Can clinicians improve Can clinicians improve implantation?implantation?

Omur Taskin,M.DOmur Taskin,M.DDept. Obstet&Gynecol, Div. Reprod. Endocr.Dept. Obstet&Gynecol, Div. Reprod. Endocr.

Akdeniz University School of MedicineAkdeniz University School of Medicine

What is meant by What is meant by “endometrial receptivity”?“endometrial receptivity”???

Is it a transient stage of endometrial Is it a transient stage of endometrial function following initiation of function following initiation of

implantation??implantation??

OrOr

a biologically autonomous condition of a biologically autonomous condition of endometrium that can be recognized in the absence endometrium that can be recognized in the absence

of implanting blastocyst??of implanting blastocyst??

Primed endometrium may be more receptive and Primed endometrium may be more receptive and associated with surface receptivity may only associated with surface receptivity may only

induced by suitable induced by suitable embryonic stimulus embryonic stimulus

Under the influence of steroid Under the influence of steroid hormones, endometrium:hormones, endometrium:

Powerful paracrine tissuePowerful paracrine tissue Bidirectional signals between embryo/stromaBidirectional signals between embryo/stroma Initiating embryo/uterine crosstalkInitiating embryo/uterine crosstalk Primary of activating implanting Primary of activating implanting

blastocystthrough the local action of cytokines or blastocystthrough the local action of cytokines or eicosanoids eicosanoids

so not just an adhesive surfaceso not just an adhesive surface

Three-stage processThree-stage process

AppositionAppositionAdhesionAdhesion InvasionInvasion

Involving synchronized crosstalk between Involving synchronized crosstalk between a receptive endometrium and a functional a receptive endometrium and a functional blastocystblastocyst

Implantation involvesImplantation involves

Complex squence of signalling eventsComplex squence of signalling events

consistingconsisting

Acquisition of adhesion ligandsAcquisition of adhesion ligands

With loss of inhibitory componentsWith loss of inhibitory components

Implantation is a complicated process that Implantation is a complicated process that requires the orchestration of a series of requires the orchestration of a series of events involving both the embryo and the events involving both the embryo and the endometriumendometrium

implantation rates remain around 25-35%implantation rates remain around 25-35%(European IVF-Monitoring Program,2005).(European IVF-Monitoring Program,2005).

Problems?Problems?

High rates of implantation failure High rates of implantation failure early pregnancy loss in IVF early pregnancy loss in IVF increase the transfer of multiple embryos.increase the transfer of multiple embryos.

Then what are the important players of the feto-maternal crosstalk in appositıon and adhesion

phases?

Endometrial morphology

Histologypinopods

Cellular adhesion molecules

IntegrinsSelectinsCadherins Immunoglobulinsmucins

cytokines

LIF IL-1

prostaglandins

prostaglandins

Ways in which a clinician could try to improve implantation rate.

Embryo transfer techniqueEmbryo transfer technique

cervical manipulation may result in an increase of cervical manipulation may result in an increase of contractions of the uterus, which has been contractions of the uterus, which has been observed to severe IVF outcome, possibly by observed to severe IVF outcome, possibly by expelling embryos from the uterine cavity expelling embryos from the uterine cavity

Fanchin et al.,1998Fanchin et al.,1998

stiff vs soft embryo transfer cathetersstiff vs soft embryo transfer catheters

In a recent meta-analysis of seven randomized In a recent meta-analysis of seven randomized controlled trails (RCT) comparing stiff and soft controlled trails (RCT) comparing stiff and soft embryo transfer catheters, significantly embryo transfer catheters, significantly increased pregnancy rates were observed with increased pregnancy rates were observed with the latter (odds ratio(OR) 1.34, 95% confidence the latter (odds ratio(OR) 1.34, 95% confidence intervals(CI)1,18-1,54) intervals(CI)1,18-1,54)

(Buckett,2006)(Buckett,2006)

Traditionally, embryo transfer after IVF has Traditionally, embryo transfer after IVF has been performed ‘blindly’, with the aim of been performed ‘blindly’, with the aim of placing the embryos 1 cm below the placing the embryos 1 cm below the fundus of the uterus (Schoolcraft, 2001)fundus of the uterus (Schoolcraft, 2001)

