*2.1. Polar Body Biopsy*

Polar bodies (PB) are by-products of the meiotic divisions of the oocyte. They have no reproductive function and can be easily removed without affecting embryo development. PB biopsy can be applied in a single step or two step method: the first consist in the simultaneous removal of the first and second PB sixteen h after insemination, whereas the two steps method entails two different biopsies, one prior to intracitoplasmatic sperm injection (ICSI), with the removal of the first PB, and the other sixteen h after insemination, with the removal of the second PB. The single step procedure would seem to be more convenient, since pronuclei detection allows for analyzing only fertilized oocytes, reducing costs and time wasting. Furthermore, combining the first and second PB biopsy could result in an improved abnormalities detection rate [25].

However, the PB biopsy only provides maternal genetic information and does not consider parental or mitotic division abnormalities [26]. European Society of Human Reproduction and Embryology started in 2012 a multicenter randomized clinical trial to establish the effectiveness of PGT-A performed with PB biopsy. The aim of the study was to evaluate whether the analysis of 23 chromosomes in the first and second polar body, and the selection of euploid embryos for transfer, increased live birth rate within one year, in women in advanced maternal age as compared to cycles without PGT-A. From June 2012 to December 2016, 205 women were assigned to cycles with PGT-A, and 191 to cycles without PGT-A (control group). However, the LB rate was not different among the two groups: 50 out of 205 (24%) in the PGT-A group and 45 out of 191 (24%) in the control group [27].

PB biopsy has the benefit of providing a long time to perform genetic testing without requiring embryo cryopreservation despite being time-consuming [25,26] and less cost-effective per LB rate [28] and, therefore, it is the only genetic testing strategy available in many countries with legal restriction on embryo genetic assessment and cryopreservation.
