rarity of the condition or because affected males are reproductively disadvantaged. therefore, medical uses of pgd for selection of a female offspring to prevent the transmission of x-linked mendelian recessive disorders are often applied. preimplantation genetic diagnosis applied for gender selection can be used for non-mendelian disorders that are significantly more prevalent in one sex. three assessments are made prior to the initiation of the pgd process for the prevention of these inherited disorders. in order to validate the use of pgd, gender selection is based on the seriousness of the inherited condition, the risk ratio in either sex, or the options for disease treatment. minor disabilities a 2006 survey reveals that pgd has occasionally been used to select an embryo for the presence of a particular disease or disability, such as deafness, in order that the child would share that characteristic with the parents. classification we distinguish three types of pgt depending on the defects evaluated: pgt-a: also called preimplantational genetic screening (pgs). improves pregnancy rates by allowing the discard of aneuploids and the selection of euploid embryos for transfer. euploid embryos are more likely to implant and develop into a healthy pregnancy. ngs and fish are the most frequently techniques used for the diagnosis of monosomies, trisomies and poliploidies. pgt-m: in this case, monogenic diseases are being evaluated in the embryo. monogenic disorders are caused by single-gene mutations (autosomal recessive, autosomal dominant, x-linked). previously, pcr, was used for pgt-m. however, more recently pgt-m uses array and ngs technology. pgt-sr: every structural abnormality in the chromosome is taken into account (translocations, inversions, duplications, insertions, deletions). various techniques used: pcr, fish and ngs. technical aspects pgd is a form of genetic diagnosis performed prior to implantation. this implies that the patient's oocytes should be fertilized in vitro and the embryos kept in culture until the diagnosis is established. it is also necessary to perform a biopsy on these embryos in order to obtain material on which to perform the diagnosis. the diagnosis itself can be carried out using several techniques, depending on the nature of the studied condition. generally, pcr-based methods are used for monogenic disorders and fish for chromosomal abnormalities and for sexing those cases in which no pcr protocol is available for an x-linked disease. these techniques need to be adapted to be performed on blastomeres and need to be thoroughly tested on single-cell models prior to clinical use. finally, after embryo replacement, surplus good quality unaffected embryos can be cryopreserved, to be thawed and transferred back in a next cycle. obtaining embryos currently, all pgd embryos are obtained by assisted reproductive technology, although the use of natural cycles and in vivo fertilization followed by uterine lavage was attempted in the past and is now largely abandoned. in order to obtain a large group of oocytes, the patients undergo controlled ovarian stimulation (coh). coh is carried out either in an agonist protocol, using gonadotrophin-releasing hormone (gnrh) analogues for pituitary desensitisation, combined with human menopausal gonadotrophins (hmg) or recombinant follicle-stimulating hormone (fsh), or an antagonist protocol using recombinant fsh combined with a gnrh antagonist according to clinical assessment of the patient's profile (age, body mass index (bmi), endocrine parameters). hcg is administered when at least three follicles of more than 17 mm mean diameter are seen at transvaginal ultrasound scan. transvaginal ultrasound-guided oocyte retrieval is scheduled 36 hours after hcg administration. luteal phase supplementation consists of daily intravaginal administration of 600 µg of natural micronized progesterone. oocytes are carefully denudated from the cumulus cells, as these cells can be a source of contamination during the pgd if pcr-based technology is used. in the majority of the reported cycles, intracytoplasmic sperm injection (icsi) is used instead of ivf. the main reasons are to prevent contamination with residual sperm adhered to the zona pellucida and to avoid unexpected fertilization failure. the icsi procedure is carried out on mature metaphase-ii oocytes and fertilization is assessed 16–18 hours after. the embryo development is further evaluated every day prior to biopsy and until transfer to the woman's uterus. during the cleavage stage, embryo evaluation is performed daily on the basis of the number, size, cell-shape and fragmentation rate of the blastomeres. on day 4, embryos were scored in function of their degree of compaction and blastocysts were evaluated according to the quality of the throphectoderm and inner cell mass, and their degree of expansion. biopsy procedures as pgd can be performed on cells from different developmental stages, the biopsy procedures vary accordingly. theoretically, the biopsy can be performed at all preimplantation stages, but only three have been suggested: on unfertilised and fertilised oocytes (for polar bodies, pbs), on day three cleavage-stage embryos (for blastomeres) and on blastocysts (for trophectoderm cells). the biopsy procedure always involves two steps: the opening of the zona pellucida and the removal of the cells. there are different approaches to both steps, including mechanical, chemical, and physical (tyrode's acidic solution) and laser technology for the breaching of the zona pellucida, extrusion or aspiration for the removal of pbs and blastomeres, and herniation of the trophectoderm cells. polar body biopsy main article: polar body biopsy a polar body biopsy is the sampling of a polar body, which is a small haploid cell that is formed concomitantly as an egg cell during oogenesis, but which generally does not have the ability to be fertilized. compared to a blastocyst biopsy, a polar body biopsy can potentially be of lower costs, less harmful side-effects, and more sensitive in detecting abnormalities. the main advantage of the use of polar bodies in pgd is that they are not necessary for successful fertilisation or normal embryonic development, thus ensuring no deleterious effect for the embryo. one of the disadvantages of pb biopsy is that it only provides information about the maternal contribution to the embryo, which is why cases of maternally inherited autosomal dominant