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Recurrent Implantation Failure

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1 Recurrent Implantation Failure

2 Schematic representation of the early stages of human implantation
Schematic representation of the early stages of human implantation. The pre-implantation embryo signals its presence to the mother by both endocrine mediators, such as human chorionic gonadotrophin, and paracrine growth factors, which act locally on the endometrium to facilitate attachment. Following attachment the embryo penetrates the luminal epithelium (LE), breaches the basement membrane (BM) and invades into the underlying stromal cells. These begin to decidualise in earnest, although in humans some pre-decidual change is evident prior to embryo attachment. A detailed description of early human implantation stages by Professor Allen Enders, together with photomicrographs, is available to view from the website of the Centre for Trophoblast Research (http://www.trophoblast.cam.ac.uk/info/enders.shtml). Sharkey, RBMonline, 2013 Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

3

4 surface of the endometrium, but the process becomes
Figure 1 Early implantation failure -during the attachment or migration stages(negative blood pregnancy test. Late implantation failure- following successful migration of the embryo through the luminal surface of the endometrium, but the process becomes disrupted prior to the formation of an intrauterine gestational sac. (biochemical pregnancy). The initial stage of implantation, when the embryo is invading the epithelial layer of the endometrium to be embedded in the stroma compartment. Source: Reproductive BioMedicine Online 2014; 28:14-38 (DOI: /j.rbmo ) Copyright © 2014

5 Definition (Tan et al., 2005): Failure to achieve a pregnancy following 2–6 IVF cycles, in which more than 10 high- grade embryos were transferred to the uterus was defined by various clinicians as RIF. In the era of limited embryos transfer, there is no consensus. Propose: RIF be defined as the failure to achieve a clinical pregnancy after transfer of at least 4 good-quality embryos in a minimum of three fresh or frozen cycles in a woman under the age of 40(Coughlan, 2014).

6 Uterine Causes of RIF Uterine Receptivity Fibroids Uterine anomalies
Thin endometrium(asherman’s syndrome) Altered expression of adhesion molecules, transcriptome, proteome Immune factors, thromophillias

7 Decreased endometrial receptivity
Undiagnosed uterine pathology: In 18–27% of women with a normal initial hysteroscopy or hysterosalpingogram, repeated hysteroscopic visualization after RIF revealed uterine abnormalities, mainly hyperplasia, polyps, endometritis, synechiae and leiomyomata (Demirol and Gurgan, 2004). The effect of leiomyomata on implantation is uncertain (Surrey, 2003). The impact of intramural lesions without cavity distortion (Eldar-Geva et al., 1998) or myomas of <4 cm (Oliveira et al., 2004) on RIF remain controversial. The presence of a thin endometrium did not influence the cumulative PRs in a prospective large cohort studies (De Geyter et al., 2000), particularly when high-quality embryos were transferred (Zhang et al., 2005). Thin or hyperechogenic endometrium or persistent endometrial fluid impaired the outcome in tubal factor, but not in polycystic ovary syndrome (PCOS) (Akman et al., 2005) or ICSI (Rinaldi et al., 1996). However, the concept that a minimum thickness (4–8 mm) is required to establish a clinical pregnancy is still arguable and should be considered in RIF.

8 Pregnancy rate and implantation rate following IVF for controls
without fibroids and subjects with fibroids stratified by their uterine position. ∗P<.05 for IM vs. controls or SS; ∗∗P<.005 for IM vs. controls. Pregnancy rate and implantation rate following IVF for controls without fibroids and subjects with fibroids stratified by their uterine position. SS = subserosal; IM = intramural; SM = submucosal. ∗P<.05 for IM vs. controls or SS; ∗∗P<.005 for IM vs. controls. Eldar-Geva T, Effect of intramural, subserosal, and submucosal uterine fibroids on the outcome of assisted reproductive technology treatment. Fertil Steril. 1998;70:687–691

9 The impact of intramural leiomyomata on IVF outcome Surrey, Fertility and Sterility, April 2000
Intramural myoma No myoma Age 36.1 36.8 Pregnancy Rate 55 65 Implantation 23 37* * <.05

10 Forest plot of studies of non-cavity-distorting intramural fibroids versus no fibroids in women undergoing IVF treatment for outcome of live birth rates. Forest plot of studies of non-cavity-distorting intramural fibroids versus no fibroids in women undergoing IVF treatment for outcome of live birth rates. Aa

11 Congenital Uterine Anomalies
Septate uterus may contribute to RIF-untreated septate uteri had a poor outcome following IVF treatment in comparison to women who had undergone hysteroscopic metroplasty prior to IVF (Lavergne et al., 1996). Ban-Frangez et al. (2009) showed that the presence of a septum, whether large or small, was associated with a miscarriage rate of about 80%, which was reduced to 30% or so after surgical removal of the septum Bicornuate uteri, a relatively common anomaly and most women have no difficulty conceiving (Grimbizis et al., 2001). The main risk for the woman with a bicornuate uterus is mid- trimester pregnancy loss and preterm birth (Grimbizis et al., 2001).

