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 : ACROSOME REACTION : release of acrosomal content (enzymes) Spermatozoon passes through ZP to the PVS : fusion of sperm plasma- and oocyt plasmamembranes Ca+ waves in the oocyt : release of cortical granules > spermblock Resumption of meiose : extrusion of second polar body, formation of female pronucleus Sperm nucleus decondendes, formation of male pronucleus and spindle The oocyt is fertilized

Normal Fertilization

pronuclei

nucleoli

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 embryos sufficient , but only 40-50% of fertilized oocytes grow to blastocysts and only 30-50% of the blastocysts implant

THUS: scoring of oocytes and fertilized oocytes (pronuclei) combined with embryo 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 implantation potential - predictive value through correlations with chromosome constitution and incidence of zygotic arrest (Gianaroli et al.,2003; Balaban et al., 2004; ..) - other reports questioned predictive value (Nicoli et al., 2010; Weitzman et al., 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 nuclei 16 – 18h after fertilization / insemination -

juxtaposed: side by side

-

centrally positioned

-

approximately same size

Aberrant PN size and position correlates with developmental arrest, and aneuploidy, disruption in raster and microtubule formation (>4µm, far apart or peripherally, presence of fragmented or additional micronuclei (Munné and Cohen, 1998) )

Nucleoli

•

human nucleus has 3 – 7 ; develop on the NucleolusOrganizingRegions of the 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 : only 10-15% reach blastocyst • Not : unequal → reduced developmental potential • Alignment or polarized distribution of chromatin is correlated with the metabolic status and ability of the nuclei to fuse and form unique embryonic genome

Abnormal fertilization

1 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 with the 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, aligned or scaterred • Asymmetrical: all other patterns • Abnormal: single nucleoli or none

But: Timing of assesment is critical, as pronuclear development is a dynamic process and zygote scoring should therefore be used with caution and only in conjunction with other methods of 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