and x-linked disorders that are exclusively maternally transmitted can be diagnosed, and autosomal recessive disorders can only partially be diagnosed. another drawback is the increased risk of diagnostic error, for instance due to the degradation of the genetic material or events of recombination that lead to heterozygous first polar bodies. cleavage-stage biopsy (blastomere biopsy) cleavage-stage biopsy is generally performed the morning of day three post-fertilization, when normally developing embryos reach the eight-cell stage. the biopsy is usually performed on embryos with less than 50% of anucleated fragments and at an eight-cell or later stage of development. a hole is made in the zona pellucida and one or two blastomeres containing a nucleus are gently aspirated or extruded through the opening. the main advantage of cleavage-stage biopsy over pb analysis is that the genetic input of both parents can be studied. on the other hand, cleavage-stage embryos are found to have a high rate of chromosomal mosaicism, putting into question whether the results obtained on one or two blastomeres will be representative for the rest of the embryo. it is for this reason that some programs utilize a combination of pb biopsy and blastomere biopsy. furthermore, cleavage-stage biopsy, as in the case of pb biopsy, yields a very limited amount of tissue for diagnosis, necessitating the development of single-cell pcr and fish techniques. although theoretically pb biopsy and blastocyst biopsy are less harmful than cleavage-stage biopsy, this is still the prevalent method. it is used in approximately 94% of the pgd cycles reported to the eshre pgd consortium. the main reasons are that it allows for a safer and more complete diagnosis than pb biopsy and still leaves enough time to finish the diagnosis before the embryos must be replaced in the patient's uterus, unlike blastocyst biopsy. of all cleavage-stages, it is generally agreed that the optimal moment for biopsy is at the eight-cell stage. it is diagnostically safer than the pb biopsy and, unlike blastocyst biopsy, it allows for the diagnosis of the embryos before day 5. in this stage, the cells are still totipotent and the embryos are not yet compacting. although it has been shown that up to a quarter of a human embryo can be removed without disrupting its development, it still remains to be studied whether the biopsy of one or two cells correlates with the ability of the embryo to further develop, implant, and grow into a full-term pregnancy. not all methods of opening the zona pellucida have the same success rate because the well-being of the embryo and/or blastomere may be impacted by the procedure used for the biopsy. zona drilling with acid tyrode's solution (zd) was looked at in comparison to partial zona dissection (pzd) to determine which technique would lead to more successful pregnancies and have less of an effect on the embryo and/or blastomere. zd uses a digestive enzyme like pronase which makes it a chemical drilling method. the chemicals used in zd may have a damaging effect on the embryo. pzd uses a glass microneedle to cut the zona pellucida which makes it a mechanical dissection method that typically needs skilled hands to perform the procedure. in a study that included 71 couples, zd was performed in 26 cycles from 19 couples and pzd was performed in 59 cycles from 52 couples. in the single-cell analysis, there was a success rate of 87.5% in the pzd group and 85.4% in the zd group. the maternal age, number of oocytes retrieved, fertilization rate, and other variables did not differ between the zd and pzd groups. it was found that pzd led to a significantly higher rate of pregnancy (40.7% vs 15.4%), ongoing pregnancy (35.6% vs 11.5%), and implantation (18.1% vs 5.7%) than zd. this suggests that using the mechanical method of pzd in blastomere biopsies for preimplantation genetic diagnosis may be more proficient than using the chemical method of zd. the success of pzd over zd could be attributed to the chemical agent in zd having a harmful effect on the embryo and/or blastomere. currently, zona drilling using a laser is the predominant method of opening the zona pellucida. using a laser is an easier technique than using mechanical or chemical means. however, laser drilling could be harmful to the embryo and it is very expensive for in vitro fertilization laboratories to use especially when pgd is not a prevalent process as of modern times. pzd could be a viable alternative to these issues. blastocyst biopsy in an attempt to overcome the difficulties related to single-cell techniques, it has been suggested to biopsy embryos at the blastocyst stage, providing a larger amount of starting material for diagnosis. it has been shown that if more than two cells are present in the same sample tube, the main technical problems of single-cell pcr or fish would virtually disappear. on the other hand, as in the case of cleavage-stage biopsy, the chromosomal differences between the inner cell mass and the trophectoderm (te) can reduce the accuracy of diagnosis, although this mosaicism has been reported to be lower than in cleavage-stage embryos. te biopsy has been shown to be successful in animal models such as rabbits, mice and primates. these studies show that the removal of some te cells is not detrimental to the further in vivo development of the embryo. human blastocyst-stage biopsy for pgd is performed by making a hole in the zp on day three of in vitro culture. this allows the developing te to protrude after blastulation, facilitating the biopsy. on day five post-fertilization, approximately five cells are excised from the te using a glass needle or laser energy, leaving the embryo largely intact and without loss of inner cell mass. after diagnosis, the embryos can be replaced during the same cycle, or cryopreserved and transferred in a subsequent cycle. there are two drawbacks to this approach, due to the stage at which it is performed. first, only approximately half of the preimplantation embryos reach the blastocyst stage. this can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the pgd. mcarthur and coworkers report that 21% of the started pgd cycles had no embryo suitable for te biopsy. this figure is approximately four times higher than the average presented by the eshre pgd consortium data, where pb and cleavage-stage biopsy are the predominant reported methods. on the other hand, delaying the biopsy to this late