12 Endometrial Markers Lack of integrin –αVβ3 in the endometrium at the time of implantation was suggested as a cause of implantation failure (Tei et al., 2003; Thomas et al., 2003). High levels of aromatase p450 mRNA (Brosens et al., 2004), changes in pinopode expression (Pantos et al., 2004) and high levels of matrix metalloproteinases (Inagaki et al., 2003) have been suggested to be associated with RIF.

13 Gene clustering by Pearson's correlation.
Use of microarray technology to compare endometrial gene expression profiles at the window of implantation according to the levels of circulating progesterone Gene clustering by Pearson's correlation. Gene clustering by Pearson's correlation. An: antagonist; ag: agonist, CON: P >1.5 ng/ml; SIN: P <1.5 ng/ml. he objective of this study was to use microarray technology to compare endometrial gene expression profiles at the window of implantation according to the levels of circulating progesterone. METHODS For this prospective cohort study, microarray data were obtained from endometrial biopsies from 12 young healthy oocyte donors undergoing COS with pituitary suppression by either gonadotrophin-releasing hormone (GnRH) agonists or antagonists, and recombinant FSH. On the day of recombinant chorionic gonadotrophin (rCG) administration, six women had serum progesterone levels (P) >1.5 ng/ml (study group) and six had serum P levels <1.5 ng/ml (control group). Endometrial samples were collected using a Pipelle catheter 7 days after the rCG injection. RESULTS Using the parametric test, we identified 140 genes significantly dysregulated (64 up- and 76 down-regulated) in the study group endometria compared with the control endometria, regardless of the GnRH analogue employed. These genes are related to cell adhesion, developmental processes, the immune system and others, which are all required for normal endometrial function development. Of the 25 gene targets previously proposed as markers for endometrial receptivity, 13 appeared over-regulated in the study group. CONCLUSIONS Our results reveal that elevated progesterone levels on the day of rCG administration can induce significant alterations in the gene expression profile of the endometrium. Labarta E et al. Hum. Reprod. 2011;26:

14 Identified 140 genes significantly dysregulated (64 up- and 76 down-regulated) in the high P(>1.5). These genes are related to cell adhesion, developmental processes, the immune system and others, which are all required for normal endometrial function development. CONCLUSIONS Our results reveal that elevated progesterone levels on the day of rhCG administration can induce significant alterations in the gene expression profile of the endometrium.

15 Venn diagram of transcripts up-regulated and down-regulated during endometrial receptivity in the natural cycle compared with the stimulated cycle. Venn diagram of transcripts up-regulated and down-regulated during endometrial receptivity in the natural cycle compared with the stimulated cycle.Each list of genes was determined with the SAM software with a FDR < 5% and an FC > 2. we compared the endometrium gene expression profiles, from the same patients, in a natural cycle and in a subsequent COS cycle. METHODS For the same normal-responder patients (n = 21), endometrial biopsies (n = 84) were collected during the pre-receptive (LH + 2) and receptive stages (LH + 7) of a natural cycle and, subsequently, on oocyte retrieval day (hCG + 2) and on transfer day (hCG + 5) of a stimulated cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved in endometrial receptivity were analyzed. RESULTS Although endometrium transition profiles from pre-receptive to receptive phases are similar between patients, COS regimens alter endometrial receptivity in comparison with natural cycle. Under COS conditions, two endometrial profiles were identified and were associated either with a moderately altered receptivity profile for the majority of the patients or a strongly altered profile for a sub-category of patients. The receptive endometrium transcription profile under COS was defective for biological functions such as TGFβ signaling, leukocyte transendothelial migration and the cell cycle. CONCLUSIONS Gonadotrophin treatments in COS cycles led to disruptions of the transcriptional activation of genes involved in normal endometrial receptivity. We propose that when the receptiveness of the endometrium is seriously compromised by the COS protocol, fresh embryo replacement should be cancelled, the embryo frozen and thawed embryo replacement should be performed under natural cycles. Haouzi D et al. Hum. Reprod. 2009;24:

16 The transcriptomic pattern of endometrial cells in natural and stimulated cycles in the same patients reveals either moderate or strong alterations of endometrial receptivity under COS protocols. A strongly altered profile during COS protocols could explain multiple implantation failures, and suggest the use of FET during a natural cycle.

