Lale Karakoc Sokmensuer, M.D. Recurent ART Failure in Evidence Based-Medicine: Embryologist Perspective Lale Karakoc Sokmensuer, M.D.

Recurrent ART Failure If all laboratory and stimulation parameters are controlled, underlying cause of failure is attributable to either of the gametes or the embryo. careful observation and embryo selection, to aid in selecting the right embryo to overcome this failure. The morphology of the zygote, the state of the cleaving embryos development, and the blastocyst can influence implantation rates.

Without functional gametes it is unlikely that success can be achieved. An early indicator of this functionality is the morphology of the zygote, which can be influenced by either the oocyte or the spermatozoon, Zygote morphology can be altered by either improving oocyte quality during stimulation or by using donor sperm Recurrent implantation failure > 3 embryo transfers with high quality embryos or the transfer of ≥ 10 embryos in multiple transfers ESHRE PGD Consortium, Hum Reprod 2005

treatment options intratubal transfer of gametes and embryos, blastocyst stage transfer, sequential embryo transfer, assisted hatching, co-cultures preimplantation genetic diagnosis for aneuploidy screening.

Intratubal transfer of zygotes or embryos more physiological embryos benefit from endosalpingeal secretions and reach the endometrium at an appropriate period that coincides more with the window of implantation

Intratubal transfer of zygotes or embryos A meta-analysis of randomized trials of tubal versus uterine embryo transfer in couples undergoing fresh, frozen, or donor embryos failed to show any beneifit of tubal transfer in the general IVF population (Habana, 2001) (Tanbo et al., 1990; Balmeceda et al.,1992; Tournaye et al., 1992; Fluker et al ., 1993; Preutthipan et al., 1994; van Voorhis et al., 1995) disadvantages→ need for laparoscopy under general anaesthesia with its associated complications

Intratubal transfer of zygotes or embryos Intratubal embryo transfer should be seriously considered in women who recurrently undergo very difficult and traumatic transcervical embryo transfers despite dilatation.

Blastocyst transfer In women who do not conceive with repeated day 3 transfers, blastocyst transfer may be considered Blastocyst stage transfer is more physiological, as the endometrium is synchronized with the developmental stage of the embryo. To assess the true viability of an embryo (post-embryonic genome activation) the embryo must be cultured up to the blastocyst stage (Braude, 1988; Taylor,1997; Urman, 2001).

Blastocyst transfer blastocyst culture has been reported to substantially increase the implantation rate per embryo transferred (Gardner, 1998; Huisman, 2000; Milki, 2000). Highest implantation rates reported for selective cleavage stage embryo transfers are lower than those reported for blastocyst transfers (Van Royen, 1999; Gardner, 2000).

Blastocyst transfer Similar to cleavage stage embryos not all blastocysts are equally implantation competent. Blastocyst grade is closely associated with the success of blastocyst transfer with good quality blastocysts giving rise to higher implantation rates compared with poor quality blastocysts. Expanded blastocysts with a normal ICM yielded higher implantation rates (Kovacevic, 2004).

The value of blastocyst transfer in RIF couples who failed to conceive with previous 3- to 4-cleavage stage embryo transfers were offered the alternative of blastocyst transfer in their subsequent cycle. 22 women underwent day 3 and 15 women day 5 embryo transfers. Despite transferring significantly fewer embryos on day 5, implantation (3.4 versus 11.3%) and pregnancy (9.1 versus 40%) rates were signicantly improved. (Cruz, 1999)

54 patients who failed to conceive with 2–3 cycles of cleavage stage embryo transfers were randomly allocated to day 2–3 or day 5 embryo transfers A significantly higher implantation rate (21.2 versus 6%) was recorded for blastocyst transfers compared with day 2–3 transfers. Pregnancy rates per oocyte retrieval and embryo transfer, although higher in the blastocyst group did not reach statistical significance. Embryo transfer cancellation rate was higher (26 versus 6.4%) in the blastocyst transfer group. Levitas, 2004

In patients with RIF, in the presence of good quality embryos, blastocyst transfer may take place The application of blastocyst transfer will permit the observation of embryonic growth well to its advanced stages and enable better embryo selection. Blastocyst transfer may be particularly useful in couples who produce many embryos.

Sequential embryo transfer outcome of sequential embryo transfers on days 2 and 4 or 5 in patients who repeatedly failed to conceive despite the transfer of good quality embryos A 38 and 60% clinical pregnancy rate was achieved in women who had day 2 and 4 and day 2 and 5 sequential embryo transfers Whether sequential embryo transfer can serve as a viable option in women recurrent implantation failure, however, remains to be determined in prospective randomized trials. Loutradis, 2004

Assisted hatching Elasticity and thinning of the zona pellucida are essential for the hatching process both of which can be adversely influenced by advancing maternal age and in-vitro culture conditions (Cohen, 1992; Schiewe, 1995; Mandelbaum, 1996). embryos with a thin zona and embryos that have been subjected to microsurgical dissection resulting in artificial gaps in their zona were shown to implant more efficiently (Cohen et al., 1989; Cohen, 1991).

