Recurent ART Failure in Evidence Recurent ART Failure in Evidence Based-Medicine: Embryologist Based-Medicine: Embryologist

PerspectivePerspective

Lale Karakoc Sokmensuer, M.D. Lale Karakoc Sokmensuer, M.D.

If all laboratory and stimulation parameters are If all laboratory and stimulation parameters are controlled, underlying cause of failure is controlled, underlying cause of failure is attributable to either of the gametes or the attributable to either of the gametes or the embryo. embryo.

careful observation and embryo selection, to aid in careful observation and embryo selection, to aid in selecting the right embryo to overcome this selecting the right embryo to overcome this failure. failure.

The morphology of the zygote, the state of the The morphology of the zygote, the state of the cleaving embryos development, and the blastocyst cleaving embryos development, and the blastocyst can influence implantation rates. can influence implantation rates.

Recurrent ART Failure

WWithout functional gametes it is unlikely that success can be ithout functional gametes it is unlikely that success can be achieved. achieved.

An early indicator of this functionality is the morphology of the An early indicator of this functionality is the morphology of the zygote, which can be influenced by either the oocyte or the zygote, which can be influenced by either the oocyte or the spermatozoon, spermatozoon,

Zygote morphologyZygote morphology can be altered by either improving oocyte can be altered by either improving oocyte quality during stimulation or by using donor spermquality during stimulation or by using donor sperm

Recurrent implantation failureRecurrent implantation failure

> 3 embryo transfers with high quality embryos or the transfer > 3 embryo transfers with high quality embryos or the transfer of of ≥ ≥ 10 embryos in multiple transfers 10 embryos in multiple transfers

ESHRE PGD Consortium, Hum Reprod 2005ESHRE PGD Consortium, Hum Reprod 2005

treatment optionstreatment options

intratubal transfer of gametes and intratubal transfer of gametes and embryos,embryos,

blastocyst stage transfer, blastocyst stage transfer, sequential embryo transfer, sequential embryo transfer, assisted hatching, assisted hatching, co-culturesco-cultures preimplantation genetic diagnosis for preimplantation genetic diagnosis for

aneuploidy screening.aneuploidy screening.

Intratubal transfer of zygotes orIntratubal transfer of zygotes orembryosembryos

more physiologicalmore physiological

embryos benefit from endosalpingeal embryos benefit from endosalpingeal secretions and reach the endometrium secretions and reach the endometrium at an appropriate period that at an appropriate period that coincides more with the window of coincides more with the window of implantationimplantation

Intratubal transfer of zygotes orIntratubal transfer of zygotes orembryosembryos

A meta-analysis of randomized trials of tubal versus A meta-analysis of randomized trials of tubal versus uterine embryo transfer in couples undergoing fresh, uterine embryo transfer in couples undergoing fresh, frozen, or donor embryos failed to show any beneifit frozen, or donor embryos failed to show any beneifit of tubal transfer in the general IVF population of tubal transfer in the general IVF population (Habana, 2001)(Habana, 2001)

((Tanbo Tanbo et alet al., 1990; Balmeceda ., 1990; Balmeceda et alet al.,1992; Tournaye .,1992; Tournaye et alet al., 1992; Fluker ., 1992; Fluker et al et al ., 1993; Preutthipan ., 1993; Preutthipan et alet al., 1994; van Voorhis ., 1994; van Voorhis et alet al., 1995., 1995))

disadvantagesdisadvantages → → need for laparoscopy under general need for laparoscopy under general anaesthesia with its associated complicationsanaesthesia with its associated complications

Intratubal transfer of zygotes orIntratubal transfer of zygotes orembryosembryos

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

Blastocyst transferBlastocyst transfer

In women who do not conceive with repeated day 3 In women who do not conceive with repeated day 3 transfers, blastocyst transfer may be consideredtransfers, blastocyst transfer may be considered

Blastocyst stage transfer is more physiological, as Blastocyst stage transfer is more physiological, as the endometrium is synchronized with the the endometrium is synchronized with the developmental stage of the embryo.developmental stage of the embryo.

To assess the true viability of an embryo (post-To assess the true viability of an embryo (post-embryonic genome activation) the embryo must be embryonic genome activation) the embryo must be cultured up to the blastocyst stage (Braude, 1988; cultured up to the blastocyst stage (Braude, 1988; Taylor,1997; Urman, 2001).Taylor,1997; Urman, 2001).