17 Ultrasound diagnosed adenomyosis has a negative impact on successful implantation following GnRH antagonist IVF treatment Thslluri, Hum. Reprod. (2012) 27 (12): The presence of ultrasound diagnosed adenomyosis was associated with a significant reduction in successful implantation of good quality embryos in patients undergoing GnRH antagonist stimulation for IVF treatment (viable clinical pregnancy rate 23.6% versus 44.6%, P= 0.017). WHAT IS KNOWN AND WHAT THIS PAPER ADDS There is currently no consensus regarding the impact of adenomyosis on implantation potential. Although some studies have identified alterations in the endometrial milieu in adenomyosis patients that may impact implantation, several papers have reported no associated reproductive deficit. However, these pregnancy outcome studies have primarily investigated patients undergoing long down-regulation IVF protocols, where low levels of serum estrogen (before commencing the ovarian stimulation) may inactivate the adenomyosis and potentially negate its effect on implantation. Given that the majority of fertility clinics are now moving towards the more ‘patient-friendly’ antagonist protocol, where patients are not placed in a hypo-estrogen state before commencing ovarian stimulation, the question of whether adenomyosis has an impact on IVF success rates in GnRH antagonist-stimulated IVF treatment needs to be examined. DESIGN This is a retrospective cohort study of 748 patients who, between April 2010 and March 2012, underwent a screening transvaginal ultrasound to identify possible pelvic pathology before commencing their IVF treatment. From this screening group, 213 patients were eligible to be included in the study as they had no obvious underlying uterine or embryonic factors that could have interfered with successful implantation (aged ≤39 years, good quality Day 4/5 embryo for single-embryo transfer, no uterine fibroids/hydrosalpinx or endometrial polyps). PARTICIPANTS AND SETTING There were 213 patients in a private IVF unit eligible to be included in the study, with 38 patients (17.84%) having ultrasound diagnosed adenomyosis and 175 patients having no adenomyosis on the scan. Only the first treatment cycle for each patient was included. MAIN RESULTS AND THE ROLE OF CHANCE The adenomyosis group had a viable clinical pregnancy rate of 23.6% compared with 44.6% in the non-adenomyosis group (P =0.017). However, the median maternal age and duration of infertility of the adenomyosis group was 2 years older and 4 months greater, respectively, than that of the non-adenomyosis group. A logistic regression analysis was performed to account for these differences between the two groups, with the adjusted results still showing a statistically significant decline in viable pregnancy rate in the adenomyosis group (OR = 0.408, CI = 0.181–0.922, P =0.031 when adjusting for maternal age; OR = 0.417, CI = 0.175–0.989, P =0.047 when adjusting for duration of infertility)

18 Hydrosalpinx Patients with hydrosalpinges have lower implantation and PRs (Zeyneloglu et al., 1998). Hydrosalpinx fluid is commonly slightly alkaline and may contain cytokines, prostaglandins or other inflammatory compounds. These compounds may have either direct embryo- toxicity or adversely affect the endometrium (Meyer et al., 1997). Reflux of hydrosalpinx fluid into the uterine cavity may result in diminishing embryonic endometrial apposition.

19 Salpingectomy of hydrosalpinges
Strandell and co-investigators were the first to show in an RCT that salpingectomy of hydrosalpinges increased PR (Strandell et al., 1999). In a recent meta-analysis (Johnson et al., 2004) of three RCTs involving prophylactic salpingectomy in 295 patients with hydrosalpinges, the pregnancy and live birth rates doubled following prophylactic salpingectomy. Laparoscopic salpingectomy is now recommended in all women with hydrosalpinx before IVF treatment, certainly following RIF.

20 Recurrent IVF failure: other factors
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

21 Recurrent IVF failure: other factors IMPACT OF OBESITY
Figure 1 Recurrent IVF failure: other factors IMPACT OF OBESITY Implantation, pregnancy, and live birth rates in IVF-ICSI cycles according to the women's BMI. Each point represents percentages and 95% CI. Penzias, Fertility and Sterility 2012; 97: OI: /j.fertnstert )

22 Smoking  Associated with an increased gonadotrophin requirement for ovarian stimulation, fewer oocytes retrieved, higher numbers of cancelled cycles, lower implantation rates and more cycles with failed fertilization in those undergoing IVF treatment (Sterzik et al., 1996, Van Voorhis et al., 1996). Male partners of women with RIF should also be advised to abstain from smoking due to its adverse effect on sperm counts and motility, increase in abnormal sperm morphology and sperm DNA damage (Potts et al., 1999).

23 Oocyte quality Suggested by:
poor response to ovarian stimulation (Ferraretti et al., 2011), with fewer numbers of oocytes retrieved, a high proportion of immature oocytes, reduced fertilization rate and low embryo utilization rate. Often associated with low antral follicle counts, high FSH and low anti-Müllerian hormone Age-related decline in oocyte quality is associated with increased chromosomal non-disjunction, resulting in aneuploid embryos, decrease in mitochondrial membrane potential and increase of mitochondrial DNA damage (Wang et al., 2009).

24 Abnormal Embryonic Development
Chromosomal abnormalities of the male or female partner, such as translocations, inversions, deletions Increased incidence of sperm chromosomal abnormalities in patients with normal karyotype and RIF was also observed (Rubio et al., 2001). The disruption of the normal sequence of chromosome replication and segregation in early human embryos might be a common cause for RIF. Increased zona thickness: Associated with lower implantation rates (Cohen et al., 1989). Zona hardening, which may be induced by in vitro culture or by in vivo ageing, can also affect hatching (De Vos and Van Steirteghem, 2000). Embryonic Stress: Several quality control methods have been suggested for identifying suboptimal components of a culture system (Gardner et al., 2005).