Deposition of embryos

In a prospective investigation significantly better results were In a prospective investigation significantly better results were obtained when the catheter tip was positioned close to the middle of obtained when the catheter tip was positioned close to the middle of the endometrial cavity (Oliveira et al.,2004)the endometrial cavity (Oliveira et al.,2004)

However, another randomized study revealed significantly higher However, another randomized study revealed significantly higher implantaion rates when embryos were deposited 1,5 or 2 cm from implantaion rates when embryos were deposited 1,5 or 2 cm from the fundus, compared with 1 cm (Coroleu et al.,2002)the fundus, compared with 1 cm (Coroleu et al.,2002)

suggesting that for every additional 1 mm that embryos are deposited away from the fundus, the odds of clinical pregnancy increased by 11% (Pope et al.,2004)

Role of USG

The blind nature of traditional ‘clinical touch’ embryo The blind nature of traditional ‘clinical touch’ embryo transfer had led to the suggestion of a role for ultrasound transfer had led to the suggestion of a role for ultrasound in improving IVF outcomes.in improving IVF outcomes.

A meta-analysis of four RCT comparing ultrasound-A meta-analysis of four RCT comparing ultrasound-guided embryo transfer versus clinical touch showed a guided embryo transfer versus clinical touch showed a significant higher pregnancy rate and implantation rate significant higher pregnancy rate and implantation rate after ultrasound-guided transfer (1,38 , 95% CI 1,20-after ultrasound-guided transfer (1,38 , 95% CI 1,20-1,60) (Buckett,2003)1,60) (Buckett,2003)

BV colonization

During embryo transfer, it is likely that bacteria from the During embryo transfer, it is likely that bacteria from the cervix may be introduced into the uterine cavity. cervix may be introduced into the uterine cavity. Bacterial vaginosis is characterized by an overgrowth of Bacterial vaginosis is characterized by an overgrowth of anaerobic organisms; the prevalence among women anaerobic organisms; the prevalence among women undergoing IVF is approximately 25% (Liversedge et undergoing IVF is approximately 25% (Liversedge et al.,1999).al.,1999).

1)1) Careful evaluation of the uterusCareful evaluation of the uterus

2)2) Avoiding the initiation of uterine Avoiding the initiation of uterine contractilitycontractility

3)3) Removing the cervical mucusRemoving the cervical mucus

4)4) Proper placement of the embryosProper placement of the embryos

5)5) Minimizing embryo expulsionMinimizing embryo expulsion

Adjuvant pharmaceutical therapies

AspirinAspirin

Randomized controlled trials investigating the use of aspirin

as an empirical therapy in non-selected IVF populations.

Low-dose aspirin for in vitro fertilization: a systematic review and

meta-analysis

T.A.Gelbaya et al., human reproduction, 2007T.A.Gelbaya et al., human reproduction, 2007

Clinical pregnancy (CP) rate per embryo transfer (ET) was not found to be significantly different between patients who received low-dose aspirin and those who received placebo or no treatment (RR 1.09 95% CI 0.92-1.29). None of the other outcomes, including CP per cycle, spontaneous abortion or ectopic pregnancy per CP and LB rate per cycle or ET was found to differ significantly between the compared groups. On the basis of up-to-date evidence, low-dose aspirin has no substantial positive effect on likelihood of pregnancy and, therefore, it should not be routinely recommended for women undergoing IVF/ICSI.

A large body of evidence shows that APL antibodies, occasionally A large body of evidence shows that APL antibodies, occasionally found in low-risk obstetric populations (Lockwood et al.,1989; Stern et found in low-risk obstetric populations (Lockwood et al.,1989; Stern et al., 1998) , are commonly found in women with reproductive al., 1998) , are commonly found in women with reproductive dysfunction such as recurrent pregnancy loss (RPL) (Cowchock et al., dysfunction such as recurrent pregnancy loss (RPL) (Cowchock et al., 1986 ;Matzner et al., 1994; Yetman and Kutteh, 1996) and idiopathic 1986 ;Matzner et al., 1994; Yetman and Kutteh, 1996) and idiopathic infertility (Gleicher et al., 19891994; Birdsall et al., 1996)infertility (Gleicher et al., 19891994; Birdsall et al., 1996)

Nevertheless, there is much controversy with regard to the association between APL antibodies and IVF outcome. Unlike the studies showing benefits after the use of heparin and aspirin in women with RPL (Kutteh, 1996; Rai et al., 1997; Tulppala et al., 1997) , there is no consensus regarding its use in IVF patients.