Blastocyst transferBlastocyst transfer

blastocyst culture has been reported to blastocyst culture has been reported to substantially increase the implantation rate substantially increase the implantation rate per embryo transferred (Gardner, 1998; per embryo transferred (Gardner, 1998; Huisman, 2000; Milki, 2000). Huisman, 2000; Milki, 2000).

Highest implantation rates reported for Highest implantation rates reported for selective cleavage stage embryo transfers are selective cleavage stage embryo transfers are lower than those reported for blastocyst lower than those reported for blastocyst transfers (Van Royen, 1999; Gardner, 2000).transfers (Van Royen, 1999; Gardner, 2000).

Blastocyst transferBlastocyst transfer Similar to cleavage stage embryos not all Similar to cleavage stage embryos not all

blastocysts are equally implantation blastocysts are equally implantation competent. competent.

Blastocyst grade is closely associated with Blastocyst grade is closely associated with the success of blastocyst transfer with good the success of blastocyst transfer with good quality blastocysts giving rise to higher quality blastocysts giving rise to higher implantation rates compared with poor quality implantation rates compared with poor quality blastocysts.blastocysts.

Expanded blastocysts with a normal ICM Expanded blastocysts with a normal ICM yielded higher implantation rates (Kovacevic, yielded higher implantation rates (Kovacevic, 2004).2004).

The value of blastocyst transfer in The value of blastocyst transfer in RIFRIF

couples who failed to conceive with previous 3- to 4-couples who failed to conceive with previous 3- to 4-cleavage stage embryo transfers were offered the cleavage stage embryo transfers were offered the alternative of blastocyst transfer in their subsequent alternative of blastocyst transfer in their subsequent cycle.cycle.

22 women underwent day 3 and 15 women day 5 22 women underwent day 3 and 15 women day 5 embryo transfers. embryo transfers.

Despite transferring significantly fewer embryos on Despite transferring significantly fewer embryos on day 5, implantation (3.4 versus 11.3%) and pregnancy day 5, implantation (3.4 versus 11.3%) and pregnancy (9.1 versus 40%) rates were signicantly improved. (9.1 versus 40%) rates were signicantly improved.

(Cruz, 1999)(Cruz, 1999)

54 patients who failed to conceive with 2–3 cycles of cleavage 54 patients who failed to conceive with 2–3 cycles of cleavage stage embryo transfers were randomly allocated to day 2–3 or stage embryo transfers were randomly allocated to day 2–3 or day 5 embryo transfers day 5 embryo transfers

A significantly higher implantation rate (21.2 versus 6%) was A significantly higher implantation rate (21.2 versus 6%) was recorded for blastocyst transfers compared with day 2–3 recorded for blastocyst transfers compared with day 2–3 transfers.transfers.

Pregnancy rates per oocyte retrieval and embryo transfer, Pregnancy rates per oocyte retrieval and embryo transfer, although higher in the blastocyst group did not reach statistical although higher in the blastocyst group did not reach statistical significance. significance.

Embryo transfer cancellation rate was higher (26 versus 6.4%) Embryo transfer cancellation rate was higher (26 versus 6.4%) in the blastocyst transfer group. in the blastocyst transfer group.

Levitas, 2004Levitas, 2004

In patients with RIF, in the presence of good In patients with RIF, in the presence of good quality embryos, blastocyst transfer may take quality embryos, blastocyst transfer may take placeplace

The application of blastocyst transfer will The application of blastocyst transfer will

permit the observation of embryonic growth permit the observation of embryonic growth well to its advanced stages and enable better well to its advanced stages and enable better embryo selection.embryo selection.

Blastocyst transfer may be particularly useful Blastocyst transfer may be particularly useful in couples who produce many embryos.in couples who produce many embryos.

Sequential embryo transferSequential embryo transfer

outcome of sequential embryo transfers on days 2 and 4 or 5 in outcome of sequential embryo transfers on days 2 and 4 or 5 in patients who repeatedly failed to conceive despite the transfer patients who repeatedly failed to conceive despite the transfer of good quality embryosof good quality embryos

A 38 and 60% clinical pregnancy rate was achieved in women who A 38 and 60% clinical pregnancy rate was achieved in women who had day 2 and 4 and day 2 and 5 sequential embryo transfershad day 2 and 4 and day 2 and 5 sequential embryo transfers

Whether sequential embryo transfer can serve as a viable option Whether sequential embryo transfer can serve as a viable option

in women recurrent implantation failure, however, remains to be in women recurrent implantation failure, however, remains to be determined in prospective randomized trials.determined in prospective randomized trials.

