Assisted microfertilization techiques Prof. Maria Elena Dell’Aquila Department of Biosciences Biotechnologies and Biopharmaceutics University of Bari Aldo Moro - Italy

Summary Introduction Historical background Techniques to overcome the ZP barrier Techniques to artificially combine the sperm cell and the oocyte Oocyte and sperm preparation for ICSI ICSI procedure ICSI in human reproductive medicine ICSI in farm animals ICSI for research on the fertilization process

Historical background T Ueara & R Yanagimaki, 1976 Biol Reprod. 1976 Nov;15(4):467-70. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Abstract Isolated nuclei of hamster spermatozoa develop into male pronuclei when injected into hamster eggs. The nuclei of fresh, frozen-thawed and freeze-dried human spermatozoa are equally capable of developing into male pronuclei when injected into hamster eggs. These results indicate that sperm nuclei are stable organelles and the egg cytoplasmic factors controlling the transformation of sperm nuclei into male pronuclei are not strictly species-specific. C Keefer & B Brackett, 1987 G Palermo et al., 1992 Van Steirteghem et al., 1993

The micromanipulation apparatus

The micromanipulation apparatus Abbreviations: lem, left electric micromanipulator; rem, right electric micromanipulator; lmm, left manual micromanipulator; rmm, right manual micromanipulator; sb, supporting block; hpi, holding pipette injector; mni, microinjection needle injector; mb, metal base; dc, digital camera (optional); air, air-cushioned platform; im, inverted microscope; st, microscope stage; hp, holding pipette; mn, microinjection needle; p, piezo.

Evolution of assisted microfertilization techniques SCHEMA

Indications for ICSI AZOOSPERMIA (absence of sperm in the ejaculate). It can be due to: - Obstruction in ejaculatory ducts - Retrograde ejaculation - where the ejaculate goes back into the bladder. - Absence of vas deferens. - Anejaculation - when the patient has problems ejaculating. - Failure of testes to produce mature sperm. SEVERE OLIGOSPERMIA - sperm count less than one million/mL ASTHENOSPERMIA - very poor sperm motility TERATOZOOSPERMIA - less than 1% normal sperm. Refractory UNEXPLAINED INFERTILITY OOCYTE ABNORMALITY - the outer layer (zona-pellucida) of oocyte is resistant to the entry of sperm. ANTISPERM ANTIBODIES

Semen samples for ICSI EJACULATED SPERM EPIDYDIMAL SPERM SPERM SAMPLES OBTAINED BY: - TESA (Testicular Sperm Aspiration) This procedure is applied in azoospermic men. The few sperm needed for oocyte fertilization are retrieved from the testes via needle aspiration. - TESE (Testicular Sperm Extraction) Sperm are obtained through microsurgery. - MESA (Microsurgical Epididymal Sperm Aspiration) Here the desired sperm are obtained from the epididymis via microsurgery. - PESA (Percutaneous Epididymal Sperm Aspiration) Here a needle is introduced into the epididymis through the skin, and the epidiyman contents aspirated to procure some sperm.

Oocyte and sperm preparation for ICSI Oocyte preparation - cumulus cell removal (80 UI/ml hyaluronidase in IVM medium) - morphology selection (PB, ZP, perivitelline space, cytoplasm) Sperm preparation - washing (Tyr-HEPES; EBSS) - motility reduction (+10% PVP)

Set up of the ICSI dish Microdrop of 7% PVP medium for sperm holding Mineral oil overlay 1 microdrops of a fertilization medium for oocyte holding 4 2 3 Sperm smear to focus the bottom of the petri dish 60 mm Ø petri dish lid

ICSI Procedure Sperm immobilization and aspiration tail first Oocyte orientation The sperm is pushed at the tip of the injection pipette Breaking oocyte membrane Aspiration of some ooplasm into the injection pipette Release of the sperm cell into the ooplasm Release of the oocyte Transfer of the oocyte into culture medium

Possible problems Excessive cytoplasmic aspiration Excessive PVP medium release within the oocyte Difficulty in breaking the oocyte membrane Difficult sperm release Risk of breaking the oolemma on the opposite side

ICSI in human with defective sperm ICSI with: Very few sperm cells Sperm head Immotile sperm Dead sperm Grossly defective sperm cells Round/elongated spermatids (ROSI/ELSI)

ICSI in human with defective sperm ICSI with: Very few sperm cells Sperm head Immotile sperm Dead sperm Grossly defective sperm cells Round/elongated spermatids (ROSI/ELSI)

ICSI in farm animals ICSI is used for clinical and commercial applications in those species in which conventional IVF does not provide suitable and reproducible results (the horse) ICSI with sexed sperm ICSI for saving high quality sperm samples ICSI with vitrified oocytes With non-motile sperms With very poor quality sperm samples

ICSI for research on the fertilization process System of unique utility for investigating the sequence of events occurring when the sperm enters the oocyte and interacts with the ooplasm Tools such as Laser Scanning Confocal Microscopy

Advanced technologies TO REDUCE STRESS DUE TO INJECTION PIEZO-ASSISTED ICSI LASER-ASSISTED ICSI TO IMPROVE SPERM SELECTION IMSI (ICSI with morphologically selected sperm) BIREFRINGENCY TO IMPROVE OOCYTE SELECTION POLSCOPE

References Transgenic Animal Technology (chap 20) Dell’Aquila et al., Hum Reprod 1997 Sanchez-Calabuig et al., Frontiers in public health 2014;2:1-7 Salamone et al., Reproduction 2017; 154:F111-F124