Assisted hatching five studies reported on the impact of assisted hatching in patients with previous implantation failures (Stein, 1995; Antinori, 1996; Chao, 1997; Nakayama, 1997; Magli, 1998). In four of these studies mechanical means, and in one study a piezoelectric technique, was used to breach the zona pellucida. assisted hatching resulted in statistically higher implantation, clinical pregnancy and ongoing pregnancy rates (Sallam, 2003). An odds ratio of 1.63 overall with a greater benefit for older women and prior assisted reproduction failure can be expected of assisted hatching (Edi-Osagie, 2003).

Assisted hatching It is current policy to perform assisted hatching using the laser in the subgroup of patients with advanced age (>37 years), one or more previous implantation failures, and with poor embryo characteristics such as suboptimal embryo quality and thick zona pellucida.

Co-cultures Co-cultures have been advocated in assisted reproduction due to inadequacy of simple media to support embryo development beyond the cleavage stage. Suggested benefits of co-cultures include secretion of trophic factors such as nutrients, substrates, growth factors, and cytokines and removal of potentially toxic substances by the cultured cells.

Randomized controlled trials that compared co-cultures in unselected patient populations undergoing IVF–embryo transfer failed to demonstrate a significant benefit (Van Blerkom, 1993; Sakkas et al., 1994).

In couples with recurrent implantation failure, granulose cell co-culture increased the progression of cleavage stage embryos to the blastocyst stage. However, these embryos did not implant more efficiently than their non co-cultured counterparts. Plachot, 1993

a total of 127 couples with an average 3 a total of 127 couples with an average 3.8 failed IVF–embryo transfer cycles, AECC (autologous endometrial co-culture) and blastocyst transfer failed to improve implantation and pregnancy rates compared with a control group undergoing IVF and day 2 embryo transfers. Simon, 1999 women with repeated implantation failures and were unable to demonstrate significant differences in implantation and pregnancy rates between AECC and simple media when embryos were transferred on day 3 Barmat, 1999

whether co-cultures are beneficial in patients with repeated implantation failures should be investigated in randomized trials.

Preimplantation genetic diagnosis for aneuploidy screening (PGD–AS) PGD for aneuploidy screening by polar body or blastomere biopsy detection of chromosomal aberrations in the embryo thus eliminating those with reduced or no implantation potential and those who are destined to abort or result in abnormal offspring. When all other factors leading to RIF have been eliminated embryonic aneuploidy is usually blamed for non-implantation.

PGD Choosing chromosomally normal embryos among a cohort may also improve embryo selection and increase pregnancy rates. advanced age, couples with recurrent implantation failure, and recurrent aborters (Gianaroli, 1999; Munné , 1999; Kahraman, 2000; Pehlivan, 2003; Caglar, 2004; Kuliev, 2004).

PGD 66 couples who after failing to conceive with 3 or more IVF cycles subsequently underwent IVF combined with PGD high rates of chromosomal abnormalities (60%) for an average maternal age of 32 years. The major chromosomal abnormality was not aneuploidy but mosaicism, polyploidy and haploidy (Gianaroli 1999, 2002).

PGD The risk of chromosomal abnormalities in embryos directly increases with the number of previous IVF failures. Whereas the chromosomal abnormality rate is around 40% in patients with two failed IVF cycles, the abnormality rate increases to 50% in patients with three failed IVF cycles and to 67% in those with more than five failures (Gianaroli, 1997).

PGD similar rates of aneuploid embryos when they compared couples who failed >2 IVF cycles with those undergoing their first or second IVF cycle (31 and 33%) Munné, 2003

significantly higher aneuploidy rates (50. 2 versus 26 significantly higher aneuploidy rates (50.2 versus 26.3%) in embryos from couples who experienced RIFs compared with controls the incidence of mosaic embryos also was significantly higher (19.9 versus 10.8%). PGD resulted in higher success rates in the RIF group (34.0% pregnancy and 19.8% implantation rates) that were comparable to young fertile controls. Pehlivan, 2002

similar implantation rates to a control group after PGD for patients with more than two failed IVF attempts (14.3 versus 11.5%) Munné, 2003 differences between these reports relies on differences in mean ages of recurrent implantation failure groups and complex underlying pathophysiology of RIF.

62.1% normoploid embryos in patients with more than two previous failed attempts using three-probe fluorescent in-situ hybridization (FISH) analysis Implantation rate was improved in the PGD group compared with controls (21.0 versus 10.0%). Wilding, 2004

using comparative genomic hybridization (CGH) for screening preimplantation embryos, chromosomal abnormalities in 60% of single blastomeres obtained from women with repeated IVF failures (Voullaire et al., 2002).

The data of the ESHRE PGD consortium, which includes PGD cycles from 25 centres, reported that implantation rate in recurrent implantation failure was only 11%, compared with 32–36% in aneuploidy screening for other indications (ESHRE PGD Consortium Steering Committee, 2002).

Whether the application of newer and more complex methods such as comparative genomic hybridization, and the use of microarrays and multiplex fluorescent polymerase chain reaction on whole genome amplified DNA will benefit the couple with recurrent implantation failure remain to be shown (Wilton, 2005).