Loutradis, 2004Loutradis, 2004

Assisted hatchingAssisted hatching

Elasticity and thinning of the zona pellucida are Elasticity and thinning of the zona pellucida are essential for the hatching process both of which can essential for the hatching process both of which can be adversely influenced by advancing maternal age be adversely influenced by advancing maternal age and in-vitro culture conditions (Cohen, 1992; Schiewe, and in-vitro culture conditions (Cohen, 1992; Schiewe, 1995; Mandelbaum, 1996).1995; Mandelbaum, 1996).

embryos with a thin zona and embryos that have been embryos with a thin zona and embryos that have been subjected to microsurgical dissection resulting in subjected to microsurgical dissection resulting in artificial gaps in their zona were shown to implant artificial gaps in their zona were shown to implant more efficiently (Cohen more efficiently (Cohen et alet al., 1989; Cohen, 1991). ., 1989; Cohen, 1991).

Assisted hatchingAssisted hatching five studies reported on the impact of assisted hatching in five studies reported on the impact of assisted hatching in

patients with previous implantation failures patients with previous implantation failures

(Stein, 1995; Antinori, 1996; Chao, 1997; Nakayama, 1997; Magli, 1998). (Stein, 1995; Antinori, 1996; Chao, 1997; Nakayama, 1997; Magli, 1998).

In four of these studies mechanical means, and in one study a In four of these studies mechanical means, and in one study a piezoelectric technique, was used to breach the zona pellucida. piezoelectric technique, was used to breach the zona pellucida.

assisted hatching resulted in statistically higher implantation, clinical assisted hatching resulted in statistically higher implantation, clinical pregnancy and ongoing pregnancy rates (Sallam, 2003). pregnancy and ongoing pregnancy rates (Sallam, 2003).

An odds ratio of 1.63 overall with a greater benefit for older women An odds ratio of 1.63 overall with a greater benefit for older women and prior assisted reproduction failure can be expected of assisted and prior assisted reproduction failure can be expected of assisted hatching (Edi-Osagie, 2003).hatching (Edi-Osagie, 2003).

Assisted hatchingAssisted hatching

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

Co-culturesCo-cultures

Co-cultures have been advocated in assisted Co-cultures have been advocated in assisted reproduction due to inadequacy of simple media reproduction due to inadequacy of simple media to support embryo development beyond the to support embryo development beyond the cleavage stage.cleavage stage.

Suggested benefits of co-cultures include Suggested benefits of co-cultures include secretion of trophic factors such as nutrients, secretion of trophic factors such as nutrients, substrates, growth factors, and cytokines and substrates, growth factors, and cytokines and removal of potentially toxic substances by the removal of potentially toxic substances by the cultured cells.cultured cells.

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

In couples with recurrent implantation failure, In couples with recurrent implantation failure, granulose cell co-culture increased the granulose cell co-culture increased the progression of cleavage stage embryos to the progression of cleavage stage embryos to the blastocyst stage. blastocyst stage.

However, these embryos did not implant more However, these embryos did not implant more efficiently than their non co-cultured efficiently than their non co-cultured counterparts. counterparts.

Plachot, 1993Plachot, 1993

a total of 127 couples with an average 3.8 failed IVF–a total of 127 couples with an average 3.8 failed IVF–embryo transfer cycles, AECC (autologous endometrial embryo transfer cycles, AECC (autologous endometrial co-culture) and blastocyst transfer failed to improve co-culture) and blastocyst transfer failed to improve implantation and pregnancy rates compared with a implantation and pregnancy rates compared with a control group undergoing IVF and day 2 embryo control group undergoing IVF and day 2 embryo transfers. transfers.

Simon, 1999Simon, 1999 women with repeated implantation failures and were women with repeated implantation failures and were

unable to demonstrate significant differences in unable to demonstrate significant differences in implantation and pregnancy rates between AECC and implantation and pregnancy rates between AECC and simple media when embryos were transferred on day 3simple media when embryos were transferred on day 3

Barmat, 1999Barmat, 1999

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

Preimplantation genetic diagnosisPreimplantation genetic diagnosisfor aneuploidy screening (PGD–AS)for aneuploidy screening (PGD–AS)

PGD for aneuploidy screening by polar body or PGD for aneuploidy screening by polar body or blastomere biopsy blastomere biopsy

detection of chromosomal aberrations in the embryo detection of chromosomal aberrations in the embryo thus eliminating those with reduced or no implantation thus eliminating those with reduced or no implantation potential and those who are destined to abort or result potential and those who are destined to abort or result in abnormal offspring.in abnormal offspring.