Characteristics of controlled trials on low-dose aspirin and IVF outcome included in the systematic review

Forest plot of the effect of aspirin versus placebo or no treatment on clinical pregnancy rate per embryo transfer.

Review: aspirin versus nothing in IVF.Comparison: aspirin versus placebo or no treatment.

Outcome: clinical pregnancy/embryo transfer.

Forest plot of the effect of aspirin versus placebo or no treatment on miscarriage rate per clinical pregnancy.

Review: aspirin versus nothing in IVF.Comparison: aspirin versus placebo or no treatment.

Outcome: miscarriage/clinical pregnancy.

Forest plot of the effect of aspirin versus placebo or no treatment on cycle cancellation rate.

Review: aspirin versus nothing in IVF.Comparison: aspirin versus placebo or no treatment.

Outcome: no. of cycles cancelled/cycles.

Main results of the two RCTs comparing aspirin versus placebo in poor responders (Lok et al., 2004)

or oocytes recipients (Weckstein et al., 1997)

Nitric oxide donorsNitric oxide donors

Effect of vaginal sildenafil on the outcome of in vitro fertilization(IVF) after multiple IVF failures attributed to poor

endometrial development

Geoffrey S. et al.,fertility sterility,2002Geoffrey S. et al.,fertility sterility,2002

Patients: A cohort of 105 infertile women aged <40 years, with normal ovarian reserve and at least two consecutive prior IVF failures attributed to inadequate endometrial development

Interventions: patients underwent IVF using a long GnRH-a protocol with the addition of sildenafil vaginal suppositories (25 mg, 4 times per day) for 3-10 days

Although subgroups of women may be identified who benefit from NO Although subgroups of women may be identified who benefit from NO donor therapy, at present the available data demand caution in its use, donor therapy, at present the available data demand caution in its use, which at present the available data demand caution in its use, which at which at present the available data demand caution in its use, which at present should be restricted to well-designed studies.present should be restricted to well-designed studies.

Aromatase inhibitorsAromatase inhibitors

Ascorbic acid Ascorbic acid appears to be involved in normal folliculogenesis

(Luck et al.,1995), ovulation (Igarashi,1977) and luteal formation and regression (Luck and Zhao,1993).

An imbalance of oxidative stress and antioxidant defence has been implicated in the pathogenesis of several diseases, including recurrent abortion, unexplained infertility and defective embryo development.

However, a RCT investigating the effect of 1,5 or 10 mg of ascorbic However, a RCT investigating the effect of 1,5 or 10 mg of ascorbic acid versus a placebo during the luteal phase in 620 women acid versus a placebo during the luteal phase in 620 women undergoing IVF showed no difference in implantation rates undergoing IVF showed no difference in implantation rates (Griesinger et al.,2002).(Griesinger et al.,2002).

Prolonged progesteroneProlonged progesterone

An important regulator of endometrium receptivity is the corpus luteum, the An important regulator of endometrium receptivity is the corpus luteum, the primary function of which is the production of progesterone.primary function of which is the production of progesterone.

The optimal duration of progesterone administration remains to be clarified. The optimal duration of progesterone administration remains to be clarified. Many centers continue with progesterone supplementation throughout the Many centers continue with progesterone supplementation throughout the first trimester of pregnancy. However, the rationale for this approach is first trimester of pregnancy. However, the rationale for this approach is unclear. Proponents point to the uterine relaxing properties of progesterone, unclear. Proponents point to the uterine relaxing properties of progesterone, elegantly demonstrated by a reported negative correlation between uterine elegantly demonstrated by a reported negative correlation between uterine contractility frequency and progesterone concentrations (Fanchin et contractility frequency and progesterone concentrations (Fanchin et al.,1998)al.,1998)