25 Assisted Hatching and Removal of Degenerate Material Significantly Improves Implantation of Frozen/Thawed Blastocysts Schlenker, Fertility and Sterility September 2005 , Number Ongoing PR Implantation Hatching 54 52 27 No Hatching 58 36 16 NS <.05

26 Hum Reprod Update. 2011;17:438 A recent meta-analysis of randomized control trials (five trials with 761 participants), assisted hatching was reported to be associated with a significant improvement in clinical pregnancy when performed in fresh embryos transferred to women with RIF (relative risk [RR] = 1.73

27 Human Reproduction & Chromosome Aneuploidy
Aneuploidy, the loss or gain of an entire chromosome, is the most common abnormality in human conceptions Chromosome aneuploidy is the leading cause of both spontaneous miscarriages and congenital birth defects Aneuploid embryos that are transferred will either fail to implant, result in pregnancy loss or an affected infant Trisomy 21 Fetus

28 Meeting the Requirements of the Embryo
Gardner (1998) Theriogenology, 49:

29 mM mM 0.32 Pyruvate 0.10 10.5 Lactate 5.87 0.5 Glucose 3.15
Gardner et al. (1996) Fertil. Steril., 65:

30 Changing Physiology of the Embryo During the Preimplantation Period
Glucose During the pre-implantation period, the embryo undergoes significant changes in its physiology, metabolism and genetic control. These changes are so dramatic that the starting point of development, the zygote, and the final stage, the blastocyst, can be likened to two very different somatic cell types. Pyruvate

31 Role of Amino Acids in Embryo Development
biosynthetic precursors energy substrates regulators of energy metabolism pHi buffers osmolytes antioxidants chelators

32 Role of Amino Acids in Embryo Development
biosynthetic precursors energy substrates regulators of energy metabolism pHi buffers osmolytes antioxidants chelators Amino acids minimize the stress within the embryo by facilitating cell function and maintaining homeostasis

33 Oxygen Atmospheric concentration is ~ 20%
Physiological concentration is ~ 5%

34 Clinical Data on the Effects of Oxygen
Meintjes M et al. (2009)A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Fertil Steril 24: 300-7 Nanassy L et al., (2009) Comparison of 5% and ambient oxygen during days 3-5 of in vitro culture of human embryos. Fertil Steril

35 Sensitivity of the mouse embryo assay (MEA) is significantly increased by in vitro maturation Paik, Schoolcraft, Krisher, Fertility and Sterility, September 2013 IVM embryos are more sensitive to culture media contaminants than zygotes. Use of an outbred strain further increases this sensitivity. Determination of cell number can improve one-cell MEA sensitivity. The IVM MEA provides a significantly more sensitive method of detecting toxins, thus preventing harmful materials from entering the human ART laboratory.

36 Other Etiologies of RIF
Karyotype abnormalities: 15.4% abnormal in patients with RIF1 Male factor Sperm DNA damage advanced paternal age Quality of embryo transfer 1Raziel, September 2002 Fertility and Sterility Vol. 78, Issue 3, Pages

37 Fertility and Sterility
Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization Emre Seli, M.D., David K Gardner, Ph.D., William B Schoolcraft, M.D., Odette Moffatt, Ph.D. and Denny Sakkas, Ph.D. Fertility and Sterility (August 2004) A prospective comparative study. Setting A university IVF clinic and a private IVF clinic. Patient(s) Men (n = 49) undergoing infertility treatment with IVF. Intervention(s) After density gradient centrifugation preparation, part of the sperm sample was used for infertility treatment, and the rest was fixed in paraformaldehyde. Strand breaks in DNA that are indicative of apoptosis were detected by the in situ DNA nick end labeling (TUNEL) technique. A total of 15,000 spermatozoa from each sample were evaluated for TUNEL reactivity by flow cytometry. Main outcome measure(s) Percentage of ejaculated spermatozoa with DNA strand breaks indicative of apoptosis, blastocyst development rate, and pregnancy rate. Result(s) Blastocyst development showed a significant negative correlation with percentage TUNEL positivity in spermatozoa. When 20% was used as a cutoff for TUNEL positivity in sperm samples, the percentage of blastocyst development was 50% higher in the <20% TUNEL-positivity group (n = 27) compared with those with ≥20% TUNEL positivity (n = 22; 44.7% blastocyst development vs. 29.8%). Clinical pregnancy rates in these two groups were 52% vs. 44%, respectively. Conclusion(s) The extent of nuclear DNA fragmentation in prepared ejaculated spermatozoa used in IVF negatively correlates with blastocyst development. A larger series of patients needs to be assessed to determine whether this paternal effect on blastocyst development may also affect pregnancy outcome. Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

38 Correlation between percentage blastocyst development
and TUNEL positivity in the spermatozoa Correlation between percentage blastocyst development and TUNEL positivity in the spermatozoa. Spearman's ρ = −0.4 (P=.004). Seli. DNA damage in prepared ejaculated spermatozoa affects blastocyst development. Fertil Steril 2004. Seli, Fertil Steril 2004

39 Fertility and Sterility
Development to the blastocyst stage of patients assessed for low (<20%) and high (>20%) TUNEL positivity. Fertility and Sterility August 2004

