PREVENTS PREMATURE FERTILIZATION OF LATE-MATURING OOCYTES PharmDr . - PowerPoint PPT Presentation

EGG MATURITY ASSESSMENT PRIOR TO ICSI PREVENTS PREMATURE FERTILIZATION OF LATE-MATURING OOCYTES PharmDr . Zuzana Holubcová, Ph.D.

MATURE EGG?

MATURE EGG DNA (Hoechst) microtubules ( a -tubulin)

MI/MII TRANSITION DNA (Hoechst) microtubules ( a -tubulin)

MI/MII TRANSITION

POLARIZED LIGHT MICROSCOPY • Based on interaction of polarized light with anisotropic materiál . • Allows quick, non-invasive imaging of birefringent structures in living cells.

POLARIZED LIGHT MICROSCOPY IN IVF PRACTICE • Used from late 1990 ´ . • Examination: 1) Meiotic spindle. 2) Zona Pellucida. 3) Sperm head. • Association between presence of MII spindle birefringence and oocyte ´ s developmental competence. Montag, Reproductive Biomedicine Online 2008 Wang et al 2001a,b Moon et al 2003, Montag 2006 Van der Velde 1998 Wang et al 2001c Montag 2006 Hyun et al 2007 Montag 2008 Rama Raju et al 2007 Montag and van der Ven 2008 Kilani et al. 2011 Kilani et al 2014 Tilia et al 2016.

MATURE EGG spindle Spindle birefringence is orientation-dependent

POLARIZED LIGHT MICROSCOPY EXAMINATION spindle Spindle signal is orientation-dependent

OPTIMAL TIME OF ICSI Can the PLM be used to adjust timing of ICSI with respect to the egg ´ s developmental stage? . time

CLINICAL STUDY • Patient ´ s informed consent. • Ethical approval. • Free of charge. • May 2016 - May 2018: 234 cycles / 916 oocytes • Poor prognosis cycles: • Slow/poor responders. (<6 MII oocytes at OPU) • 25 – 48 years, average 36.96 ± 4.28. • Severe male infertility factor in 14.96 % of cycles.

DENUDATION late OPU MI→MII or MII? denudation MI or MII ? MII MII MII early MII MI MII time

STUDY DESIGN 35 - 36 hours Hormonal Oocyte - slow/poor responders retrieval (<6 MII oocytes) stimulation + 3-4 hours Biochemical/clinical pregnancy Live birth Polarized light microscopy ~2 hours Grade D Grade B Grade A Grade C ICSI - individual culture Fertilization - single-step media - 5% 0 2 , 6% CO 2 Utilization Blastulation Cryopreservation

INCIDENCE OF SPINDLE GRADES

MATURATION DELAY Grade B Grade D Grade A Grade C time MII spindle present MII spindle absent Cycle with majority Sperm injection (ICSI) of spindle-negative oocytes postponed

POSTPONING ICSI Grade B Grade D Grade A Grade C time MII spindle present MII spindle absent 59,39 % 98/165 ~2 hours

POSTPONING ICSI

PREIMPLANTATION DEVELOPMENT Chi-squared test, p<0.001 FR ……. Fertilization Rate, BR …... Blastulation Rate, UR …… Utilization Rate

PREIMPLANTATION DEVELOPMENT Holubcová et al, J Assist Reprod Genet. 2019

CLINICAL OUTCOME • 90.74 % transferred embryos derived from spindle-positive oocytes. • 47/49 live births from embryo transfers involving spindle-positive oocytes. MI MII • 28.24 % transferred embryos 11 babies from MI/MII oocytes.

LIMITATIONS Egg maturity Chromosome status Mitochondria Mitotic errors Sperm Endometrium Embryonic genome activation Epigenetics Presence of the spindle does not warrant successfull pregnancy.

CONCLUSIONS time • Spindle signal may appear during in vitro maturation as oocyte progresses through physiological MI/MII transition during which meiotic spindle reassembles.

CONCLUSIONS MII oocytes UR: 45.12 % = Oocyte retrieval MI→MII („l ate-maturing “ oocytes) UR: 30.94 % UR: 30.94 % „ Rescue in vitro maturation “

For further details: Holubcová et al, Journal of Assisted Reproduction and Genetics 2019

ACKNOWLEDGEMENTS • Embryology: Aleš Hampl Drahom í ra Kyjovská Radka Štěpánová Darja P á ralov á PLM study team Martina Martonová Tereza Klenková • IVF specialists. • Nurses. Reprofit

MII MII MII MII MI MI MI GV THANK YOU FOR YOUR ATTENTION GV