(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).
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.
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. 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
The impact of intramural leiomyomata on IVF outcome Surrey, Fertility and Sterility, April 2000 Intramural myomaNo myoma Age Pregnancy Rate5565 Implantation2337* * <.05
Aa Forest plot of studies of non-cavity-distorting intramural fibroids versus no fibroids in women undergoing IVF treatment for outcome of live birth rates.
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).
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.
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. Labarta E et al. Hum. Reprod. 2011;26:
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.
Venn diagram of transcripts up-regulated and down-regulated during endometrial receptivity in the natural cycle compared with the stimulated cycle. Haouzi D et al. Hum. Reprod. 2009;24:
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.
Ultrasound diagnosed adenomyosis has a negative impact on successful implantation following GnRH antagonist IVF treatment Thslluri, Hum. Reprod. (2012) 27 (12):
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.
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.
Figure 1 Penzias, Fertility and Sterility 2012; 97: Penzias, Fertility and Sterility 2012; 97: OI : /j.fertnstert ) Recurrent IVF failure: other factors IMPACT OF OBESITY
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).
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).
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).
Assisted Hatching and Removal of Degenerate Material Significantly Improves Implantation of Frozen/Thawed Blastocysts Schlenker, Fertility and Sterility September 2005, NumberOngoing PRImplantation Hatching No Hatching NS<.05
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
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
Meeting the Requirements of the Embryo Gardner (1998) Theriogenology, 49:
mM 0.32Pyruvate Lactate Glucose3.15 Gardner et al. (1996) Fertil. Steril., 65:
Changing Physiology of the Embryo During the Preimplantation Period Pyruvate Glucose
Role of Amino Acids in Embryo Development biosynthetic precursors energy substrates regulators of energy metabolism pHi buffers osmolytes antioxidants chelators
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
Oxygen Atmospheric concentration is ~ 20% Physiological concentration is ~ 5%
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: Nanassy L et al., (2009) Comparison of 5% and ambient oxygen during days 3-5 of in vitro culture of human embryos. Fertil Steril
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.
Other Etiologies of RIF Karyotype abnormalities: 15.4% abnormal in patients with RIF 1 Male factor Sperm DNA damage advanced paternal age Quality of embryo transfer 1 Raziel, September 2002Fertility and Sterility Vol. 78, Issue 3, Pages
Correlation between percentage blastocyst development and TUNEL positivity in the spermatozoa Seli, Fertil Steril 2004
Development to the blastocyst stage of patients assessed for low ( 20%) TUNEL positivity. Fertility and Sterility August 2004
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.
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
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.
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)
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)
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
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.
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.
Gardner et al. (2004) Fertil Steril 81: ImplantationOPRTwins (%) 1 Bc, n=23 2 Bc, n=25 Prospective Randomized Trial of 1 vs 2 Blastocyst Transfer Gardner, F&S, 2004
Single Cleavage Stage vs Single Blastocyst Transfer Papanikolaou et al. (2006) N Engl J Med, 354: Day 3 Day 5 2 monozygotic twins on day 3, No monozygotics were seen following day 5 transfer women were <36 years olds ImplantationPregnantDelivery * * **
Vitrification, BC FETD5, Slow Freeze, FET # Cyclesn=441n=272 % Blastocysts Survival98.3%83% (P<0.05) # Blastocysts Transferred Clinical Pregnancy (fht)71.9%57% (P<0.05) Implantation Rate (fht)54.6%35% (P<0.05) CCRM:Vitrification versus Slow Freeze
Fresh vs Frozen SET Shapiro, Fertility & Sterility, February 2013;99,2; ,
Shapiro, Fertility and Sterility 2013; 99: Matched-cohort comparison of single-embryo transfers in fresh and frozen-thawed embryo transfer cycles
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 FET Results in Better Outcomes and Healthier Babies
Unexplained RIF Group (n=130) ≥3 consecutive IVF failures Unexplained Repeated Miscarriage (RM) (n=77) ≥3 consecutive pregnancy losses Frozen Embryo Transfer Euploid embryos only All embryos are grown to the blastocyst stage for trophectoderm biopsy CCS using qPCR (RMA-NJ) RIF and RM patients had no other infertility indications
Ovarian Reserve and Blastocyst Development No significant differences between the groups
*P< * * Patients in the RM group are 1.35 times more likely to have an aneuploid blastocyst 43.6% 56.4% 58.1% 41.9%
More likely to be aneuploid in RM group; * P < 0.05; *** P < 0.01
No significant difference in blastocyst development, blastocyst quality or embryo gender between the RIF and RM groups
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.
CCRM RCT of CCS vs Blastocyst transfer in women >35
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 Infertile patients of maternal age >35 years were computer randomized at oocyte retrieval into either: 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
100% Survival from Vitrification (n=74) Prior to Transfer Post Warming
IVF Cycle and Transfer Outcome Fishers Exact Test; *Significance = P<0.05 * * * %
Fertility and Sterility 2012; 97: Forest plot of studies of clinical touch embyro transfer (CTET) versus ultrasound-guided embryo transfer (UGET) for outcome of clinical pregnancy rate.
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.
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.
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.
Immunotherapy IVIF 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).
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.
Outcome of highly purified menotropin (HP-hMG) vs recombinant follicle-stimulating hormone (rFSH) in high responders Arce, Gynecol Endocrinol, Early Online: 1–7 2014
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
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