The intricate "ART" of ICSI

Abstract

This manuscript explores the intricacies and nuances of the Intracytoplasmic Sperm Injection (ICSI) procedure, drawing on insights from three decades of experience at a specialized center managing numerous cases of male factor infertility. Our center is comprised of an embryology laboratory, an andrology and assisted fertilization laboratory, and a Preimplantation Genetic Testing for Aneuploidy (PGT-A) laboratory, each fostering specialized expertise independently. Collaboration among these laboratories, alongside reproductive physicians and urologists, ensures comprehensive feedback and optimal care for patients undergoing infertility treatment. The manuscript specifically focuses on the andrology laboratory's pivotal role in evaluating and treating infertile patients, highlighting critical preparations for the ICSI procedure, and the key considerations essential to its successful implementation, including the selection of the ideal spermatozoon, oocyte dysmaturity, proper equipment, and most importantly the execution of the procedure itself.

Keywords: ART; Andrology; Embryology; ICSI; Reproductive genetics.

Fig. 1

ICSI: A single sperm is injected into an oocyte. The injected oocyte is left with a proper funnel in the equatorial plane

Fig. 2

Specialized team structure: Our laboratory operates uniquely, comprising three specialized teams, each dedicated to a distinct aspect of ART. This organization enables focused expertise and greatly enhances the success of our ART program. SCF (sperm chromatin fragmentation), PLCζ (phospholipase C zeta), AOA (assisted oocyte activation), PGT (preimplantation genetic testing), ICSI (intracytoplasmic sperm injection)

Fig. 3

TUNEL staining for detection of total SCF: Blue fluorescence indicates sperm nucleus stained by 4′,6-diamidino-2-phenylindole (DAPI). Green fluorescence indicates sperm with DNA fragmentation. Normal value, ≥ 15%

Fig. 4

FISH assessment: Examples of individual spermatozoa carrying disomies on chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Sperm nuclei were stained with DAPI (blue), and chromosomes were labeled with distinct fluorophores for a multicolor visual display. In some cases, DAPI was omitted to allow for the assessment of chromosomes labeled with a blue fluorophore

Fig. 5

Centrosome assessment: Blue fluorescence indicates sperm nucleus stained by DAPI. Green fluorescence staining for centrin, a centrosomal protein, as a marker for centrosome presence. Normal value, ≥ 60%

Fig. 6

TEM analysis: Transmission electron micrographs of a normal proximal centriole (left), indicated by a green arrow, and an abnormal proximal centriole (right) with disorganized microtubular structures and missing arms, indicated by a red arrow

Fig. 7

TEM analysis: Transmission electron micrographs of a single spermatozoon from a patient with complete globozoospermia (right), compared to a normal spermatozoon (left)

Fig. 8

Immunofluorescence staining for the PLCζ protein: Blue fluorescence indicates sperm nucleus stained by (DAPI). Green fluorescence in the acrosomal, equatorial (middle), or post-acrosomal area of the sperm head indicates the presence of PLCζ. Normal value, ≥ 30%

Fig. 9

Clinical outcome in historical ICSI cycles vs subsequent cycles with assisted oocyte activation (AOA): AOA, whether by ionomycin or recombinant human PLZζ protein, was able to significantly improve clinical outcome in a subsequent cycle within in same couples (n, 51)