Fertilization of human oocytes : what's needed?

Fertilization of human oocytes : what’s needed?- A mature oocyt- A mature spermatozoon capable to fertilize

Spermatozoa- In vivo spermatozoa attain their fertilizing capacity in the female tract= CAPACITATION ; in vitro this is done by gradient preparation , at 37°-39°C- Two properties : - hyperactive motility- change in surface membrane properties- Spermatozoa bind to receptors on the ZP- ZP induces morphological transformation of the spermatozoon : ACROSOMEREACTION : release of acrosomal content (enzymes)- Spermatozoon passes through ZP to the PVS : fusion of sperm plasma- andoocyt plasmamembranes- Ca+ waves in the oocyt : release of cortical granules > spermblock- Resumption of meiose : extrusion of second polar body, formation of femalepronucleus- Sperm nucleus decondendes, formation of male pronucleus and spindle- The oocyt is fertilized

Normal Fertilizationpronucleinucleoli

Importance of scoring of fertilized oocytes?In ART success rates stay low :- < 30% of all embryos develop completely- Transfer of 2 – 6 embryos: higher success BUT multiple pregnancies- Extended culture to day 5: transfer of 1 to 2 embryossufficient , but only 40-50% of fertilized oocytes growto blastocysts and only 30-50% of the blastocysts implantTHUS: scoring of oocytes and fertilized oocytes (pronuclei) combined withembryo and blastocyst scoring, could help select the BEST embryo?

PN-score : scoring of the male and female pronucleus- contain the genetic material : parental chromosomes- indicator of both gamete quality and subsequent embryo implantationpotential- predictive value through correlations with chromosome constitution andincidence of zygotic arrest (Gianaroli et al.,2003; Balaban et al., 2004; ..)- other reports questioned predictive value (Nicoli et al., 2010; Weitzman etal., 2010)

PN-score : proposed scoring systems• Scott et al.,2000: Z1, Z2, Z3 and Z4• Tesarik & Greco (1999)• Ludwig et al : Scott + Tesarik

First step : Position, size and morphology of the nuclei16 – 18h after fertilization / insemination- juxtaposed: side by side- centrally positioned- approximately same sizeAberrant PN size and position correlates with developmental arrest, andaneuploidy, disruption in raster and microtubule formation (>4µm, far apartor peripherally, presence of fragmented or additional micronuclei (Munné and Cohen,1998) )

Nucleoli• human nucleus has 3 – 7 ; develop on the NucleolusOrganizingRegions ofthe chromosomes (coded for rDNA)• equal number in two daughter cells• nucleoli fuse during mitosis, but always synchrony in number and form• asynchrony : abnormal cells (cancer cells)• abnormal NOR morphology : fragmentation, aging →more dense bodies• involved in cell cycle control : growth regulating and mitogenic proteins• transcription of ribosomal genes• during oocyte maturation : RNA synthesis low : nucleoli small and scattered• at time of fertilization : rRNA synthesis high, nucleoli reform and grow

Second step :Scoring of nucleoli• 3 – 7/ nucleus, equal number, aligned• Not : pinpoints → delayed nuclear events, slow development : only10-15% reach blastocyst• Not : unequal → reduced developmental potential• Alignment or polarized distribution of chromatin is correlated withthe metabolic status and ability of the nuclei to fuse and form uniqueembryonic genome

Abnormal fertilization1 PN : 1% after IVF and ICSI : parthenogenetic (haploid) easy in ‘aged’oocytes or error in fertilization (diploid, but high risk of aneuploidy)3 PN: 1% after ICSI: failure in extrusion of second polar body; 5% in IVF

Cytoplasm : halo → high quality embryos, center is site withthe highest metabolic activity

Istanbul Consensus Workshop (2011)– 17 ± 1 h post insemination– Normal fertilization : 2 centrally, juxtaposed, equal in size nuclei,with equal number of nucleoli– Score :• Symmetrical : equal number and size of nucleoli, alignedor scaterred• Asymmetrical: all other patterns• Abnormal: single nucleoli or noneBut: Timing of assesment is critical, as pronuclear developmentis a dynamic process and zygote scoring should therefore beused with caution and only in conjunction with other methodsof evaluation.

References• Essential Reproduction : Martin H. Johnson, 6th ed.• Textbook of Assisted Reproduction Techniques : Gardner, Weismann, Howles• Atlas of Human Gametes and Conceptus : L. Veeck• In Vitro Fertilization : Kay Elder and Brian Dale, 3th ed• Human Reproduction Vol 27 : Embryo Atlas