40 Differential sperm RNA profiles are associated with subsequent blastocyst development Janesch, Fertility and Sterility, September 2010 The sperm nucleus contains diverse populations of RNA that are potentially transmitted to the oocyte at the time of fertilization. The functional role of these transcripts could include contribution to embryogenesis and/or transcriptional gene silencing. Infertile couples (n=16) undergoing IVF using donor oocytes (female factor standardized), donated normozoospermic samples with consent. Cycles were divided into 2 groups relative to blastocyst quality: Group A (Good) = ≥25% of D5 blastocysts ≥Grade 3BB, and Group B (Poor) = <15% of D5 blastocysts ≥Grade 3BB. Total RNA was isolated from about 1 million sperm (ICSI preparation, Group A=8 and Group B=8) and reverse transcribed for quantitative real-time PCR. Three genes, AKAP4, CLU and HSBP, exhibited significantly decreased expression in Group B sperm samples compared with Group A (P<0.05). Both AKAP4 and CLU participate in biological processes related to development. Following D5 blastocyst transfer, implantation rates indicated a trend towards greater competence of Group A blastocysts (A=66.7% v. B=43.8%, ns). CONCLUSION: Differential sperm RNA profiles from donor oocyte IVF cycles reflected subsequent D5 blastocyst quality. In particular, genes related to development showed a decrease in expression in association with poor blastocyst development and competence. Further studies are required to determine if these sperm transcripts indeed play a functional role during embryogenesis.

41 ET Technique ET technique is critical to a successful pregnancy outcome. The avoidance of blood, mucus, bacterial contamination, trauma to the endometrium, touching the fundus, and excessive uterine contractions are all associated with better PRs and implantation rates. Utilization of a trial transfer, full bladder, ultrasonographic guidance, and use of soft catheters, all appear to facilitate a successful ET

42 New Methods of Embryo Assessment
time-lapse observations using an incubator with an integrated optical microscope may minimize the changes in the culturing environment by integrating the culture, observation, and time-lapse recording of cells into one system. Metabolomic analysis of follicular fluid (FF) can provide valuable information about individual oocyte maturation and developmental potential. Measurement of oxygen, pyruvate, and glucose consumption by the embryo in the culture medium has been correlated with viability. Amino acid turnover, which appears to be correlated to blastocyst development.

43 Management options Lifestyle modification: BMI, smoking, alchohol
Review stimulation: Dose of gonadotrophin may be increased or decreased. There is no firm evidence that antagonist protocol is better than agonist protocol or vice versa. For poor responders to FSH stimulation in down-regulated cycles may benefit from the addition of LH (Surrey and Schoolcraft, 2000). Evidence also points to a possible benefit from the addition of LH to the cycles of women older than 35years of age (Balasch et al., 2001, Marrs et al., 2004, Phelps et al., 1999). In women with endometriosis and adenomyosis, the use of GnRH agonists for a few months prior to IVF or ICSI may increase the pregnancy rate (Sallam, Surrey)

44 Management options Sperm DNA fragmentation
Medical treatment- oral antioxidant treatment has been shown to reduce the incidence of sperm DNA fragmentation (Greco et al., 2005b). Select spermatozoa with low levels of DNA damage (Sakkas and Alvarez, 2010). use of annexin-V columns which has been shown to significantly reduce the percentage of spermatozoa with DNA fragmentation as measured by the TUNEL test sperm selection with hyaluronic acid binding (Jakab et al., 2005, Said et al.). Intracytoplasmic morphologically selected sperm injection (IMSI) utilizes spermatozoa selected under high-power magnification with a defined set of morphological criteria. A recent meta-analysis comparing ICSI and IMSI outcome demonstrated a statistically significant improvement in implantation and pregnancy rates and a significant decrease in miscarriage rates with use of IMSI (Souza Setti et al., 2010) It has been suggested that men with high levels of DNA damage in ejaculated spermatozoa have spermatozoa removed surgically from the testis for ICSI (Greco et al., 2005a). The use of testicular spermatozoa in couples with repeated implantation failure associated with high sperm DNA fragmentation in semen has been reported to result in a significant increase in pregnancy rate (Weissman et al., 2008) and reduction of miscarriage rate (Borini et al., 2006)

45 Management options Optimal culture media-Blastocyst transfer
Zona hardening-Assisted hatching Screen for Chromosomal abnormalities: CCS Assessment of embryo quality and viability-Time-lapse imaging, Metabolomics,Proteomics Improving ET technique Fertility and Sterility Volume 97, Issue 5 , Pages , May 2012

46 Co-culture The suggested beneficial effects of the co-culture include the secretion of embryotrophic factors such as nutrients, growth factors and cytokines and detoxifying of free radicals and potentially harmful substances (Simon et al., 1999). The most promising co-culture method seems to be homologous endometrial cells (Jayot et al., 1995). Using this method, Spandorfer et al. (2004) reported 49% PR in 1030 patients with RIF.

47 Blastocyst transfer Transfer of embryos at the blastocyst stage is a more physiological approach because the human embryos enter the endometrial cavity only 5 days after fertilization, at the morula-blastocyst stage. Culturing the embryos to the blastocyst stage evaluates embryos post embryonic genome activation. Two large RCTs have shown that blastocyst transfer after RIF following day 2–3 transfer carried significantly higher implantation and live birth rates (Guerif et al., 2004; Levitas et al., 2004). Improved embryo selection and uterine receptivity may explain the benefit of embryo transfer at the blastocyst stage for couples with RIF.

