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

2. 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

3. Historical background
T Ueara & R Yanagimaki, 1976 Biol Reprod Nov;15(4):
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

4. The micromanipulation apparatus

5. 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.

6. Evolution of assisted microfertilization techniques
SCHEMA

7. 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

8. 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.

9. 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)

10. 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

11. 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

12. 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

13. 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)

14. 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)

15. 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

16. 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

17. 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

18. 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