When all other factors leading to RIF have been When all other factors leading to RIF have been eliminated embryonic aneuploidy is usually blamed for eliminated embryonic aneuploidy is usually blamed for non-implantation.non-implantation.

PGDPGD

Choosing chromosomally normal embryos Choosing chromosomally normal embryos among a cohort may also improve embryo among a cohort may also improve embryo selection and increase pregnancy rates. selection and increase pregnancy rates.

advanced age, couples with recurrent advanced age, couples with recurrent implantation failure, and recurrent aborters implantation failure, and recurrent aborters

(Gianaroli, 1999; Munné , 1999; Kahraman, 2000; Pehlivan, 2003; Caglar, (Gianaroli, 1999; Munné , 1999; Kahraman, 2000; Pehlivan, 2003; Caglar, 2004; Kuliev, 2004).2004; Kuliev, 2004).

66 couples who after failing to conceive with 3 or 66 couples who after failing to conceive with 3 or more IVF cycles subsequently underwent IVF more IVF cycles subsequently underwent IVF combined with PGD combined with PGD

high rates of chromosomal abnormalities (60%) for an high rates of chromosomal abnormalities (60%) for an average maternal age of 32 years.average maternal age of 32 years.

The major chromosomal abnormality was not The major chromosomal abnormality was not aneuploidy but mosaicism, polyploidy and haploidy aneuploidy but mosaicism, polyploidy and haploidy

(Gianaroli 1999, 2002).(Gianaroli 1999, 2002).

PGD

The risk of chromosomal abnormalities in The risk of chromosomal abnormalities in embryos directly increases with the number of embryos directly increases with the number of previous IVF failures.previous IVF failures.

Whereas the chromosomal abnormality rate is Whereas the chromosomal abnormality rate is around 40% in patients with two failed IVF around 40% in patients with two failed IVF cycles, the abnormality rate increases to 50% in cycles, the abnormality rate increases to 50% in patients with three failed IVF cycles and to 67% patients with three failed IVF cycles and to 67% in those with more than five failures (Gianaroli, in those with more than five failures (Gianaroli, 1997).1997).

PGDPGD

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

Munné,Munné, 20032003

PGDPGD

significantly higher aneuploidy rates (50.2 versus 26.3%) significantly higher aneuploidy rates (50.2 versus 26.3%) in embryos from couples who experienced RIFs in embryos from couples who experienced RIFs compared with controlscompared with controls

the incidence of mosaic embryos also was significantly the incidence of mosaic embryos also was significantly higher (19.9 versus 10.8%). higher (19.9 versus 10.8%).

PGD resulted in higher success rates in the RIF group PGD resulted in higher success rates in the RIF group (34.0% pregnancy and 19.8% implantation rates) that (34.0% pregnancy and 19.8% implantation rates) that were comparable to young fertile controls. were comparable to young fertile controls.

Pehlivan, 2002 Pehlivan, 2002

similar implantation rates to a control group similar implantation rates to a control group after PGD for patients with more than two after PGD for patients with more than two failed IVF attempts (14.3 versus 11.5%) failed IVF attempts (14.3 versus 11.5%)

Munné, 2003 Munné, 2003 differences between these reports relies on differences between these reports relies on

differences in mean ages of recurrent differences in mean ages of recurrent implantation failure groups and complex implantation failure groups and complex underlying pathophysiology of RIF.underlying pathophysiology of RIF.

62.1% normoploid embryos in patients with 62.1% normoploid embryos in patients with more than two previous failed attempts using more than two previous failed attempts using three-probe fluorescent in-situ hybridization three-probe fluorescent in-situ hybridization (FISH) analysis (FISH) analysis

Implantation rate was improved in the PGD Implantation rate was improved in the PGD group compared with controls (21.0 versus group compared with controls (21.0 versus 10.0%). 10.0%).

Wilding, 2004 Wilding, 2004

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

The data of the ESHRE PGD consortium, The data of the ESHRE PGD consortium, which includes PGD cycles from 25 centres, which includes PGD cycles from 25 centres, reported that implantation rate in recurrent reported that implantation rate in recurrent implantation failure was only 11%, comparedimplantation failure was only 11%, compared

with 32–36% in aneuploidy screening for other with 32–36% in aneuploidy screening for other indicationsindications

(ESHRE PGD Consortium Steering Committee, 2002).(ESHRE PGD Consortium Steering Committee, 2002).

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