48 Prospective Randomized Trial of 1 vs 2 Blastocyst Transfer
90 80 70 60 1 Bc, n=23 50 (%) 2 Bc, n=25 40 Gardner et al. (2004) Fertil Steril 81:551-5. 30 20 10 Implantation OPR Twins Gardner, F&S, 2004

49 * * ** Single Cleavage Stage vs Single Blastocyst Transfer
Papanikolaou et al. (2006) N Engl J Med, 354: 50 * * ** 40 Day 3 30 Day 5 20 2 monozygotic twins on day 3, No monozygotics were seen following day 5 transfer 10 Implantation Pregnant Delivery women were <36 years olds

50 Clinical pregnancy rate per randomized couple.
The probability of clinical pregnancy was significantly higher in patients with a blastocyst-stage embryo transfer compared with patients in which a cleavage-stage embryo transfer was performed (OR: 1.27, 95% CI: 1.03–1.55; P = 0.02; heterogeneity: P = 0.22; fixed effects model) (Fig. 4) Papanikolaou E G et al. Hum. Reprod. 2008;23:91-99 © The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please

51 CCRM:Vitrification versus Slow Freeze
Vitrification, BC FET D5, Slow Freeze, FET # Cycles n=441 n=272 % Blastocysts Survival 98.3% 83% (P<0.05) # Blastocysts Transferred 1.9 2.2 Clinical Pregnancy (fht) 71.9% 57% Implantation Rate (fht) 54.6% 35%

52 Fresh vs Frozen SET Shapiro, Fertility & Sterility, February 2013;99,2; 389-392,
These findings show for the first time that a day 6 blastocyst transferred in a freeze-thaw cycle is more likely to result in pregnancy and ongoing pregnancy than a similar-quality day 6 blastocyst transferred in a fresh autologous cycle. This clinical evidence suggests that ovarian stimulation inhibits embryonic implantation consistent with impaired endometrial receptivity. It also suggests that the impairment is primarily through embryo-endometrium asynchrony, with little or no impairment of implantation of rapidly developing (day 5) blastocysts but substantial impairment of slower (day 6) blastocysts.

53 Matched-cohort comparison of single-embryo transfers in fresh and frozen-thawed embryo transfer cycles Comparison of rates of ongoing pregnancy following fresh (red) and freeze-thaw (blue) transfers of day 5 or day 6 blastocysts. Shapiro, Fertility and Sterility 2013; 99:

54 FET Results in Better Outcomes and Healthier Babies
Roque et al, 2012 Meta-analysis revealed significantly higher clinical pregnancy rates following FET versus fresh transfer Pinborg et al, 2010 Singletons from FETs have significantly better neonatal outcome than offspring from fresh transfers Henningsen et al, 2011 Birth weight was significantly higher in siblings born after FETs compared with fresh embryos

55 CCS

56 Frozen Embryo Transfer
Unexplained RIF Group (n=130) ≥3 consecutive IVF failures Unexplained Repeated Miscarriage (RM) (n=77) ≥3 consecutive pregnancy losses All embryos are grown to the blastocyst stage for trophectoderm biopsy CCS using qPCR (RMA-NJ) Frozen Embryo Transfer Euploid embryos only RIF and RM patients had no other infertility indications

57 Ovarian Reserve and Blastocyst Development
There were no significant differences between patient groups for maternal age, D3 FSH, AMH, AFC, blastocyst development, number of blastocysts biopsied, blastocyst survival post vitrification, or number of euploid blastocysts transferred. However, the proportion of euploid blastocysts was significantly greater for RIF compared with RM patients (58.1% vs. 43.6%; P<0.0001). Transfer of identified euploid blastocysts also resulted in significant differences in IVF outcome between the two groups: Implantation with fetal cardiac activity (RIF=37.6% vs. RM=76.4%; P<0.0001) Clinical pregnancy (RIF=42.1% vs. RM=84.5%; P<0.0001) and live birth (RIF=36.0% vs. RM=82.8%; P<0.0001).  No significant differences between the groups

58 Patients in the RM group are 1.35 times more
likely to have an aneuploid blastocyst * 58.1% * 56.4% 43.6% 41.9% This was significant on the individual biopsy days as well = 1.26x on D5 and 1.42x on D6. *P<0.0001

59 More likely to be aneuploid in RM group;
Individual Chromosome Aneuploidy – There were highly significant differences in the distribution of aneuploidy on individual chromosomes for chromosomes 4 (P-Value ), 10 (P-Value ), 19 (P-Value ), 22 (P-Value ), and X (P-Value ). All of these chromosomes were more likely to be aneuploid in the RM group with these likelihoods: Chromosome 4 – 4.46x more likely in RM Chromosome 10 – 2.87x more likely in RM Chromosome 19 – 1.7x more likely in RM Chromosome 22 – 1.52x more likely in RM Chromosome X – 3.2x more likely in RM More likely to be aneuploid in RM group; * P < 0.05; *** P < 0.01

60 No significant difference in blastocyst development, blastocyst quality or embryo gender between the RIF and RM groups

61 IVF BC-CCS Cycle Outcome
*P<0.05; **P<0.001 ** ** ** * * There were no significant differences between patient groups for maternal age, D3 FSH, AMH, AFC, blastocyst development, number of blastocysts biopsied, blastocyst survival post vitrification, or number of euploid blastocysts transferred. However, the proportion of euploid blastocysts was significantly greater for RIF compared with RM patients (58.1% vs. 43.6%; P<0.0001). Transfer of identified euploid blastocysts also resulted in significant differences in IVF outcome between the two groups: Implantation with fetal cardiac activity (RIF=37.6% vs. RM=76.4%; P<0.0001) Clinical pregnancy (RIF=42.1% vs. RM=84.5%; P<0.0001) and live birth (RIF=36.0% vs. RM=82.8%; P<0.0001). 