Secondly, progesterone has been shown to have potentially beneficial Secondly, progesterone has been shown to have potentially beneficial immunomodulatory properties. Studies in mice demonstrated that immunomodulatory properties. Studies in mice demonstrated that progesterone administration abrogated the abortigenic effects of stress progesterone administration abrogated the abortigenic effects of stress exposure by decreasing the frequency of Th1 cytokines (Blois et al., 2004). exposure by decreasing the frequency of Th1 cytokines (Blois et al., 2004). Previous studies suggested that succesful pregnancy is more likely when Previous studies suggested that succesful pregnancy is more likely when Th2 rather than Th1 cytokines are predominant (Wegmann et al., 1993)Th2 rather than Th1 cytokines are predominant (Wegmann et al., 1993)

Although supplementation of progesterone is widely used to Although supplementation of progesterone is widely used to improve implantation rates, the application of luteal oestradiol improve implantation rates, the application of luteal oestradiol supplementation remains controversial.supplementation remains controversial.

A meta-analysis of three RCT (Smitz et al., 1993; Lewin et al.,1994; A meta-analysis of three RCT (Smitz et al., 1993; Lewin et al.,1994; Farhi et al.,2000) using a long GnRH agonist protocol, reported no Farhi et al.,2000) using a long GnRH agonist protocol, reported no difference in pregnancy rates when oestrogen was added to difference in pregnancy rates when oestrogen was added to progesterone in the luteal phase (Pritts and Atwood,2002).progesterone in the luteal phase (Pritts and Atwood,2002).

In contrast, a recent RCT of 166 women undergoing ICSI reported In contrast, a recent RCT of 166 women undergoing ICSI reported significantly higher pregnancy and implantation rates after oestradiol significantly higher pregnancy and implantation rates after oestradiol supplementation (Lukaszuk et al.,2005).supplementation (Lukaszuk et al.,2005).

Glucocorticoids

Uterine receptivity is controlled by locally acting growth factors, Uterine receptivity is controlled by locally acting growth factors, cytokines and uterine natural killer (uNK) cells (Dey et al.,cytokines and uterine natural killer (uNK) cells (Dey et al., 2004). It 2004). It has been shown that uNK cells may have an important role in early has been shown that uNK cells may have an important role in early implantation, since they accumulate around arteries supplying the implantation, since they accumulate around arteries supplying the implantation site (Croy et al.,2002). implantation site (Croy et al.,2002).

A defect in the integrity of the number of uNK cells has also been A defect in the integrity of the number of uNK cells has also been implicated in implaniation failure. Ledee-Batailie et al. reported implicated in implaniation failure. Ledee-Batailie et al. reported higher numbers of NK cells in endomelrial biopsies from women higher numbers of NK cells in endomelrial biopsies from women with implantation failure versus fertile controls (Ledee-Bataille et with implantation failure versus fertile controls (Ledee-Bataille et al.,2005).al.,2005).

An RCT of 206 patients, investigating the use of glucocorticoids An RCT of 206 patients, investigating the use of glucocorticoids from oocyte retrieval onwards, reported no differences in embryo from oocyte retrieval onwards, reported no differences in embryo implantation or pregnancy rates (Moffitt et al.,implantation or pregnancy rates (Moffitt et al., 1995). These results 1995). These results are in line with another RCT addressing the effect of adjuvant are in line with another RCT addressing the effect of adjuvant glucocorticoids (Mottia et al.,2005).glucocorticoids (Mottia et al.,2005).

Peri-implantation glucocorticoid administration for assisted reproductive technology cycles

Boomsma C. et al., cochrane dtabase syst rev, 2007Boomsma C. et al., cochrane dtabase syst rev, 2007

Objectives: Objectives:

To investigate whether the administration of glucocorticoids To investigate whether the administration of glucocorticoids around the time of implantation improves clinical outcomes in around the time of implantation improves clinical outcomes in subfertile women undergoing IVF or ICSI, compared to no subfertile women undergoing IVF or ICSI, compared to no glucocorticoid administration.glucocorticoid administration.

Thirteen studies (1759 couples) were included.Thirteen studies (1759 couples) were included. Overall, there is no clear evidence that administration of Overall, there is no clear evidence that administration of

peri-implantation glucocorticoids in ART cycles peri-implantation glucocorticoids in ART cycles significantly improves clinical outcome. The use of significantly improves clinical outcome. The use of glucocortcoids in women undergoing IVF (rather than glucocortcoids in women undergoing IVF (rather than ICSI) was associated with an improvement in pregnancy ICSI) was associated with an improvement in pregnancy rates of borderline statistical significance.rates of borderline statistical significance.

