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

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

If all laboratory and stimulation parameters If all laboratory and stimulation parameters are controlled, underlying cause of failure is are 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, careful observation and embryo selection, to aid in selecting the right embryo to to aid in selecting the right embryo to overcome this failure. overcome this failure.

The morphology of the zygote, the state of The morphology of the zygote, the state of the cleaving embryos development, and the the cleaving embryos development, and the blastocyst can influence implantation rates. blastocyst 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 quality can be altered by either improving oocyte quality during stimulation or by using donor spermduring stimulation or by using donor sperm

Recurrent implantation failureRecurrent implantation failure

> 3 embryo transfers with high quality embryos or the transfer of > 3 embryo transfers with high quality embryos or the transfer 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 embryos benefit from endosalpingeal secretions and endosalpingeal secretions and reach the endometrium at an reach the endometrium at an appropriate period that appropriate period that coincides more with the window coincides more with the window of implantationof implantation

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; ., 1992; Fluker 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 traumatic transcervical embryo transfers despite dilatation.transfers despite dilatation.

Blastocyst transferBlastocyst transfer

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

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 embryonic genome activation) the embryo must be cultured up to the blastocyst stage (Braude, be cultured up to the blastocyst stage (Braude, 1988; Taylor,1997; Urman, 2001).1988; Taylor,1997; Urman, 2001).

Blastocyst transferBlastocyst transfer

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

Highest implantation rates reported for Highest implantation rates reported for selective cleavage stage embryo transfers selective cleavage stage embryo transfers are lower than those reported for are lower than those reported for blastocyst transfers (Van Royen, 1999; blastocyst transfers (Van Royen, 1999; Gardner, 2000).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 the success of blastocyst transfer with good quality blastocysts giving rise to good quality blastocysts giving rise to higher implantation rates compared with higher implantation rates compared with poor quality blastocysts.poor quality blastocysts.

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

The value of blastocyst The value of blastocyst transfer in RIFtransfer in RIF

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

(Cruz, 1999)(Cruz, 1999)

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

A significantly higher implantation rate (21.2 versus 6%) A significantly higher implantation rate (21.2 versus 6%) was recorded for blastocyst transfers compared with day 2–was recorded for blastocyst transfers compared with day 2–3 transfers.3 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 although higher in the blastocyst group did not reach statistical significance. statistical significance.

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

Levitas, 2004Levitas, 2004

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

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

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

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

Sequential embryo transferSequential embryo transfer

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

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

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

option in women recurrent implantation failure, however, option in women recurrent implantation failure, however, remains to be determined in prospective randomized trials.remains to be 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 essential for the hatching process both of which can be adversely influenced by advancing can be adversely influenced by advancing maternal age and in-vitro culture conditions maternal age and in-vitro culture conditions (Cohen, 1992; Schiewe, 1995; Mandelbaum, (Cohen, 1992; Schiewe, 1995; Mandelbaum, 1996).1996).

embryos with a thin zona and embryos that have embryos with a thin zona and embryos that have been subjected to microsurgical dissection been subjected to microsurgical dissection resulting in artificial gaps in their zona were resulting in artificial gaps in their zona were shown to implant more efficiently (Cohen shown to implant 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, (Stein, 1995; Antinori, 1996; Chao, 1997; Nakayama, 1997; Magli, 1998). 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, assisted hatching resulted in statistically higher implantation, clinical pregnancy and ongoing pregnancy rates (Sallam, 2003). clinical pregnancy and ongoing pregnancy rates (Sallam, 2003).

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

Assisted hatchingAssisted hatching

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

Co-culturesCo-cultures

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

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

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

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

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

Plachot, 1993Plachot, 1993

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

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

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

Barmat, 1999Barmat, 1999

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

Preimplantation genetic Preimplantation genetic diagnosisdiagnosis

for aneuploidy screening for aneuploidy screening (PGD–AS)(PGD–AS)

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

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

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

PGDPGD

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

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

(Gianaroli, 1999; Munné , 1999; Kahraman, 2000; (Gianaroli, 1999; Munné , 1999; Kahraman, 2000; Pehlivan, 2003; Caglar, 2004; Kuliev, 2004).Pehlivan, 2003; Caglar, 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 high rates of chromosomal abnormalities (60%) for an average maternal age of 32 years.an 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, (Gianaroli 1999, 2002).2002).

PGD

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

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

PGDPGD

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

Munné,Munné, 20032003

PGDPGD

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

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

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

Pehlivan, 2002 Pehlivan, 2002

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

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

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

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

Wilding, 2004 Wilding, 2004

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

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

with 32–36% in aneuploidy screening with 32–36% in aneuploidy screening for other indicationsfor other indications

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

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