62 Conclusion: Overall, RIF patients did experience some benefit from the transfer of a euploid blastocyst but not as significant as was observed for RM patients of equivalent maternal age. Even though embryo euploidy is essential for healthy fetal development, other factors including flaws in endometrial receptivity, embryonic function, and embryo-endometrium dialogue should be further investigated in unexplained RIF. RIF patients defined as implantation failure in ≥3 consecutive IVF cycles (n=130) and unexplained recurrent miscarriage (RM) patients defined as ≥3 consecutive clinical pregnancy losses (n=77; control group), underwent biopsy at the blastocyst stage for chromosome numeration using either quantitative PCR or SNP microarray, prior to vitrification. Euploid blastocysts were warmed and transferred in a subsequent FET.   There were no significant differences between patient groups for maternal age, D3 FSH, AMH, AFC, blastocyst development, number of blastocysts biopsied, blastocyst survival post vitrification, or number of euploid blastocysts transferred. However, the proportion of euploid blastocysts was significantly greater for RIF compared with RM patients (58.1% vs. 43.6%; P<0.0001). Transfer of identified euploid blastocysts also resulted in significant differences in IVF outcome between the two groups: Implantation with fetal cardiac activity (RIF=37.6% vs. RM=76.4%; P<0.0001) Clinical pregnancy (RIF=42.1% vs. RM=84.5%; P<0.0001) and live birth (RIF=36.0% vs. RM=82.8%; P<0.0001).  Overall, RIF patients did experience some benefit from the transfer of a euploid blastocyst but not as significant as was observed for RM patients of equivalent maternal age. Even though embryo euploidy is essential for healthy fetal development, other factors including flaws in endometrial receptivity, embryonic function, and embryo-endometrium dialogue should also be considered in future investigation.

63 RCT of CCS vs Blastocyst transfer in women >35
CCRM RCT of CCS vs Blastocyst transfer in women >35

64 Frozen Embryo Transfer
Infertile patients of maternal age >35 years were computer randomized at oocyte retrieval into either: All embryos are grown to the blastocyst stage Surplus blastocysts biopsied for CCS prior to vitrification Day 5 Fresh Transfer Embryo selection based on morphology Control Group Frozen Embryo Transfer Euploid embryos only Blastocyst biopsy for CCS on either D5 or D6 (mean = 5.2) Test Group CCS using SNP microarray technology

65 100% Survival from Vitrification (n=74)
Post Warming Prior to Transfer

66 IVF Cycle and Transfer Outcome
% * * Mean paternal age = 40.3 years * Fishers Exact Test; *Significance = P<0.05

67 Lipocalin-1: a potential marker for noninvasive aneuploidy screening
Concentrations (mean ± SEM) of lipocalin-1 in spent culture media were measured by ELISA and statistically analyzed by a Student t-test. Control media (n = 5); combined microdrops (×3) from euploid (n = 12) and aneuploid (n = 12) blastocysts (∗∗P<.01); individual microdrops from euploid (n = 4) and aneuploid (n = 4) blastocyst (∗P<.05). Gray bar, Control media; blue bars, euploid spent culture media; red bars, aneuploid spent culture media. Source: Fertility and Sterility 2011; 95: (DOI: /j.fertnstert ) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

68 Forest plot of studies of clinical touch embyro transfer (CTET) versus ultrasound-guided embryo transfer (UGET) for outcome of clinical pregnancy rate. Forest plot of studies of clinical touch embyro transfer (CTET) versus ultrasound-guided embryo transfer (UGET) for outcome of clinical pregnancy rate. Fertility and Sterility 2012; 97:

69 Improving endometrial receptivity
Hysteroscopic correction of cavity pathology(Demirol and Gurgan, 2004) Patients with RIF who had a normal hysterosalpingogram were prospectively randomized into office hysteroscopic evaluation (n = 210) or nothing (n = 211). Patients who had abnormal hysteroscopic findings (n = 56) were operated on during the procedure. Clinical PR was significantly higher in the treatment group (30.4% following normal hysteroscopy and 32.5% following hysteroscopic operation) compared to that in the controls (21.6%). Hence, treatment of intrauterine pathologies found by hysteroscopic evaluation improved the pregnancy outcome. Myomectomy The favourable PRs obtained after myomectomy lead many clinicians to believe that removal of myomas increases pregnancy and live-birth rates (review Donnez and Jadoul, 2002). However, no appropriate prospective studies have been performed. No information on the value of myomectomy in RIF is available, although most clinicians recommend hysteroscopic removal of submucous fibroids distorting the uterine cavity.