Treatment of repeated unexplained in vitro Treatment of repeated unexplained in vitro fertilization failure with intravenous fertilization failure with intravenous

immunoglobulin: a randomized, placebo-controlled immunoglobulin: a randomized, placebo-controlled Canadian trialCanadian trial

Mary D. S. et al.,fertility and sterility, 2000Mary D. S. et al.,fertility and sterility, 2000

Fifty-one couples with a history of repeated Fifty-one couples with a history of repeated unexplained IVF failure who were unexplained IVF failure who were

preparing for another fresh IVF cycle or preparing for another fresh IVF cycle or replacement of cryopreserved embryos.replacement of cryopreserved embryos.

Conclusion(s): In this randomized clinical Conclusion(s): In this randomized clinical trial, IVIG did not improve the live-birth trial, IVIG did not improve the live-birth rate in couples with repeated unexplained rate in couples with repeated unexplained IVF failure, stringently defined by known IVF failure, stringently defined by known determinants of IVF outcome.determinants of IVF outcome.

Insulin sensitizing drugsInsulin sensitizing drugs

A meta-analysis of eight RCT investigating metformin in women with A meta-analysis of eight RCT investigating metformin in women with PCOS demonstrated no significant differences in pregnancy rates, PCOS demonstrated no significant differences in pregnancy rates, although the risk of ovarian hyperstimulation syndrome (OHSS) was although the risk of ovarian hyperstimulation syndrome (OHSS) was significantly reduced by metformin (Costeilo et al.significantly reduced by metformin (Costeilo et al., , 2006).2006).

One recent RCT of 101 women with PCOS undergoing IVF, One recent RCT of 101 women with PCOS undergoing IVF, included in this meta-analysis, demonstrated lower rates of included in this meta-analysis, demonstrated lower rates of miscarriage and OHSS in the group receiving metformin (Tang et miscarriage and OHSS in the group receiving metformin (Tang et al.,al., 2006).2006).

GnRH agonistGnRH agonist

Ovarian stimulation Ovarian stimulation regimensregimens

Assisted hatching on assisted conception (IVF & ICSI)Assisted hatching on assisted conception (IVF & ICSI)

Objectives: to determine whether assisted hatching (ah) of embryos Objectives: to determine whether assisted hatching (ah) of embryos facilitates live births and clinical pregnancy and whether it impacts on facilitates live births and clinical pregnancy and whether it impacts on negative outcomes (such as multiple pregnancy and miscarriage)negative outcomes (such as multiple pregnancy and miscarriage)

Results: twenty-three randomised controlled trials consisting of 2668 Results: twenty-three randomised controlled trials consisting of 2668 women reported on 849 pregnancy outcomeswomen reported on 849 pregnancy outcomes

Conclusions: despite significantly improved odds of clinical pregnancy, Conclusions: despite significantly improved odds of clinical pregnancy, there is insufficient evidence to determine any effect of AH on live birth there is insufficient evidence to determine any effect of AH on live birth rates. The increased multiple pregnancy rate is of concern although it rates. The increased multiple pregnancy rate is of concern although it likely that with a policy of single embryo transfer this may be lowered. likely that with a policy of single embryo transfer this may be lowered. Currently, there is insufficient evidence to recommend assisted Currently, there is insufficient evidence to recommend assisted hatching.hatching.

Seif MM, Edi-Osagie EC. et al., cochrane database syst rev, 2005Seif MM, Edi-Osagie EC. et al., cochrane database syst rev, 2005

Hyaluronic acid can successfully replace as the sole macromolecule in a human embryo transfer

medium

Alex S. et al., fertility and sterility, 2003Alex S. et al., fertility and sterility, 2003

Favorable influence of local injury to the endometrium in Favorable influence of local injury to the endometrium in intracytoplamic sperm injection patients with high-order intracytoplamic sperm injection patients with high-order

implantation failureimplantation failure

Local injury to the endometrium prior to controlled ovarian stimulation Local injury to the endometrium prior to controlled ovarian stimulation may considerably improve implantation rates and pregnancy outcomes may considerably improve implantation rates and pregnancy outcomes

in intracytoplasmic sperm injection patients with high-order implantation in intracytoplasmic sperm injection patients with high-order implantation failure (>=4 IVF trials and >=12 transferred embryos).failure (>=4 IVF trials and >=12 transferred embryos).

