Cell Sleeper (Nipro, Japan) consists of an outer vial, an inner tray, and screw cap (A). The inner tray is removed from the vial, placed in the lid of a large culture dish, and a 2-μL droplet of cryopreservation solution is pipetted into the tray, in a central position (B). Spermatozoa are aspirated and ejected into the droplet on the tray with the aid of a microinjection pipette (C). Immediately thereafter, the tray is returned to the vial and the vial is closed with the screw cap. The vial is placed in a horizontal position 4–5 cm above the surface of liquid nitrogen (D). After 2 min, the vial is submerged in LN2 and secured into a cryopreservation cane for storage (E)