70 Treatment of thin endometrium
Low-dose aspirin (Weckstein et al., 1997) and vaginal sildenafil (Sher and Fisch, 2002) were suggested in cases of RIF with thin endometrium. High-dose estrogens. Vaginal administration of micronized estradiol to maximize estrogenic effect (Tourgeman et al., 2001) Antifibrotic treatment with pentoxifylline and high-dose vitamin E (Ledee-Bataille et al., 2002) has been shown to increase PR in cases with a thin endometrium. Endometrial stimulation Barash et al. (2003) performed repeated endometrial biopsies in 45 cases. Pregnancy and live birth rates in the IVF cycle following the biopsy were doubled. They concluded that local injury to the endometrium increased the incidence of implantation. There is a need for a prospective controlled study to prove the value of this procedure.

71 in patients undergoing in vitro fertilization
Local injury to the endometrium doubles the incidence of successful pregnancies in patients undergoing in vitro fertilization Outcome of IVF-ET treatments in the control and biopsy-treated patients. Implantation rate was calculated as the ratio between the number of embryonal sacs diagnosed by sonography and the total number of embryos transferred into the uterus (P = ). The rate of clinical pregnancy is expressed as the ratio between the number of patients in whom clinical pregnancy was diagnosed and the total number of patients that underwent embryo transfer (P = ). The rate of live births is expressed as the ratio between the number of births and the total number of patients that underwent embryo transfer (P = .016). Barash. Endometrial injury increases its receptivity.Fertil Steril 2003. Barasch, Fertility and Sterility (June 2003) Copyright © 2003 American Society for Reproductive Medicine Terms and Conditions

72 Local endometrial injury and IVF outcome: a systematic review and meta- analysis
Tarek El-Toukhy, SeshKamal Sunkara and Yakoub Khalaf Reproductive BioMedicine Online (October 2012) Summary of the live birth/ongoing pregnancy rate for the three studies which included patients with prior IVF failure. Copyright © Terms and Conditions

73 Local injury by endometrial biopsy promotes an inflammatory response.
Local injury of the endometrium induces an inflammatory response that promotes successful implantation Gnainsky, Fertility and Sterility Volume 94, Issue 6 , Pages , November 2010 Local injury by endometrial biopsy promotes an inflammatory response. Proinflammatory cytokines such as TNF-α, produced by the wounded endometrium, stimulate the secretion of other chemokines/cytokines which, in turn, recruit macrophages/DCs to the site of implantation. These immune cells enhance the inflammatory reaction and may trigger the uterine epithelium to produce molecules that interact with the blastocyst, facilitating its apposition and attachment to the uterine wall.

74 reasons for exclusion of women from analysis.
Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF Flow-chart outlining the Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF reasons for exclusion of women from analysis. Flow-chart outlining the reasons for exclusion of women from analysis. Boomsma C et al. Hum. Reprod. 2009;24: © The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please

75 Receiver operating characteristic curve showing the area under the curves (AUC) to predict pregnancy by the concentration of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and embryo quality (top quality embryo vs. suboptimal quality embryo). Receiver operating characteristic curve showing the area under the curves (AUC) to predict pregnancy by the concentration of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and embryo quality (top quality embryo vs. suboptimal quality embryo). Boomsma C et al. Hum. Reprod. 2009;24: © The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please

76 Immunotherapy IVIF Heparin and aspirin
Stephenson and Fluker (2000) in a double-blind, placebo-RCT including 51 couples with RIF found that IVIG did not improve the live birth rate. Thus, the effectiveness of IVIG treatment in RIF is still unresolved. Heparin and aspirin Two large RCTs indicated that heparin and aspirin did not improve pregnancy or implantation rates in RIF (Urman et al., 2000), even for autoantibody-positive patients (Stern et al., 2003). Similarly, immunotherapy using partner’s leukocytes was not shown to affect RIF (Carp et al., 1994).

77 Seshadri, Reproductive BioMedicine Online(December 2012)
Effect of heparin on the outcome of IVF treatment: a systematic review and meta-analysis Seshadri, Reproductive BioMedicine Online(December 2012) The effect of heparin versus placebo on the implantation rate in women with recurrent implantation failure without coagulation defects Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

78 Treating endometriosis
The administration of GnRH agonists for 3–6 months before ART in women with endometriosis significantly increases the ongoing PR (Surrey et al., 2002). No deleterious effect on ovarian response was observed. A recent meta-analysis of three RCTs indicated that this treatment increased the odds of clinical pregnancy by (Sallam et al., 2006). Most investigators agree that there is no benefit in the removal of endometriomas before IVF (Garcia-Velasco et al., 2004; Wong et al., 2004). Furthermore, surgery might be deleterious for ovarian reserve.

79 Outcome of highly purified menotropin (HP-hMG) vs recombinant follicle-stimulating hormone (rFSH) in high responders Arce, Gynecol Endocrinol, Early Online: 1–

80 Recommendations: Review prior cycles
Optimize stimulation Optimize embryology: Day 5, AHA, CCS, FET R/O hydrosalpinx, Uterine pathology, abnormal karyotype, DNA fragmentation If endometriosis or adenomyosis-Lupron or Letrozole If history of difficult transfer-laminaria

81 Recommendations: If high rate of CCS normals, consider GC
If all abnormal on CCS, egg donation If male factor severe: age>60, high DNA fragmentation, NOA, with good day 3 embryos but poor blastocyst development, consider Sperm donation

82 THANK YOU


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