Fertility and Sterility,2007Fertility and Sterility,2007

Preimplantation genetic diagnosis and human implantation- A review

By selecting chromosomally normal embryos for replacement, By selecting chromosomally normal embryos for replacement, PGD for aneuploidy can increase implantation rates, reduce PGD for aneuploidy can increase implantation rates, reduce

spontaneous abortion rates, and avoid aneuploid conceptions. spontaneous abortion rates, and avoid aneuploid conceptions. When eight chromosomes are analysed, a significant increase When eight chromosomes are analysed, a significant increase in implantation is achieved. PGD is also found to significantly in implantation is achieved. PGD is also found to significantly

reduce the incidence of spontaneous abortion and reduce the incidence of spontaneous abortion and chromosomally abnormal conceptionschromosomally abnormal conceptions

S. Munne et al., The Institute for Reproductive Medicine and Science of Saint Barnabas, 2003S. Munne et al., The Institute for Reproductive Medicine and Science of Saint Barnabas, 2003

The effect of serum concentration of leukamia inhibitory factor on

in vitro fertilization treatment outcome

To evaluate the association of peripheral leukaemia inhibitory factor (LIF) levels on implantation and miscarriage rates after in vitro fertilization (IVF) treatment.

The systemic levels of LIF concentration have no association with implantation rate or miscarriage rate in women undergoing IVF treatment. Measuring serum LIF concentration prior to embryo transfer in IVF treatment has no predictive value of implantation rate or miscarriage rate.

Meen-yau T. et al., reprod immunol., 2007 Meen-yau T. et al., reprod immunol., 2007

The effect of intercourse on pregnancy rates during The effect of intercourse on pregnancy rates during assisted human reproductionassisted human reproduction

intercourse during an IVF cycle has the potential to improve pregnancy rates since exposure to semen is intercourse during an IVF cycle has the potential to improve pregnancy rates since exposure to semen is reported to promote embryo development and implantation in animals. Conversely, coitus-induced uterine reported to promote embryo development and implantation in animals. Conversely, coitus-induced uterine contractions or introduction of infection may have a detrimental effect.contractions or introduction of infection may have a detrimental effect.

A multicentre prospective randomized control trial was conducted to determine if intercourse during the peri-A multicentre prospective randomized control trial was conducted to determine if intercourse during the peri-transfer period of an IVF cycle has any influence on pregnancy success. Participants undergoing thawed embryo transfer period of an IVF cycle has any influence on pregnancy success. Participants undergoing thawed embryo transfer (Australian centre) or fresh embryo transfers (Spanish centres) were randomized either to abstain or transfer (Australian centre) or fresh embryo transfers (Spanish centres) were randomized either to abstain or to engage in vaginal intercourse around the time of embryo transferto engage in vaginal intercourse around the time of embryo transfer

There was no significant difference between the intercourse and abstain groups in relation to the pregnancy There was no significant difference between the intercourse and abstain groups in relation to the pregnancy rate (23.6 and 21.2% respectively), but the proportion of transferred embryos that were viable at 6-8 weeks was rate (23.6 and 21.2% respectively), but the proportion of transferred embryos that were viable at 6-8 weeks was significantly higher in women exposed to semen compared to those who abstained (11.01 versus 7.69 viable significantly higher in women exposed to semen compared to those who abstained (11.01 versus 7.69 viable embryos per 100 transferred embryos, P = 0.036, odds ratio 1.48, 95% confidence interval 1.01-2.19). embryos per 100 transferred embryos, P = 0.036, odds ratio 1.48, 95% confidence interval 1.01-2.19).

Hence exposure to semen around the time of embryo transfer increases the likelihood of successful early embryo Hence exposure to semen around the time of embryo transfer increases the likelihood of successful early embryo implantation and development.implantation and development.

Tremellen et al, Hum Reprod. 2000 Dec;15(12):2653-8